CSIR Unit 4 - Full Notes

UNIT 4: CELL COMMUNICATION AND CELL SIGNALING
A. Host parasite interaction:
Bacteria are consistently associated with the body The normal flora is acquired rapidly during and shortly
surfaces of animals. There are many more bacterial cells after birth and changes continuously throughout life
on the surface of a human (including the gastrointestinal
tract) than there are human cells that make up the animal. The organisms present at any given time reflect the age,
The bacteria and other microbes that are consistently nutrition and environment of the individual. It is
associated with an animal are called the normal flora, or therefore difficult to define the normal flora very
more properly the "indigenous microbiota", of the precisely because it is to a large extent environmentally
animal. These bacteria have a full range of symbiotic determined. This is well illustrated by data from NASA
interactions with their animal hosts. astronauts who were rendered relatively bacteriologically
sterile by antibiotic treatment before their space flights. It
Why is it called the normal flora? took only 6 weeks after the flight for their flora to
repopulate, and the repopulating species were precisely
The term flora is used because the majority of the those of their immediate neighbors. The bowel flora of
organisms concerned are bacteria. It has been estimated children in developing countries is quite different from
that humans have approximately 1013 cells in the body that of children in developed countries. In addition,
and something like 1014 bacteria associated with them, breastfed infants have lactic acid streptococci and
the majority in the large bowel. Members of groups such lactobacilli in their gastrointestinal tract, whereas
as viruses, fungi and protozoa are also regularly found in bottlefed children show a much greater variety of
healthy individuals, but form only a minor component of organisms.
the total population of resident organisms.
Different regions of the skin support different flora
The organisms occur in those parts of the body that are
exposed to, or communicate with, the external Exposed dry areas have relatively few resident organisms
environment, namely the skin, nose and mouth, and on the surface, whereas moister areas (axillae, perineum,
intestinal and urinogenital tracts. Internal organs and between the toes, scalp) support much larger populations.
tissues are normally sterile. The main organisms found in Staphylococcus epidermidis is one of the commonest
these sites are shown in Figure 1. species, making up some 90% of the aerobes and
occurring in densities of 103-104/cm2; S. aureus may be
present in the moister regions.
Anaerobic diphtheroids occur below the skin surface in

hair follicles, sweat and sebaceous glands,
Propionibacterium acnes being a familiar example.
Changes in the skin occurring during puberty often lead
to increased numbers of this species, which can be
associated with acne.
A number of fungi, including Candida, occur on the scalp

and around the nails. They are infrequent on dry skin, but
can cause infection in moist skin folds (intertrigo).
Both the nose and mouth can be heavily colonized by

bacteria
The majority of bacteria here are anaerobes. Common

species colonizing these areas include streptococci,
staphylococci, diphtheroids and Gram-negative cocci.
Some of the aerobic bacteria found in healthy individuals
are potentially pathogenic (e.g. Staph. aureus,
Streptococcus pneumoniae, Strep. pyogenes, Neisseria
meningitidis); Candida is also a potential pathogen.
The mucous membranes of the mouth can have the same

microbial density as the large intestine, numbers
approaching 1011/g wet weight of tissue.
Dental caries is one of the commonest infectious

diseases in developed countries
The surfaces of the teeth and the gingival crevices carry

large numbers of anaerobic bacteria. Plaque is a film of
bacterial cells anchored in a polysaccharide matrix,
which the organisms secrete. When teeth are not cleaned
regularly, plaque can accumulate rapidly and the
INSTITUTE FOR ADVANCED STUDIES, JODHPUR 1

activities of certain bacteria, notably Streptococcus The vast majority (95-99%) are anaerobes, Bacteroides
mutans, can lead to dental decay (caries), as acid being especially common and a major component of
fermented from carbohydrates can attack dental enamel. fecal material. Harmless protozoans can also occur in the
The prevalence of dental decay is linked to diet. intestine (e.g. Entamoeba coli) and these can be
considered as part of the normal flora, despite being
The pharynx and trachea carry their own normal flora animals.
The flora of the pharynx and trachea may include both α- The urethra is lightly colonized in both sexes, but the
and β-hemolytic streptococci as well as a number of vagina supports an extensive flora of bacteria and fungi
anaerobes, staphylococci (including Staph. aureus),
Neisseria and diphtheroids. The respiratory tract is The urethra in both sexes is relatively lightly colonized,
normally quite sterile, despite the regular intake of although Staph. epidermidis, Strep. faecalis and
organisms by breathing. However substantial numbers of diphtheroids may be present. In the vagina the
clinically normal people may carry the fungus composition of the bacterial and fungal flora undergoes
Pneumocystis jiroveci (previously known as P. carinii) in age-related changes: Before puberty the predominant
their lungs. organisms are staphylococci, streptococci, diphtheroids
and Escherichia coli.
In the gut the density of microorganisms increases from
the stomach to the large intestine Subsequently, Lactobacillus aerophilus predominates, its
fermentation of glycogen being responsible for the
Stomach contents harbor only transient organisms, the maintenance of an acid pH, which prevents overgrowth
acidic pH providing an effective barrier. However, the by other vaginal organisms.
gastric mucosa may be colonized by acid-tolerant
lactobacilli and streptococci. The upper intestine is only A number of fungi occur, including Candida, which can
lightly colonized (104 organisms/g), but populations overgrow to cause the pathogenic condition 'thrush' if the
increase markedly in the ileum, where streptococci, vaginal pH rises and competing bacteria diminish. The
lactobacilli, enterobacteriaceae and Bacteroides may all protozoan Trichomonas vaginalis may also be present in
be present. Bacterial numbers are very high (estimated at healthy individuals.
1011/g) in the large bowel, and many species can be
found (Fig. 2). Advantages and disadvantages of the normal flora
Some of the species of the normal flora are positively

beneficial to the host
The importance of these species for health is sometimes

revealed quite dramatically under stringent antibiotic
therapy. This can drastically reduce their numbers to a
minimum, and the host may then be overrun by
introduced pathogens or by overgrowth of organisms
normally present in small numbers.
After treatment with clindamycin, overgrowth by

Clostridium difficile, which survives treatment, can give
rise to antibiotic-associated diarrhea or, more seriously,
pseudomembranous colitis.
Ways in which the normal flora prevents colonization by

potential pathogens include the following.
Skin bacteria produce fatty acids, which
discourage other species from invading.
Gut bacteria release a number of factors with
antibacterial activity (bacteriocins, colicins) as
well as metabolic waste products that help
prevent the establishment of other species.
Vaginal lactobacilli maintain an acid
environment, which suppresses growth of other
organisms.
The sheer number of bacteria present in the
normal flora of the intestine means that almost
all of the available ecologic niches become
occupied; these species therefore outcompete
others for living space.
Gut bacteria also release organic acids, which may have

some metabolic value to the host; they also produce B
vitamins and vitamin K in amounts that are large enough
to be valuable if the diet is deficient. The antigenic
2 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

stimulation provided by the intestinal flora helps to 'symbiosis' (literally 'living together'). Symbiosis has no
ensure the normal development of the immune system. overtones of benefit or harm and includes a wide
diversity of associations. Attempts have been made to
What happens when the normal flora is absent? categorize types of association very specifically, but
these have failed because all associations form part of a
Germ-free animals tend to live longer, presumably continuum (Fig.3). Three broad categories of symbiosis-
because of the complete absence of pathogens, and commensalism, mutualism and parasitism-can be
develop no caries. However, their immune system is less identified on the basis of the relative benefit obtained by
well developed and they are vulnerable to introduced each partner. None of these categories of association is
microbial pathogens. At the time of birth, humans are restricted to any particular taxonomic group. Indeed
germ free, but acquire the normal flora during and some organisms fit into each category depending upon
immediately after birth, with the accompaniment of the circumstances in which they live (Fig. 4).
intense immunologic activity.
The disadvantages of the normal flora lie in the potential

for spread into previously sterile parts of the body
This may happen:

when the intestine is perforated or the skin is
broken;
during extraction of teeth (when viridans
streptococci may enter the bloodstream);
when organisms from the perianal skin ascend
the urethra and cause urinary tract infection.
Members of the normal flora are important causes of
hospital-acquired infection when patients are exposed to
invasive treatments. Patients suffering burns are also at
risk.
Overgrowth by potentially pathogenic members of the

normal flora can occur when the composition of the flora
changes (e.g. after antibiotics) or when:
the local environment changes (e.g. increases
in stomach or vaginal pH);
the immune system becomes ineffective (e.g.
AIDS, clinical immunosuppression).
Under these conditions, the potential pathogens take the

opportunity to increase their population size or invade
tissues, so becoming harmful to the host.
SYMBIOTIC ASSOCIATIONS
All living animals are used as habitats by other

organisms; none is exempt from such invasion-even
protozoans have their own flora and fauna. As evolution
has produced larger, more complex and better regulated
bodies, it has increased the number and variety of
habitats for other organisms to colonize. The most
complex bodies, those of birds and mammals (including
humans), provide the most diverse environments, and are
the most heavily colonized.
As the normal flora demonstrates, pathogenesis is not the

inevitable consequence of host-microbe associations.
Many factors influence the outcome of a particular
association, and organisms may be pathogenic in one
situation but harmless in another. To understand the
microbiologic basis of infectious disease, host-microbe
associations that can be pathogenic need to be placed
firmly in the context of other symbiotic (interspecies)
associations, such as commensalism or mutualism, where
the outcome for the host does not normally involve any
damage or disadvantage.
All associations in which one species lives in or on the

body of another can be grouped under the general term

Commensalism: In commensalism one species of time. It may reflect selection of an increased level of
organism uses the body of a larger species genetically determined resistance in the host population
and decreased pathogenicity in the parasite (as has
At its simplest, a commensal association is one in which happened with myxomatosis in rabbits). Alternatively, it
one species of organism uses the body of a larger species may be the evolutionary norm, and 'unbalanced
as its physical environment and may make use of that pathogenicity' may simply be the consequence of
environment to acquire nutrients. organisms becoming established in 'unnatural' (i.e. new)
hosts. So, like the other categories of symbiosis,
Like all animals, humans support an extensive parasitism is impossible to define exclusively except in
commensal microbial flora on the skin, in the mouth and the context of clearcut and highly pathogenic organisms.
in the alimentary tract. The majority of these microbes The belief that 'harmfulness' is a necessary characteristic
are bacteria, and their relationship with the host may be of a parasite is difficult to sustain in any broader view,
highly specialized, with specific attachment mechanisms and the reasons for this are discussed in more detail
and precise environmental requirements. Normally such below.
microbes are harmless, but they can become harmful if
their environmental conditions change in some way (e.g. THE CHARACTERISTICS OF PARASITISM
Bacteroides, Escherichia coli, Staphylococcus aureus).
Conversely, commensal microbes can benefit the host: Many different groups of organisms are parasitic, and
by preventing colonization by more pathogenic all animals are parasitized
species (e.g. the intestinal flora);
by producing metabolites that are used by the Parasitism as a way of life has been adopted by many
host (e.g. the bacteria and protozoa in the different groups of organisms. Some groups, such as
ruminant stomach). viruses, are exclusively parasitic, but the majority include
both parasitic and free-living representatives. Parasites
It follows that the normal definition of commensalism is occur in all animals, from the simplest to the most
merely one of convenience, as the association can merge complex, and are an almost inevitable accompaniment of
into either mutualism or parasitism. organized animal existence. We can see, then, that
parasitism has been an evolutionary success; as a way of
Mutualism: Mutualistic relationships provide reciprocal life it must confer very considerable advantages.
benefits for the two organisms involved
Parasitism has metabolic, nutritional and reproductive
Frequently the relationship is obligatory for at least one advantages
member, and may be for both. Good examples are the
bacteria and protozoa living in the stomachs of domestic The most obvious advantage of parasitism is metabolic.
ruminants, which play an essential role in the digestion The parasite is provided with a variety of metabolic
and utilization of cellulose, receiving in return both the requirements by the host, at no energy cost to itself, so it
environment and the nutrition essential for their survival. can devote a large proportion of its own resources to
The dividing line between commensalism and mutualism replication or reproduction. This one-sided metabolic
can be hard to draw. In humans, good health and relationship shows a broad spectrum of dependence, both
resistance to colonization by pathogens can depend upon within and between the various groups of parasites.
the integrity of the normal commensal enteric bacteria, Some parasites are totally dependent upon the host, while
many of which are highly specialized for life in the others are only partly dependent.
human intestine, but there is certainly no strict mutual
dependence in this relationship. Viruses are completely dependent upon the host for all
their metabolic needs
Parasitism: In parasitism the symbiotic relationship
benefits only the parasite. Viruses are at one extreme of the 'parasite dependency'
spectrum. They are obligate parasites, possessing the
The terms 'parasites' and 'parasitism' are sometimes genetic information required for production of new
thought to apply only to protozoans and worms, but all viruses, but none of the cellular machinery necessary to
pathogens are parasites. Parasitism is a one-sided transcribe or translate this information, to assemble new
relationship in which the benefits go only to the parasite, virus particles or to produce the energy for these
the host providing parasites with their physicochemical processes. The host provides not only the basic building
environment, their food, respiratory and other metabolic blocks for the production of new viruses, but also the
needs, and even the signals that regulate their synthetic machinery and the energy required (Fig. 5).
development. Although parasites are thought of as Retroviruses go one stage further in dependence,
necessarily harmful, this is a view colored by human and inserting their own genetic information into the host's
veterinary clinical medicine, and by the results of DNA in order to parasitize the transcription process.
laboratory experimentation. In fact many 'parasites' Viruses therefore represent the ultimate parasitic
establish quite innocuous associations with their natural condition and are qualitatively different from all other
hosts and are not at all pathogenic under normal parasites in the nature of their relationship with the host.
circumstances (e.g. when their natural host is in good
health); the rabies virus, for example, coexists with many The basis for the fundamental difference between viruses
wild mammals but can cause fatal disease in humans. and other parasites is the difference between virus
This state of 'balanced pathogenicity' is sometimes organization and the cellular organization of prokaryotic
explained as the outcome of selective pressures acting and eukaryotic parasites. Non-viral parasites have their
upon a relationship over a long period of evolutionary own genetic and cellular machinery, and multi-enzyme

systems for independent metabolic activity and Parasite development can be controlled by the host
macromolecular synthesis. The degree of reliance on the
host for nutritional requirements varies considerably and The advantage that parasitism confers in reproductive
follows no consistent pattern between the various groups, terms makes it vital to coordinate parasite development
nor does it follow that smaller parasites tend to be more with the availability of suitable hosts. Indeed, one of the
dependent; for example, some of the largest parasites, the characteristic features of parasites is that their
tapeworms, are wholly reliant upon the host's digestive development may be controlled partly or completely by
machinery to provide their nutritional needs. All, of the host, the parasite having lost the ability to initiate or
course, receive nutrition from the host, but whereas some to regulate its own development. At its simplest, host
use macromolecular material (proteins, polysaccharides) control is limited to providing the cell surface molecules
of host origin and digest it using their own enzyme necessary for parasite attachment and internalization.
systems, others rely on the host for the process of Many parasites, from viruses to protozoa, rely on the
digestion as well, being able to take up only low recognition of such molecular signals for their entry into
molecular weight materials (amino acids, host cells, and this process provides the trigger for their
monosaccharides). Nutritional dependence may also replicative or reproductive cycles.
include host provision of growth factors that the parasite
is unable to synthesize itself. All internal parasites rely Other parasites, primarily the eukaryotes, require more
upon the host's respiratory and transport systems to comprehensive and sophisticated signals, often a
provide oxygen, although some respire anaerobically in complex of signals, to initiate and regulate their entire
either a facultative or obligate manner. developmental cycle. The complexity of the signal
required for development is one of the factors
determining the specificity of the host-parasite
relationship. Where the availability of one of the signals
entails that parasite development can occur in only one
species, host specificity is high. Where many host
species are capable of providing the necessary signals for
a parasite, specificity is low.
Disadvantages of parasitism
The most obvious disadvantage of parasitism arises from

the fact that the host controls the development of the
parasite. No development is possible without a suitable
host, and many parasites will die if no host becomes
available. For this reason, several adaptations have
evolved to promote prolonged survival in the outside
world and so maximize the chances of successful host
contact (e.g. virus particles, bacterial spores, protozoan
cysts and worm eggs). The prolific replication of
parasites is another device to achieve the same end.
Nevertheless, where parasites fail to make contact with a
host, their powers of survival are ultimately limited.
Adaptation to host signals can therefore have a
reproductive cost (i.e. the loss of many potential
parasites).
THE EVOLUTION OF PARASITISM
As so many organisms are parasitic and every group of

animals is subject to invasion by parasites, the
development of parasitism as a way of life must have
occurred at an early stage in evolution and at frequent
intervals thereafter. How this occurred is not fully
understood, and it may well have been different in
different groups of organisms. In many, parasitism most
probably arose as a consequence of accidental contacts
between organism and host. Of many such contacts some
would have resulted in prolonged survival, and under
favorable nutritional circumstances prolonged survival
would have been associated with enhanced replication,
giving the organism a selective advantage within the
environment.
Bacterial parasites evolved through accidental contact
In the case of bacteria, it is easy to see how accidental

contact in environments rich in free-living bacteria could

lead to successful colonization of the gastrointestinal Many bacterial parasites have evolved to live inside host
tract and external orifices. Initially the organisms cells
concerned would have had to be facultative parasites,
capable of life both within or outside host organisms Bacteria that became parasitic by accidental contact
(many pathogenic bacteria still have this property, e.g. would have lived outside host cells at first and would not
Legionella, Vibrio), but selective pressures would have have had the advantages of being intracellular. The
forced others into obligatory parasitism. Such events are evolution of the intracellular habit required further
of course speculative, but are supported by the close modifications to allow survival within host cells, but
relationship of enteric bacteria such as E. coli with free- could easily have been initiated by passive phagocytic
living photosynthetic purple bacteria. uptake. Subsequent survival of the microbe would
depend upon the possession of surface or metabolic
Many bacteria and related parasites of humans and other properties that prevented digestion and destruction by the
mammals may have originated via the route of accidental host cell. The success of intracellular life can be
contact, but it is clear that others have become adapted to measured not only by the large number of bacteria that
these hosts after initially becoming parasitic in other have adopted this habit, but also by the extent to which
species. Blood-feeding arthropods provide an example of some organisms have integrated their biology with that
the most obvious route for this, as their parasites have of the host cell. The endpoint of such integration is
ready access to the tissues of the animals on which the perhaps to be seen in the evolution of the eukaryote
arthropods feed. mitochondrion, which is widely considered to be the
product of symbiotically associated heterotrophic purple
bacteria (Fig. 6).
The pathway of virus evolution is uncertain process of secondary simplification, then parasitism must
have evolved long after the evolution of prokaryotes and
Clearly, parasitism by bacteria, which are undoubtedly eukaryotes. If viruses are primitively non-cellular then it
ancient organisms (they can be traced back 3-5 billion is possible that they became parasitic at a very early
years in the fossil record), depended upon the evolution stage in the evolution of cellular life, at some point when,
of higher organisms to act as hosts. Whether the same is because of environmental change, independent existence
true of viruses is open to question, and depends upon became impossible. A third alternative is that viruses
whether viruses are considered primarily or secondarily were never anything other than fragments of the nuclear
simple. If viruses evolved from cellular ancestors by a material of other organisms and have in effect always

been parasitic. Modern viruses may, in fact, have arisen of the continuing battle between host and parasite-the
by all three pathways. former attempting to contain or destroy, the latter
attempting to evade or suppress-and why the emergence
Eukaryote parasites have evolved through accidental of new, and the return of old, infectious diseases are a
contact constant threat.
The evolution of parasitism by eukaryotes is likely to Parasites are faced not only with the problems of
have arisen much as it may have done in prokaryotes (i.e. surviving within the environment they experience
through accidental contact and via blood-feeding initially, but also of surviving in that environment as it
arthropods). Examples can be found among both changes in ways that are likely to be harmful to them.
protozoan and worm parasites to support this view: The inflammatory and immune responses that follow the
There are protozoa such as the free-living ameba establishment of infection are the most important means
Naegleria, which can opportunistically invade the by which the host can control infections by those
human body and cause severe and sometimes fatal organisms able to penetrate its natural barriers and
disease. survive within its body. These responses represent
There are several species of nematode worms that formidable obstacles to the continued survival of
can live either as parasites or as free-living parasites, forcing them to evolve strategies to cope with
organisms, Strongyloides stercoralis being the most harmful changes in their environment. The successful
important in humans. parasite is therefore one that can cope with, or evade, the
It is likely that trypanosomes (the protozoans host's response in one of the ways shown in Figure 7.
responsible for sleeping sickness) were primarily
adapted as parasites of blood-feeding flies and only
secondarily became established as parasites of
mammals.
Parasite adaptations to overcome host inflammatory and

immune responses
We can view the evolution of parasitism and the

adaptations necessary for life within another animal as
being exactly analogous to the adaptations necessary for
life within any other specialized habitat: the environment
in which parasites live is merely one of the many to
which organisms have become adapted in evolution
(comparable with life in soil, freshwater, salt water,
decaying material and so on). However, it is always
necessary to remember that in one major respect
parasitism is quite different from any other specialist
mode of life. This difference is that the environment in
which a parasite lives, the body of the host, is not
passive; on the contrary it is capable of an active
response to the presence of the parasite.
The attractiveness of animal bodies as environments for
parasites means that hosts are under continual pressures
from infection, and these pressures are increased when
hosts live:
close together;
in insanitary conditions;
in climates that favor the survival of parasite
stages in the external world.
Pressure of infection has been a major influence in

evolution
Pressure of infection has been a major selective influence

in evolution, and there is little doubt that it has been
largely responsible for the development of the
sophisticated inflammatory and immune responses we
All of these adaptations are known to exist within
see in humans and other mammals. In evolutionary terms
different groups of parasites and they are well
all infection has its costs to the host because it diverts
documented in the case of some of the major human
valuable resources from the activities of survival and
pathogens. Indeed they are often the very reason why
reproduction; there has therefore been pressure to
such organisms are major pathogens. Nevertheless,
develop means of overcoming infection whether or not it
transmission and survival of many parasites depends
causes disease. Of course, this is not the focus of clinical
upon the existence of particularly susceptible host
microbiology, which legitimately places emphasis on the
individuals (e.g. children) to provide a continuing
costs of infection in terms of frank disease, but it should
reservoir of infective stages.
be remembered because it explains more fully the nature

Changes in parasites create new problems for hosts virus have permitted extensive infections in humans. Of a
different nature, but relevant to the general theme, is the
From what has been said above, it can be appreciated that acquisition of drug resistance in bacteria and protozoa
there is no such thing as a static host-parasite (Fig. 8).
relationship, and that concepts of unchanging
'pathogenicity' or 'harmlessness' cannot be justified. Each The activity of many antibiotics can be blocked by
relationship is an 'arms race', changes in one member bacterial enzymes coded for by genes located on
being countered by changes in the other. Quite subtle cytoplasmic DNA in plasmids. The ability of bacteria to
changes in either can completely change the balance of transfer plasmids between individual organisms means
the relationship, towards greater or lesser pathogenicity that strains or species previously susceptible to an
for example. antibiotic can acquire the ability to produce such
enzymes and so gain antibiotic resistance directly from
Perhaps the most important contemporary illustration of resistant organisms. These newly resistant forms are then
this situation is the dramatic and explosive appearance of differentially selected under antibiotic treatment, the
HIV infections. This group of viruses was originally susceptible individuals being deleted from the
restricted to non-human primates, but changes in the population.
Although the underlying genetic and metabolic changes changed the frequency of certain HLA antigens in areas
do not by themselves influence pathogenicity, the where infection was severe.
expression of such changes in the face of intense and
selective chemotherapy certainly does so. Social and behavioral changes can be as important as
genetic changes in altering host-parasite relations
Host adaptations to overcome changes in parasites
Social and behavioral changes can alter host-parasite
Changes in the host can also alter the balance of a host- relations both positively and negatively (Fig. 9).
parasite relationship. A particularly dramatic example is Although many bacterial infections of the intestine have
the intense selection for resistant genotypes in rabbit declined in importance with changes in human lifestyle,
populations exposed to the myxomatosis virus, which there are other contemporary microbiologic problems in
took place concurrently with selection for reduced the developed world whose onset can be traced directly
pathogenicity in the virus itself. There are no exactly to sociologic, environmental and even medical change
equivalent examples in humans, but in evolutionary time (Fig. 9).
there have been major selective influences on
populations prompting changes to permit survival in the A particularly good example is disease arising from
face of life-threatening infections. A good example is the domestication of pets (e.g. toxoplasmosis) because it
selective pressure exerted by falciparum malaria, which illustrates that human freedom from some infections
has been responsible for the persistence of many alleles arises primarily because of lack of contact with the
associated with hemoglobinopathies (e.g. sickle cell organisms and not from any innate resistance to the
hemoglobin). establishment of the infection itself. Diseases arising
from contact with infected animals or animal products
Although these abnormalities are detrimental to a varying (zoonotic infections) constitute a constant threat that can
degree, they persist because they are (or were) associated be realized by behavioral or environmental changes that
with resistance to malarial infection. Malaria has also alter established patterns of human-animal contact.

Host-Parasite Relationships
By convention, when the word parasite is used without
If a symbiont either harms or lives at the expense of qualification, it refers specifically to a protozoan or
another organism (the host), it is a parasitic organism, helminthic (nematode, trematode, cestode) organism.
and the relationship is called parasitism. In this
relationship the body of the host can be viewed as a Several types of parasitism are recognized. If an
microenvironment that shelters and supports the growth organism lives on the surface of its host, it is an
and multiplication of the parasitic organism. The ectoparasite; if it lives in- ternally, it is an
parasitic organism is usually the smaller of the two endoparasite. The host on or in which the parasitic
partners and is metabolically dependent on the host. organism either attains sexual maturity or reproduces is
There are many parasitic agents or organisms among the the final host. A host that serves as a temporary but
viruses, bacteria, fungi, plants, and animals (Table 1). essential environment for some stages of development is
an intermediate host.
In contrast, a transfer host is not necessary for the

completion of the organism’s life cycle but is used as a
vehicle for reaching a final host. A host infected with a
parasitic organism that also can infect humans is called a
reservoir host.
Because, by definition, parasitic organisms are dependent

on their hosts, the symbiotic relationship between the an organism or its products. Any organism or agent that
host and parasite is a dynamic one (figure 1). When a produces such a disease is a pathogen [Greek patho,
parasite is growing and multiplying within or on a host, disease, and gennan, to produce]. Its ability to cause
the host is said to have an infection. The nature of an disease is called pathogenicity. A primary (frank)
infection can vary widely with respect to severity, pathogen is any organism that causes disease in a
location, and number of organisms involved (table 2). healthy host by a direct interaction. Conversely, an
An infection may or may not result in overt disease. opportunistic pathogen is an organism that is either
normally free-living, or a part of the host’s normal
An infectious disease is any change from a state of microbiota, but which may adopt a pathogenic role under
health in which part or all of the host body is not capable certain circumstances, such as when the immune system
of carrying on its normal functions due to the presence of is compromised.
resistance. Usually the greater the number of organisms

At times an infectious organism can enter a latent state in within a given host, the greater the likelihood of disease.
which there is no shedding of the organism and no
symptoms present within the host. This latency can be
either intermittent or quiescent. Intermittent latency is However, a few organisms can cause disease if they are
exemplified by the herpes virus that causes cold sores extremely virulent or if the host’s resistance is low. A
(fever blisters). After an initial infection, the symptoms host’s resistance can drop so much that its own
subside. However, the virus remains in nerve tissue and microbiota may cause disease.
can be activated weeks or years later by factors such as
stress or sunlight. In a quiescent latency the organism Such a disease is sometimes called an endogenous
persists but remains inactive for long periods of time, disease because the agent originally comes from within
usually for years. For example, the varicella-zoster virus the host’s own body. Endogenous diseases can be a
causes chickenpox in children and remains after the serious problem among hospitalized patients with very
disease has subsided. In adulthood, under certain low resistance.
conditions, the same virus may erupt into a disease called
shingles. Cold sores; Chickenpox (varicella) and shingles The term virulence [Latin virulentia, from virus, poison]
(herpes zoster). refers to the degree or intensity of pathogenicity. It is
determined by three characteristics of the pathogen:
The outcome of most host-parasite relationships is invasiveness, infectivity, and pathogenic potential.
dependent on three main factors: (1) the number of Invasiveness is the ability of the organism to spread to
organisms present in or on the host, (2) the virulence of adjacent or other tissues. Infectivity is the ability of the
the organism, and (3) the host’s defenses or degree of organism to establish a focal point of infection.

Pathogenic potential refers to the degree that the 7. Be either cleared from the body of the host, establish a
pathogen causes damage. A major aspect of pathogenic persistent infection, or kill the host
potential is toxigenicity. 8. Be shed back into the environment
The determinants of pathogenicity are now discussed in

Toxigenicity is the pathogen’s ability to produce toxins, more detail.
chemical substances that will damage the host and Entry, Contact, and Primary Replication
produce disease.
The first step in the infectious process is the attachment
Virulence is often measured experimentally by and entrance of the virus into a susceptible host and the
determining the lethal dose 50 (LD50) or the infectious host’s cells. Entrance may be accomplished through one
dose 50 (ID50). These values refer to the dose or number of the body surfaces (skin, respiratory system,
of pathogens that will either kill or infect, respectively, gastrointestinal system, urogenital system, or the
50% of an experimental group of hosts within a specified conjunctiva of the eye). Other viruses enter the host by
period (figure 2). needle sticks, blood transfusions and organ transplants,
or by insect vectors (organisms that transmit the
pathogen from one host to another). Some viruses
replicate at the site of entry, cause disease at the same
site (e.g., respiratory and gastrointestinal infections), and
do not spread throughout the body. Others spread to sites
distant from the point of entry and replicate at these sites.
For example, the enteroviruses enter through the
gastrointestinal tract but produce disease in the central
nervous system. Mucous membranes.
Viral Spread and Cell Tropism
Mechanisms of viral spread vary, but the most common

routes are the bloodstream and lymphatic system. The
presence of viruses in the blood is called viremia. In
some instances, spread is by way of nerves (e.g., rabies
virus, herpes simplex, and varicellazoster viruses).
Viruses exhibit cell, tissue, and organ specificities. These

specificities are called tropisms (Greek trope, turning).
It should be noted that disease can result from causes A tropism by a specific virus usually reflects the
other than toxin production. Sometimes a host will presence of specific cell surface receptors on the
trigger exaggerated immunological responses eucaryotic host cell for that virus.
(immunopathology) upon a second exposure or chronic
exposure to a microbial antigen. These hypersensitivity Cell Injury and Clinical Illness: Destruction of the
reactions damage the host even though the pathogen virus-infected cells in the target tissues and alterations in
doesn’t produce a toxin. Tuberculosis is a good example host physiology are responsible for the development of
of the involvement of hypersensitivity reactions in viral disease and clinical illness. Some tissue (e.g.,
disease. intestinal epithelium) can rapidly regenerate after a viral
attack and withstand extensive damage. Other tissues,
Some diseases also might be due to autoimmune such as nervous system tissues, are not able to regenerate
responses. For instance, a viral or bacterial pathogen may and may never resume normal functioning after damage
stimulate the immune system to attack host tissues has occurred.
because it carries antigens that resembled those of the
host, a phenomenon known as molecular mimicry. The potential effects of viruses on individual host cells
Streptococcal infections may cause rheumatic fever in are the result of a complex series of events. There are
this way. four generally accepted patterns of a viral infection. (1)
In lytic infections the virus multiplies and kills the host
Pathogenesis of Viral Diseases cell immediately and new virions are released. (2) In
persistent viral infections the virus lives in the host cell
The fundamental process of viral infection is the and releases small numbers of virions over a long period
expression of the viral replicative cycle in a host cell. of time. This causes little damage to the host cell. (3) In
latent infections, the virus resides in the cell but produces
The steps for the infectious process involving viruses are no virions. At some later time the virus can be activated
that a virus must and a lytic infection occurs. (4) Some viruses can
1. Enter a host transform the host cell into a cancer cell that becomes the
2. Contact and enter susceptible cells focal point for a tumor.
3. Replicate within the cells
4. Spread to adjacent cells Host Immune Response: Both humoral and cellular
5. Cause cellular injury components of the immune response are involved in the
6. Engender a host immune response control of viral infections.

Recovery from Infection: The host will either succumb

or recover from a viral infection. Recovery mechanisms In this context colonization means the establishment of a
involve nonspecific defense mechanisms and specific site of microbial reproduction on or within a host. It does
humoral and cellular immunity. The relative importance not necessarily result in tissue invasion or damage.
of each of these factors varies with the virus and the Colonization depends on the ability of the bacteria to
disease. compete successfully with the host’s normal microbiota
Virus Shedding: The last step in the infectious process for essential nutrients. Specialized structures that allow
is shedding of the infectious virus back into the bacteria to compete for surface attachment sites also are
environment. This is necessary to maintain a source of necessary for colonization.
viruses in a population of hosts. Shedding often occurs
from the same body surface used for entry. During this Bacterial pathogens and many nonpathogens adhere with
period, an infected host is infectious and can spread the a high degree of specificity to particular tissues.
virus. In some viral infections, such as a rabies infection, Adherence factors called adhesins are one reason for this
humans are dead-end hosts because virus shedding does specificity. Adhesins are specialized molecules or
not occur. structures on the bacteria’s cell surface that bind to
complementary receptor sites on the host cell surface.
Pathogenesis of Bacterial Diseases They are one type of virulence factor.
The steps for infections by pathogenic bacteria include Virulence factors are bacterial products or structural
1. Maintain a reservoir. A reservoir is a place to live components (e.g., capsules and adhesins) that contribute
before and after causing an infection. to virulence or pathogenicity.
2. Initially be transported to the host.
3. Adhere to, colonize, and/or invade the host. Invasion of the Bacterial Pathogen
4. Multiply (grow) or complete its life cycle on or in the
host or the host’s cells. Entry into host cells and tissues is a specialized strategy
5. Initially evade host defense mechanisms. used by many bacterial pathogens for survival and
6. Possess the ability to damage the host. multiplication. Pathogens often actively penetrate the
7. Leave the host and return to the reservoir or enter a host’s mucous membranes and epithelium after
new host. attachment to the epithelial surface. This may be
accomplished through production of lytic substances that
The first five factors influence the degree of infectivity alter the host tissue by (1) attacking the ground substance
and invasiveness. Toxigenicity plays a major role in the and basement membranes of integuments and intestinal
sixth. linings, (2) degrading carbohydrate protein complexes
between cells or on the cell surface (the glycocalyx), or
Maintaining a Reservoir of the Bacterial Pathogen (3) disrupting the cell surface.
All bacterial pathogens must have at least one reservoir. At times a bacterial pathogen can penetrate the epithelial
The most common reservoirs for human pathogens are surface by passive mechanisms not related to the
other humans, animals, and the environment. Since the pathogen itself. Examples include (1) small breaks,
source and/or reservoir of the pathogen is part of the lesions, or ulcers in a mucous membrane that permit
infectious disease cycle. initial entry; (2) wounds, abrasions, or burns on the
skin’s surface; (3) arthropod vectors that create small
Transport of the Bacterial Pathogen to the Host wounds while feeding; (4) tissue damage caused by other
organisms; and (5) existing eucaryotic internalization
An essential feature in the development of an infectious pathways (e.g., endocytosis and phagocytoses.
disease is the initial transport of the bacterial pathogen to
the host. The most obvious means is direct contact— Once under the mucous membrane, the bacterial
from host to host (coughing, sneezing, body contact). pathogen may penetrate to deeper tissues and continue
Bacteria also are transmitted indirectly in a variety of disseminating throughout the body of the host. One way
ways. Infected hosts shed bacteria into their the pathogen accomplishes this is by producing specific
surroundings. products and/or enzymes that promote spreading. These
products are virulence factors. Bacteria may also enter
Once in the environment bacteria can be deposited on the small terminal lymphatic capillaries that surround
various surfaces, from which they can be either epithelial cells. These capillaries merge into large
resuspended into the air or indirectly transmitted to a host lymphatic vessels that eventually drain into the
later. Soil, water, and food are indirect vehicles that circulatory system. Once the circulatory system is
harbor and transmit bacteria to hosts. Vectors and reached, the bacteria have access to all organs and
fomites (inanimate objects that harbor and transmit systems of the host.
pathogens) also are involved in the spread of many
bacteria. Growth and Multiplication of the Bacterial Pathogen
Attachment and Colonization by the Bacterial For a bacterial pathogen to be successful in growth and
Pathogen reproduction (colonization), it must find an appropriate
environment (nutrients, pH, temperature, redox potential)
After being transmitted to an appropriate host, the within the host. Those areas of the host’s body that
bacterial pathogen must be able to adhere to and colonize provide the most favorable conditions will harbor the
host cells and tissues.

pathogen and allow it to grow and multiply to produce an carried on the temperate bacteriophage λ, and its
infection. expression is regulated by iron. The toxin is produced
only by strains lysogenized by the phages. Expression of
Some bacteria invade specific cells in which they grow the virulence gene of Bordetella pertussis (the pathogen
and multiply. Many of these intracellular pathogens have that causes whooping cough) is enhanced when the
evolved such elaborate nutrient-gathering mechanisms bacteria grow at body temperature (37°C) and suppressed
that they have become totally dependent on the host’s when grown at a lower temperature. Finally, the
cells. Finally, some bacteria can actively grow and virulence factors of Vibrio cholerae (the pathogen that
multiply in the blood plasma. The presence of viable causes cholera) are regulated at various levels by many
bacteria in the bloodstream is called bacteremia. The environmental factors. Expression of the cholera toxin is
presence of bacteria or their toxins in the blood often is higher at pH 6 than at pH 8 and higher at 30 than at
termed septicemia [Greek septikos, produced by 37°C. Osmolality and available amino acids are also
putrefaction, and haima, blood]. important.
Leaving the Host Pathogenicity Islands
The last determinant of a successful bacterial pathogen is Many bacteria (Yersinia spp., Pseudomonas aeruginosa,
its ability to leave the host and enter either a new host or Shigella flexneri, Salmonella typhimurium,
a reservoir. Unless a successful escape occurs, the enteropathogenic E. coli) are pathogenic because they
disease cycle will be interrupted and the microorganism have large segments of DNA, called pathogenicity
will not be perpetuated. Most bacteria employ passive islands, that carry genes responsible for virulence. These
escape mechanisms. Passive escape occurs when a pathogenicity islands have been acquired during
pathogen or its progeny leave the host in feces, urine, evolution by horizontal gene transfer. A pathogen may
droplets, saliva, or desquamated cells. have more than one pathogenicity island. An excellent
example of virulence genes carried in a pathogenicity
The Clonal Nature of Bacterial Pathogens island are those involved in protein secretion. So far, five
pathways of protein secretion (types I to V) have been
The main mechanism bacteria use to exchange genetic described in gram-negative bacteria. A set of
information is the transfer of extrachromosomal genetic approximately 20 genes encode a pathogenicity
elements, plasmids, and phages. Many genes that code mechanism termed the type III secretion system that
for bacterial virulence factors are found on plasmids or enables gram-negative bacteria to secrete and inject
phages. These mobile genetic elements can transfer virulence proteins into the cytoplasm of eucaryotic host
virulence factors between members of the same species cells.
or different species by horizontal gene transfer. At times
the genetic elements are part of highly mobile DNA Unlike other bacterial secretory systems, the type III
(transposons), and there is a recombination between the system is triggered specifically by contact with host cells,
extrachromosomal DNA and the chromosome. When this which helps avoid inappropriate activation of host
recombination occurs, the genes coding for virulence defenses. Secretion of these virulence proteins into a host
may become chromosomal. cell initiates sophisticated “biochemical crosstalk”
between the pathogen and the host. The injected proteins
One important result of the conservation of these resemble eucaryotic factors that signal transduction
chromosomal genes is that the bacteria are clonal. Some functions and they are capable of interfering with
bacterial pathogens have only one or a few clonal types eucaryotic signaling pathways.
existing in the environment. For example, the bacterium
that causes typhoid fever (Salmonella typhi) has two Redirection of cellular signaling transduction may disarm
clonal types, whereas there are over 100 clonal types of host immune responses or reorganize the cytoskeleton,
Haemophilus influenzae, but only a small number are thus establishing subcellular niches for bacterial
associated with bacterial pneumonia. colonization and facilitating “stealth and interdiction” of
host defense communication lines.
Regulation of Bacterial Virulence Factors
Pathogenicity islands generally increase microbial
Some pathogenic bacteria have adapted to both the free- virulence and are not present in nonpathogenic members
living state and to an environment within a human host. of the same genus or species. One specific example is
In the adaptive process, these pathogens have evolved found in E. coli. The enteropathogenic E. coli possesses
complex signal transduction pathways to regulate the large DNA fragments, 35 to 170 kilobases in size, that
genes necessary for virulence. A virulence factor may be contain several virulence genes absent from commensal
present simply because the bacterium has been infected E. coli. Some of these genes code for proteins that alter
by a phage. Often environmental factors control the actin microfilaments within a host intestinal cell. As a
expression of the virulence genes. Common signals consequence, the host cell surface bulges and develops
include temperature, osmolality, available iron, pH, into a cuplike pedestal to which the bacterium tightly
specific ions, and other nutrient factors. Several binds.
examples are now presented.
The gene for diphtheria toxin from Corynebacterium

diphtheriae (the pathogen that causes diphtheria) is

A2. Cell fusion in both normal and abnormal cells
Cell fusion is a process in which two or more cells hybridization between leukocytes and somatic cells.
become one by merging their plasma membranes. The Sixty years later, this idea was expanded by proposing
ability of a cell to fuse to other cells is referred to as that hybridization of tumor cells with lymphocytes
fusogenicity. The progeny of cell fusion are known as results in metastatic cells, and that cell fusion promotes
hybrids. Perhaps the best-known hybrids are hybridomas, the phenotypic and genotypic diversity of tumors.
which are made by fusing myeloma cells with Several lines of evidence support at least some of these
lymphocytes to produce monoclonal antibodies. notions.
Although cells can be easily fused in the laboratory using
readily available chemicals, cell fusion in live organisms Many tumor cells are fusogenic
appears to be a complex, poorly understood, multistep
process that involves cell-cell recognition, cell adhesion, The propensity of various tumor cells to fuse
and membrane fusion. spontaneously in tissue culture has been a known, though
unexplained, phenomenon for many decades. In fact, this
Cell fusion is a part of normal development and tissue property of tumor cells was used to make somatic cell
homeostasis hybrids before fusion with polyethylene glycol (PEG) or
viruses had been developed. Some tumor cell lines are so
Our life begins with fusion of our parents’ gametes. A fusogenic that they fuse spontaneously more efficiently
pregnancy depends on normal functioning of the than in the presence of PEG, a difference explainable by
placental barrier, the main part of which is the the toxicity of this agent. The ability to fuse is not limited
syncytiotrophoblast, a gigantic syncytium (a cell to a particular cell or tumor type and can occur between
resulting from fusion of numerous cells) whose surface tumor cells as well as between a tumor and a normal cell.
area can reach 10 square meters. As the embryo Tumor cell fusion is not limited to tissue culture. Human
develops, its muscles are formed by fusion of myoblasts tumor cells injected into hamsters produced metastases
into syncytia through a multistep process that involves formed by hybrids of human and hamster cells. In
products of multiple genes. another study, treatment of chimeric mice with a
carcinogen resulted in tumors, in which about 1% of cells
In the adult body, the maintenance of the bones is had marker alleles from both parental strains, an
controlled in part by osteoclasts, which are multinuclear observation explainable by cell fusion.
cells formed by the fusion of mononuclear progenitors.
Macroscopic foreign objects, such as a splinter or an Interestingly, despite these observations, cell fusion is
implanted device, are encapsulated by foreign body giant rarely, if at all, considered in contemporary studies that
cells (FBGC) that attempt to dissolve the intruder and are use experimental models of cancer. Because the evidence
thought to be formed by fusion of mononuclear for cell fusion in human cancers is indirect, it is not as
precursors. Langhans cells, a variation of FBGC, are compelling as that for experimental tumors. One
found in tuberculosis patients at the sites of local observation is that premature chromosome condensation
inflammation. (PCC) is observed in tumor cells. PCC is a typical result
of a fusion between cells in different stages of the cell
The observations that embryonic stem cells may cycle, although PCC can be also induced in mononuclear
differentiate into multiple cell types through cell fusion cells, for example by caffeine.
suggested a new role of cell fusion in mammalian
development, although alternative explanations for these Cell fusion could also explain the origin of multinuclear
observations have not been ruled out. Studies in the tumor cells in which nuclei undergo asynchronous DNA
nematode C. elegans provide evidence that cell fusion synthesis or mitosis, even though alternative
can be a major part of development, at least in explanations of this phenomenon are as plausible. It is
invertebrates. About one-third of the cells that are born in unclear whether the scarcity of reports on cell fusion in
this organism are subsequently fused into 44 syncytia in human tumors is due to rarity of this event, to a difficulty
a reproducible and stereotypic way. to detect it, or to insufficient interest in this subject. The
last possibility should not be underestimated. For
Remarkably, even the side of a cell that will fuse is example, the number of reports on apoptosis that were
predetermined. In summary, cell fusion of normal published during the last ten years is about 100 times
somatic cells is a tightly controlled process that is higher than that published in the preceding twenty. With
restricted to only a few cell types in humans, and results all likelihood, this difference is due to a change in the
in terminally differentiated multinuclear cells incapable subject’s popularity rather than a change in incidence or
of proliferation. Intriguingly, tumor cells appear to role of apoptosis in studied organisms.
violate strict rules of cell fusion.
Hybrids can be more malignant than the parental
Cell fusion and cancer cells
The idea that cell fusion contributes to cancer Importantly, while physiological fusion of normal
progression was introduced almost 100 years ago with a somatic cells produces non-proliferating differentiated
proposal that malignancy is a consequence of multinuclear cells, fusion of tumor cells results in

proliferating hybrids. The survival rate of these hybrids becomes metastatic by fusion to normal cells that travel
in tissue culture can be as high as 1%. Considering that throughout the body freely, such as lymphocytes or
the rate of cell fusion in experimental tumors was also macrophages. Several studies support this idea. One
estimated at 1%, a 1 cm3 tumor of about 109 cells may study found that a parental cell line, when injected in a
harbor 105 proliferating hybrid cells. The question is, can mouse, produced hundreds of colonies in the liver, while
any of these cells be more malignant than their parents? a derivative line produced only a few. Surprisingly, when
a colony formed by the derivative line was injected into a
In fact, one of the early reports on spontaneous cell new host, it produced as many colonies as did the
fusion came from an observation that cultures containing parental cell line.
a mixture of two mouse cell lines were occasionally
overgrown by a new cell line, whose karyotype was the The analysis of histocompatibility markers and
sum of the parental karyotypes. This observation karyotypes of the host mice, the injected cells, and the
indicated that the new cell line is a hybrid, and that resulting metastases led to the conclusion that the
hybrids can grow faster than their parents. The increase in the metastatic potential was due to a fusion
subsequent studies indicated that, at least in experimental with the host cells. This conclusion was supported by the
systems, hybrids can be more drug-resistant and more finding that the high metastatic potential can be restored
metastatic than the parental cells (Figure 1). in the derivative cells by fusing them with mouse bone
marrow cells before injection.
Other studies discovered that cell fusion can determine

the tissues into which tumor cells metastasize. For
example, fusion of nonmetastatic mouse plasmacytoma
or myeloma cells with lymphocytes or splenic dendritic
cells not only resulted in metastatic hybrids, but the
target tissues of metastases varied with the type of
normal cells used as well . One can only wonder whether
implications of this finding are considered in developing
cancer vaccines, which involves injecting patients with
hybrids between tumor cells and normal dendritic cells.
Because tissues that are common sites of metastases are
normally rich in macrophages, the “wolf in sheep’s
clothing” model argues that tumor cells gravitate to these
sites because, as macrophage hybrids, they acquire
macrophage’s tropism.
Another possibility is that tumor cells can acquire the

“sheep’s clothing” by fusing to a normal cell of a tissue,
thus producing a tumor cell that can grow in the new
environment. This mechanism is not unlike that proposed
for the stem cell differentiation, where a stem cell
differentiates by fusing to a resident cell from a host
tissue.
Cell fusion can contribute to drug resistance
Cell fusion can increase tumor cell diversity
Cell fusion can consolidate resistance to various drugs by
combining genes responsible for resistance to various Perhaps the main property of cancer cells that makes
agents. For example, fusion of tumor cells that were them malignant is the ability to produce diverse progeny.
resistant to 5-fluorouracil with tumor cells resistant to Indeed, as an oncology textbook states, “…by the time of
methotrexate produced hybrids that were resistant to both initial diagnosis, cancers consist of multiple
drugs. An unexpected observation was that the hybrids subpopulations of cells with diverse genetic,
were resistant to mephalan, a drug to which both parental biochemical, immunologic, and biologic characteristics”.
lines were sensitive. This observation emphasized the How this diversity emerges and how it is maintained is
difficulty in predicting the diversity that cell fusion can not clear. The evidence that cell fusion can contribute to
create. Our own experiments indicated that fusion of tumor heterogeneity came from using cell fusion as a tool
drug-sensitive transformed cells to primary cells results for somatic cell genetics, in particular to investigate
in heterokaryons that are resistant to apoptosis. Although whether malignancy is a dominant or recessive trait.
this effect lasted only a few days, it is not unreasonable Because hybrids between a highly and a weakly
to speculate that even a temporary resistance may allow a malignant tumor cell line were highly malignant, the
small fraction of tumor cells to survive a session of initial conclusion was that malignancy is a dominant
chemotherapy. trait. However, the subsequent studies found that hybrids
between tumor cells and normal cells were not
Cell fusion can promote the ability to metastasize tumorigenic. The controversy was resolved by realizing
that hybrids become tumorigenic if they lose certain
The ability to metastasize is, arguably, the deadliest “normal” chromosomes, an observation that eventually
property of cancers. A proposed link between cell fusion led to the discovery of tumor suppressor genes.
and metastasis can be described as the “wolf in sheep’s
clothing” model. The model suggests that a tumor cell

A less appreciated outcome of these studies was the protein family, which includes both viral and cellular
conclusion that chromosomal aberrations are hallmarks proteins. The fusion proteins are identified by a
of hybrids. These aberrations include chromosome hydrophobic motif, known as the fusion peptide, which is
nondisjunction, mitotic recombination, translocations, required for the fusogenicity of these proteins.
deletions, insertions, and inversions, a list that is
remarkably similar to that observed in tumor cells. Perhaps any fusion protein or any other agent that
Although how exactly cell fusion causes these induces cell fusion should be investigated for its
abnormalities is not clear, the result is hardly surprising carcinogenicity. For example, HIV is known to induce
considering that cell fusion produces cells with a sum of syncytia, which suggests that in addition to its known
parental chromosomes and more than two centrosomes. role in disease, it might be useful to investigate whether
The aberrant chromosome segregation associated with this ability contributes to malignancies associated with
cell fusion was proposed to explain aneuploidy as a this virus, such as Kaposi’s sarcoma. Remarkably, the
hallmark of cancer cells. Although aneuplody is a feature genes that encode human endogenous retroviruses
of nearly all of more than 20,000 solid tumors analyzed (HERV) or their individual proteins, including the fusion
in human, and it was even proposed to be a cause rather proteins, comprise at least eight percent of the human
than a consequence of cancer, how and why tumor cells genome. In fact, syncytin, the fusion protein that is
become aneuploid is not clear. The observation that thought to mediate formation of the syncytiotrophoblast
tumor cells are prone to cell fusion, the fact that fusion and is specifically expressed in placenta, is the envelope
results in polyploidy cells, and the finding that cell fusion fusion protein of HERV-W. This finding indicates that
is followed by abnormal chromosome segregation, expression of HERV proteins can determine cell fate,
together argue that if cell fusion is a part of cancer and, incidentally, gives support to the hypothesis that
progression, then tumor cells should eventually become placental mammals evolved because our ancestor had the
aneuploid. However, this explanation of aneuploidy by germ line infected with a retrovirus.
no means excludes other mechanisms, such as mutations
that cause abnormal mitosis. The sheer abundance of HERV genes in the human
genome provides an ample opportunity for their
Changes in epigenetic regulation that follow cell fusion deregulated expression. Indeed, HERV particles were
are another factor that can contribute to tumor diversity. found in both normal and tumor tissues. Production of
These changes appear to be unpredictable, and gene HERV has been associated with multiple sclerosis and
expression can be selectively silenced, activated, or cancer, although whether this association is causative is
unchanged. Considering that the human body has about unclear, in part because HERV particles do not appear to
200 cell types that are different because of their gene be infectious. Perhaps a causal link between HERVs and
expression pattern, it is easy to imagine how cell fusion these diseases can be found by investigating whether
can produce monsters. In summary, cell fusion can be an these particles induce cell fusion. Cell death is a
engine of genomic and epigenetic variability that has a predominant outcome of cell fusion, which might explain
potential to make cells with new properties at a rate how expression of HERV can lead to multiple sclerosis,
exceeding that achievable by random mutagenesis. while the ability of some cells to survive cell fusion can
contribute to cancer, as we discussed.
Why are tumor cells fusogenic?
Overall, at least in experimental systems, cell fusion has
There is no definitive answer to this question, which is been linked to several fundamental features of cancer,
not surprising considering that the phenomenon lost even though molecular mechanisms that cause this fusion
whatever popularity it had before modern experimental remain obscure. Although one can argue that this link is a
approaches capable of dissecting its mechanism were peculiarity of experimental tumors, the hypothesis that
developed. Several factors were proposed to explain cell fusion contributes to cancer in humans seems to be
formation of hybrids in tumors: viruses, cholesterol equally plausible. Considering that modern tools of
crystals, and the natural fusogenicity of macrophages. experimental biology have not been applied to studies of
The ability of viruses to induce cell fusion is mediated by cell fusion in tumors, there is an abundance of
the viral proteins that mediate entry of enveloped viruses possibilities that are ready to be explored.
into the cell. These proteins are members of the fusion
B1. CELL SIGNALLING:
1. Cell-to-Cell Signaling: Hormones and Receptors: chemicals, including small molecules (e.g., amino acid
derivatives, acetylcholine), peptides, and proteins, are
No cell lives in isolation. In all multi cellular organisms, used in this type of cell-to-cell communication. The extra
survival depends on an elaborate intercellular cellular products synthesized by signaling cells can
communication network that coordinates the growth, diffuse away or be transported in the blood, thus
differentiation, and metabolism of the multitude of cells providing a means for cells to communicate over longer
in diverse tissues and organs. Cells within small groups distances than is possible by chains of direct cell-cell
often communicate by direct cell-cell contact. We will contacts.
examine how cells communicate by means of extra
cellular signaling molecules. These substances are 2. Overview of Extracellular Signaling
synthesized and released by signaling cells and produce a
specific response only in target cells that have receptors Communication by extracellular signals usually involves
for the signaling molecules. An enormous variety of six steps: (1) synthesis and (2) release of the signaling

molecule by the signaling cell; (3) transport of the signal pioneering work of Earl W. Sutherland, whose research
to the target cell; (4) detection of the signal by a specific lead to a Noble prize in 1971. Sutherland and his
receptor protein; (5) a change in cellular metabolism, colleagues were investigating how the animal hormone
function, or development triggered by the receptor-signal epinephrine stimulates the breakdown
complex; and (6) removal of the signal, which often (depolymerization) of the storage polysaccharide
terminates the cellular response. glycogen with liver cells and skeletal muscle cells.
In many eukaryotic microorganisms (e.g., yeast, slime Glycogen depolymerization releases the sugar glucose-1-
molds, and protozoans), secreted molecules coordinate phosphate, which the cell converts to glucose-6-
the aggregation of free-living cells for sexual mating or phosphate. The cell can then use this compound, an early
differentiation under certain environmental conditions. intermediate in glycolysis, for energy production. Alter-
Chemicals released by one organism that can alter the natively, the compound can be stripped of phosphate and
behavior or gene expression of other organisms of the released into the blood as glucose that can fuel cells
same species are called pheromones. throughout the body. Thus one effect of epinephrine,
which is secreted from the adrenal gland during times of
3. Signaling Molecules Operate over Various physical or mental stress, is the mobilization of fuel
Distances in Animals reserves.
In animals, signaling by extracellular, secreted molecules Sutherland's research team discovered that epinephrine
can be classified into three types endocrine, paracrine, stimulates glycogen breakdown by somehow activating a
or autocrine based on the distance over which the cytosolic enzyme, glycogen phosphorylase. However,
signal acts. In addition, certain membrane-bound proteins when epinephrine was added to a test-tube mixture
on one cell can directly signal an adjacent cell. containing the phosphorylase and its substrate, glycogen,
no depolymerization occurred.
In endocrine signaling, signaling molecules, called
hormones, act on target cells distant from their site of Epinephrine could activate glycogen phosphorylase only
synthesis by cells of endocrine organs. In animals, an when the hormone was added to a solution containing
endocrine hormone usually is carried by the blood from intact cells. This result told Sutherland two things. First,
its site of release to its target. epinephrine does not interact directly with the enzyme
responsible for glycogen breakdown; an intermediate
In paracrine signaling, the signaling molecules released step or series of steps must be occurring inside the cell.
by a cell only affect target cells in close proximity to it. Second, the plasma membrane is somehow involved in
The conduction of an electric impulse from one nerve transmitting the epinephrine signal.
cell to another or from a nerve cell to a muscle cell
(inducing or inhibiting muscle contraction) occurs via Thus Sutherland's early work suggested that the process
paracrine signaling. The role of this type of signaling, going on at the receiving end of a cellular conversation
mediated by neurotransmitters, in transmitting nerve can be dissected into three stages: reception,
impulses. Many signaling molecules regulating transduction, and response (FIGURE 1):
development in multicellular organisms also act at short
range.
In autocrine signaling, cells respond to substances that

they themselves release. Many growth factors act in this
fashion, and cultured cells often secrete growth factors
that stimulate their own growth and proliferation. This
type of signaling is particularly common in tumor cells,
many of which overproduce and release growth factors
that stimulate inappropriate, unregulated proliferation of
themselves as well as adjacent nontumor cells; this
process may lead to formation of tumor mass. 1. Reception is the target cell's detection of a signal
coming from outside the cell. A chemical signal is
Some compounds can act in two or even three types of "detected" when it binds to a cellular protein, usually at
cell-to-cell signaling. Certain small amino acid the cell's surface.
derivatives, such as epinephrine, function both as
neurotransmitters (paracrine signaling) and as systemic 2. The binding of the signal molecule changes the
hormones (endocrine signaling). Some protein hormones, receptor protein in some way, thus initiating the process
such as epidermal growth factor (EGF), are synthesized of transduction. The transduction stage converts the
as the exoplasmic part of a plasma- membrane protein; signal to a form that can bring about a specific cellular
membrane-bound EGF can bind to and signal an adjacent response. In Sutherland's system, the binding of
cell by direct contact. Cleavage by a protease releases epinephrine to the outside of a receptor protein in a liver
secreted EGF, which acts as an endocrine signal on cell's plasma membrane leads via a series of steps to
distant cells. activation of glycogen phosphorylase. Transduction
sometimes occurs in a single step but more often requires
4. The Three stages of cell signaling are Reception, a sequence of changes in a series of different molecules-a
transduction, and Response signal-transduction pathway. The molecules in the
pathway are often called relay molecules.
Our current understanding that how chemical massager
act via signal transduction pathways had its origin in

3. In the third stage of cell signaling, the transduced

signal finally triggers a specific cellular response. The Most signal molecules are water-soluble and too large to
response may be almost any imaginable cellular activity- pass freely through the plasma membrane. But, as
such as catalysis by an enzyme (such as glycogen Sutherland learned for epinephrine, they still influence
phosphorylase), rearrangement of the cytoskeleton, or cellular activity in major ways. Like yeast mating factors,
activation of specific genes in the nucleus. The cell- most water-soluble signal molecules bind to specific sites
signaling process helps ensure that crucial activities like on receptor proteins embedded in the cell's plasma
these occur in the right cells, at the right time, and in membrane. Such a receptor transmits information from
proper coordination with the other cells of the organism. the extracellular environment to the inside of the cell by
changing shape or aggregating when a specific ligand
5. Signal Reception and the Initiation of Transduction binds to it. We'll see how membrane receptors work by
looking at three major types: G-protein-linked receptors,
When we speak to someone, others nearby may hear our tyrosine kinase receptors, and ion-channel receptors.
message, sometimes with unfortunate consequences.
However, errors of this kind rarely occur between cells. G-Protein-Linked Receptors
The signals emitted by α yeast cell are "heard" only by
its prospective mates, β cells. Similarly, although A G-protein-linked receptor is a plasma-membrane
epinephrine encounters many types of cells as it receptor that works with the help of a protein called a G
circulates in the blood, only certain target cells detect and protein (FIGURE 2a).
react to the hormone. The signal receptor is the identity
tag on the target cell. Many different signal molecules use G-protein and many
other hormones, and neurotransmitters. These receptors
a. A signal molecule binds to a receptor protein, vary in their binding sites for recognizing signal
causing the protein to change shape molecules and for recognizing different G proteins inside
the cell. Nevertheless, G-protein-linked receptor proteins
A cell targeted by a particular chemical signal has are all remarkably similar in structure. They each have
molecules of a receptor protein that recognizes the signal seven a-helices spanning the membrane, as shown in
molecule. The signal molecule is complementary in FIGURE 3.
shape to a specific site on the receptor and attaches there,
like a key in a lock-or like a substrate in the catalytic site
of an enzyme. The signal molecule behaves as a ligand,
the term for a small molecule that specifically binds to a
larger one. Ligand binding generally causes a receptor
protein to undergo a change in conformation-that is, to
change shape. For many receptors, this shape change
directly activates the receptor so that it can interact with
another cellular molecule. For other kinds of receptors,
as we'll see shortly, the immediate effect of ligand
binding is more limited, mainly causing the aggregation
of two or more receptor molecules.
Loosely attached to the cytoplasmic side of the

membrane, the G protein functions as a switch that is on
or off depending on which of two guanine nucleotides is
attached, GDP or GTP. When GDP is bound, the G
protein is inactive; when GTP is bound, it is active.
(GTP, or guanosine triphosphate, is similar to ATP.)
When the appropriate chemical signal binds as a ligand

to the extracellular side of a G-protein-linked receptor,
the receptor is activated, changing conformation in such
a way that it, in turn, can activate a G protein: The
receptor binds a specific, inactive G protein and causes a
GTP to displace the GDP (FIGURE 2b). The activated G
protein then binds to another protein, usually an enzyme,
and alters its activity. These changes are only temporary,
however, for the G protein also functions as a GTPase
enzyme and soon hydrolyzes its bound GTP to GDP
(FIGURE 2c). Now inactive again, the G protein leaves
the enzyme. The GTPase function of the G protein
b. Most signal receptors are plasma-membrane allows the pathway to shut down rapidly when the extra-
proteins cellular signal molecule is no longer present.

G-protein receptor systems are extremely widespread and The receptor protein is now recognized by specific relay
diverse in their functions. In addition to the functions proteins inside the cell. Each such protein binds to a
already mentioned, they are important in embryonic specific phosphorylated tyrosine, undergoing a structural
development, as indicated by genetic studies. For change that activates it (the relay protein may or may not
instance, mutant mouse embryos lacking a certain G be phosphorylated by the tyrosine kinase). One tyrosine-
protein do not develop normal blood vessels and die in kinase receptor dimer may activate ten or more different
utero. Furthermore, G proteins ate involved in sensory intracellular protein simultaneously, triggering as many
reception; in humans, for example, both vision and smell different transduction pathways and particular cellular
depend on such proteins. Similarities in structure among responses. The ability of a single ligand-binding event to
G proteins and G-protein-linked receptors of modern trigger so many pathway is a key difference between
organisms suggest that G proteins and G-protein linked these receptors and G-protein receptors. Abnormal
receptors evolved very early, possibly as sensory recep- tyrosine-kinase receptors that aggregate even without
tors of ancient microbes. ligand cause some kinds of cancer.
G-protein systems are involved in many human diseases,

including bacterial infections. The bacteria that cause
cholera, pertussis (whooping cough), and botulism,
among others, make their victims ill by producing toxins
that interfere with G-protein function. Although drugs for
treating infections and other kinds of diseases have often
been discovered by trial and error, pharmacologists now
realize that up to 60% of all medicines used today exert
their effects by influencing G-protein pathways.
Tyrosine-Kinase Receptors
Among the chemical signals impinging on cells in an

animal's body are growth factors, the local regulators that
stimulate cells to grow and reproduce. Cell reproduction
involves a variety of activities of different parts of the
cell, including protein synthesis in the cytoplasm,
chromosome duplication in the nucleus, and the
rearrangement of elements of the cytoskeleton. Helping
the cell regulate and coordinate these activities is a type
of receptor specialized for triggering more than one
signal-transduction pathway at once.
The receptor for a growth factor is often a tyrosine-

kinase receptor, one of a major class of plasma-
membrane receptors characterized by having enzymatic
activity. Part of the receptor protein on the cytoplasmic
side of the membrane functions as an enzyme, called a
tyrosine kinase, that catalyzes the transfer of phosphate
groups from ATP to the amino acid tyrosine on a Ion-Channel Receptors
substrate protein. Thus tyrosine-kinase receptors are
membrane receptors that attach phosphates to protein Some membrane receptors of chemical signals are
tyrosines. Tyrosine-kinase receptors often have the Ligand-gated ion channels. These channels are protein
structure shown in rough schematic form in FIGURE 4. pores in plasma membrane that open or close in response
Before the signal molecule binds, the receptors exist as to chemical signal, allowing or blocking the flow of
individual polypeptides. Notice that each has an specific ions such as Na + or Ca2+. Like the other
extracellular signal binding site, an intracellular tail receptors we have discussed these channel proteins bind
containing a number of tyrosines, and a single ex helix a signal molecule as a ligand at the specific site on their
spanning the membrane. The binding of a signal extracellular side (FIGURE 5).
molecule to such a receptor does not cause enough of a
conformational change to activate the cytoplasmic side of The shape change
the protein directly. produced in the channel
protein immediately leads
Instead, the activation occurs in three steps: (1) The to a change in the
ligand binding causes two receptor polypeptides to concentration of a
aggregate, forming a dimer (a protein consisting of two particular ion inside the
polypeptides). (2) This aggregation activates the cell. Often this change
tyrosine-kinase parts of both polypeptides, each of which directly affects cell
then (3) phosphorylates the tyrosines on the tail of the functioning in some way.
other polypeptide. In summary, the effect of the signal At a synapse between
molecule a tyrosine-kinase receptor is polypeptide nerve cells, for example, it
aggregation and phosphorylation of the receptor. may trigger an electrical
signal that propagates

down the length of the receiving cell. Ligand-gated ion Keep in mind that the original signal molecule is not
channels are very important in the nervous system, as are physically passed along a signaling pathway; in most
gated ion channels that are controlled by electrical cases, it never even enters the cell. When we say that the
signals . signal is relayed along a pathway, we mean that certain
Intracellular Receptors: information is passed on. At each step the signal is
transduced into a different form, commonly a
Not all signal receptors are membrane proteins. Some are conformational change in a protein. Very often, the
proteins located in the cytoplasm or nucleus of target conformational change is brought about by
cells. To reach such a receptor, a chemical messenger phosphorylation.
must be able to pass through the target cell's plasma
membrane. A number of important signaling molecules e. Protein phosphorylation, a common mode of
can do just that, either because they are small enough to regulation in cells, is a major mechanism of signal
pass between the membrane phospholipids or because transduction
they are themselves lipids and therefore soluble in the
membrane. Chemical messengers with intercellular We have already seen how phosphorylation is involved
receptors include the steroid hormones and thyroid in the activation of tyrosine kinase receptors. In fact, the
hormones of animals, which are lipids; the small gaseous phosphorylation of proteins is a widespread cellular
molecule nitric oxide (NO)(which do not have known mechanism for regulating protein activity. The general
receptor and directly activate guanlate cyclase to produce name for an enzyme that transfers phosphate groups from
c-GMP as secondary messanger); and certain small ATP to a protein is protein kinase. Unlike receptor
signal molecules used by bacteria. tyrosine kinases, most cytoplasmic protein kinases act
not on themselves, but on other substrate proteins; also,
The behavior of testosterone is representative of steroid they phosphorylate their substrates on either of two
hormones: Secreted by cells of the testis, the hormone amino acids, serine or threonine. Such serine/threonine
travels through the blood and enters cells all over the kinases are widely involved in signaling pathways in
body. In target cells-those with testosterone receptor animals, fungi, and plants.
molecule in their cytosol – the hormone binds to the
receptor, activating it. The active form of the receptor Many of the relay molecules in signal-transduction path-
protein can then bind to and turn on genes in the nucleus ways are protein kinases, and they often act on each
that control male sex characteristics. Thus, the receptor other. FIGURE 6, shows a hypothetical pathway contain-
carries out the complete transduction of the signal. ing three different protein kinases. This sequence is
similar to many known pathways, including those
Prostaglandins, thrombaxones, prostacyclins are non- triggered in yeast by mating factors and in animal cells
polar but have membrane receptors. by many growth factors.
c. Signal Transduction Pathways: A cascade of protein phosphorylation, each, transmits the

signal bringing with it a conformational change. Each
When signal receptors are plasma-membrane proteins, shape change results from the interaction of the charged
like most of those we have discussed, the transduction phosphate groups with charged and polar amino acids.
stage of cell signaling is usually a multistep pathway. The addition of phosphates often changes a protein from
One benefit of such Pathways is signal amplification. If an inactive form to an active form (although in other
some of the molecules in a Pathway transmit the signal to cases phosphorylation decreases the activity of the
multiple molecules of the next components of in the protein).
series, the result can be a large number of activated
molecules at the end of the pathway. In other words, a
very small number of extracellular signal molecules can
produce a major cellular response. Moreover, multistep
pathways provide more opportunities for coordination
and regulation than simples systems do.
d. Pathway rely signals form receptor to cellular

responses:
The binding of a specific extracellular signal molecule to

receptor in the plasma membrane triggers the first step in
the chain of molecular interactions - the signal
transduction pathway-that leads to a particular response
within the cell. Like falling dominoes, the signal-
activated receptor activates another protein, which
activates another molecule and so on until the protein
that produces the final cellular response is activated. The
molecules that relay a signal from receptor to response,
sometimes called relay molecules, are mostly proteins.
The interaction of proteins is a major theme of cell
signaling. Indeed, protein interaction is a unifying theme
of all regulation at the cellular level.

The importance of protein kinases can hardly be over- cAMP does not persist for long in the absence of the
stated. Fully 1 % of our own genes are thought to code hormone, because another enzyme converts the cAMP to
for protein kinases. A single cell may have hundreds of an inactive product, AMP. Another surge of epinephrine
different kinds, each with specificity for a different is needed to boost the cytosolic concentration of cAMP
substrate protein Together they probably regulate a large again.
proportion of thousands of the proteins in a cell. Among
these are most of the proteins that, in turn, regulate cell
reproduction. Abnormal activity of such a kinase
frequently causes abnormal cell growth and contributes
to the development of cancer.
For a cell to respond normally to an extracellular signal it

must have mechanisms for turning off the signal-
transduction pathway when the initial signal is no longer
present. The effects of protein kinases are rapidly
reversed in the cell by protein phosphatases, enzymes
that remove phosphate groups from proteins. At any
given moment, the activity of a protein regulated by
phosphorylation depends on the balance in the cell
between active kinase and active phosphatase molecules.
When the extracellular signal molecule is not present,
active phosphatase molecules predominate, and the
signaling pathway and cellular response shut down.
f. Certain small molecules and ions are key Subsequent research revealed that epinephrine is only
components of signaling pathways (second one of many hormones and other signal molecules that
messengers) trigger pathways involving cAMP. It also brought to
light the other components of cAMP pathways, including
Not all components of signal-transduction pathways are G proteins, G-protein-linked receptors, and protein
proteins. Many signaling pathways also involve small, kinases (FIGURE 7). The relay molecule immediately
non-protein, water-soluble molecules or ions, called after cAMP in a signaling pathway is usually protein
second messengers. (The extracellular signal molecule kinase A, a serine/threonine kinase. Cyclic AMP
that binds to the membrane receptor is a pathway's “first activates this kinase. The active kinase then
messenger”) Because second messengers are both small phosphorylates various other proteins, depending on the
and water-soluble, they can readily spread throughout the cell.
cell by diffusion. For example, as we'll see shortly, it is a
second messenger called cyclic AMP that carries the Further fine-tuning of cell metabolism is provided by
signal initiated by epinephrine from the plasma other G-protein systems that inhibit adenylyl cyclase. In
membrane of a liver or muscle cell into the cell's interior, these systems, a different signal molecule activates a
where it brings about glycogen breakdown. Second mes- different receptor, which activates an inhibitory G
sengers participate in pathways initiated by both G- protein.
protein linked receptors and tyrosine-kinase receptors.
The two most widely used second messengers are cyclic Now that we know about the role of cAMP in G-protein
AMP and calcium ions, Ca2+. A large variety of relay signaling pathways, we can explain in molecular detail
proteins are sensitive to the cytosolic concentration of how certain microbes cause disease. Consider cholera, a
one or the other of these second messengers. disease that is frequently epidemic in places where the
water supply is contaminated with human feces. People
Cyclic AMP acquire the cholera bacterium, Vibrio cholerae, by
drinking contaminated water. The bacteria colonize the
Once Earl Sutherland had established that epinephrine lining of the small intestine and produce a toxin, which
somehow causes glycogen breakdown without passing in this case is an enzyme that chernically modifies a G
through the plasma membrane, the search began for the protein involved in regulating salt and water secretion.
second messenger (he coined the term) that transmits the Because the modified G protein is unable to hydrolyze
signal from the plasma membrane to the metabolic GTP to GDP, it remains stuck in its active form,
machinery in the cytoplasm. continuously stimulating adenylyl cyclase to make
cAMP. The resulting high concentration of cAMP causes
Sutherland found that the binding of epinephrine to the the intestine cells to secrete large amounts of water and
plasma membrane of a liver cell elevates the cytoplasmic salts into the intestines. An infected person quickly
concentration of a compound called cyclic adenosine develops profuse diarrhea and if left untreated can easily
monophosphate, abbreviated cyclic AMP or cAMP. An die from the loss of water and salts.
enzyme built into the plasma membrane, adenylyl
cyclase, converts ATP to cAMP in response to an Calcium Ions and Inositol Trisphosphate
extracellular signalin this case, epinephrine. Adenylyl
cyclase becomes active only after epinephrine binds to a Many signal molecules in animals, including
specific receptor protein. Thus the first messenger, the neurotransmitters, growth factors, and some hormones,
hormone, causes a membrane enzyme to synthesize induce responses in their target cells via signal-
cAMP, which broadcasts the signal to the cytoplasm. The transduction pathways that; increase the cytosolic

concentration of calcium ions (Ca2+). Calcium is even total protein). Calmodulin mediates many calcium-
more widely used than cAMP as a second messenger. regulated processes in cells. When calcium ions bind to
Increasing the cytosolic concentration of Ca2++ causes it, calmodulin changes conformation and then binds to
many responses in animal cells, including muscle cells to other proteins, activating or inactivating them. The
contraction, secretion of certain substances, and cell proteins most often regulated by calmodulin are protein
division. In plant cells, calcium functions as a second kinases and phosphatases-the most common relay
messenger in signaling pathways plants have evolved for proteins in signaling pathways.
coping with environmental stresses, such as drought or
cold. Cells use Ca2+ as a second messenger in both G- 6. Cellular Responses to Signals
protein pathways and tyrosine kinase receptor pathways.
We now take a closer look at the cell's eventual response
Although cell always contain Ca2+, this ion can function to an extracellular signal-what some researchers call the
as a second messanger because its concentration in the "output response: What is the nature of the final step in a
cytosol is normally much lower than the concentration signaling pathway?
outside the cell. In fact, the level of Ca2+ in the blood
and extracellular fluid of an animal often exceeds that in a. In response to a signal, a cell may regulate activities
the cytosol by more than 10,000 times. Calcium ions are in the cytoplasm or transcription in the nucleus
actively transported out of cell and are actively imported
from the cytosol into the endoplasmic reticulum (and, Ultimately, a signal-transduction pathway leads to the
under some conditions, into mitochondria and regulation of one or more cellular activities. The
chloroplasts). As a result, the calcium concentration in regulated activities may occur in the cytoplasm, such as a
the ER is usually much higher than in the cytosol rearrangement of the cytoskeleton, the opening or closing
(FIGURE 8). Because the cytosolic calcium level is low, of an ion channel in the plasma membrane, or some
a small change in absolute numbers of ions represents a aspect of cell metabolism. As we have discussed already,
relatively large percentage change in calcium the response of liver cells to signaling by the hormone
concentration. epinephrine helps regulate cellular energy metabolism.
The final step in the signaling pathway activates the
In response to a signal relayed by a signal-transduction enzyme that catalyzes the breakdown of glycogen.
Pathway, the cytosolic calcium level may rise, usually by
a mechanism that release Ca2+ from the cell’s ER. The Many other signaling pathways ultimately regulate not
pathway leading to calcium release involve still other the activity of an enzyme but the synthesis of enzymes or
second messengers, Diacyl glycerol (DAG) and inositol other proteins, usually by turning specific genes on or
triphosphate (IP3). These two messengers are produced off. Recall that the genes in a cell's DNA function by
by cleavage of a certain kind of phospholipid in the being transcribe into an RNA version called messenger
plasma membrane. FIGURE shows how this occurs and RNA (mRNA), which leaves the nucleus and is
how IP3 stimulates the release of calcium from the ER. translated into a specific protein by ribosomes in the
Because IP3 acts before calcium in the pathway, calcium cytoplasm. Special protein called transcription factors
could be considered a "third messenger:' However, control which genes are turned on-that is, transcribed into
scientists use the term second messenger for all small, mRNA-in a particular cell at a particular time. The
non-protein components of signal-transduction pathways. activity of a transcription factor may, itself be regulated
by a signaling pathway that extends into the cell nucleus.
All the different kinds of signal receptors and relay
molecules introduced in this chapter participate in gene-
regulating pathways, as well as in pathway leading to
other kinds of responses. The molecular messengers that
produce gene-regulation responses include grow factors
and certain plant and animal hormones.
b. Elaborate Pathways amplify and specify the cell’s

response to signals:
Why are there often so many steps between the original

signaling event at the cell surface and the cell’s
response? As Mentioned earlier, signaling pathways with
a multiplicity of steps have to important benefits: They
amplify the signal (and thus response), and they
contribute to the specificity of response.
c. Signal Amplification
Elaborate enzyme cascade amplifies the cell’s response

to signal. At each catalytic step in the cascade, the
In some cases, calcium ions activate a signal- number of activated products is much greater then in the
transduction protein directly, but often they function by preceding step. For example, in the epinephrine-
means of calmodulin, a Ca2+ -binding protein present at triggered pathway in Figure, each adenylate cyclase
high levels in eukaryotic cells (in an animal cell, for molecule catalyzes the formation of many c-AMP
example, calmodulin may represent as much as 1% of the molecules, each molecule of protein kinase A

phosophorylate many molecule of next kinase in the protein interacts both with microfilaments of the
pathway, and soon. The amplification effects depends the cytoskeleton and with several different components of
fact that these protein persists in active form long enough signaling pathways that relay information from the cell
to process numerous molecules of the substrate before surface, including pathways regulating immune cell
they become inactive again. As a result signal proliferation. This multifunctional relay protein is thus
amplification, a small number of epinephrine molecule both a branch point and an important intersection point in
binding to receptors on the surface of liver cell or a complex signal transduction network that controls
skeletal muscle can lead to a release of hundreds of immune cell behavior. When the WAS protein is absent,
millions of glucose molecule from glycogen. the cytoskeleton is not properly organized and signaling
pathways are disrupted, leading to the WAS symptoms.
d. The specificity of cell signaling:
Consider two different cells in your body-a liver cell and

heart muscle cell, for example. Both are in contact with
your bloodstream and are therefore constantly exposed to
many different hormone molecules, as well as to local
regulators secreted by nearby cells. Yet the liver cell
responds to some signals but ignores others, and the
same is true for the heart cell. And some kinds of signals
trigger responses in both cells but different responses.
For instance, epinephrine stimulates the liver cell to
break down glycogen, but the main response of the heart
cell to epinephrine is contraction, leading to a more rapid
heartbeat. How do we account for this difference?
The explanation for the specificity exhibited in cellular

responses to signals is the same as the basic explanation
for virtually all differences between cells: Different kinds
of cells have different collections of proteins (FIGURE
9).
The response of a particular cell to a signal depends on

its particular collection of signal receptor proteins, relay
proteins, and proteins needed to carry out the response. A
liver cell, for example, is poised to respond appropriately
to epinephrine by having the proteins, as well as those
needed to manufacture glycogen.
To keep FIGURE 9 simple, we have not indicated the
Thus two cells that respond differently to the same signal inactivation mechanisms that are an essential aspect of
differ in one or more of the proteins that handle and cell signaling. For a cell of a multicellular organism to
respond to the signal. Notice in FIGURE 9 that pathways remain alert and capable of responding to incoming
may some molecules in common. For example, cells A, signals, each molecular change in its signaling pathways
B, C all use the same receptor protein for the triangular must last only a short time. As we saw in the cholera
signal molecule; differences in other proteins account for example, if a signaling pathway components become
their differing responses. locked into one state, whether active or inactive, dire
consequences for the organism can result.
Also note that in cell B, a pathway triggered by a single
kind of signal diverges to produce two responses, and Thus a key to a cell’s continuing receptiveness to
that in cell C, two pathways triggered by separate signals regulation is the reversibility of the changes that signals
converge to modulate a single response. Branching of produce. The binding of signal molecules to receptors is
pathways and "cross-talk" between pathways are reversible, which means that the lower the concentration
important in regulating and coordinating a cell's of signal molecules the fewer will be bound at any given
responses to incoming information. Moreover, the use of moment. When they leave the receptor, the receptor
some of the same proteins in more. than one pathway reverts to its inactive form Then, by a variety of means,
allows the cell to recognize on number of different the relay molecules return to their inactive forms: The
protein it must make. The importance of relay proteins GTPase activity intrinsic to a G protein hydrolyzes its
that serve as points of branching or intersection in bound GTP; the enzyme phosphodiesterase converts
signaling pathways is highlighted by the problems cAMP to AMP; protein phosphatases inactivates
arising when these proteins are defective or missing. For phosphorylated kinases and other proteins; and so forth.
instance, in an inherited disorder called Wiskott Aldrich As a result, the cell is soon ready to respond to a fresh
syndrome (WAS), the absence of a single relay protein signal.
leads to such diverse effects as abnormal bleeding,
eczema, and a predisposition to infections and leukemia.
These symptoms are thought to arise primarily from the
absence of the protein in cells of the immune system. By
studying normal cells, scientists found that the WAS
protein is located just beneath the cell surface. The

A3. SIGNAL TRANSDUCTION IN BACTERIA & BACTERIAL CHEMOTAXIS
Most prokaryotic signal-transduction systems and a few Accepting Chemotaxis Proteins (MCPs), because they
eukaryotic pathways use phosphotransfer schemes are reversibly methylated. Repellents and some
involving two conserved components, a histidine protein attractants bind to periplasmic proteins, which then
kinase and a response regulator protein. The histidine interact with the receptors. For eg. the attractant aspartate
protein kinase, which is regulated by environmental binds directly to tar protein, which is also known as
stimuli, autophosphorylates at a histidine residue, aspartate receptor.
creating a high-energy phosphoryl group that is
subsequently transferred to an aspartate residue in the The four chemoreceptors have the same design. Each
response regulator protein. Phosphorylation induces a ~60 KD protein contains four regions:
conformational change in the regulatory domain that 1) A periplasmic domain that binds an attractant or a
results in activation of an associated domain that effects repellent,
the response. The basic scheme is highly adaptable, and 2) A transmembrane segment consisting of two α helices,
numerous variations have provided optimization within 3) A cytosolic system and
specific signaling systems. The domains of two- 4) A transmembrane region that can be reversibly
component proteins are modular and can be integrated methylated at several sites.
into proteins and pathways in a variety of ways, but the
core structures and activities are maintained. Thus Their cytosolic domains are highly conserved because
detailed analyses of a relatively small number of they interact with the same components of the processing
representative proteins provide a foundation for system. In fact, the cytosolic domains of tsr and tar
understanding this large family of signaling proteins. contain an identical 48 residue sequence. In contrast,
their periplasmic domains are distinctive because they
Chemotaxis bind different attractants, repellents, and attractant-
protein complexes. A chimeric receptor formed from the
Chemoreceptor on bacteria detects attractant and amino-terminal half of tar and the carboxy-terminal half
repellent and send signal to flagella. Some of these of tsr is triggered by aspartate and other attractant that
molecules as aspartate, bind directly to chemoreceptors, are normally sensed by tar. Thus, these chemoreceptors
whereas others are carried by binding proteins. transmit signals across the membrane in essentially same
way.
The direction of rotation of flagella determines whether
bacteria swim smooth or tumble. Bacterium flagella is an
extra cellular appendage that is rotated by a motor
located at the junction of flagellum and cell envelope. It
is powered by the proton-motive force across the plasma
membrane. An intriguing feature of this motor is that it
can rotate clockwise or counterclockwise, and switch
between them almost instantaneously.
When the flagella rotate counterclockwise, the helical

filaments form a coherent bundle and cell swims
smoothly. When the flagella rotate clockwise, in contrast,
the bundle flies apart because the screw sense of the
helical flagella does not match the direction of rotation.
Each flagellum then pulls in a different direction, and so
the bacterium tumbles. The three dimensional structure of periplasmic domain of
aspartate receptor dimerizes to form a highly elongated
When a bacterium moves toward an increasing structure. Each subunit is four helix bundle that is 20 Ao
concentration of an attractant, tumbling becomes less in diameter and more than 70 Ao long. The binding sites
frequent. In contrast, when it moves away from the for aspartate are at the interface between subunits, more
attractant, tumbling becomes more frequent. Repellent than 60 Ao from the membrane surface.
have opposite effect on tumbling frequency. Tumbling
serves as reorient a misdirected bacterium. Bacteria A receptor triggered phosphorylation cascade controls
possesses a temporal sensing mechanism i.e a bacterium the direction of flagellar movement: Genetic analysis
detects a spatial gradient of attractant not by comparing revealed the existence of common processing genes and
the concentration at its head and tail, but by traveling provided valuable clues concerning its molecular
through space and comparing its observations through circuitry. Eight che loci have been identified: A, B, C, D,
time. W, R, Y and Z. mutation in these genes impair
chemotaxis to all attractant and repellent, in contrast with
Chemotaxis begins with the binding of molecules to four mutation in a particular kind of receptor will block
kinds of receptors in the plasma membrane. In E.coli and responses to a subset of these agents. Bacteria devoid of
other gram negative bacteria, the PM is surrounded by an all cytosolic che proteins (known as gutted strains)
outer membrane that is permeable to small molecules; a swims smoothly all the time; because their flagella rotate
periplasmic space separates the PM from the outer counterclockwise(CCW) continuously. The addition of
membranes. The chemoreceptors are encoded by the tsr, cheY proteins to gutted stains, by controlling expression
tar, trg and tap genes and were originally called Methyl of cheY gene, lead to increased clockwide(CW) rotation.

Furthermore, flagella rotates exclusively CW in mutant histidine residue. This auto phosphorylation is greatly
that express abnormally high levels of cheY protein in accelerated by the binding of cheA, together with che W,
the presence of normal amounts of the other che proteins. to an unoccupied receptor or a repellent receptor
These finding indicates that the direction of flagellar complex but not to an attractant receptor complex. The
rotation is controlled by the che Y protein. phosphoryl group attached to cheA can then be
transferred to cheY. Hence, repellent increase the level
The essentiality of ATP for both the excitation and of phosphorylated cheA and, in turn, of phosphorylated
adaptation phases of chemotaxis provides a tantalizing cheY. Attractant have the opposite effect. In this way,
hint as to how che Y might be controlled. A specific repellent increase CW rotation and tumbling, whereas
aspatrate of che Y can become phosphorylated. Most attractants increase CCW rotation and smooth
significant, phosphorylated cheY induces tumbling, swimming.
whereas dephosphorylated cheY has virtually no effect
on flagellar switch. Phosphorylated cheY is transient It is interesting to note that cheA belongs to a family of
species. These attached phosphoryl group is regulatory protein that enables the bacteria to sense their
simultaneously hydrolyzed in a reaction that is increased environment and internal metabolic state. For eg. NtrB,
100 fold by che Z. which is a homologue of cheA, controls the expression
of genes that respond to nitrogen balance in cell. All
How does cheY acquire phosphate group and how its homologues of cheA are auto phosphorylating kinases
phosphorylation controlled by chemoreceptor? Melsimon that regulate the activity of target proteins by
found that purified cheA protein undergoes a slow auto phosphorylating them. These sensor effector pairs are
phosphorylation. The site of phosphorylation is N-3 of called two component regulatory systems.

Quorum sensing in Bacteria and other Organisms:

Quorum sensing is a type of decision-making process mutualistic symbiont in the photophore (or light-
used by decentralized groups to coordinate behavior. producing organ) of the Hawaiian bobtail squid. When V.
Many species of bacteria use quorum sensing to fischeri cells are free-living (or planktonic), the
coordinate their gene expression according to the local autoinducer is at low concentration and thus cells do not
density of their population. Similarly, some social insects luminesce. However, when they are highly concentrated
use quorum sensing to make collective decisions about in the photophore (about 1011 cells/ml) transcription of
where to nest. In addition to its function in biological luciferase is induced, leading to bioluminescence.
systems, quorum sensing has several useful applications
for computing and robotics. Escherichia coli: In the Gram-negative bacteria
Escherichia coli, cell division may be partially regulated
Quorum sensing can function as a decision-making by Autoinducer (AI-2)-mediated quorum sensing. This
process in any decentralized system, as long as individual species uses AI-2, which is produced and processed by
components have (a) a means of assessing the number of the lsr operon. Part of it encodes an ABC transporter
other components they interact with and (b) a standard which imports AI-2 into the cells during the early
response once a threshold number of components is stationary (latent) phase of growth. AI-2 is then
detected. phosphorylated by the LsrK kinase, and the newly
produced phospho-AI-2 can either be internalized or used
Bacteria to suppress LsrR, a repressor of the lsr operon (thereby
activating the operon). Transcription of the lsr operon is
Some of the best-known examples of quorum sensing also thought to be inhibited by dihydroxyacetone
come from studies of bacteria. Bacteria use quorum phosphate (DHAP) through its competitive binding to
sensing to coordinate certain behaviors based on the local LsrR. Glyceraldehyde 3-phosphate has also been shown
density of the bacterial population. Quorum sensing can to inhibit the lsr operon through cAMP-CAPK-mediated
occur within a single bacterial species as well as between inhibition. This explains why when grown with glucose
diverse species, and can regulate a host of different E. coli will lose the ability to internalize AI-2 (because of
processes, essentially serving as a simple communication catabolite repression). When grown normally, AI-2
network. A variety of different molecules can be used as presence is transient.
signals.
Pseudomonas aeruginosa: The opportunistic bacteria
Pseudomonas aeruginosa uses quorum sensing to
Mechanism coordinate the formation of biofilms, swarming motility,
exopolysaccharide production, and cell aggregation.
Bacteria that use quorum sensing constantly produce and These bacteria can grow within a host without harming
secrete certain signaling molecules (called autoinducers it, until they reach a certain concentration. Then they
or pheromones). These bacteria also have a receptor that become aggressive, their numbers sufficient to overcome
can specifically detect the signaling molecule (inducer). the host's immune system and form a biofilm, leading to
When the inducer binds the receptor, it activates disease. In this species, AI-2 was found to increase
transcription of certain genes, including those for inducer expression of sdiA, a transcriptional regulator of
synthesis. There is a low likelihood of a bacterium promoters which promote ftsQ, part of the ftsQAZ
detecting its own secreted Acetyl Homoserine Lactone operon essential for cell division. Another form of gene
(AHL) . Thus, in order for gene transcription to be regulation which allows the bacteria to rapidly adapt to
activated, the cell must encounter signaling molecules surrounding changes is through environmental signaling.
secreted by other cells in its environment. When only a Recent studies have discovered that anaerobiosis can
few other bacteria of the same kind are in the vicinity, significantly impact the major regulatory circuit of QS.
diffusion reduces the concentration of the inducer in the This important link between QS and anaerobiosis has a
surrounding medium to almost zero, so the bacteria significant impact on production of virulence factors of
produce little inducer. However, as the population grows this organism. It is hoped that the therapeutic enzymatic
the concentration of the inducer passes a threshold, degradation of the signaling molecules will prevent the
causing more inducer to be synthesized. This forms a formation of such biofilms and possibly weaken
positive feedback loop, and the receptor becomes fully established biofilms. Disrupting the signalling process in
activated. Activation of the receptor induces the up this way is called quorum quenching.
regulation of other specific genes, causing all of the cells
to begin transcription at approximately the same time. Social Insects
This coordinated behavior of bacterial cells can be useful
in a variety of situations. For instance, the Social insect colonies are an excellent example of a
bioluminescent luciferase produced by V. fischeri would decentralized system, because no individual is in charge
not be visible if it were produced by a single cell. By of directing or making decisions for the colony. Several
using quorum sensing to limit the production of groups of social insects have been shown to use quorum
luciferase to situations when cell populations are large, sensing when making collective decisions.
V. fischeri cells are able to avoid wasting energy on the
production of useless products. Examples
Examples Ants: Colonies of the ant Temnothorax albipennis nest in

Vibrio fischeri: Quorum sensing was first observed in small crevices between rocks. When the rocks shift and
Vibrio fischeri, a bioluminiscent bacterium that lives as a the nest is broken open, these ants must quickly choose a

new nest to move into. During the first phase of the options, quorum sensing enables the colony as a whole to
decision-making process, a small portion of the workers quickly make good decisions about where to move.
leave the destroyed nest and search for new crevices.
When one of these scout ants finds a potential nest, she Honeybees: Honey bees (Apis mellifera) also use
assesses the quality of the crevice based on a variety of quorum sensing to make decisions about new nest sites.
factors including the size of the interior, the number of Large colonies reproduce through a process called
openings (based on light level), and the presence or budding, in which the queen leaves the hive with a
absence of dead ants. The worker then returns to the portion of the workers to form a new nest elsewhere.
destroyed nest, where she will wait for a short period After leaving the nest, the workers form a swarm that
before recruiting other workers to follow her to the nest hangs from a branch or overhanging structure. This
she found using a process called tandem running. The swarm persists during the decision-making phase until a
waiting period is inversely related to the quality of the new nest site is chosen.
site- for instance a worker that has found a poor site will
wait longer than a worker that encountered a good site. The quorum sensing process in honey bees is similar to
As the new recruits visit the potential nest site and make the method used by Temnothorax ants in several ways. A
their own assessment of its quality, the number of ants small portion of the workers leave the swarm to search
visiting the crevice increases. During this stage ants may out new nest sites, and each worker assesses the quality
be visiting many different potential nests. However, of the cavity she finds. The worker then returns to the
because of the differences in the waiting period the swarm and recruits other workers to her cavity using the
number of ants in the best nest will tend to increase at the honey bee waggle dance. However, instead of using a
greatest rate. Eventually, the ants in this nest will sense time delay, the number of dance repetitions the worker
that the rate at which they encounter other ants has performs is dependent on the quality of the site. Workers
exceeded a particular threshold, indicating that the that found poor nests stop dancing sooner, and can
quorum number has been reached. Once the ants sense a therefore be recruited to the better sites. Once the visitors
quorum, they return to the destroyed nest and begin to a new site sense that a quorum number (usually 10 to
rapidly carrying the brood, queen, and fellow workers to 20 bees) has been reached, they return to the swarm and
the new nest. Scouts that are still tandem-running to begin using a new recruitment method called piping. This
other potential sites are also recruited to the new nest and vibration signal causes the swarm to take off and fly to
the entire colony moves.Thus although no single worker the new nest location. In an experimental test this
may have visited and compared all of the available decision-making process enabled honey bee swarms to
choose the best nest site in four out of five trials.
Plant Two Component Signaling system
The "Prokaryotic" Lifestyle of Plants conserved His residue (Fig. 1A). The phophoryl group is
then transferred to an invariant Asp within a conserved
At first glance, a 100-year-old tree and a bacterium do domain of the receiver (Fig. 1A). This His-to-Asp
not have much in common. However, both plants and phosphorelay controls the activity of the output domain
bacteria face a similar problem: They are confined to of the response regulator, which results, for example, in
their environment and have to endure and adapt to a interaction with other proteins or binding to DNA.
broad array of environmental conditions. To manage this
complex task, prokaryotic organisms rely on the two-
component signaling system, which is involved in such
diverse processes as chemotaxis, osmotic sensing, and
light perception (Stock et al., 2000). As shown in recent
years, all canonical elements of two-component signaling
systems are also found in plants (Schaller et al., 2001).
This Update focuses on recent advances in our
understanding of plant two-component systems, with
particular emphasis on the function of plant response
regulators.
Protein Elements and Biochemistry of Two-

Component Signaling Systems
In its simplest form, the two-component signaling system

is composed of two elements: a His kinase and a
response regulator (Fig. 1A). Although the majority of
prokaryotic two-component signaling systems are
composed of just these two proteins, there also exist Figure 1. Schematic representation and basic features
more complex signaling systems that include additional of the simple (A) and the multistep (B) two-component
elements, which are referred to as multistep, two- signaling systems. Signal perception by the input domain
component systems (Fig. 1B). The biochemistry of two- of the His or hybrid kinase induces autophosphorylation
component signal transduction is a phosphorelay: After a of the transmitter at a conserved His residue (H). The
signal-induced activation by the input-domain, the phosphate (P) is relayed to a conserved Asp residue (D)
transmitter of a His kinase autophosphorylates at a that is localized either in the receiver of the cognate

response regulator (A) or, in the case of hybrid kinases, two-hybrid system. However, it has not yet been
in an attached receiver domain (B). In the multistep, two- demonstrated that ETR1 and the other members of the
component signaling system, His-containing ethylene receptor family act as ethylene-triggered hybrid
phosphotransfer (HPt) domain proteins serve as kinases in plants.
phospho-His intermediates between the hybrid kinase
and the response regulator.
The outlined one-step, two-component signaling system

can be extended into a multistep system by including
additional protein modules containing phosphorylatable
Asp or His residues. The second Asp is again included in
a receiver module. In plants, the additional receiver
module is often carboxyterminally fused to the His
kinase, thereby creating a hybrid type of sensor kinase
(Fig. 1B). The second His is embedded in a domain
known as the HPt domain. The HPt domain enables the
transfer of a phosphoryl group between two receiver
modules. Many of the prokaryotic HPt domains are
physically linked to other two-component signaling
elements, but the Arabidopsis HPt domains are all
individual proteins.
Figure 2. Structure of representative members of two-
Why are some two-component systems of the simple and component signaling elements from Arabidopsis. A,
others of the complex type? The reason for extending the Hybrid kinases: CRE1 and ETR1. The function of the
complexity of two-component systems does not GAF-related domain of ETR1 is unknown. B, ARR:
apparently lie in signal amplification. Within a two- ARR2, type-B response regulator; ARR4, type-A
component system, the signal is relayed as a phosphoryl response regulator. The GARP motif of type-B response
group in a strict one-to-one manner. Thus, the signaling regulators is responsible for DNA binding and the
output of the simple and the multistep, two-component nuclear localization sequences (NLS) for nuclear
system is the same. If it is not the gain in signal targeting. The Pro- and Gln-rich (P/Q-rich) domain
amplification, what could be the reason behind the serves as transactivation domain, which may interact
apparent preference of multistep systems in plants? The with the basal transcription machinery. C, AHP: AHP1
answer to this question will become clear in subsequent and AHP2. The conserved amino acid residues (H, His;
sections. D, Asp) of the different two-component elements are
indicated.
Two-Component Signaling Elements of Arabidopsis
If ethylene is signaling through a two-component system,
In the early 1990s, the cloning of three genes led to the one would expect to identify mutants affected in genes
breakdown of a dogma. Until then, it was believed that coding for HPt domain proteins and response regulators.
two-component signaling systems were restricted to Such mutants have not been reported, potentially because
prokaryotes. However, with the realization that the of functional redundancy within these signaling
Arabidopsis ethylene receptor ETR1, the phytochrome elements. The element shown to act downstream of the
photoreceptors, and the yeast (Saccharomyces ethylene receptors and found capable of physically
cerevisiae) osmonsensor SLN1 were all related to His interacting with ETR1and ERS1 is CTR1 (constitutive
kinases, it became clear that some genera of eukaryotes triple response 1), which is thought to function as a RAS-
depended on two-component systems as well. With like mitogen-activated protein kinase kinase kinase.
sequencing of the Arabidopsis genome now completed,
17 His and hybrid kinase-like proteins (AHK), five HPt Thus, it appears that the hybrid kinase-like ethylene
proteins (AHP), and 23 response regulators (ARR) have receptors can function in a unique way linking the
been identified in Arabidopsis. Although analysis has perception of the hormone to the activity of a mitogen-
just begun, it is evident that, with the exception of activated protein kinase cascade. However, although
phytochromes, most AHKs, AHPs, and ARRs in CTR1 is a central component in ethylene signal
principle function as phosphotransfer proteins. transduction, there are several indications for the
existence of additional signaling elements: (a) ctr1-null
Ethylene Perception And Signaling mutants are still slightly responsive to ethylene, and (b)
the phenotype of quadruple etr1-etr2-ein4-ers2 receptor
The ethylene perception and transduction system is one mutants is more pronounced in comparison with that of
of the best understood plant signaling pathways. The ctr1. With AHP and ARR knockout lines now in hand, it
initial identification of ETR1 (Fig. 2A) was followed by should soon be possible to determine whether two-
the description of four additional ethylene receptors component proteins represent these additional ethylene
either belonging to the hybrid kinase type (ETR2 and signaling components.
EIN4) or the His kinase type (ERS1 and ERS2). By
generating ethylene-binding sites in a heterologous yeast The recent advances in our understanding of cytokinin
system and in genetic studies, it was elegantly shown that signaling offer important insights into plant two-
ETR1 is a membrane-associated ethylene receptor (Fig. component systems. In 1996, a putative hybrid kinase
2A). ETR1 exhibits His autophosphorylation activity in (CKI1) was described by Kakimoto, which, when
vitro and interacts with several AHP proteins in the yeast overexpressed, resulted in cytokinin-independent growth

of calli. However, most of the important advances came type-B ARRs localized to the nucleus and were
just recently. By screening for the loss of cytokinin- transcriptionally active. The first hint pointing to
dependent greening and shoot formation of hypocotyl potential target promoters came with the histochemical
explants from Arabidopsis, the cre1 (cytokinin response analysis of the expression pattern of an ARR2
1) mutant was identified. The CRE1 gene codes for a promoter::uidA construct in transgenic Arabidopsis
hybrid kinase with an aminoterminal cytokinin-binding plants. Although the ARR2 gene was expressed in almost
domain flanked by two transmembrane stretches (Fig. all Arabidopsis organs, particularly high -glucuronidase
2A). In contrast to ETR1, CRE1 contains a second activity was observed in pollen grains. A similar
receiver-like domain downstream of the transmitter (Fig. expression pattern is described for the nuclear genes for
2A). When expressed in yeast or fission yeast components of the mitochondrial respiratory complex I
(Schizosaccharomyces pombe) strains, in which the (nCI genes). The pollen-specific activity of the nCI genes
endogenous hybrid kinases were knocked out, the CRE1 is mediated by a conserved promoter element called the
gene product rescued the altered growth phenotypes of pollen box. Lohrmann and colleagues (2001)
these mutants in the presence of cytokinin. These results demonstrated sequence-specific binding of ARR2 to the
indicate that (a) CRE1 can bind cytokinin, (b) cytokinin pollen box element and narrowed down the binding
binding induces the His kinase activity of CRE 1, and (c) sequence to a 5'-W/GAT/W-3' core, where W is either A
CRE1 can transfer the phosphoryl group to the intrinsic or T. This sequence matches the consensus binding site
HPt domain proteins of yeast and fission yeast initiating identified independently by Sakai et al. (2000) using the
multistep, two-component signaling cascades. The GARP domains of ARR1 and ARR2 in an oligo-
function of CRE1 as a sensor His kinase in planta is also selection approach. With the identification of the nCI
suggested by recent studies performed in Arabidopsis genes as targets and with the demonstration of
protoplasts. However, although CRE1 is a cytokinin transactivation capability, it is evident that ARR1,
receptor with His kinase activity, it is probably not ARR2, and presumably the other type-B ARRs function
responsible for mediating all of the plant cytokinin as transcription factors inside the nucleus (Fig. 3).
responses. Two additional CRE1-like His kinases in
Arabidopsis, AHK2 and AHK3, presumably also
function as cytokinin receptors.
Arabidopsis Response Regulators And Hpt Domain

Proteins
Although His kinase-like proteins were described in the

early 1990s, it required almost a decade for other two-
component signaling elements to be identified in higher
plants, namely response regulators and HPt domain
proteins. According to sequence homology, domain
composition, and transcriptional regulation, most
Arabidopsis ARRs can be described as either type A or Figure 3. Model of an Arabidopsis multistep
B. The type-A response regulators are relatively small phosphorelay, demonstrating signal transduction from
and contain a receiver domain along with short C- the extranuclear membranes to the nucleus. Signal
terminal extensions (Fig. 2B). The mRNA of type-A perception by the input domain of a membrane-bound
ARRs is rapidly accumulating to varying extent after sensor His kinase (e.g. CRE1) induces
cytokinin treatment. The type-B ARRs have receiver autophosphorylation of the transmitter. The phosphate
domains along with an extended C-terminal output (P) is relayed to the cognate AHP (e.g. AHP2) interacting
domain and are not induced by cytokinin (Fig. 2B). with the cytosolic receiver module of the kinase. The
Recent microarray studies, however, indicate that the phosphorylated AHP shuttles to the nucleus and transfers
expression of the type-B response regulator ARR2 is the phosphate to the receiver domain of its cognate type-
down-regulated by the phytochrome-A photoreceptor in B response regulator (e.g. ARR1 and ARR2). The
seedlings. We will now highlight recent insights into the dephosphorylated AHP shuttles back to the cytosol
function of type-A and type-B ARRs. where it can be rephosphorylated. Phosphorylation of the
receiver domain of the response regulator results in a
Type-B ARRs: Response Regulators With conformational change in its output domain. This
Transcription Factor Properties conformational change may either induce binding of
type-B ARRs to their target promoter sequence (e.g. the
The comparison of the amino acid sequences of the GAT box in the case of ARR1 and ARR2) and/or
output domains of type-B ARRs revealed a highly activation of their transcriptional capacity. The promoter-
conserved stretch of about 60 amino acids. This stretch associated, active response regulators triggers the
was given the name GARP motif according to the transcription of its target genes (e.g. nCI genes, type-A
founding members Golden2, ARR, and Psr1 (Fig. 2B). ARR genes). The conserved amino acid residues (H, His;
The GARP motif is predicted to fold into an -helical D, Asp) of the different two-component moduls are
structure and is related to the DNA-binding motifs of the indicated. POLII, RNA polymerase II-dependent basal
basic helix-loop-helix and the MYB classes of transcription machinery.
transcription factors. Thus, it was proposed that the
GARP motif represented a novel DNA-binding domain, Recent evidence suggests that the type-B response
and that type-B ARRs might function as transcription regulators ARR1 and ARR2 are involved in cytokinin
factors. By studying the subcellular distribution and signaling. An arr1-null mutant was found to be partially
transactivation capability in vivo, it became evident that resistant to cytokinin in shoot regeneration and root

elongation, whereas ARR1-overexpressing lines suppression. This release could potentially be induced by
displayed the opposite, hypersensitive phenotype. phosphorylation of the highly conserved Asp in the
Similar alterations in sensitivity to cytokinin were receiver domain of ARR1.
observed when the expression of several type-A ARR
genes including ARR4 were analyzed in the arr1 mutant The finding that type-B ARRs are localized to the
and ARR1-overexpressing lines. A comparable nucleus raises the question as to how the signal
cytokinin-inducible effect of ARR1 and ARR2 on the information perceived by the extranuclear and
expression of an ARR6 (type-A ARR)::reporter construct membrane-bound sensor His kinases is transduced to the
was observed in Arabidopsis mesophyl protoplasts. nuclear compartment. For instance, one of the cognate
These results suggest that ARR1 and ARR2 mediate the sensor His kinases of ARR1 and ARR2 is very likely the
cytokinin-induced expression of type-A ARRs (Fig. 4). It cytokinin receptor CRE1, which is predicted to be
is of interest to note that ARR2 very strongly induced the present on an extranuclear membrane. Moreover,
ARR6::reporter gene expression in Arabidopsis protein-protein interaction studies revealed that the
protoplasts even in the absence of cytokinin. This sensor His kinases CKI1, CRE1, and ETR1 cannot
observation is indicative for the presence of a further directly interact with ARR1 and ARR2. As outlined in
stimulus that enables the activation of ARR2 and, in turn, Figure 1B, the Arabidopsis HPt domain proteins could
ARR2-regulated target genes in protoplasts. perform the information transfer as part of a multistep,
two-component phosphorelay. His kinases (ETR1, CKI1,
and CRE1) as well as response regulators (ARR2) can
interact with AHPs, indicating that HPt domain proteins
may serve as molecular adaptors. AHPs are localized to
the cytosol as well as the nucleus of the plant cell, and
thus have the ability to perform functions in both
compartments. Although the AHP proteins have a size of
around 12 kD and may enter the nucleus by diffusion, at
least some AHPs transiently accumulate in the nucleus of
Arabidopsis protoplasts in response to cytokinin
application. These observations support the following
model of AHP action (Fig. 3). After activation by the
specific signal the sensor His-kinase autophosphorylates
and then transfers the phosphoryl group to the cognate
Figure 4. Model for the action of an ARR4-comprising AHP. The AHP then shuttles into the nucleus and relays
two-component signaling system on phytochrome B the phosphate to nuclear type-B ARRs. Phosphorylation
signal transduction. Red light (R) induces the of the receiver module triggers the transcription factor
photoconversion of the inactive Pr form of phytochrome properties of type-B ARRs, which results in an altered
B (phyB) in the physiologically active Pfr form. activity of their target genes. The dephosphorylated AHP
Phosphorylated ARR4 then stabilizes the active Pfr form then moves back to the cytoplasmic compartment to be
by inhibition of phyB dark reversion (dark rev.). Pfr rephosphorylated. This model would indicate that higher
stabilization enhances the sensitivity of plants to red light plants have adapted the ancestral two-component
and induces more intense photomorphogenic responses systems to a novel type of eukaryotic intracellular
(phyB signaling). The phosphorylation state and the signaling mechanism. Because of the necessity for a
activity of ARR4 is potentially regulated by a multistep shuttling HPt domain protein, this adaptation could only
phosphorelay comprising of the HPt protein AHP1 and be achieved by use of multistep, two-component
presumably CRE1-like hybrid kinases (two-component systems.
signaling). Therefore, ARR4 enables cross talk and thus
integration of signal information provided by hormone TYPE-A ARRs: Response Regulators Modulating
(cytokinin) and the red-light signaling cascades (cross Other Signal Transduction Pathways
talk). The expression of ARR4 and the protein
composition of the two-component system are regulated As mentioned above, the transcription of type-A
by cytokinin (transcriptional regulation). The conserved response regulator genes like ARR4 is rapidly induced
amino acid residues (H, His; D, Asp) of the different by cytokinin. This and some additional criteria
two-component modules are indicated. FR, Far-red light; established the hypothesis that type-A ARRs are primary
P, phosphate residue. response genes of cytokinin. Recent studies suggest that
the cytokinin-regulated transcriptional control of type-A
From the study in Arabidopsis protoplasts, it is not clear ARR genes is mediated by type-B response regulators
whether the activities of ARR1 and ARR2 are regulated Fig. 4). An effector protein recently was identified for a
by Asp phosphorylation. This raises the question of type-A response regulator, thereby lending insight into
whether cytokinin signals through a canonical the signaling mechanisms that operate downstream of
phosphorelay to regulate the transcription factor these response regulators. Sweere and colleagues (2001)
properties of type-B response regulators. However, the demonstrated that ARR4 specifically interacts with the
conservation of the canonical phosphorylation sites in the plant photoreceptor phytochrome B. It is interesting that
type-B ARRs (and also the type-A ARRs) are strongly ARR4 did not interact with the COOH terminus of
indicative of the functional relevance of these sites. phytochrome B, which is related to transmitter domains
Moreover, Sakai and colleagues (2001) provided in of His kinases, but with the extreme NH2-terminus.
planta evidence that the phospho-accepting receiver
domain suppresses the function of ARR1 in the absence Phytochrome B is a red/far-red reversible chromoprotein,
of cytokinin and that the cytokinin signal releases this which regulates red light-specific photomorphogenetic

responses in plants. It is synthesized in the inactive Pr A Complex Plant Two-Component Signaling Network
form and photoconverted by red light into the Emerges: Conclusions And Perspectives
physiologically active Pfr form. Far-red light is able to
induce the photoconversion of the active Pfr form back to As described above, there is now the first experimental
the inactive Pr form. In addition, active Pfr converts back evidence that hybrid kinases like CRE1, AHK2, and
to inactive Pr in a light-independent manner known as AHK3 signal through multistep phosphorelays
dark reversion, which is thought to be an additional containing AHPs and ARRs. In addition, a wealth of
switch-off mechanism for phytochromes. Independent yeast two-hybrid and in vitro protein-protein interaction
approaches in yeast and in Arabidopsis indicated that the studies indicate the possibility of a complex two-
interaction of ARR4 with phytochrome B resulted in the component signaling network in cells of higher plants. A
stabilization of the physiologically active Pfr form by detail of such a potential network is shown in Figure 5.
inhibition of Pfr-to-Pr dark reversion (Fig. 4). Specificity to some of these interactions can be
recognized. For example, ETR1 and CRE1 interact with
The functional relevance of the phytochrome B:ARR4 both AHP1 and AHP2, whereas CKI1 interacts with only
interaction and Pfr stabilization was examined in AHP2 (Fig. 5). Specificity is also apparent for the
transgenic plants overexpressing ARR4. These plants interaction of ARRs with AHPs. ARR2 interacts with
displayed a hypersensitivity to red light in respect to all AHP1 and AHP2, whereas the interaction of ARR4 is
photomorphogenic responses tested (e.g. hypocotyl restricted to AHP1 (Fig. 5). For instance, as indicated in
elongation, root growth, and flowering time). Moreover, previous sections, two-component signaling elements
transgenic Arabidopsis plants expressing a mutant form from Arabidopsis can participate in phosphorelays from
of ARR4, which was not phosphorylated at the biologically irrelevant fungal systems. However, even
conserved Asp residue in a plant cell-free phosphorelay given the preliminary nature of this emerging picture,
system, displayed hyposensitive phenotypes to red light cross talk between different two-component signaling
in hypocotyl elongation and flowering time. These data pathways would have major functional implications.
suggests that the mutant protein exerts a dominant- Two-component systems may form a complex signaling
negative effect on the function of endogenous ARR4 and network capable of sensing, computing, and integrating a
that Asp phosphorylation of ARR4 plays an important broad range of signals. The sessile lifestyle of plants
role in modulating phytochrome B activity. It is makes it necessary that every cell monitors and integrates
interesting that the above-mentioned wild-type and a multitude of inputs to reach a finely balanced decision
mutant ARR4-overexpressing lines also displayed weak on how to react at the cellular and the whole-plant level.
hyposensitive and hypersensitive, respectively, The increased potential for cross talk potentially
phenotypes in response to cytokinin treatment. Recent represents an additional reason as to why multistep two-
studies in protoplasts have shown that the overexpression component systems rather than simple two-component
of type-A ARRs represses their own expression, systems were utilized in plants. We have already
suggesting that type-A ARRs may also provide a received our first glance at this complexity from studies
negative feedback regulation in cytokinin signaling. revealing cross talk between the ARR4-comprising two-
component system with phytochrome B-dependent signal
Because no His kinase activity of phytochrome B has transduction. Overlapping sets of synergistic and
been reported and because of the primary cytokinin antagonistic physiological responses are also described
response properties of ARR4, the photoreceptor may be a for many other stimuli such as ethylene and cytokinin.
target of a cytokinin-modulated two-component signaling Complexly interconnected two-component systems could
system (Fig. 4). CRE1-like cytokinin receptors might participate in the generation of these overlapping
regulate the activity of ARR4 by a His-Asp phosphorelay physiological responses.
and thereby modulate phytochrome-B signaling. Such a
phosphorelay may involve the HPt domain protein
AHP1, which has the ability to function in a
phosphorelay with CRE1 and also acts upstream of
ARR4. AHP1 and ARR4 are found in both the cytosol
and nucleus. Thus, the putative AHK-AHP1-ARR4
phosphorelay, as well as the interaction of ARR4 with
phytochrome B, may occur in the cytosol to modulate
cytosolic, phytochrome B-dependent processes. On the
other hand, AHP1 may also be able to transfer the
phosphoryl group from the cytosol to the nucleus, where
ARR4 may then modulate nuclear, phytochrome B-
dependent responses. Regardless of which sensor His
kinase regulates ARR4 activity, the studies by Sweere
Figure 5. Detail from the emerging network of two-
and colleagues suggest a novel type of cross talk between
component signaling systems in Arabidopsis. Lines
an ancestral two-component system and the phytochrome
indicate direct protein-protein interactions detected in the
B-dependent light-signaling pathway (Fig. 4). This cross
yeast two-hybrid system and in vitro. Proteins that are
talk may enable the integration of signal information
not connected do not display physical interaction. ETR1,
provided by hormone (cytokinin) and red light
CRE1, and CKI hybrid kinase suggested to be involved
(phytochrome B) signal transduction cascades. It is now
cytokinin signaling. AHP1 and AHP2, HPt domain
of major interest to identify the effector proteins of the
proteins. ARR2 and ARR4, Response regulators. The
remaining type-A response regulators and to clarify the
conserved amino acid residues (H, His; D, Asp) of the
underlying cross talk mechanisms.
different two-component moduls are indicated.

C. Cell-Cell Interactions
The Extracellular Matrix Extracellular matrices are composed of tough fibrous

proteins embedded in a gel-like polysaccharide ground
Although animal cells are not surrounded by cell walls, substance. In addition to fibrous structural proteins and
many of the cells in tissues of multicellular organisms are polysaccharides, the extracellular matrix contains
embedded in an extracellular matrix consisting of adhesion proteins that link components of the matrix
secreted proteins and polysaccharides. The extracellular both to one another and to attached cells
matrix fills the spaces between cells and binds cells and
tissues together. One type of extracellular matrix is The major structural protein of the extracellular matrix is
exemplified by the thin, sheetlike basal laminae, or collagen, which is the single most abundant protein in
basement membranes, upon which layers of epithelial animal tissues. The collagens are a large family of
cells rest (Figure.). In addition to supporting sheets of proteins, containing at least 19 different members. They
epithelial cells, basal laminae surround muscle cells, are characterized by the formation of triplet alpha helices
adipose cells, and peripheral nerves. Extracellular matrix, in which three polypeptide chains are wound tightly
however, is most abundant in connective tissues. For around one another in a ropelike structure. Collagen
example, the loose connective tissue beneath epithelial molecules assemble in a regular staggered array to form
cell layers consists predominantly of an extracellular fibrils. The molecules overlap by one-fourth of their
matrix in which fibroblasts are distributed. Other types of length, and there is a short gap between the N terminus
connective tissue, such as bone, tendon, and cartilage, of one molecule and the C terminus of the next. The
similarly consist largely of extracellular matrix, which is assembly is strengthened by covalent cross-links between
principally responsible for their structure and function. side chains of lysine or hydroxylysine residues, primarily
at the ends of the molecules
Connective tissues also contain elastic fibers, which are

particularly abundant in organs that regularly stretch and
then return to their original shape eg. Lungs. Elastic
fibers are composed principally of a protein called
elastin, which is crosslinked into a network by covalent
bonds formed between the side chains of lysine residues
(similar to those found in collagen).
The fibrous structural proteins of the extracellular matrix

are embedded in gels formed from polysaccharides called
glycosaminoglycans, or GAGs, which consist of
repeating units of disaccharides. One sugar of the
disaccharide is either N-acetylglucosamine or N-
acetylgalactosamine and the second is usually acidic
(either glucuronic acid or iduronic acid). Hyaluronan is
the only GAG that occurs as a single long polysaccharide
chain. All of the other GAGs are linked to proteins to
form proteoglycans, which can consist of up to 95%
polysaccharide by weight. In addition to being
components of the extracellular matrix, some
proteoglycans are cell surface proteins that function in
cell adhesion.
Adhesion proteins, the third class of extracellular matrix

constituents, are responsible for linking the components
of the matrix both to one another and to the surfaces of
cells. The prototype of these molecules is fibronectin,
which is the principal adhesion protein of connective
tissues. Fibronectin is a dimeric glycoprotein consisting
of two polypeptide chains, each containing nearly 2500
amino acids. In the extracellular matrix, fibronectin is
further crosslinked into fibrils by disulfide bonds.
Fibronectin has binding sites for both collagen and
GAGs, so it crosslinks these matrix components. A
distinct site on the fibronectin molecule is recognized by
cell surface receptors and is thus responsible for the
attachment of cells to the extracellular matrix.
Basal laminae contain a distinct adhesion protein called

laminin. Laminins can self-assemble into meshlike
polymers. Such laminin networks are the major structural

components of the basal laminae synthesized in very

early embryos, which do not contain collagen.
The major cell surface receptors responsible for the

attachment of cells to the extracellular matrix are the
integrins. The integrins bind to short amino acid
sequences present in multiple components of the
extracellular matrix, including collagen, fibronectin, and
laminin.
Figure: Stable cell-cell junctions mediated by the
Direct Cell-Cell Interactions cadherins Homophilic interactions between
cadherins mediate two types of stable cell-cell
Direct interactions between cells, as well as between adhesions. In adherens junctions, the cadherins are
cells and the extracellular matrix, are critical to the linked to bundles of actin filaments via the catenins.
development and function of multicellular organisms. In desmosomes, desmoplakin links members of the
Some cell-cell interactions are transient, such as the cadherin superfamily (desmogleins and
interactions between cells of the immune system and the desmocollins) to intermediate filaments.
interactions that direct white blood cells to sites of tissue
inflammation. In other cases, stable cell-cell junctions
play a key role in the organization of cells in tissues. For
example, several different types of stable cell-cell
junctions are critical to the maintenance and function of
epithelial cell sheets. Plant cells also associate with their
neighbors not only by interactions between their cell
walls, but also by specialized junctions between their
plasma membranes.
Cell Adhesion Proteins: Cell-cell adhesion is a selective

process, such that cells adhere only to other cells of
specific types. Such selective cell-cell adhesion is
mediated by transmembrane proteins called cell adhesion
molecules, which can be divided into four major groups:
the selectins, the integrins, the immunoglobulin (Ig)
superfamily (so named because they contain structural
domains similar to immunoglobulins), and the cadherins.
The selectins mediate transient interactions between
leukocytes and endothelial cells or blood platelets. The
selectins recognize cell surface carbohydrates. One of
their critical roles is to initiate the interactions between
leukocytes and endothelial cells during the migration of Tight Junctions: Tight junctions, which are usually
leukocytes from the circulation to sites of tissue associated with adherens junctions and desmosomes
inflammation. The selectins mediate the initial adhesion in a junctional complex, are critically important to
of leukocytes to endothelial cells. This is followed by the the function of epithelial cell sheets as barriers
formation of more stable adhesions, in which integrins between fluid compartments. Tight junctions play
on the surface of leukocytes bind to intercellular two roles-- First, tight junctions form seals that
adhesion molecules (ICAMs), which are members of the prevent the free passage of molecules (including
Ig superfamily expressed on the surface of endothelial ions) between the cells of epithelial sheets. Second,
cells. The firmly attached leukocytes are then able to tight junctions separate the apical and basolateral
penetrate the walls of capillaries and enter the underlying domains of the plasma membrane by preventing the
tissue by migrating between endothelial cells. free diffusion of lipids and membrane proteins
between them. Consequently, specialized transport
The fourth group of cell adhesion molecules, the systems in the apical and basolateral domains are
cadherins, also display homophilic binding specificities. able to control the traffic of molecules between
They are not only involved in selective adhesion between distinct extracellular compartments. Tight junctions
embryonic cells but are also primarily responsible for the are the closest known contacts between adjacent
formation of stable junctions between cells in tissues. For cells. They were originally described as sites of
example, E-cadherin is expressed on epithelial cells, so apparent fusion between the outer leaflets of the
homophilic interactions between E-cadherins lead to the plasma membranes, although it is now clear that the
selective adhesion of epithelial cells to one another. It is membranes do not fuse. Instead, tight junctions
noteworthy that loss of E-cadherin can lead to the appear to be formed by a network of protein strands
development of cancers arising from epithelial cells, that continues around the entire circumference of
illustrating the importance of cell-cell interactions in the cell. Each strand in these networks is thought to
controlling cell behavior. Different members of the be composed of transmembrane proteins (claudins
cadherin family, such as N-cadherin (neural cadherin) and occludin) that bind to similar proteins on
and P-cadherin (placental cadherin), mediate selective adjacent cells, thereby sealing the space between
adhesion of other cell types. their plasma membranes.

Gap Junctions: Gap junctions, which are found in connexin extracellular domains hence the term
most animal tissues, serve as direct connections "gap junction," which was coined by electron
between the cytoplasms of adjacent cells. They microscopists.
provide open channels through the plasma
membrane, allowing ions and small molecules (less
than approximately a thousand daltons) to diffuse
freely between neighboring cells, but preventing the
passage of proteins and nucleic acids. Consequently,
gap junctions couple both the metabolic activities
and the electric responses of the cells they connect.
Most cells in animal tissues including epithelial
cells, endothelial cells, and the cells of cardiac and
smooth muscle communicate by gap junctions. In
electrically excitable cells, such as heart muscle
cells, the direct passage of ions through gap
junctions couples and synchronizes the contractions
of neighboring cells. Gap junctions also allow the
passage of some intracellular signaling molecules,
such as cAMP and Ca2+, between adjacent cells,
potentially coordinating the responses of cells in
tissues.
Gap junctions are constructed of transmembrane

proteins called connexins. Six connexins assemble
to form a cylinder with an open aqueous pore in its
center. Such an assembly of connexins in the plasma
membrane of one cell then aligns with the
connexins of an adjacent cell, forming an open
channel between the two cytoplasms. The plasma
membranes of the two cells are separated by a gap
corresponding to the space occupied by the
D1. Uncontrolled Cell division leads to Cancer:
In the normal circumstances, the proliferation of body About 100 different types of human cancers have been
cells is under strict control. The cells differentiate, divide recognized. Cancers arising from epithelial cells are
and die in a sequential manner in a healthy organism. referred to as carcinomas while that from connective
Cancer is characterized by loss of control of cellular tissues are known as sarcomas. Methods for the early
growth and development leading to excessive detection and treatment of cancers have been developed.
proliferation and spread of cells. However, little is known about the biochemical basis of
cancer.
Cancer is derived from a Latin word meaning crab. It is
presumed that the word cancer originated from the Metastasis: Metastasis refers to the spread of cancer
character of cancerous cells which can migrate and cells from the primary site of origin to other tissues of
adhere and cause pain (like a crab) to any part of the the body where they get deposited and grow as secondary
body. tumors. Metastasis is the major cause of cancer related
morbidity and mortality. The biochemical basis of
Neoplasia literally means new growth. Uncontrolled metastasis is not clearly known. It is believed that the
growth of cells results in tumors (a word originally used morphological changes in tumor cells, loss of contact
to represent swelling). Oncology (Greek: oncos-tumor) inhibition, loss of anchorage dependence and alterations
deals with the study of tumors. in the structure of certain macromolecules are among the
important factors responsible for metastasis.
The tumors are of two types.
Metastasis is Genetically Controlled: Cancer cells
a. Benign tumors: They usually grow by expansion and cannot regulate their growth and division. As a result,
remain encapsulated in a layer of connective tissue. they develop into malignant tumors that may potentially
Normally benign tumors are not Life threatening e.g. invade neighboring tissue. Sometimes, cancer cells de-
moles, warts. These types of benign tumors are not tach from the primary tumor and settle elsewhere in the
considered as cancers. body, where they grow and divide, producing secondary
b. Malignant tumors or cancers: They are tumors. This process, called metastasis, is often the cause
characterized by uncontrolled proliferation and spread of of death in cancer patients.
cells to various parts of the body, a process referred to as
metastasis. Malignant tumors are invariably life A metastatic cancer cell spreads from the primary tumor
threatening e.g. lung cancer, leukemia. by entering the blood or lymphatic circulatory system.

These cells are carried in the circulation until they lodge 1. Chemical Carcinogens: It is estimated that almost
in a capillary bed. Normally, more than 99 percent of the 80% of the human cancers are caused by chemical
cells die. The surviving cells invade tissue adjacent to the carcinogens in nature. The chemicals may be organic
capillary bed and begin dividing to fom1 a secondary (e.g. dimethyl benzanthracene, benzo (a) pyrene,
tumor. To reach a new site, the tumor cells pass through dimethyl nitrosamine) or inorganic (arsenic, cadmium) in
the layer of epithelial cells lining the interior wall of the nature. Entry of the chemicals into the body may occur
capillary (or lymph vessel) and penetrate the surrounding by one of the following mechanisms.
extracellular matrices. The extracellular matrix is a 1. Occupation e.g. asbestos, benzene.
meshwork of proteins and carbohydrate molecules 2. Diet e.g. Aflatoxin B produced by fungus
separating tissues; it is a scaffold for tissue growth and (Aspergillus flavus) contamination of
inhibits the migration of cells. To establish a secondary foodstuffs, particularly peanuts.
tumor, metastatic cells secrete enzymes that digest this 3. Drugs-certain therapeutic drugs can be
meshwork, creating holes through which they can carcinogenic e.g. diethylstibesterol.
migrate. 4. Life style e.g. cigarette smoking (includes
The ability to invade extracellular matrix is also a proper- benzo(a)pyrene and polonium).
ty of some normal cell types. Implantation of the embryo
in the uterine wall during pregnancy, for example, a. Mechanism of action: Although a few of the
requires cell migration across the extracellular matrix. chemicals are directly carcinogenic, majority of them
White blood cells reach the site of infection by require prior metabolism to become carcinogenic. The
penetrating capillary walls. The mechanism of invasion enzymes such as cytochrome P450 responsible for the
is probably the same in these cells as in cancer cells. The metabolism of xenobiotics are involved in dealing with
difference is that in normal cells, the invasive ability is the chemical carcinogens. In general, a chemically non-
controlled and tightly regulated, while in tumor cells, reactive procarcinogen is converted to an ultimate
regulation has been lost. carcinogen by a series of reactions.
Metastasis is regulated by genes that encode or regulate The carcinogens can covalently bind to purines,
matrix-cutting enzymes. Protein-cutting enzymes called pyrimidines and phosphodiester bonds of DNA, often
metallo-proteinases are necessary for cell invasion. causing unrepairable damage. The chemical carcinogens
Tumor cell lines with high metastatic capacity produce frequently cause mutations (a change in the nucleotide
more metallo-proteinases than do tumor cell lines with sequence of DNA), which may finally lead to the
low invasive properties. Activated metallo-proteinases development of cancer; hence they are regarded as
are inhibited by the presence of a tissue inhibitor of mutagens.
metalloproteinase (TIMP). Normal cells produce TIMP
and thereby suppress inappropriate activity of b. Ames assay: This is a laboratory test to check the
metalloproteinase. In tumor cells, TIMP inhibits carcinogenecity of chemicals. Ames assay employs the
metalloproteinase only when the number of TIMP use of a special mutant strain of bacterium, namely
molecules is greater than the number of Salmonella typhimurium (His-). This organism cannot
metalloproteinase molecules. On the basis of its action synthesize histidine; hence the same should be supplied
and regulation, TIMP can be considered as a protein that in the medium for its growth. Addition of chemical
suppresses metastasis. carcinogens causes mutations (reverse mutation)
restoring the ability of the bacteria to synthesize histidine
Incidence: Cancer is the second largest killer disease (His+). By detecting the strain of Salmonella (His+) in
(the first being coronary heart disease) in the developed the colonies of agar plates, the chemical mutagens can be
countries. It is estimated that cancer accounts for more identified. The Ames assay can detect about 90% of the
than 20% of the deaths in United States. Based on the chemical carcinogens. This test is regarded as a
current rate of incidence, it is believed that one in every 3 preliminary screening procedure. Animal experiments
persons will develop cancer at sometime during his life. are conducted for the final assessment of
carcinogenecity.
Although humans of all age develop cancer, the
incidence increases with advancement of age. More than c. Promoters of carcinogenesis: Some of the chemicals
70% of the new cancer cases occur in persons over 60 on their own are not carcinogenic. Certain substances
years. Surprisingly; cancer is a leading cause of death in known as promoting agents make them carcinogenic.
children in the age group 3-13 years, half of them die due The application of benzo (a) pyrene to the skin, as such,
to leukemia. does not cause tumor development. However, if this is
followed by the application of croton oil, tumors will
Etiology: In general, cancers are multifactorial in origin. develop. In this case, benzo (a) pyrene is the initiating
The causative agents include physical, chemical, genetic agent while croton oil acts as a promoting agent or
and environmental factors. A survey in USA has shown promoter. Several compounds that act as promoting
that about 90% of all cancer deaths are due to avoidable agents in various organs of the body have been
factors such as tobacco, pollution, occupation, alcohol identified. These include saccharin and phenobarbital.
and diet.
2. Radiation energy: Ultraviolet rays, X-rays and y-rays
Most of the cancers are caused by chemical carcinogens, have been proved to be mutagenic in nature causing
radiation energy and viruses. These agents may damage cancers. These rays damage DNA, which is the basic
DNA or interfere with its replication or repair. mechanism to explain the carcinogenecity of radiation
energy. For instance, exposure to UV rays results in the
formation of pyrimidine dimers in DNA while X-rays

cause the production of free radicals. These types of The presence of viral particles and the enzyme reverse
molecular damages are responsible for the carcinogenic transcriptase, besides the occurrence of base sequence
effects of radiations. in the DNA of malignant cells, complementary to tumor
viruses indicate the involvement of viruses in cancer. The
3. Carcinogenic Viruses: The involvement of viruses in viruses involved in the development of cancer,
the etiology of cancer was first reported by Rouse in commonly known as oncogenic viruses, may contain
1911. He demonstrated that the cell-free filtrates from either DNA or RNA. A selected list of tumor viruses is
certain chicken sarcomas (tumors of connective tissues) as follows:
promote new sarcomas in chickens. Unfortunately, this
epoch-making discovery of Rouse was ignored for DNA-the ultimate in carcinogenesis: DNA is the
several years. This is evident from the fact that Rous was ultimate critical macromolecule in carcinogenesis.
awarded the Nobel Prize in 1966 at the age of 85 for his Several evidences support this fact.
discovery in 1911. 1. Cancers are transmitted from mother to
daughter cells. In other words, cancer cells
Table 1: Selected tumor viruses beget cancer cells.
2. Chromosomal abnormalities are observed in
Class Members many tumor cells.
3. Damage to DNA caused by mutations often
DNA Viruses results in carcinogenesis.
Adenovirus Adenovirus 12 and 18 4. Laboratory experiments have proved that
Herpesvirus Epstein-Barr virus, Herpes Simplex virus purified oncogenes can transform normal cells
Papovirus Papilloma virus, Polyoma Virus into cancer cells.
RNA Viruses
Reterovirus type B Mammary tumor virus of mouse
Reterovirus type C Leukemia, Sarcoma
D3. Molecular Basis of Cancer
It is clear that the two main properties of cancer,

uncontrolled cell division and metastasis, result from 31.3.1. Tumor-Suppressor Genes Normally Suppress
mutations. As mentioned earlier, these mutations can Cell Division
involve large-scale genomic instability, chromosome
loss, chromosome rearrangement, or the insertion of Studies have identified a number of genes that when mu-
foreign (often viral) DNA sequences into loci. Smaller tated, confer a predisposition to specific cancers (Table
scale mutations, including nucleotide substitutions or 2). One example of an inherited predisposition to cancer
deletions, or more subtle modifications that alter only the is retinoblastoma, an inherited cancer of the retinal cells
amount of a gene product or the time over which it is of the eye.
active may also be involved. Retinoblastoma: Retinoblastoma (RB) occurs with a
frequency that ranges from 1 in 14,000 to 1 in 20,000,
In general, control of cell division is regulated in two and most often appears between the ages of 1 and 3
ways: (l) by genes that normally function to suppress cell years. Two forms of retinoblastoma are known. In the
division and (2) by genes that normally function to familial form (about 40 percent of all cases), individuals
promote cell division. The first group of regulatory genes who carry one mutant RB allele are far more susceptible
is called tumor suppressor genes. When expressed, these to developing retinoblastoma than those with two normal
genes halt passage through the cell cycle and prevent wild-type RB alleles.
mitosis. For cell division to occur, these genes or their
gene products (or both) must be inactive or absent. If
tumor suppressor genes become permanently inactivated
or deleted through mutation, control over cell division is
lost, and the mutant cells begin to proliferate in an
uncontrolled fashion.
Genes that normally promote cell division are called

protooncogenes. These genes can be "on" or "off," and
when they are "on," they promote cell division. To stop
mitosis, these genes or their gene products (or both) must
be inactivated. If these genes become permanently
switched on, then uncontrolled cell division occurs,
leading to tumor formation. Mutant forms of
protooncogenes are called oncogenes. In the next
sections, we examine how mutations in tumor suppressor
genes can lead to a loss of cell cycle control and lead to
cancer formation. Then, we consider the role of
Predisposition to this form of cancer is inherited as an
protooncogenes and oncogenes. In recent years, third
autosomal dominant trait although the mutation itself is
categories of genes that control the cell death or
actually recessive, as we will explain later. About 90
apoptosis are also believed to be involved in
percent of those who inherit the mutant RB allele will
carcinogenesis.

develop retinal tumors, usually in both eyes. In addition, retina [Fig 4 (a)]. If the second RB allele becomes
these individuals are predisposed to other forms of mutated in a retinal cell, retinoblastoma will develop.
cancer, including osteosarcoma, a bone cancer, even if Therefore, individuals carrying an inherited mutation of
they do not develop retinoblastoma. the RB gene are predisposed to develop retinoblastoma,
as only one additional mutational event is required to
The second form of retinoblastoma (the remaining 60 cause tumor formation. This does not happen in all cases.
percent) is not familial, and tumors develop About 10 percent of those inheriting a mutant RB allele
spontaneously. This sporadic form is characterized by the do not develop cancer. Presumably, the wild-type allele
appearance of tumors only in one eye, and onset occurs of the RB gene does not mutate in any retinal cells.
at a much later age than in the familial form.
In the nonfamilial sporadic cases [Fig 4 (b)], inde-
By studying both types of retinoblastoma, Alfred pendent mutations in both normal RB alleles must occur
Knudson and his colleagues developed a model that in the same retinal cell for a tumor to develop. As might
requires the presence of two mutated copies of the RB be expected, these events are far less frequent and occur
gene in the same retinal cell for tumor formation; in other at a much later age. As predicted by Knudson's model,
words, tumor development is a recessive trait. In the such sporadic forms of retinoblastoma are more likely to
familial form, one mutant RB allele is inherited and is occur in a single eye.
carried by all cells of the body, including cells of the
The Retinoblastoma (RB) Gene: The retinoblastoma when pRB is inactivated by phosphorylation. pRB is
(RB) gene, located on chromosome 13, encodes a protein regulated by the action of CDKs. When CDK4 binds to
designated as pRB. This protein is found in the nuclei of cyclin D, the kinase phosphorylates pRB. pRB is
all cell and tissue types examined so far and also is found phosphorylated in the S and the G2/M stages of the cell
in both resting (GO) cells and actively dividing cells. cycle, but is not phosphorylated in GO and G1. When
Moreover, pRB is present at all stages of the cell cycle pRB is unphosphorylated (its active state), it binds to
and is part of a regulatory pathway that is important in members of the E2F family of transcription factors.
cell cycle progression. It acts as a molecular switch that Transcription factors are proteins that bind to the
controls the passage of cells from G1 into S phase. promoter region of genes and regulate transcription. E2F
Because the normal gene is expressed ubiquitously in the transcription factors control the expression of some 20-
body, cells can progress through the G/S transition only 30 genes required to move the cell from G1 to S. In its

unphosphorylated form, pRB binds to E2F, blocking Knudson's model, familial cases inherit one mutant allele
transcription, and keeping the cell in G1. (Fig 5) The through the germ line and develop a mutation in the
phosphorylation of pRB by CDK4 takes place in late G1. remaining normal allele in a somatic cell. Sporadic cases,
When phosphate groups are added, pRB releases E2F, on the other hand, require two independent mutations of
allowing transcription of genes that move the cell from G the WT gene within the same cell, and tumors more often
1 into S. involve only one kidney.
The WT gene is on the short arm of chromosome 11 and

encodes a transcription factor. This protein has four
contiguous zinc finger domains, motifs characteristic of
DNA-binding proteins that regulate transcription. WT has
a restricted pattern of expression; it is normally active
only in cells of the fetal kidney and only during the brief
time when the nephron (the basic filtration unit of the
kidney) is being formed. The structure of the WT protein,
its pattern and time of expression, and the resultant
mutant phenotype have led to the hypothesis that the WT
Direct evidence for the role of pRB in regulating the cell
gene encodes a nuclear protein which turns off genes that
cycle comes from several lines of experimental work.
maintain cell division. Alternatively, the gene product
Osteosarcoma (bone cancer) cells are homozygous for
may switch on genes that begin differentiation of the
mutant RB alleles and do not produce pRB. When
fetal cells into components of the kidney. In tumor cells,
osteosarcoma cells are injected into a cancer-prone strain
the mutant gene is switched on at the appropriate time in
of mice, tumors are formed. If a normal RB gene is
the appropriate cells, but the altered gene product is
transferred to the cancer cells, pRE is produced and no
unable to regulate its target genes, resulting in continued
tumors are formed when these genetically modified cells
cell division, aberrant differentiation, and tumor
are injected into mice.
formation.
In a separate two-step experiment, a normal RB gene was
Although both RB and WT genes encode nuclear proteins
transferred into osteosarcoma cells. These cells produced
that suppress tumor formation, there are some differences
pRB and stopped cell division. When D or E cyclins
in their properties and modes of action. pRb does not
were added to these pRB-blocked cells, cell division
bind to DNA; instead, it interacts with E2F, as described
resumed. The addition of cyclins resulted in
in the previous section. WT has all the characteristics of
phosphorylation of pRb, presumably by activating CDK4
a DNA-binding transcription factor. pRb is expressed in
or another kinase. The results demonstrate that active
all dividing cells, whereas the WT protein is found only
pRb stops cell division, that pRB is a target of a G1
in a restricted period of kidney development. pRb is a
CDK/cyclin complex, and that inactivating pRB allows
general regulator of cell division; the WT protein is a
passage through the cell cycle and subsequent division,
cell- or tissue-specific regulator of gene activity during
confirming the role of pRB in G1 control.
fetal or neonatal development.
In normal retinal cells, pRB is active and prevents
p53: Guardian of the Genome
passage into S by interacting with the transcription factor
E2F. In retinoblastoma cells, both copies of the RB gene
Another tumor suppressor gene p53, has been implicated
are mutated, and no functional pRB is present. As a
in human cancer. p53 encodes a nuclear transcription
result, E2F activates the genes required for passage
factor. p53 mutations are found in a wide range of
through the G/S checkpoint into the S phase. This
cancers, including breast, lung, bladder, and colon
important checkpoint is continually overridden in these
cancers. It is estimated that 50-60 percent of all cancers
cells, resulting in uncontrolled growth and tumor
are associated with mutations in the p53 gene, suggesting
formation.
that it controls one or more key events in the proliferation
of all cells and is not involved in a cell- or tissue-specific
The Wilms Tumor Gene: Wilms tumor (WT) is a
form of regulation. Inherited mutations of p53 are
kidney cancer found primarily in children. It has a
associated with the Li-Fraumeni syndrome, an autosomal
frequency of about 1 in 10,000 births, and, like
dominant condition with a predisposition to develop
retinoblastoma, it is found in two forms, a noninherited
cancers in several tissues at a high frequency.
sporadic form and a familial form conferring a
predisposition to WT. The familial predisposition is
Normally the p53 protein is present in cells at a low
inherited as an autosomal dominant trait. According to
level, in a rapidly degraded, inactive form. Several types

of signals can shut down p53 degradation, causing a predisposition to breast cancer; this predisposition is
rapid increase in p53 protein levels. Chemical damage to inherited as an autosomal dominant trait. About 85
DNA, double-stranded breaks in DNA induced by percent of women carrying one mutant BRCA1 allele will
ionizing radiation, or the presence of DNA repair develop a mutation in the second allele and get breast
intermediates generated by exposure to ultraviolet light cancer. These women also have an increased risk of
all cause a shutdown ofp53 turnover. Activation of p53 ovarian cancer.
protein can lead to several responses, including (1) DNA
repair; (2) cell cycle arrest; and (3) apoptosis, a A second breast cancer gene, BRCA2, on the long arm of
genetically programmed pathway of cell death. These chromosome 13, is also inherited as an autosomal
tasks are accomplished by target genes activated by p53 dominant predisposition to breast cancer, but is not
in its role as a transcription factor. The expression of associated with an increased risk of ovarian cancer.
more than 20 target genes are controlled by the action of Together, these genes account for a large majority of
p53. breast cancer associated with a genetic predisposition
(about 10 percent of all cases of breast cancer). The
Cell cycle arrest by p53 can occur at several points in the mutant forms of these genes play no role in sporadic
cycle, including G1. To arrest the cycle at this phase, p53 cases of breast cancer, which make up 90 percent of all
activates transcription of a CDK inhibitor called p21. The cases.
p21 protein targets a number of CDK/cyclin complexes,
including the CDK4/cyclin D complex (which was Both BRCA1 and BRCA2 encode large proteins that are
discussed earlier). confined to the nucleus and expressed in many tissues.
Expression is at its highest during S phase of the cell
Inhibiting the CDK4/cyclin D complex keeps pRB in its cycle. Circumstantial evidence, including their common
active configuration, repressing the transcription of genes pattern of expression, similar phenotype of the mutant
needed to move the cell from G 1 into S phase. Cells alleles (breast cancer), cellular localization, and
without functional p53 protein cannot arrest in G 1 experimental evidence from studies in mice, suggests that
following irradiation and move immediately from G 1 both genes have similar functions and are involved in
into S. These cells do not repair the DNA damage caused DNA repair. Direct evidence for the role of the BRCAI
by the radiation and, as a result, have a high rate of protein in DNA repair also exists. In cells exposed to
mutation. Thus, the p53 gene is often referred to as the ionizing radiation (which produces double stranded DNA
"guardian of the genome." breaks), BRCAI is phosphorylated (a sign of molecular
activation). Current efforts center on identifying the
As just mentioned, more than 50 percent of all human kinase proteins that carry out this phosphorylation, in an
cancers carry a mutation in the p53 gene. The active p53 attempt to place the BRCA 1 and the BRCAl proteins in
protein is a tetramer, so mutation of one p53 allele a pathway associated with DNA repair.
usually abolishes all p53 activity, because almost all
tetramers will contain at least one defective subunit. As a Recent evidence suggests that the BRCAI protein can be
result, p53 mutations act as dominant negative mutations, phosphorylated by two different kinases and that either
and individuals heterozygous for a p53 mutation will kinase is activated by double-stranded DNA breaks (Fig
develop cancer with a frequency of 90-95 percent. The 7). In this model, DNA damage activates the ATM
central role of the p53 gene in cell cycle control kinase or the Chkl kinase (or both). These kinases
emphasizes the relationships between cancer and the cell phosphorylate the BRCA1 and p53 proteins. The
cycle and between genes that regulate cell growth and activated p53 protein arrests replication during S phase to
cancer. allow DNA repair. The activated BRCA 1 protein
participates in DNA repair with the BRCA2 protein, the
Breast Cancer Genes: Mutations in BRCAl, a gene on mRAD51 protein, and other nuclear proteins involved in
the long arm of chromosome 17, are associated with a DNA repair.

The role of BRCA1 and BRCA2 proteins in DNA repair

may also explain why they are not associated with
sporadic cases of breast cancer. In sporadic cases, at least
three mutations would have to accumulate in a breast cell
for it to become cancerous: Both copies of the BRCAl or
BRCA2 genes would have to be mutant, and at least one
other mutation in a cell-cycle regulation gene would have
to occur. On the other hand, those who inherit a mutant
copy of either the BRCAl or BRCA2 gene already have
one mutation and need only two more to trigger breast
cancer.
31.3.2. Oncogenes induce uncontrolled cell division:
We now discuss the second category of genes involved in

cell cycle regulation: those that normally promote cell
division, called protooncogenes. When expressed, these
genes promote or maintain cell division. To stop
division, these genes or their gene products (or both)
must be inactivated. As mutant alleles called oncogenes,
they induce or maintain the uncontrolled cell division ii. Chromosomal translocation: Some of the tumors
associated with cancer. The protein products of exhibit chromosomal abnormalities. This is due to the
protooncogenes are found throughout the cell, including rearrangement of genetic material (DNA) by
the plasma membrane, cytoplasm, and nucleus. In spite chromosomal translocation i.e. splitting off a small
of their wide-ranging locations within the cell, fragment of chromosome which is joined to another
suggesting varying functions, all protooncogene proteins chromosome. Chromosomal translocation usually results
characterized to date alter gene expression either directly in over-expression of protooncogenes.
or indirectly.
Unlike tumor suppressor genes, where mutations in both

alleles of a gene are necessary to promote the
development of cancer, only one of the two copies of a
protooncogene needs to mutate to induce malignancy,
resulting in a dominant phenotype.
Thus genes capable of causing cancer are known as

oncogenes (Greek : oncos-tumor or mass). Oncogenes
were originally discovered in tumor causing viruses.
These viral oncogenes were found to be closely similar to
certain genes present in the normal host cells, which are
referred to as protooncogenes. Now, about 40 viral and
cellular protooncogenes have been identified.
Protooncogenes encode for growth-regulating proteins.
The activation of protooncogenes to oncogenes is an
important step in the causation of cancer.
a. Activation of protooncogenes to oncogenes:

There are several mechanisms for converting the Burkitt's lymphoma, a cancer of human B-lymphocytes,
protooncogenes to oncogenes; some of the important is a good example of chromosomal translocation. In this
ones are described here under. case, a fragment from chromosome 8 is split off and
joined to chromosome 14 containing myc gene (Fig. 9).
i. Viral insertion into chromosome: When certain This results in the activation of inactive myc gene leading
retroviruses (genetic material RNA) infect cells, a to the increased synthesis of certain proteins, which make
complementary DNA (cDNA) is made from their RNA the cell malignant.
by the enzyme reverse transcriptase. The cDNA so
produced gets inserted into the host genome (Fig.8). The iii. Gene amplification: Several-fold amplifications of
integrated double-stranded cDNA is referred to as certain DNA sequences are observed in some cancers.
provirus. This pro-viral DNA takes over the control of Administration of anticancer drugs Methotrexate (an
the transcription of cellular chromosomal DNA and inhibitor of the enzyme dihydrofolate reductase) is
transforms the cells. Activation of protooncogene myc to associated with gene amplification. The drug becomes
oncogene by viral insertion ultimately causing inactive due to gene amplification resulting in a several-
carcinogenesis is well known (e.g. avian leukemia). fold (about 400) increase in the activity of dihydrofolate
Some DNA viruses also get inserted into the host reductase.
chromosome and activate the protooncogenes.
iv. Point mutation: The ras protooncogene is the best
example of activation by point mutation (change in a
single base in the DNA). The mutated ras oncogene

produces a protein (GTPase), which differs in structure oncoproteins. These proteins are the altered version of
by a single amino acid. This alteration diminishes the their normal counterparts and are involved in the
activity of GTPase, a key enzyme involved in the control transformation and multiplication of cells. Some of the
of cell growth. products of oncogenes are below discussed below.
The presence of ras mutations is detected in several i. Growth factors: Several growth factors stimulating
human tumors-90% of pancreatic, 50% of colon and 30% the proliferation of normal cells are know. They regulate
of lung. However, ras mutations have not been detected cell division by transmitting the message across the
in the breast cancer. plasma membrane to the interior of the cell
(transmembrane signal transduction). It is believed that
b. Classification and Mechanism of oncogenes: growth factors play key role in carcinogenesis.
Oncogenes encode for certain proteins, namely
A selected list of polypeptide growth factors, and their sources and major functions are:
Epidermal growth factor (EGF) Salivary gland, Fibrobast Stimulates the growth of Epidermal & Epithelial cells
Platelet derived growth factor (PDGF) Platelets Stimulates growth of mesenchymal cells,
promotes wound healing
Transforming Growth factor (TGF-α) Epithelial cells Similar to EGF
Transforming Growth factor (TGF-β) Platelets, Kidney, placenta Inhibitory effect on tumor cells
Tumor Necrosis factor (TNF-α) Monocytes Necrosis of tumor cells
Interleukin-2 Helper T-Lymphocytes Stimulates growth & maturation of T-Cells
The cell proliferation is stimulated by growth factor. In activating proteins (GAP) which accelerate the
general, a growth factor binds to a protein receptor on the hydrolysis of GTP of ras P21. Thus, in normal cells, the
plasma membrane. This binding activates cytoplasmic activity of ras P21 is well regulated.
protein kinases Ieading phosphorylation of intracellular
target protein. The Phosphorylated proteins in turn, act as
intracellular messengers to stimulate cell division, the
mechanism of which is not clearly known.
Transforming growth factor (TGF-α) is a protein

synthesized and required for the growth of epithelial
cells. TGF-α is produced in high concentration in
individuals suffering from psoriasis, a disease
characterized by excessive proliferation of epidermal
cells.
ii. Growth factor receptors: Some oncogenes encoding

growth factor receptors have been identified. Over
expression and/or structural alterations in growth factor
receptors are associated with carcinogenesis. For
instance, the over expression of gene erb-B, encoding
EGF-receptor is observed in lung cancer.
iii. GTP-binding proteins: These are a group of signal

transducing proteins. Guanosine triphosphate (GTP)-
binding proteins are found in about 30% of human
cancers. The mutation of ras protooncogene is the single-
most dominant cause of many human tumors.
The involvement of ras protein (product of ras Gene)

with molecular weight 21 Kd (P21) in multiplication is
illustrated in Fig. 10. The inactive ras is in a bound state
with GDP. When the cells are stimulated by growth
factors, ras P21 gets activated by exchanging GDP for
GTP.
This exchange process is catalysed by guanine nucleotide

releasing factor (GRF). The active ras P21 stimulates
regulators such as cytoplasmic kinases, ultimately
causing DNA replication and cell division. In normal Point mutations in ras gene result in the production of
cells, the activity of ras P21 is short-lived. The GTPase altered ras P21, lacking GTPase activity. This leads to the
activity, which is an integral part (intrinsic) of ras P21, occurrence of ras P21 in a permanently activated state,
hydrolyses GTP to GDP, reverting ras 21 to the original causing uncontrolled multiplication of cells.
state. There are certain proteins, namely GTPase

iv. Non-receptor tyrosine kinases: These proteins are

found on the interior of the inner plasma membrane. Two forms of genetic predisposition to colon cancer are
They phosphorylate the cellular target proteins (involved known: (1) an autosomal dominant trait, known as
in cell division) in response to external growth stimuli. familial adenomatous polyposis (FAP) and (2) a
Mutations in the protooncogenes (e.g. ab/) encoding non- genetically complex trait, known as hereditary
receptor tyrosine kinases increase the kinase activity and, nonpolyposis colorectal cancer (HNPCC). FAP is present
in turn, phosphorylation of target proteins causing in about 1 percent of all cases, and HNPCC is
unlimited cell multiplication. responsible for 2-4 percent of all colon cancers. The
remaining 95 percent of all cases are sporadic.
Colon Cancer Results from a Series of Mutations:
Cancer is clearly a multistep process resulting from a FAP and Colon Cancer: In the FAP-associated model,
number of specific mutational events. Studies of tumors spontaneous cases of colon cancer begin with a mutation
such as retinoblastoma have established that in some in the APC gene, which maps to the long arm of
cases only a few steps are required to transform a normal chromosome 5. This mutation occurs in a normal
cell into a malignant one. However, most cancers epithelial cell in the lining of the colon. In spontaneous
develop in several steps, with intermediate levels of cases, an APC mutation causes epithelial cells to partially
genetic and cellular transformation. escape cell cycle control. These cells divide to form a
small cluster called a polyp. Those who inherit a mutant
Colorectal cancer has been used to obtain detailed infor- APC allele have the mutation in all cells of their body. As
mation about the nature and order of genetic and cellular a result, APC heterozygotes form hundreds or thousands
events that result in a malignant growth. The malignant of polyps. In either case, polyps are clones, all of which
form of colorectal tumors develops from preexisting carry an APC mutation. The loss of the corresponding
benign tumors. Several discrete precancerous stages allele on the homologous copy of chromosome 5 is not
occur, and these stages can be isolated and studied. necessary for polyp formation. The first step and the
Furthermore, both hereditary and sporadic forms of relative order of subsequent mutations are shown in
colorectal cancer exist. As a result, colorectal cancer is a Figure 11.
useful model for studying the interaction of genetic and
environmental factors in tumor formation.
Subsequent mutations in polyp cells cause intermediate genome. The gene on chromosome 2 is a DNA repair
stages of tumor formation. Mutations in the ras oncogene gene called MSH2. Inactivation of this gene causes the
on chromosome 12 (combined with a mutant APC allele) rapid accumulation of mutations and the development of
cause the polyp to grow larger and to develop fingerlike colorectal cancer. A second DNA repair gene, MLH 1,
(villous) outgrowths. This is the intermediate adenoma associated with HNPCC has been mapped to
stage. To progress further, a polyp cell must acquire chromosome 3. At least two other DNA repair genes
mutations in the DCC (deleted in colon cancer) gene on related to MLH 1 and MSH2 have been identified and
chromosome 18. Mutations in both DCC alleles results in remain to be mapped. These genes are called mismatch
late-stage adenomas. Finally, mutation of the p53 gene repair genes (MMR) and generate a condition called
causes the transition to a cancerous cell. As described microsatellite instability. Mutations in any MMR genes
earlier, mutations in p53 are pivotal to the development promote genome wide genetic instability, accelerating
of a number of cancers, including lung, brain, and breast the rate at which mutations accumulate, with colon
cancers as well as colon cancer. Metastasis occurs after cancer. as one outcome. It may turn out that mutations in
the formation of colon cancer, and it involves an anyone of these genes is enough to cause HNPCC.
unknown number of mutational steps.
Mutations leading to HNPCC are similar to FAP-
HNPCC and Colon Cancer: HNPCC-associated colon associated colon cancer, but have several important
cancer develops after a small number of polyps are differences. FAP begins with a mutation in APC, and the
formed, rather than the hundreds or thousands associated formation of hundreds or thousands of polyps. Each
with FAP. Genes associated with HNPCC have been polyp progresses slowly toward cancer by accumulating
mapped to loci at 2p 16 and 3p21. Mutations at these loci mutations in other genes (ras, DCC, etc.) However,
generate a cascade of mutations in short, tandemly because there are so many polyps, there is a high
repeated microsatellite sequences located throughout the probability that at least one polyp will accumulate all the

mutations necessary to cause colon cancer. HNPCC predisposed to a specific form of cancer inherit one
begins with mutation in DNA repair genes in an mutant copy of such a gatekeeper and need only one
epithelial cell of the colon, generating many genome additional mutation to initiate tumor formation. In
wide mutations, one of which may include APC. spontaneous cases, both copies of the gatekeeper must
Mutation of APC is a relatively rare event, so only a few mutate for cancer to develop.
polyps will form. However, even though there are few Caretaker genes maintain the integrity of the genome, in-
polyps, the continuing high mutation rate ensures that at cluding DNA repair genes such as MSH2 and MLH 1.
least one polyp will acquire all the mutations necessary These genes repair DNA damage caused by
to become cancerous. FAP-associated colon cancer is environmental agents such as ultraviolet light, or
therefore a disease of rapid tumor initiation, with slow mismatches that occur during DNA replication. Mutation
progression to cancer, while HNPCC is a disease with of a caretaker gene does not promote tumor formation
slow initiation of tumors, but rapid progression to cancer. directly, but leads to genetic instability that increases the
Both forms of colon cancer are mediated by mutations in mutation rate of all genes, including gatekeeper genes. If
the APC gene. a gatekeeper gene mutates and begins tumor formation,
the process is accelerated by the high rate of mutation in
Gatekeeper and Caretaker Genes: The two pathways the cell.
to colon cancer in FAP and HNPCC offer an insight into
the nature of genes that control cancer predisposition. 3. Genes that regulate apoptosis
These differences indicate that mutations in two types of
genes cause predisposition to cancer: gatekeeper genes A new category of genes that regulate programmed cell
and caretaker genes. The FAP gene is a gatekeeper; it death (apoptosis) have been discovered. These genes are
normally inhibits cell growth. In general, tumor suppres- also important in the development of tumors.The gene,
sor genes are gatekeepers. In different cell types, only namely bcl-2, causes B-cell lymphoma by preventing
one or a few genes serve as gatekeepers, and if both programmed cell death. It is believed that over-
copies of the gatekeeper mutate, a specific cancer, such expression of bcl-2 allows other mutations of
as retinoblastoma or colon cancer, develops. Individuals protooncogenes that, ultimately, leads to cancer.
D4 Viruses and Cancer
Tumor cells also can arise by non-genetic means through termed oncogenes. The normal cellular gene in its
the actions of specific tumor viruses. Tumor viruses are unmodified, non-transduced form is termed a proto-
of two distinct types. There are viruses with DNA oncogene since it has the capacity to transform cells if
genomes (e.g. papilloma and adenoviruses) and those altered in some way or expressed in an uncontrolled
with RNA genomes (termed retroviruses). RNA tumor manner. Numerous oncogenes have been discovered in
viruses are common in chickens, mice and cats but rare the genomes of transforming retroviruses.
in humans. The only currently known human retroviruses
are the human T-cell leukemia viruses (HTLVs) and the The second mechanism by which retroviruses can
related retrovirus, human immunodeficiency virus (HIV). transform cells relates to the powerful transcription
promoting effect of the LTRs. When a retrovirus genome
Retroviruses can induce the transformed state within the integrates into a host genome it does so randomly. At
cells they infect by two mechanisms. Both of these some frequency this integration process leads to the
mechanisms are related to the life cycle of these viruses. placement of the LTRs close to a gene that encodes a
When a retrovirus infects a cell its RNA genome is growth regulating protein. If the protein is expressed at
converted into DNA by the viral encoded RNA- an abnormally elevated level it can result in cellular
dependent DNA polymerase (reverse transcriptase). The transformation. This is termed retroviral integration
DNA then integrates into the genome of the host cell induced transformation. It has recently been shown that
where it can remain being copied as the host genome is HIV induces certain forms of cancers in infected
duplicated during the process of cellular division. individuals by this integration induced transformation
Contained within the sequences at the ends of the process.
retroviral genome are powerful transcriptional promoter
sequences termed long terminal repeats (LTRs). The Cellular transformation by DNA tumor viruses, in most
LTRs promote the transcription of the viral DNA leading cases, has been shown to be the result of protein-protein
to the production of new virus particles. interaction. Proteins encoded by the DNA tumor viruses,
termed tumor antigens or T antigens, can interact with
At some frequency the integration process leads to cellular proteins. This interaction effectively sequesters
rearrangement of the viral genome and the consequent the cellular proteins away from their normal functional
incorporation of a portion of the host genome into the locations within the cell. The predominant types of
viral genome. This process is termed transduction. proteins that are sequestered by viral T antigens have
Occasionally this transduction process leads to the virus been shown to be of the tumor suppressor type. It is the
acquiring a gene from the host that is normally involved loss of their normal suppressor functions that results in
in cellular growth control. Because of the alteration of cellular transformation.
the host gene during the transduction process as well as
the gene being transcribed at a higher rate due to its Unified hypothesis of carcinogenesis: The
association with the retroviral LTRs the transduced gene multifactorial origin of cancer can be suitably explained
confers a growth advantage to the infected cell. The end by oncogenes. The physical and chemical agents, viruses
result of this process is unrestricted cellular proliferation and mutations all lead to the activation of oncogenes
leading to tumorigenesis. The transduced genes are causing carcinogenesis. The antioncogenes and the genes

regulating apoptosis are intimately involved in of a protein, namely vinculin, is said to be

development of cancer. A simplification of a unified responsible for the loss of anchorage property in
hypothesis of carcinogenesis is depicted in fig 12. cancer cells.
e) Alteration in permeability properties: The
tumor cells have altered permeability and
transport across the membranes.
2. Biochemical changes
a) Increased replication and transcription: The

synthesis of DNA and RNA is increased in cancer
cells, indicating an increase in anabolic processes.
b) Increased glycolysis: The fast growing tumor
cells are characterized by elevation in aerobic and
anaerobic glycolysis. This truly reflects the
increased energy demands multiplying cells.
Characteristics of growing tumor cells: c) Increased pyrimidine metabolism: A reduction
in the catabolic reactions such as degradation of
The morphological and biochemical changes in the pyrimidines is observed in tumor cells.
growing tumor cells are briefly described here. These d) Enzyme alterations: The activities of certain
observations are mostly based on the in vitro culture enzymes are changed e.g. proteases.
studies. Knowledge on the alterations in the biochemical e) Reduced requirement of growth factors: The
profile of tumor cells guides in the selection of tumor cells require much less quantities of
chemotherapy of cancers. growth factors. Despite this fact, there is an
increased production of growth factors by these
1. General and morphological changes cells.
f) Synthesis of fetal proteins: During fetal life,
a) Shape of cells: The tumor cells are much rounder certain genes are active, leading to the synthesis
in shape compared to normal cells. of specific proteins. These genes are suppressed
b) Alterations in cell structures: The cytoskeletal in adult cells. However, the tumor cells
structure of the tumor cells with regard to actin synthesize the fetal proteins e.g.:
filaments is different. carcinoembryonic antigen, α-fetoprotein.
c) Loss of contact inhibition: The normal cells are g) Alterations in the structure of molecules:
characterized by contact inhibition i.e. they form Changes in the structure of glycoproteins and
monolayers. Further, they cannot move away glycolipids are observed
from each other. The cancer cells form multi h) Reduced synthesis of certain molecules: A
layers due to loss of contact inhibition. As a diminished synthesis of specialized proteins is
result, the cancer cells freely move and get seen in tumor cells.
deposited in any part of the body, a property i) Changes in isoenzymes: The isoenzyme profile
referred to as metastasis. of cancer cells is close to the fetal pattern.
d) Loss of anchorage dependence: The cancer cells j) Alterations in antigens: A loss of regularly
can grow without attachment to the surface. This occurring antigens coupled with the appearance
is in contrast to the normal cells, which firmly of new antigens in tumor cells is reported.
adhere to the surface. Alteration in the structure
EXTRA CONCEPTS
• The incidence of cancer increases as age advances, more than 70% of the new cases occurring in persons
over 60.
• Chemical carcinogens cause about 80% of the human cancers.
• The products of oncogenes (growth factors, GTP-binding proteins) have been implicated in the development
of cancer. Antioncogenes apply breaks and regulate the cell proliferation.
• The physical and chemical agents, viruses and mutations result in the activation of oncogenes causing
carcinogenesis.
• The abnormal products of tumor cells, referred to as tumor markers, (CEA AFP, PSA) are useful for the
diagnosis and prognosis of cancer.
• Knowledge on the alterations in the biochemical profile of tumor cells is helpful for the chemotherapy of
cancers.

E1. Cells, Tissues, and Organs of the Immune System
The immune system is an organization of cells, tissues, more organelles (especially lysosomes and
organs, and molecules with specialized roles in phagolysosomes), and have a plasma membrane covered
defending against viruses, microorganisms, cancer cells, with ruffles or microvilli. Macrophages have receptors
and nonself proteins (e.g., organ transplants). for antibodies and complement; these can coat
microorganisms or foreign material and enhance
Cells of the Immune System phagocytosis. This enhancement is termed opsonization.
Macrophages spread throughout the animal body and
The cells responsible for both nonspecific and specific take up residence in specific tissues where they are given
immunity are the white blood cells called leukocytes. special names. Since macrophages are highly phagocytic,
[Greek leukos, white, and kytos, cell]. All of the their function in nonspecific resistance will be discussed
leukocytes originate from pluripotent stem cells in the in more detail in the context of phagocytosis.
fetal liver and in the bone marrow of the animal host
(figure 1), from which they migrate to other body sites,
undergo further development, and perform their various
functions. These cells of the immune system are present
throughout the host’s body. Some become residents
within tissues, where they respond to local trauma and
sound the alarm; others circulate in body fluids and are
recruited to the sites of infection. In defending the host
against pathogenic microorganisms, leukocytes cooperate
with each other first to recognize the pathogen as an
invader and then to destroy it. These different leukocytes
are now briefly examined.
Lymphoid Cells
Lymphocytes [Latin lympha, water, and cyte, cell] are

the major cells of the specific immune system.
Lymphocytes can be divided into three populations: T
cells, B cells, and natural killer cells. B cells or B
lymphocytes reach maturity within the bone marrow,
circulate in the blood, and also settle in various lymphoid
organs. T cells or T lymphocytes mature in the thymus
gland; they can remain in the thymus, circulate in the
blood, or reside in lymphoid organs such as the lymph
nodes and spleen. B cells and T cells will be discussed
further along with their roles in specific immunity.
Natural killer cells are important in killing cells infected
with either viruses or intracellular bacteria and
destroying cancer cells.
Mononuclear Cells
There are two types of mononuclear (i.e., a single large Granulocytes

nucleus) cells—monocytes and macrophages. Both types
are highly phagocytic and make up the monocyte- Granulocytes have irregular-shaped nuclei with two to
macrophage system. Recall that during phagocytosis five lobes, and the cytoplasmic matrix has granules
(figure 1) that contain reactive substances that kill
large particles and even other microorganisms are
engulfed and enclosed in a phagocytic vacuole or microorganisms and enhance inflammation. Because of
phagosome. the irregular-shaped nuclei, granulocytes are also called
polymorphonuclear leukocytes or PMNs. Three types
of granulocytes exist: basophils, eosinophils, and
Monocytes [Greek monos, single, and cyte, cell] are
mononuclear phagocytic leukocytes with an ovoid or neutrophils.
kidney-shaped nucleus and granules in the cytoplasm that
stain gray-blue (figure 1). They are produced in the bone Basophils [Greek basis, base, and philein, to love] have
an irregular-shaped nucleus with two lobes, and the
marrow and enter the blood, circulate for about eight
hours, enlarge, migrate to the tissues, and mature into granules stain bluish-black with basic dyes. Basophils are
macrophages. nonphagocytic cells that function by releasing histamine,
prostaglandins, serotonin, and leukotrienes from their
granules upon appropriate stimulation. Because these
As just noted, macrophages [Greek macros, large, and
phagein, to eat] are derived from monocytes and are also physiological mediators influence the tone and diameter
of blood vessels, they are termed vasoactive agents.
classified as mononuclear phagocytic leukocytes.
However, they may be larger than monocytes, contain Basophils (and mast cells) possess high-affinity receptors
for an immunoglobulin(IgE) and thereby become coated

with IgE antibodies. Once coated, antigens can trigger Unlike basophils, eosinophils are mobile cells that can
the cell to secrete vasoactive mediators. These migrate from the bloodstream into tissue spaces. Their
inflammatory mediators play a major role in certain role is important only in the defense against protozoan
allergic responses such as eczema, hay fever, and asthma. and helminth parasites, mainly by releasing cationic
proteins and reactive oxygen metabolites into the
Eosinophils [Greek eos, dawn, and philien] have a two- extracellular fluid to damage the parasite’s plasma
lobed nucleus connected by a slender thread of membranes.
chromatin, and the granules stain red with acidic dyes.
Neutrophils [Latin neuter, neither, and philien] stain substances and defensins to kill ingested
readily at a neutral pH, have a nucleus with three to five microorganisms. Neutrophils are described in more detail
lobes connected by slender threads of chromatin, and in the contexts of the inflammatory response and
contain fine primary and secondary inconspicuous phagocytosis.
granules. Like macrophages, neutrophils have receptors
for antibodies and complement
proteins and are highly phagocytic
cells. However, unlike the
macrophage, neutrophils do not
reside in healthy tissue but rapidly
migrate to the site of tissue damage
and infection where they are the
principal phagocytic and
microbicidal cells. The lytic
enzymes and bactericidal
substances in neutrophils are contained within large Mast Cells
primary and smaller secondary granules. Primary
granules contain peroxidase, lysozyme, and various Mast cells are bone marrow–derived cells found in
hydrolytic enzymes, whereas secondary granules have connective tissue. They contain granules with histamine
collagenase, lactoferrin, and lysozyme. Both of these and other pharmacologically active substances that
granules help accomplish intracellular digestion. contribute to the inflammatory response. Mast cells,
Neutrophils also use oxygen-dependent and oxygen- along with basophils, play an important role in the
independent pathways that generate antimicrobial development of allergies and hypersensitivities.

Dendritic Cells
Dendritic cells can recognize specific pathogen-

associated molecular patterns on microorganisms and
play an important role in nonspecific resistance. They
can differentiate between potentially harmful
microorganisms and “self” molecules. After the pathogen
is recognized, it binds to the dendritic cell’s pattern
recognition receptors and then is phagocytosed. These
cells are also stimulated by endogenous activators such
as interferon-γ, heat-shock proteins, and tumor necrosis
factor that are released in response to microbial infection.
After stimulation, dendritic cells migrate to the
bloodstream or lymphatic system and present antigens to
T cells. Thus dendritic cells also play an important role in
the specific immune response.
Organs and Tissues of the Immune System

The spleen is the large secondary lymphoid organ
Based on function, the organs and tissues of the immune located in the abdominal cavity. Whereas lymph nodes
system can be divided into primary or secondary are specialized for trapping microorganisms and antigens
lymphoid organs or tissues. The primary organs or from local tissues, the spleen specializes in filtering the
tissues are where immature lymphocytes mature and blood and trapping blood-borne microorganisms and
differentiate into antigen-sensitive mature B and T cells. antigens. Once trapped by macrophages and dendritic
The thymus is the primary lymphoid organ and bone cells, the pathogen is phagocytosed and antigens are
marrow is the primary lymphoid tissue. The secondary presented to B and T cells, which become activated to
organs and tissues serve as areas where lymphocytes may carry out their immune functions.
encounter and bind antigen, whereupon they proliferate
and differentiate into fully mature, antigen-specific
effector cells. The spleen is a secondary lymphoid organ
and the lymph nodes and mucosal-associated tissues
(GALT, gut-associated lymphoid tissue and SALT, skin
associated lymphoid tissues) are the secondary lymphoid
tissues.
Primary Lymphoid Organs and Tissues
Immature undifferentiated lymphocytes are generated in

the bone marrow, and mature and become committed to a
particular antigenic specificity within the primary
lymphoid organ/tissues. The two most important of these
in mammals are the thymus and bone marrow. Lymph nodes lie at the junctions of lymphatic vessels
where they filter out harmful microorganisms and
The thymus is a lymphoid organ located above the heart. antigens from the lymph; pathogens and antigens are
Precursor cells from the bone marrow migrate into the trapped by phagocytic and dendritic cells. Fixed
thymus to the outer cortex where they proliferate. As macrophages then phagocytose the foreign material.
they mature and acquire T cell surface markers, they
move to the inner cortex where approximately 90% die, It is within the lymph nodes that B cells proliferate into
possibly as part of the acquisition of immune tolerance. antibody- secreting plasma cells. Dendritic and T cells
The other 10% move into the medulla, become mature T are also found here; dendritic cells serve as antigen-
cells, and enter the bloodstream (figure). In birds, presenting cells and T helper cells promote the B-cell
undifferentiated lymphocytes move from the bone immune response.
marrow to the bursa of Fabricius where B cells mature.
In mammals, the bone marrow is the site of B-cell
maturation. Like thymic selection during T-cell
maturation, a selection process within the bone marrow
eliminates B cells with self-reactive antibody receptors
(the acquisition of tolerance).
Secondary Lymphoid Organ/Tissue
The spleen is the most highly organized secondary

lymphoid organ and the lymph nodes the most highly
organized tissue.

E2. Antigens
The immune system distinguishes between “self” and Most antigens, however, have more than one determinant
“nonself” through an elaborate recognition process. Prior site or more than one copy of the same epitope, and are
to birth the body inventories the proteins and various termed multivalent. Multivalent antigens generally elicit
other large molecules present (self) and removes most T a stronger immune response than do monovalent
cells specific for self-determinants. ubsequently self- antigens.
substances can be distinguished from nonself substances,
and lymphocytes can produce specific immunologic
reactions against the latter, leading to their removal.
Substances, such as proteins, nucleoproteins,
polysaccharides, and some glycolipids, that elicit an
immune response and react with the products of that
response are called antigens (antibody generator). Most
antigens are large, complex molecules with a molecular
weight generally greater than about 10,000 dalton. The
ability of a molecule to function as an antigen depends
on its size and structural complexity.
Antigenicity and Immunogenicity
A. Antigenicity is defined as the property of a substance

(antigen) that allows it to react with the products of a
specific immune response (antibody or T-cell receptor).
B. Immunogenicity is defined as the property of a

substance (immunogen) that endows it with the capacity
to provoke a specific immune response.
C. All immunogens are antigens, but the reverse is

not true.
1. Immunogens are usually complex, large molecules,
that are able to induce an immune response by
themselves. Immunogens are also antigens in that
specific antibodies or specific T cells recognizing them
will be formed as a consequence of the immune response
they elicit.
2. Certain low molecular weight substances, known as
haptens, are unable to induce an immune response by
themselves. However, if haptens are coupled to an
immunogenic carrier molecule, the immune system will
recognize them as separate epitopes and produce
antihapten antibodies that react with soluble hapten
molecules, free of carrier protein. Thus, a hapten is an
antigen, but not an immunogen.
3. Unfortunately, the terms antigen and immunogen are
often interchanged.
While B lymphocytes recognize epitopes expressed by
Antigenic Determinants native antigens, T lymphocytes recognize small peptides
generated during antigen processing or derived from
Each antigen can have several antigenic determinant newly synthesized proteins. Studies with x-ray
sites or epitopes (figure 1). Epitopes are the regions or crystallography and two-dimensional nuclear magnetic
sites in the antigen that bind to the antigen-binding site of resonance imaging have resulted in the detailed
a specific antibody or with a T cell receptor. Antibodies characterization of epitopes presented by some small
are formed most readily in response to determinants that proteins, such as lysozyme, in their native configuration.
project from the foreign molecule or to terminal residues
of a specific polymer chain. Chemically, determinants From such studies the following rules have been derived
include sugars, organic acids and bases, amino acid side for antibody-antigen recognition:
chains, hydrocarbons, and aromatic groups.
1. Most epitopes are composed of a series of 15 to 22
The number of antigenic determinant sites on the surface amino acids located on discontinuous segments of the
of an antigen is its valence. The valence determines the polypeptide chain, forming a roughly flat area with peaks
number of antibody molecules that can combine with the and valleys that establish contact with the folded
antigen at one time. If one determinant site is present, the hypervariable regions of the antibody heavy and light
antigen is monovalent. chains.

2. Specific regions of the epitope constituted by a few coupled to a protein to form a nucleoprotein. The
amino acids bind with greater affinity to specific areas of autoimmune responses characteristic of some of the so-
the antibody binding site, and thus are primarily called autoimmune diseases (e.g., systemic lupus
responsible for the specificity of antigen-antibody erythematosus) are often directed to DNA and RNA that
interaction. On the other hand, the antibody binding site may have stimulated the immune system as
has some degree of flexibility, which contributes to the nucleoproteins.
good fit with the corresponding epitope. 4. Polypeptides. Hormones such as insulin and other
3. A polypeptide with 100 amino acids may have as polypeptides, although relatively small in size (M.W.
many as 14 to 20 non-overlapping determinants. 1500), are usually immunogenic when isolated from one
However, a typical 100 amino acid globular protein is species and administered over long periods of time to an
folded over itself, and most of its structure is hidden individual of a different species.
from the outside. Only surface determinants on
molecules will usually be accessible for recognition by B Haptens and Carriers
lymphocytes and for interactions with antibodies.
A. Landsteiner, Pauling, and others discovered in the
Characteristics of Immunogenicity 1930s and 1940s that small aromatic groups, such as
aminobenzene sulphonate, amino-benzene arsenate, and
Many different substances can induce immune responses. amino-benzene carboxylate could be chemically coupled
The following characteristics have an important to immunogenic proteins (carriers) and induce
influence in the ability that a substance has to behave as antihapten antibodies in this form. These authors used
an immunogen. hapten-carrier conjugates to study the specificity of the
immune response.
A. Foreigness. As a rule, only substances recognized as
“non-self” will trigger the immune response. Microbial Many small organic molecules are not antigenic by
antigens and heterologous proteins are obviously “non- themselves but can become antigenic if they bond to a
self” and are strongly immunogenic. larger carrier molecule such as a protein (figure 2).
B. Molecular Size. The most potent immunogens are

macromolecular proteins [molecular weight (M.W.) >
100,000]. Molecules smaller than 10,000 daltons are
weakly immunogenic.
C. Chemical Structure and Complexity. Proteins and

polysaccharides are among the most potent immunogens,
although relatively small polypeptide chains, nucleic
acids, and even lipids can, given the right circumstances,
be immunogenic.
1. Proteins. Large heterologous proteins expressing a
wide diversity of antigenic determinants are potent
immunogens.
a. The immunogenicity of a protein is strongly
influenced by its chemical composition. Positively
charged (basic) amino acids, such as lysine, arginine,
and histidine are repeatedly present in the antigenic
sites of lysozyme and myoglobin, while aromatic
amino acids (such as tyrosine) are found in two of
albumin's six antigenic sites. Therefore, it appears that
basic and aromatic amino acids may contribute more
strongly to immunogenicity than other amino acids;
basic proteins with clusters of positively charged
amino acids are strong immunogens.
b. There appears to be a direct relationship between
antigenicity and chemical complexity: aggregated or
chemically polymerized proteins are much stronger
immunogens than their soluble monomeric
counterparts.
2. Polysaccharides. Polysaccharides are among the most
important natural antigens, since either pure
polysaccharides or the sugar moieties of glycoproteins,
lipopolysaccharides, glycolipid-protein complexes, etc.,
are immunogenic. Many microorganisms have
polysaccharide-rich capsules or cell walls, and a variety
of mammalian antigens, such as the erythrocyte antigens
(A, B, Le, H), are short-chain polysaccharides They cannot stimulate antibody formation or T-cell
(oligosaccharides). responses by themselves, but can react with antibodies
3. Nucleic acids. Nucleic acids usually are not once formed. Such small molecules are termed haptens
immunogenic, but can induce antibody formation if [Latin haptein, to grasp]. When lymphocytes are

stimulated by the combined molecule, they can react to antigen is trapped on the surface of those dendritic cells,
either the hapten or the larger carrier molecule. which migrate to the lymph node follicles, where the
Conjugation of a hapten to a carrier protein makes the initial stages of the immune response take place.
hapten immunogenic because the carrier protein can be
processed and presented to specific T cells. As a result, C. Use of Adjuvants. Adjuvants are agents that, when
both hapten-specific and carrier-specific antibodies can administered along with an antigen, enhance the specific
be made. One example of a hapten is penicillin. By itself response. Several factors seem to contribute to this
penicillin is not antigenic. However, when it combines enhancement, including delayed release of antigen,
with certain serum proteins of sensitive individuals, the nonspecific inflammatory effects, and the activation of
resulting molecule does initiate a severe and sometimes monocytes and macrophages.
fatal allergic immune reaction. In these instances the 1. One of the most effective adjuvants is Complete
hapten is acting as an antigenic determinant on the carrier Freund's Adjuvant (CFA), a water-in-oil emulsion with
molecule. killed mycobacteria in the oil phase. Bacillus Calmette-
Guérin (BCG) is an attenuated strain of Mycobacterium
B. In the last two decades the hapten-carrier concept has bovis used as vaccine against tuberculosis and is also an
found significant applications in medicine, particularly to effective adjuvant. Muramyl-dipeptide (MDP), the
enhance immunization protocols, and has also been active moiety of Mycobacterium tuberculosis and of
demonstrated to be the pathological basis for some BCG, also has adjuvant properties.
abnormal immune reactions: 2. Several other microbial and inorganic compounds
1. Poorly immunogenic polysaccharides have been have been used as adjuvants. Some of these adjuvants
shown to induce strong immune responses when have been used therapeutically with the aim of boosting
conjugated to immunogenic proteins, and this the immune response of compromised patients. Many of
observation has resulted in vaccine development for these compounds cause intense inflammatory reactions
immunoprophylaxis of infectious diseases. and discomfort.
2. Using drug-protein conjugates it has been possible to 3. Aluminum hydroxide, an inert compound that
produce antibodies to a wide variety of drugs, which are absorbs the immunogen, stimulates phagocytosis, and
used in numerous drug immunoassays (e.g., plasma delays removal from the inoculation site, is the adjuvant
digoxin levels). most frequently used with human vaccines. Aluminum
3. Some hypersensitivity reactions to some drugs, hydroxide is not as effective as most of the adjuvants
chemicals, and metals is believed to result from listed above, but is considerably less toxic.
spontaneous coupling of these compounds to endogenous
proteins, creating hapten-carrier combinations. One Exogenous and Endogenous Antigens
example of this mechanism is the spontaneous coupling
of the penicilloyl derivative of penicillin to a host A. Most of the antigens to which we react are of
protein, believed to be the first step toward developing exogenous origin, and include microbial antigens,
hypersensitivity to penicillin. environmental antigens (such as pollens and pollutants),
and medications. The objective of the immune response
Factors Associated with the Induction of an Immune is the elimination of foreign antigens, but, in some
Response instances, the immune response itself may have a
deleterious effect (hypersensitivity states).
Besides the chemical nature of the immunogen, other
factors strongly influence the development of an immune B. “Alloantigens,” i.e. antigenic determinants that
response. distinguish one individual from another within the same
species, are unique exogenous antigens. These are alleles
A. Genetic Constitution of the Animal. Different of highly polymorphic systems, which define the
animal species or different strains of one given species antigenic makeup of the cells and tissues of an
show different degrees of responsiveness to a given individual. A classic example is the A, B, O blood group
antigen. In humans, different individuals can behave as antigens: some individuals carry the A specificity, some
“high responders” or “low responders” to any given are B positive, some are AB positive, and some express
antigen. The genetic control of the immune response neither A nor B (O). Other alloantigenic systems are the
seems mainly related to the repertoire of MHC histocompatibility (MHC or in the human, HLA)
molecules, which bind antigen fragments and present antigens of nucleated cells and tissues, the platelet (P1)
them to the immune system, as we will discuss later. The antigens, and the immunoglobulin allotypes. Examples of
animal will respond well to those antigens that are sensitization to exogenous alloantigens include:
processed into peptides or oligopeptides with high 1. Women who may become sensitized to fetal red cell
affinity for the binding sites of the MHC molecules. antigens or immunoglobulin alloantigens during
pregnancy.
B. Method of Antigen Administration. A given dose of 2. Repeated blood transfusions that can induce
antigen may elicit no detectable response when injected sensitization against cellular or immunoglobulin
intravenously, but may elicit a strong immune response if alloantigens from the donor(s).
injected intradermally. This last route of administration 3. Organ transplantation that usually results in
results in slow removal from the site of injection and sensitization against histocompatibility alloantigens
prolonged antigenic stimulation. The presence of expressed in the transplanted organ.
dendritic cells in the dermis (where they are known as
Langerhans cells) is believed to be a significant factor C. Endogenous antigens, by definition, are part of self,
determining the vigorous immune responses obtained and the immune system should not react against them.
when antigens are injected intradermally. The injected The response to self antigens can be the cause of severe

pathological situations (autoimmune diseases), although delivers strong activating signals that apparently override
it may also have an important role in normal catabolic the need for co-stimulatory signals.
processes (i.e., antibodies to denatured IgG may help in
eliminating antigen-antibody complexes from C. Special Characteristics of the Immune Response to
circulation; antibodies to oxidized LDL may help in T-Independent Antigens. The antibody produced in
eliminating a potentially toxic lipid). response to stimulation with T-independent antigens is
predominantly IgM. The switch to IgG production
T-Dependent and T-Independent Antigens requires T-cell help or cytokine production; this switch
does not occur, therefore, with T-independent antigens.
A. Functional Definition. Early studies on the Immunological memory for T independent antigens is
physiological role of T lymphocytes included either not evident or very weak.
experiments in which inbred rodents were sublethally
irradiated to render them immunoincompetent, and their
immune systems were then reconstituted with T
lymphocytes, B lymphocytes, or mixtures of T and B
lymphocytes obtained from normal animals of the same
strain. After reconstitution of the immune system, the
animals were challenged with a variety of antigens, and
their antibody responses were measured.
1. For most antigens, including complex proteins,

heterologous cells, and viruses, a measurable antibody
response was only observed in animals reconstituted with
mixtures of T and B lymphocytes. Since T lymphocytes
do not synthesize antibody, their role must be one of
assisting the proliferation and/or differentiation of B
lymphocytes. The antigens that can elicit antibody
responses exclusively in animals reconstituted with both
T and B cells are known as T-dependent antigens.
Structurally, T-dependent antigens are usually complex
proteins with large numbers of different antigenic
determinants with little repetition among themselves.
2. Other antigens, particularly polysaccharides, can
induce antibody synthesis in animals depleted of T
lymphocytes, and are known as T-independent antigens.
3. It should be noted that there appears to be a continuous
gradation from T-dependence to T-independence, rather
than two discrete groups of antigens in many species.
However, this differentiation is useful as a “working
classification” of antigens.
B. Biological Basis of T-Independence. Two theories

Superantigens
concerning the signaling by T-independent antigens have
been proposed:
Certain antigens provoke such a drastic immune response
that they are termed superantigens. Superantigens are
1. Some T-independent antigens, such as bacterial
bacterial proteins. These superantigens nonspecifically
lipopolysaccharides (LPS), have mitogenic properties
stimulate T cells to proliferate by interacting with both
and can deliver dual signals to B cells, one by occupying
class II major histocompatibility complex products on
the antigen-specific receptor (membrane
antigen-presenting cells and the T-cell receptor (figure
immunoglobulin), and the other by a nonspecific
3). Good examples of superantigens are the
mitogenic signal involving the lipid moiety of LPS,
staphylococcal enterotoxins that cause food poisoning
whose nature has not been well characterized. The
and the toxin that causes toxic shock syndrome.
association of these two signals would be sufficient to
Superantigens cause symptoms by stimulating the release
stimulate B cells and promote their differentiation into
of massive quantities of cytokines from T cells and
antibody-producing cells.
should be considered possible chronic associates in such
2. Other T-independent antigens (such as
diseases as rheumatic fever, arthritis, Kawasaki
polysaccharides) do not have mitogenic properties, but
syndrome, atopic dermatitis, and one type of psoriasis.
are constituted by multiple repeats of a limited number of
sugar molecules, allowing extensive cross-linking of
membrane immunoglobulins. Receptor cross-linking

E3. Immunoglobulin Structure
I. General Structure of Immunoglobulins technique that separates proteins by charge in a first step,
allowing their antigenic characterization in a second step.
A. Information concerning the precise structure of the
antibody molecule started to accumulate as technological
developments were applied to the study of the general
characteristics of antibodies. By the early 1940s
antibodies had been characterized electrophoretically as
gamma globulins (Fig. 1) and also classified into large
families by their sedimentation coefficient determined
by analytical ultracentrifugation (7S and 19S antibodies).
It also became evident that plasma cells were
responsible for immunoglobulin synthesis and that a
malignancy known as multiple myeloma was a
malignancy of immunoglobulin-producing plasma cells.
B. As protein fractionation techniques became available, 2. Reduction of disulfide bonds. If the IgG molecule is
complete immunoglobulins and their fragments were incubated with a reducing agent containing free SH
isolated in large amounts, particularly from the serum groups and fractionated by gel filtration (a technique that
and urine of patients with multiple myeloma. These separates proteins by size) in conditions able to
proteins were used both for studies of chemical structure dissociate noncovalent interactions, two fractions are
and for immunological studies that led to the definition obtained. The first fraction corresponds to polypeptide
of antigenic differences between proteins from different chains of M.W. 55,00 (heavy chains); the second
patients; this was the basis for the initial identification of corresponds to polypeptide chains of M.W. 23,000 (light
the different classes and subclasses of immunoglobulins chains) (Fig. 3).
and the different types of light chains.
B. The IgG Structural Model. The sum of data

obtained by proteolysis and reduction experiments
resulted in the conception of a diagrammatic two-
dimensional model for the IgG molecule (Fig. 4).
II. Immunoglobulin G (IgG): The Prototype
Immunoglobulin Molecule
A. General Considerations. IgG, a 7S immunoglobulin,

is the most abundant immunoglobulin in human serum
and in the serum of most mammalian species. It is also
the immunoglobulin most frequently detected in large
concentrations in multiple myeloma patients. For this
reason, it was the first immunoglobulin to be purified in
large quantities and to be extensively studied from the
structural point of view. The basic knowledge about the
structure of the IgG molecule was obtained from two
types of experiments:
1. Proteolytic digestion. The incubation of purified IgG

with papain, a proteolytic enzyme extracted from the
latex of Carica papaya, results in the splitting of the
molecule into two fragments that differ both in charge
and antigenicity. These fragments can be easily
demonstrated by immuno-electrophoresis (Fig. 2), a

C. Proteolytic Fragments and Functional Topography III. The Structural and Antigenic Heterogeneity of
of the Molecule Heavy and Light Chains
1. Papain digestion, splitting the heavy chains in the
hinge region (so designated because this region of the A. Immunoglobulin Classes. Five classes of
molecule appears to be stereoflexible) results in the immunoglobulins were identified due to antigenic
separation of two Fab fragments and one Fc fragment differences of the heavy chains and designated as IgG
per IgG molecule (Fig. 5). (the classic 7S immunoglobulin), IgA, IgM (the classic
a. The Fab fragments are so designated because they 19S immunoglobulin), IgD, and IgE. IgG, IgA, and IgM
contain the antigen binding site. together constitute over 95% of the whole
b. The Fc fragment is so designated because it can be immunoglobulin pool in a normal human being and are
easily crystallized. designated as major immunoglobulin classes. Because
c. If the disulfide bond joining heavy and light chains they are common to all humans, the immunoglobulin
in the Fab fragments is split, a complete light chain classes can also be designated as isotypes. The major
can be separated from a fragment that comprises about characteristics of the five immunoglobulin classes are
half of one of the heavy chains, the NH2 terminal half. summarized in Table 1.
This portion of the heavy chain contained in each
Fab fragment has been designated the Fd fragment.
2. A second proteolytic enzyme, pepsin, splits the heavy

chains at the carboxyl side of the disulfide bonds that B. Light-Chain Isotypes. The light chains also proved to
join them at the hinge region, producing a double Fab be antigenically heterogeneous and two main isotypes
fragment or F(ab')2 (Fig. 6), while the Fc portion of the were defined: kappa and lambda. Each immunoglobulin
molecule is digested into peptides. molecule is constituted by a pair of identical heavy
chains and a pair of identical light chains; hence, a given
immunoglobulin molecule can have either kappa or
lambda chains but not both. A normal individual will
have a mixture of immunoglobulin molecules in his
serum, some with kappa chains (e.g., IgGk), and others
with lambda chains (e.g., IgGλ). Normal serum IgG has a
2:1 ratio of kappa chain- over lambda chain-bearing IgG
molecules. In contrast, monoclonal immunoglobulins,
the results of the synthetic activity of malignant
proliferations of plasma cells, such as multiple myeloma,
have one single heavy-chain isotype and one single light-
chain isotype, since they are the product of large number
of cells all derived from a single mutant, constituting one
large clone of identical cells.
3. The comparison of Fc, Fab, F(ab')2, and whole IgG
molecules shows both important similarities and C. Immunoglobulin Subclasses. Antigenic differences
differences between the whole molecule and its between the heavy chains of IgG and IgA exist and
fragments. define subclasses of those immunoglobulins. The most
a. Both Fab and F(ab')2 contain antibody binding sites, important structural and biological characteristics of IgG
but while the intact IgG molecule and the F(ab')2 are and IgA subclasses are listed in Tables 2 and 3.
bivalent, the Fab fragment is monovalent. Therefore,
a Fab fragment can bind to an antigen, but cannot 1. IgG subclasses. Some interesting biological and
cross-link two antigen molecules. structural differences have been demonstrated for IgG
b. An antiserum raised against the Fab fragment reacts proteins of different subclasses.
mostly against light-chain determinants; the a. From the functional point of view IgG1 and IgG3 are
immunodominant antigenic markers for the heavy more efficient in terms of complement fixation and have
chain are located in the Fc fragment. greater affinity for monocyte receptors. Those properties
c. The F(ab')2 fragment is identical to the intact can be correlated with a greater degree of biological
molecule as far as antigen-binding properties, but activity, both in normal antimicrobial responses, in
lacks the ability to fix complement, bind to cell which these properties have direct consequences in
membranes, etc., which are determined by the Fc opsonization and bacterial killing, and in pathological
region of the molecule. conditions, in which the formation of immune complexes

containing IgG1 and IgG3 antibodies is more likely to 2. The amino acid sequence of the light-chain variable
have pathogenic consequences. regions is different even in proteins of the same
b. From the structural point of view the IgG3 subclass antigenic type, and early workers thought that this
has the greatest number of structural and biological sequence would be totally individual to any single
differences relative to the remaining IgG subclasses. protein. With increasing data, it became evident that
Most differences appear to result from the existence of an some proteins shared similarities in their variable
extended hinge region (which accounts for the greater regions, and it has been possible to classify variable
M.W.), with a large number of disulfide bonds linking regions into three groups, Vk, Vλ, and VH. Each group
the heavy chains together (estimates of their number vary has been further subdivided into several subgroups.
between 5 and 15). This extended hinge region seems to
be easily accessible to proteolytic enzymes, and this
lability of the molecule is likely to account for its
considerably shorter half-life.
3. The light-chain V-region subgroups (Vk, Vλ) are

“type” specific (i.e., Vk subgroups are only found in k
proteins and Vλ subgroups are always associated with λ
chains). In contrast, the heavy-chain V-region subgroups
(VH) are not “class” specific. Thus, any given VH
subgroup can be found in association with the heavy
chains of any of the known immunoglobulin classes and
subclasses.
4. The heavy chain of IgG is about twice as large as a
light chain; it is composed of approximately 450 amino
2. IgA subclasses. Of the two subclasses known, it is acids, and a variable and a constant region can also be
interesting to note that a subpopulation of IgA2 identified. The variable region is composed of the first
molecules carrying the A2m(1) allotype is the only 113 to 121 amino acids (counted from the amino terminal
example of a human immunoglobulin molecule lacking end), and subgroups of these regions can also be
the disulfide bond joining heavy and light chains. The identified. The constant region is almost three times
IgA2 A2m(1) molecule is held together through larger; for most of the heavy chains, it starts at residue
noncovalent interactions between heavy and light chains. 116 and ends at the carboxyl terminus (Fig. 7). The
maximal degree of homology is found between constant
IV. Immunoglobulin Regions and Domains regions of IgG proteins of the same subclass.
A. Constant and Variable Regions. The light chains of B. Immunoglobulin Domains. The immunoglobulin
human immunoglobulins are composed of 211 to 217 molecule contains several disulfide bonds formed
amino acids. As mentioned above, there are two major between contiguous cysteine residues. Some of them join
antigenic types of light chains (k and λ); when the amino two different polypeptide chains (interchain disulfide
acid sequences of light chains of the same type were bonds), keeping the molecule together. Others
compared, it became evident that two regions could be (intrachain bonds) join different areas of the same
distinguished in the light-chain molecules: a variable polypeptide chain, leading to the formation of “loops.”
region, comprising the portion between the amino These “loops” and adjacent amino acids constitute the
terminal end of the chain and residues of 107 to 115, and immunoglobulin domains, which are folded in a
a constant region, extending from the end of the characteristic β-pleated sheet structure (Fig. 8).
variable region to the carboxyl terminus (Fig. 7). 1. Variable regions of both heavy and light chains have a
single domain, which is involved in antigen binding.
1. The light-chain constant regions were found to be 2. Light chains have one single constant region domain
almost identical in light chains of the same type, but (CL), while heavy chains have several constant region
differ markedly in k and λ chains. It is assumed that the domains (three in the case of IgG, IgA, and IgD; four in
difference in antigenicity between the two types of light the case of IgM and IgE). The constant region domains
chains is directly correlated with the structural are generically designated as CH1, CH2, and CH3, or, if
differences in constant regions. one wishes to be more specific, they can be identified by

the class of immunoglobulins to which they belong by 1. The T-cell antigen receptor molecule, the major
adding the symbol for each heavy chain class (γ, α, µ, δ, histocompatibility antigens, the polyimmunoglobulin
ε). For example, the constant region domains of the IgG receptor on mucosal cells (see below), and the CD2
molecule can be designated as Cγ1, Cγ2, and Cγ3. molecule on T lymphocytes are some examples of
proteins included in the immunoglobulin superfamily.
2. The majority of the membrane proteins of the
immunoglobulin superfamily seem to be functionally
involved in recognition of specific ligands that may
determine cell-cell contact phenomena and/or cell
activation.
3. Different functions have been assigned to the different

domains and regions of the heavy chains. For instance,
Cγ2 is the domain involved in complement fixation,
while both Cγ2 and Cγ3 are believed to be involved in
the binding to phagocytic cell membranes.
4. The “hinge region” is located between CH1 and CH2,
and its name is derived from the fact that studies by a
variety of techniques, including fluorescence VI. The Antibody Combining Site
polarization, spin-labeling, electron microscopy, and x-
ray crystallography, have shown that the Fab fragments The binding of antigens by antibody molecules takes
can rotate and waggle, coming together or moving apart. place in the Fab region, and is basically a noncovalent
As a consequence, IgG molecules can change their shape interaction that requires a good fit between the antigenic
from a “Y” to a “T” and vice versa, using the region determinant and the antigen binding site on the
intercalated between Cγ1 and Cγ2 as a “hinge.” The immunoglobulin molecule. The antigen binding site
length and primary sequence of the hinge regions play an appears to be formed by the variable regions of both
important role in determining the segmental flexibility of heavy and light chains folded in close proximity, forming
IgG molecules. For example, IgG3 has a 12 amino acid a pouch where an antigenic determinant or epitope will
hinge amino terminal segment and has the highest fit.
segmental flexibility. The hinge region is also the most
frequent point of attack by proteolytic enzymes. In A. Hypervariable Regions. Certain sequence stretches
general, the resistance to proteolysis of the different IgG of the variable regions vary widely from protein to
subclasses is inversely related to the length of the hinge protein, even among proteins sharing the same type of
amino terminal segments—IgG3 proteins are the most variable regions. For this reason, these highly variable
easily digestible, while IgG2 proteins, with the shortest stretches have been designated as hypervariable
hinge region, are the most resistant to proteolytic regions.
enzymes.
B. The structure of hypervariable regions is believed to
V. The Immunoglobulin Superfamily of Proteins play a critical role in determining antibody specificity
since these regions are believed to be folded in such a
The existence of globular “domains” (Fig. 8) is way that they form a “pouch” where a given epitope of
considered as the structural hallmark of immunoglobulin an antigen will fit. In other words, the hypervariable
structure. A variety of other proteins that exhibit amino regions will interact to create a binding site whose
acid sequence homology with immunoglobulins also configuration is complementary to that of a given
contain Ig-like domains (Fig. 9). Such proteins are epitope. Thus, these regions can be also designated as
considered as members of the immunoglobulin complementarity-determining regions.
superfamily, based on the assumption that the genes
which encode them must have evolved from a common VII. Immunoglobulin M: A Polymeric Molecule
ancestor gene coding for a single domain, much likely
the gene coding for the Thy-1 molecule found on murine A. The Pentameric Nature of IgM. Serum IgM is
lymphocytes and brain cells. basically composed of five subunits (monomeric
subunits, IgMs), each one of them composed of two light

chains (k or λ) and two heavy chains (µ). The heavy This is a small polypeptide chain of 15,000 daltons, also
chains are larger than those of IgG by about 20,000 found in polymeric IgA molecules. One single J chain is
daltons, corresponding to an extra domain on the found in any polymeric IgM or IgA molecule, regardless
constant region (CH4). of how many monomeric subunits are involved in the
B. The J chain, a third polypeptide chain, can be polymerization. It has been postulated that this chain
revealed by adequate methodology in IgM molecules. plays some role in the polymerization process.
VIII. Immunoglobulin A: A Molecularly

Heterogeneous Immunoglobulin
A. Serum IgA is molecularly heterogeneous, composed

of a mixture of monomeric, dimeric, and larger
polymeric molecules. In a normal individual, over 70–
90% of serum IgA is monomeric. Monomeric IgA is
similar to IgG, and composed of two heavy chains (α)
and two light chains (k or λ). The dimeric and polymeric
forms of IgA found in circulation are covalently bonded
synthetic products containing J chains. C. Secretory component is constituted by a single
polypeptide chain of approximately 70,000 daltons with
B. IgA is the predominant immunoglobulin in secretions. five homologous immunoglobulin-like domains. It is
Secretory IgA molecules are most frequently dimeric, synthesized by epithelial cells in the mucosa and by
contain J chains as do all polymeric immunoglobulin hepatocytes, initially as a larger membrane molecule
molecules, and, in addition, contain a unique polypeptide known as a polyimmunoglobulin receptor, from which
chain, designated as secretory component (SC) (Fig.11). SC is derived by proteolytic cleavage separating SC from
the intramembrane and cytoplasmic segments of its
membrane form.

IX. The Minor Immunoglobulin Classes: IgD and IgE C. IgE is an extremely important immunoglobulin
because of its biological properties. Its biological role
A. General Concepts. IgD and IgE were the last appears to be predominantly related to antiparasitic
immunoglobulins to be identified due to their low responses, but its main clinical relevance is related to
concentrations in serum. Both are monomeric allergic reactions.
immunoglobulins, similar to IgG, but their heavy chains 1. IgE has the unique property of binding to Fcε
are larger than γ chains. IgE has five domains in the receptors on the membranes of mast cells and basophils.
heavy chain (one variable and four constant); IgD has The binding of IgE to those receptors has an extremely
four heavy-chain domains (as most other monomeric high affinity (7.7 × 109 1/M-1), about 100-fold greater
immunoglobulins). than the affinity of IgG binding to monocyte receptors.
2. The high affinity binding of IgE to basophil membrane
B. IgD and IgM are the predominant immunoglobulin receptors is the basis for its designation as
classes in the B lymphocyte membrane, where they are homocytotropic antibody, and is responsible for its role
the antigen-binding molecules in the antigen-receptor in allergic reactions. In allergic individuals, if those IgE
complex. The B-cell antigen complex is composed of molecules have a given antibody specificity and react
membrane Ig and several other membrane proteins with the antigen while attached to the basophil or mast
including Igα and Igβ, which have sequence motifs in cell membranes, they will trigger the release of histamine
their cytoplasmic portions that are required for signal and other substances that cause the symptoms of allergic
transduction. reactions.
3. IgE is the most thermolabile immunoglobulin and
1. Membrane IgD and IgM are monomeric. The heavy loses biological activity (i.e., the ability to bind to high
chains of membrane IgD and IgM (δm, µm) differ from affinity Fcε receptors) after heating at 56°C for 30
that of the secreted forms at their carboxyl termini, where minutes. This binding depends on the tertiary structure of
the membrane forms have a hydrophobic transmembrane the C-terminal portion of Cε2 and the N-terminal portion
section and a short cytoplasmic tail that are lacking in the of the Cε3 domain. Circular dichroism studies
secreted forms. In contrast, a hydrophilic section is found demonstrated that heating changes the configuration of
at the carboxyl termini of heavy chains of secreted Igs. Cε3 and Cε4 domains, and the changes in configuration
2. The biological role of circulating IgD is not clear. of Cε3 are likely to be critical in preventing proper
binding to the receptor.
E4. Diversity of Antibodies
One unique property of antibodies is their remarkable

diversity. According to current estimates each human can
synthesize more than 1011 (100 billion) different kinds of
antibodies. How is this diversity generated? The answer
is threefold: rearrangement of antibody gene segments,
somatic mutations, and generation of different codons
during antibody gene splicing.
Immunoglobulin genes are split or interrupted genes with

many gene segments. Embryonic B cells contain a small
number of gene segments, close together on the same
chromosome, that determine the constant (C) region of
the light chains (figure 1).
Separated from them, but on the same chromosome, is a

larger cluster of segments that determines the variable
(V) region of the light chains. During B-cell
differentiation, one segment for the constant region is
joined by a process of recombination mediated by
specific proteins to one segment for the variable region.
This splicing produces a complete light-chain antibody
gene. A similar splicing mechanism also occurs to join
the constant and variable segments of the heavy chains.
Because the light-chain genes actually consist of three
parts, and the heavy-chain genes consist of four, the
formation of a finished antibody molecule is slightly
more complicated than previously outlined (figure 2).

(which is variable in length) occurs that joins one V gene

segment with one J segment. This DNA joining process
is termed combinatorial joining because it can create
many combinations of the V and J regions. When the
light-chain gene is transcribed, transcription continues
through the DNA region that encodes for the constant
portion of the gene. RNA splicing subsequently joins the
V J and C regions, creating mRNA. Combinatorial
joining in the formation of a heavy-chain gene occurs by
means of DNA splicing of the heavy-chain counterparts
of V and J along with a third set of D (diversity)
sequences (figure 3a). Initially, all heavy chains have the
µ type of constant region. This corresponds to antibody
class IgM (figure 3b). Another DNA splice joins the VDJ
region with a different constant region that can
subsequently change the class of antibody produced by
The germ line DNA for the light chain gene contains the B cell (figure 3c).
multiple coding sequences called V and J (joining)
regions. During the differentiation of a B cell, a deletion
different λ chains). The heavy chains have approximately

250 to 1,000 VH, 10 to 30 D, and 4 JH regions, giving a
maximum of 120,000 different combinations. Because
any light chain can combine with any heavy chain, there
will be a maximum of 2x108 possible k chain antibody
types.
The value of 2x 108 different antibodies is actually an

underestimate because antibody diversity is further
augmented by two processes:
1. The V regions of germ line DNA are susceptible to a
high rate of somatic mutation during B-cell development
in response to an antigen challenge. These mutations
allow B-cell clones to produce antibodies with somewhat
different polypeptide sequences.
2. The junction for either VJ or VDJ splicing in
combinatorial joining can occur between different
nucleotides and thus generate different codons in the
spliced gene. For example, one VJ splicing event can join
The amount of antibody diversity in the mouse that can the V sequence CCTCCC with the J sequence TGGTGG
be generated by combinatorial joining is shown in table in two ways: CCTCCC TGGTGG --- CCGTGG, which
1. In this animal the k light chains can be formed from codes for the amino acids proline and tryptophan; and
any combination of about 250 to 350 Vk and 4 Jk regions CCTCCC TGGTGG --- CCTCGG, which codes for
giving a maximum of approximately 1,400 different k proline and arginine. Thus the same VJ joining could
chains. The λ chains have their own Vλ and Jλ regions produce polypeptides differing in a single amino acid.
but smaller in number than their k counterparts (6

E.5 Specific and Non-Specific Immune Response
The vertebrate body possesses two mechanisms which ⇒ Comprise 60% - 70% of total white cells plays
protect it from potentially dangerous viruses, bacteria, main role is in inflammation..
other pathogens, and abnormal cells which could develop ⇒ They are the first cells to arrive at the site of
into cancer. inflammation by leaving the blood, through the
endothelium into the tissue (extravasation).
1. One of these mechanisms is nonspecific, that is, it does ⇒ Attracted by chemical signals (chemotaxis), they
not distinguish between infective agents. enter infected tissues by amoeboid movement
2. The second mechanism is specific in that it responds in ⇒ Only live a few days as they destroy themselves
a very specific manner (e.g., production of antibodies) to when destroying pathogens
the particular type of infective agent. ⇒ Neutrophils are attracted into the tissue by
chemo tactic factors that include complement
I. Nonspecific Defense against Infection proteins, clotting proteins (stimulated to be
produced by tissue damage) and T cell derived
Nonspecific defense mechanisms help prevent entry and cytokines. In the tissues, neutrophils are active
spread of invading microbes in an animal's body. phagocytic cells, like macrophages. Neutrophils,
⇒ An invading microbe must cross the external however, do not act as antigen presenting cells.
barrier formed by the skin and Neutrophils, instead, are most effective at killing
mucous membranes. ingested microorganisms and can do this by
⇒ If the external barrier is penetrated, the microbe oxygen dependent or independent pathways.
encounters a second line of defense: interacting
mechanisms of phagocytic white blood cells, Monocytes comprise only about 5% of the white blood
antimicrobial proteins, and the inflammatory cells, but they provide an even more effective phagocytic
response. defense. They mature, circulate for a few hours, then
migrate to the tissues where they enlarge and become
A. The skin and mucous membranes provide first-line macrophages.
barriers to infection
Macrophages are large amoeboid cells that use
The skin and mucous membranes act as physical barriers pseudopodia to phagocytize microbes, which are then
preventing entry of pathogens, and as chemical barriers of destroyed by digestive enzymes and reactive forms of
anti-pathogen secretions. oxygen within the cell. Macrophages are long lived.
⇒ In humans, oil and sweat gland secretions acidify the ⇒ Most wander through interstitial fluid
skin (pH 3 to 5), which discourages microbial phagocytosing bacteria, viruses, and cell debris
growth. ⇒ Some reside permanently in organs and
⇒ Saliva, tears, and mucous secretions contain connective tissues
antimicrobial proteins and wash away potential ⇒ Fixed macrophages are especially numerous in
invading microbes. the lymph nodes and spleen.
⇒ An enzyme (lysozyme) in perspiration, tears, and
saliva attacks the cell walls of many bacteria and Eosinophils represent about 1.5% of the total white cell
destroys other microbes entering the respiratory count and have limited phagocytic activity.
system and eyes.
⇒ In the respiratory tract, nostril hairs filter inhaled ⇒ Contain destructive enzymes which are
particles and mucus traps microorganisms that are discharged against the outer covering of the
then swept out of the upper respiratory tract by cilia, invading pathogen. Eosinophils are granulocytes
thus preventing their entrance into the lungs. named because of their intense staining with
⇒ In the digestive tract, stomach acid kills many 'eosin'.
bacteria that enter with foods or those trapped in ⇒ Under the microscope, eosinophils typically
swallowed mucus from the upper respiratory system. have a bilobed nucleus and contain many basic
crystal granules in their cytoplasm. The granules
B. Phagocytic cells, inflammation, and antimicrobial are eosinophil mediators that are toxic to many
proteins function early in infection organisms and also to tissues as in asthma
Asthma.
1. Microbes that penetrate the skin or mucous membranes ⇒ Eosinophils are motile and phagocytic and are
encounter amoeboid white blood cells capable of particularly active in parasitic infection. In fact,
phagocytosis or cell lysis. blood work showing an increase in eosinophils
is often caused by parastic diseases.
Neutrophils are cells that become phagocytic in infected ⇒ Main contribution is defense against larger
tissue. Neutrophils are produced in the bone marrow from invaders such as parasitic worms.
the granulocyte-monocyte stem cell. These cells are often
called poly morpho nuclear cells (PMN's). This is because Mast cells are formed in the tissue from undifferentiated
of the polymorphic shape of the nucleus. precursor cells released into the blood from the bone
marrow. They are not the tissue counterparts of basophils

but they have a similar importance in allergic reactions ⇒ Increased blood flow to the site of injury
and are only found in tissues. delivers clotting elements that help block the
spread of pathogenic microbes and begin the
⇒ There are two types of mast cells found either in the repair process.
connective tissues or in mucosal sites. Both types
contain numerous granules with preformed mediators Migration of phagocytic cells into the injured area is also a
which can be released from mast cells after result of increased blood flow and increased leakage from
stimulation. The preformed mediators include the capillaries.
histamine and other pharmacologically active
substances. ⇒ Phagocytes are attracted to the damaged tissues
⇒ Stimulation also results in the production of newly by several chemicals given off.
formed mediators by mast cells such as ⇒ Neutrophils arrive first, followed closely by
prostaglandins and leukotrienes. Stimulation of mast monocytes which develop into macrophages.
cells occurs in several ways such as by the ⇒ The neutrophils eliminate microorganisms and
anaphylatoxins (C3a, and C5a) of the complement then die.
system or by the crosslinking of surface IgE. Mast ⇒ Macrophages destroy pathogens and clean up
cells have high affinity Fc receptors for the IgE that the remains of damaged tissue cells and dead
is produced against an allergen. As a result, mast cell neutrophils.
release is most significant in either acute ⇒ Dead cells and fluid leaked from the capillaries
inflammation or in allergic responses. may accumulate as pus in the area before it is
absorbed by the body.
Natural killer (NK) cells destroy the body's own infected
cells, especially those harboring viruses. These cells are 3. Antimicrobial proteins
sometimes called large granular lymphocytes (LGL's). NK
cells have some surface markers in common with T cells, A number of proteins function in nonspecific defense by
and they are also functionally similar to cytotoxic T either directly attacking microorganisms or impeding their
lymphocytes (CTL's). Like CTL's, NK cells are reproduction. The three most important nonspecific
particularly important in the killing of cellular targets protein groups are complement proteins, interferon and
(usually tumor cells). Unlike CTL's, however, the killing the Cytokines.
by NK cells is nonspecific, they do not need to recognize
antigen/MHC on the target cell. NK cells do not have a T The complement system is a group of at least 20 serum
cell receptor and are not T cells. An NK cell kills a target proteins that interact with other defense mechanisms.
cell by releasing perforin (and other molecules) which These proteins interact in a series of steps that result in
damages the target cell membrane leading to death. NK lysis of the invading microbes. Complement activation is a
cells also cause death by inducing apoptosis in the target. cascade of events which resembles in some ways the
The cytokine TNF alpha is released by the NK cells and coagulation pathway. It consists of a series of enzymes
may be involved in this process. that become activated by each other in a cascade fashion.
It's confusing and hard to remember, but still important
⇒ Also assault abnormal cells that could form because complement has an important role in clearing
tumors antigen. This is done by the four major functions of
⇒ Are not phagocytic, but attack the membrane, complement activation:
causing cell lysis
⇒ local vasodilation
2. The inflammatory response ⇒ attraction of immune cells, especially
phagocytes (chemotaxis)
A localized inflammatory response occurs when there is ⇒ opsonization (or tagging) of foreign organisms
damage to a tissue due to physical injury or entry of for phagocytosis
microorganisms. ⇒ destruction of invading organisms by the
membrane attack complex (MAC attack)
⇒ Vasodilation of small vessels near the injury
increases the blood supply to the area, which The interferons are substances produced by virus-
produces the characteristic redness. infected cells that help other cells resist infection by the
⇒ The dilated vessels become more permeable virus.
allowing fluids to move into surrounding tissues
resulting in localized edema (swelling). ⇒ They are secreted by infected cells as a
nonspecific defense earlier than specific
Chemical signals are important in initiating an antibodies appear.
inflammatory response ⇒ Cannot save the infected cell, but their diffusion
to neighboring cells stimulates production of
⇒ Histamine is released from injured circulating proteins in those cells that inhibit viral
basophils (type of white blood cell) and mast replication.
cells (type of cell found in connective tissue). ⇒ Not a virus-specific defense; interferon
⇒ Released histamine causes localized produced to infection by one strain of virus
vasodilation, and the capillaries in the area produces resistance in cells to other unrelated
become leakier. viruses.

⇒ Most effective against short-term infections ⇒ They also form in the bone marrow, and then
(colds and influenza). migrate to the thymus gland to mature.
⇒ Interferons are now being mass produced using
recombinant DNA technology and are being Diversity refers to the immune system's ability to respond
tested as treatments for viral infections and to numerous kinds (millions) of invaders, which are
cancer. recognized by their antigenic markers. Each population of
antibody-producing lymphocytes is stimulated by a
Cytokines are the chemicals produced by these cells in specific antigen; the stimulated lymphocytes synthesize
order to communicate and orchestrate the attack. Just as and secrete the appropriate antibody.
hormones in the endocrine system can produce an effect
on other cells, so cytokines can act on other immune cells, Antigens interact with specific lymphocytes, inducing
especially cells that are close by.Cytokines have several immune responses and immunological memory. The
important characteristics: ability of the immune system to respond to the wide
variety of antigens which enter the body is based in the
⇒ The same cytokine may be made by a number of enormous diversity of antigen-specific lymphocytes
different cells. present in the system. Each lymphocyte will recognize and
⇒ The same cytokine may have different effects in respond to only one antigen. This specificity is determined
different circumstances. (This is called during embryonic development before any antigens are
'pleotropy') encountered, and is the consequence of the antigen
⇒ Different cytokines may have the same activity receptor on the lymphocyte's surface.
depending on the situation ('redundancy').
⇒ Cytokines often act together and increase the When an antigen enters the body and binds to receptors on
effects of one another ('synergy'). the specific lymphocytes, those lymphocytes are activated
⇒ They may also act as antagonists. and begin to divide and to differentiate.
⇒ Most cytokines have either paracrine or
autocrine effects. Paracrine means they act on ⇒ The divisions produce a large number of
cells near to them or that they are actually identical effector cells (clones), which bind to
touching. The autocrine function of IL-2 is well the antigen that stimulated the response.
known because, when a T cell is stimulated to ⇒ If, for example, a B cell is activated, it will
make IL-2, it stimulates itself via the IL-2 proliferate to produce a large number of plasma
receptor to proliferate. An example of an cells that will each secrete an antibody that
uncommon endocrine function for cytokines is functions as an antigen receptor for the specific
IL-1 which can cause fever by stimulating the antigen that activated the original B cell.
hypothalamus.
Thus, each antigen activates only a small number of the
II. How Specific Immunity Arises diverse group of lymphocytes. The activated cells
proliferate to produce a clone of millions of effector cells
The Adaptive immune system is the body's third line of which are specific for the original antigen (clonal
defense and is very specific in its response. selection).
It is distinguished from nonspecific defenses by: ⇒ Clonal selection = Antigenic-specific selection

of a lymphocyte that activates it to produce
¾ Specificity and Diversity clones of effector cells dedicated to eliminating
¾ Self/non-self recognition the antigen that provoked the initial immune
¾ Memory response. One clone of cells consists of effector
cells. Another clone of cells consists of memory
Specificity refers to the immune system's ability to cells.
recognize and eliminate particular microorganisms and ⇒ Effector cells are the short-lived cells that
foreign molecules Lymphocytes provide the specificity actually defend the body during an immune
and diversity of the immune system. Lymphocytes (a type response.
of white blood cell) are responsible for both humoral and ⇒ Effector cells are populations of cells resulting
cell-mediated immunity. The different responses are due from division of lymphocytes that were
to the two main classes of lymphocytes in the body: B activated by the binding of antigens to their
cells and T cells. antigen receptors.
⇒ Early B and T cells (as well as other

lymphocytes) develop from stem cells in the
bone marrow and are very much alike. They
only differentiate after reaching their site of
maturation.
⇒ B cells (B lymphocytes) are responsible for the
humoral immune response.
⇒ They form in the bone marrow and remain there
to complete their maturation.
⇒ T cells (T lymphocytes) are responsible for the
cell-mediated immune response.

E6. INTERACTION RESPONSIBLE FOR ANTIGEN-ANTIBODY INTERACTION:
Strength of Antigen-Antibody Interactions Antibody Affinity Is a Quantitative Measure of

Binding Strength
The noncovalent interactions that form the basis of
antigenantibody (Ag-Ab) binding include hydrogen The combined strength of the noncovalent interactions
bonds, ionic bonds, hydrophobic interactions, and van between a single antigen-binding site on an antibody and
der Waals interactions (Figure 1). Because these a single epitope is the affinity of the antibody for that
interactions are individually weak (compared with a epitope. Low-affinity antibodies bind antigen weakly and
covalent bond), a large number of such interactions are tend to dissociate readily, whereas high-affinity
required to form a strong Ag-Ab interaction. antibodies bind antigen more tightly and remain bound
Furthermore, each of these noncovalent interactions longer. The association between a binding site on an
operates over a very short distance, generally about 1 x antibody (Ab) with a monovalent antigen (Ag) can be
10 -7 mm (1 angstrom, Å); consequently, a strong Ag- Ab described by the equation
interaction depends on a very close fit between the
antigen and antibody. Such fits require a high degree of
complementarity between antigen and antibody, a
requirement that underlies the exquisite specificity that
characterizes antigen-antibody interactions. Antibody Avidity
The affinity at one binding site does not

always reflect the true strength of the
antibody-antigen interaction. When
complex antigens containing multiple,
repeating antigenic determinants are
mixed with antibodies containing
multiple binding sites, the interaction of
an antibody molecule with an antigen
molecule at one site will increase the
probability of reaction between those
two molecules at a second site. The
strength of such multiple interactions
between a multivalent antibody and
antigen is called the avidity. The
avidity of an antibody is a better
measure of its binding capacity within
biological systems (e.g., the reaction of
an antibody with antigenic determinants
on a virus or bacterial cell) than the
affinity of its individual binding sites.
High avidity can compensate for low
affinity. For example, secreted
pentameric IgM often has a lower affinity than IgG, but
the high avidity of IgM, resulting from its higher
valence, enables it to bind antigen effectively.
Cross-Reactivity
Although Ag-Ab reactions are highly specific, in some

cases antibody elicited by one antigen can cross-react
with an unrelated antigen. Such cross-reactivity occurs
if two different antigens share an identical or very similar
epitope. In the latter case, the antibody’s affinity for the
cross-reacting epitope is usually less than that for the
original epitope.
Cross-reactivity is often observed among polysaccharide

antigens that contain similar oligosaccharide residues.
The ABO blood-group antigens, for example, are
glycoproteins expressed on red blood cells. Subtle
differences in the terminal residues of the sugars attached
to these surface proteins distinguish the A and B blood-
group antigens.An individual lacking one or both of
these antigens will have serum antibodies to the missing
antigen(s). The antibodies are induced not by exposure to

red blood cell antigens but by exposure to cross-reacting type B individual has anti-A; and a type O individual
microbial antigens present on common intestinal thus has anti-A and anti-B.
bacteria. These microbial antigens induce the formation
of antibodies in individuals lacking the similar blood- A number of viruses and bacteria have antigenic
group antigens on their red blood cells. (In individuals determinants identical or similar to normal host-cell
possessing these antigens, complementary antibodies components. In some cases, these microbial antigens
would be eliminated during the developmental stage in have been shown to elicit antibody that cross-reacts with
which antibodies that recognize self epitopes are weeded the host-cell components, resulting in a tissue-damaging
out.) The blood-group antibodies, although elicited by autoimmune reaction. The bacterium Streptococcus
microbial antigens, will cross-react with similar pyogenes, for example, expresses cell-wall proteins
oligosaccharides on foreign red blood cells, providing the called M antigens. Antibodies produced to streptococcal
basis for blood typing tests and accounting for the M antigens have been shown to cross-react with several
necessity of compatible blood types during blood myocardial and skeletal muscle proteins and have been
transfusions. A type A individual has anti-B antibodies; a implicated in heart and kidney damage following
streptococcal infections.
E 7. PRIMARY AND SECONDARY IMMUNE RESPONE:
The primary immune response is the proliferation of memory cells are activated and rapidly
lymphocytes to form clones of effector cells specific to an proliferate to form a new clone of effector cells
antigen during the body's first exposure to the antigen. and memory cells.
⇒ These new clones of effector and memory cells
⇒ There is a 10- to 17-day lag period between are the secondary immune response.
initial exposure and maximum production of ⇒ This acquired immunity has long been
effector cells. recognized as a resistance to some infections
⇒ The lymphocytes selected by the antigen are encountered earlier in life (e.g., chicken pox).
differentiating into effector B cells and T cells
during the lag period. Lymphocyte development gives rise to an immune system
⇒ Activated B cells give rise to effector cells that distinguishes self from non-self
called plasma cells, which secrete antibodies
(humoral response) that eliminate the activating ⇒ Like all blood cells, lymphocytes derive from
antigen. stem cells in bone marrow or liver of a
developing embryo. Initially, all lymphocytes
A secondary immune response occurs when the body is are alike, but depending on the site of
exposed to a previously encountered antigen. maturation, they develop into T cells or B cells.
⇒ Cells that remain in the bone marrow develop
⇒ The response is faster (2 to 7 days) and more into B cells The "B" stands for bursa of
prolonged than a primary response. Fabricius an organ found in birds where they
⇒ The antibodies produced are more numerous, were first discovered. However, you can equate
and they are more effective at binding the the "B" with bone.
antigen. ⇒ Cells that migrate to the thymus
develop/matures into T cells. The "T" stands for
thymus.
Immune tolerance for self
Self/non-self recognition is the ability of the immune

system to distinguish between the body's own molecules
and foreign molecules (antigens).
⇒ Develops before birth when T and B

lymphocytes begin to mature in the thymus and
bone marrow of the embryo.
⇒ Antigen receptors on the surfaces of
This ability to recognize a previously encountered antigen lymphocytes are responsible for detecting
is known as immunological memory. foreign molecules that enter the body. There are
no lymphocytes reactive against the body's own
⇒ Based on memory cells, which are produced molecules under normal conditions.
during clonal selection for effectors in a ⇒ Self-tolerance = the lack of a destructive
primary immune response. immune response to the body's own cells.
⇒ Memory cells are not active during the primary ⇒ Failure of this system leads to autoimmune
response and survive in the system for long disorders that destroy the body's own tissues.
periods. (Effector cells produced in the primary
response are active, and thus, short-lived.)
⇒ When the same antigen that caused a primary
immune response again enters the body, the

E8. The Complement Components
The proteins and glycoproteins that compose the enzyme that dissolves the fibrin of blood clots) may
complement system are synthesized mainly by liver activate C1 directly without antibody participation.
hepatocytes, although significant amounts are also Following binding of antigen to antibody, the C1
produced by blood monocytes, tissue macrophages, and complement component, which is composed of three
epithelial cells of the gastrointestinal and genitourinary proteins (q, r, and s), attaches to the Fc portion of the
tracts. These components constitute 5% (by weight) of antibody molecule through its C1q subcomponent.
the serum globulin fraction.
In the presence of calcium ions, a trimolecular complex
Most circulate in the serum in functionally inactive forms (C1qrs - Ag - Ab) that has esterase activity is rapidly
as proenzymes, or zymogens, which are inactive until formed.
proteolytic cleavage, which removes an inhibitory
fragment and exposes the active site. The activated C1’s subcomponent attacks and cleaves its
natural substrates in serum (C2 and C4).
The complement-reaction sequence starts with an
enzyme cascade. Complement components are This leads to binding of a portion of each molecule (C2b
designated by numerals (C1–C9), by letter symbols (e.g., and C4b) to the antigen-antibody-complement complex
factor D), or by trivial names (e.g., homologous with the release of small C4a and C2a fragments. (The
restriction factor). released C2 fragment traditionally has been called C2a.
Peptide fragments formed by activation of a component For consistency, this text will label all larger complement
are denoted by small letters. In most cases, the smaller fragments that are bound to the target cell as b
fragment resulting from cleavage of a component is fragments.) With the binding of C2b to C4b, an enzyme
designated “a” and the larger fragment designated “b” with trypsinlike proteolytic activity is generated. The
(e.g., C3a, C3b; note that C2 is an exception: C2a is the natural substrate for this enzyme is C3; thus it is termed a
larger cleavage fragment). C3 convertase.
The larger fragments bind to the target near the site of Through the activity of C 4b2b (the bar indicates an
activation, and the smaller fragments diffuse from the active enzyme complex), C3 is cleaved into a bound
site and can initiate localized inflammatory responses by subcomponent C3b and a C3a soluble component.
binding to specific receptors.
C3b then absorbs to bound C4b2b, forming the complex
The complement fragments interact with one another to C 4b2b 3b, which acts as a C5 convertase and cleaves
form functional complexes. Those complexes that have C5 into fragments C5a and C5b. C6 and C7 rapidly bind
enzymatic activity are designated by a bar over the to C5b, forming a C 5b67 complex that possesses an
number or symbol (e.g., C4b2a, C3bBb). unstable membrane-binding site; once bound to a
membrane, this complex is stable.
Complement Activation
C8 and C9 then bind, forming the membrane attack
Figure 1 outlines the pathways of complement activation. complex (C 5b67 89) that creates a pore in the plasma
The early steps, culminating in formation of C5b, can membrane of the target cell. It is believed that the actual
occur by the classical pathway, the alternative pore is a doughnut-shaped polymer of C9. (The perforin
pathway, or the lectin pathway. The final steps that lead pores generated by cytotoxic T lymphocytes are
to a membrane attack are the same in all pathways. somewhat similar to complement pores.)
The Classical Complement Pathway If the cell is eucaryotic, Na and H2O enter through the
pore leading to osmotic lysis of the cell. If the cell is a
Activation of the classical complement pathway gram-negative bacterium, lysozyme from the blood
requires initiation by the interaction of antibodies with an enters through the pore and digests the peptidoglycan cell
antigen that is usually cell bound (figure 1). wall causing the bacterium to lyse osmotically. In
contrast, gram-positive bacteria are resistant to the
The order of effectiveness in activating complement is as cytolytic action of the membrane attack complex because
follows: IgM Æ IgG3 Æ IgG1Æ IgG2. they lack an exposed outer membrane and the thick
peptidoglycan prevents an attack on the plasma
However, some microbial products (lipid A of endotoxin membrane.
and staphylococcal protein A) or plasmin (a proteolytic

E9. MAJOR HISTOCOMPATIBILITY COMPLEX (HLA in HUMANS):
The major histocompatibility complex (MHC; of antigen is on surface of cell and, bound to a molecule
sometimes referred to as human leukocyte antigens or known as MHC gene product.
HLA in humans) is a group of glycoproteins (proteins
with attached sugar chains) embedded in the plasma Thus the role of MHC molecules coded by the MHC is
membranes of cells. to bind to peptide fragments derived from the protein
antigens and interact with TCR. The Binding of MHC
As T cells interacts with the antigen that come form the molecule with peptide is selective.
inside of the cells they use an antigen recognition system ⇒ MHC molecules may be viewed as third set of
which is distinct from the antigen recognition recognition molecules for the antigen in the
system.The T cell receptor (TCR) for antigen interacts immune response, in addition to antigen
with the antigen almost exclusively only when fragment specific T cells and B cells.

⇒ Every vertebrate species has MHC genes and

products which are cause of rejection of tissues
between different members of same species
and are dominated by T cells.
⇒ These important "self-markers" are coded for
by a family of genes
⇒ There are at least 20 MHC genes and at least
100 alleles for each gene.
⇒ This results in millions of different MHC
molecules.
⇒ The probability that two individuals will have
matching MHC sets is virtually zero unless
they are identical twins.
There are two main classes of MHC molecules in the

body:
¾ Class I MHC molecules are located on all
nucleated cells of the body.
¾ Class II MHC molecules are found only
on specialized cells, such as
macrophages, B cells, and activated T Although MHC molecules give identity to the cell as
cells. being "self" the major function of MHC molecules is to
bind to an antigen, then present this antigen on the
surface of the cell, thereby facilitating antigen binding to
a T cell
⇒ Class MHC I molecules facilitate antigen
binding to cytotoxic T
cells (CD 8 Cells).
⇒ Class MHC II molecules facilitate antigen
binding to helper T cells
(CD 4 cells).
Each MHC-antigen combination forms a unique

complex that is recognized by specific antigen receptors
on certain T cells
E10. HUMORAL AND CELL MEDIATED Immune Responses
The body will mount either a humoral response or a cell- In humoral immunity or cell-mediated immunity, there
mediated response, depending on the antigen which are several general steps involved in the immune
stimulates the system. responses.
⇒ Humoral immunity produces antibodies in
response to toxins (ex bee venom), free 1. The antigen must encounter the B-lymphocytes, T-
bacteria, and viruses present in the body fluids. lymphocytes, and antigen-presenting cells (APCs)
⇒ "Humor" is medieval term for body fluids. capable of carrying out an adaptive immune
Here it refers to the fluid of the blood and the response.
lymph. 2. Naive lymphocytes must recognize epitopes of an
⇒ Antibodies to these types of antigens are antigen by means of antigen-specific receptor
synthesized by certain lymphocytes and then molecules on their surface and become activated.
secreted as soluble proteins which circulate This is known as clonal selection.
through the body in blood plasma and lymph. 3. The large clones of identical B-lymphocytes, T4-
⇒ Cell-mediated immunity is the response to lymphocytes, and T8-lymphocytes now differentiate
intracellular bacteria and viruses, fungi, into effector cells capable of directing body
protozoans, worms, transplanted tissues, and defenses against the original antigen resulting in its
cancer cells. destruction or neutralization.
⇒ Depends on the direct action of certain types 4. Some of the B-lymphocytes, T4-lymphocytes, and
of T- lymphocytes rather than antibodies. T8-lymphocytes differentiate into long-lived,
circulating memory cells.

In the humoral response, B cells produce antibodies The antibodies produced during humoral immunity
against extracellular pathogens ultimately defend the body through a variety of different
⇒ The humoral response occurs when an antigen means. These include:
binds to B cell receptors that are specific for
the antigen. 1. Opsonization
⇒ The B cells differentiate into a clone of plasma
cells which begin to secrete antibodies that are Opsonization, or enhanced attachment, refers to the
most effective against pathogens circulating in antibody molecules IgG, IgE and the complement
the blood or lymph. proteins C3b and C4b attaching antigens to phagocytes.
⇒ Memory cells are also produced and form the This results in much more efficient phagocytosis.
basis for secondary immune responses.
The selective activation of a B cell results from one of

two mechanisms:
1. T-dependent antigens: Antigens that evoke the

cooperative response of macrophages, helper T cells, and
B cells
o These antigens cannot stimulate antibody
production without helper T cell
involvement.
o Most antigens are T-dependent. Memory
cells are produced in T-dependent
responses.
2. T-independent antigens: Antigens that trigger

humoral immune responses without macrophages or T
cell involvement
o These antigens usually are long chains of 2. Antibody-Dependent Cellular Cytotoxicity
repeating units, such as polysaccharides or (ADCC) by NK cells
protein subunits often found in bacterial
capsules and flagella. NK cells are capable of antibody-dependent cellular
o B cells are stimulated directly by the cytotoxicity or ADCC. NK cells have receptors on their
antigen, which probably binds surface for the Fc portion of IgG. When IgG is made
simultaneously to several antigen receptors against epitopes on "foreign" membrane-bound cells,
on the B cell surface. e.g., virus-infected cells and cancer cells, the Fab
o The antibody production (humoral response) portions of the antibodies react with the "foreign" cell.
is usually much weaker than that of T- The NK cells then bind to the Fc portion of the antibody.
dependent antigens.
o No memory cells are generated in T- The NK cell then releases pore-forming proteins called
independent responses. perforins, proteolytic enzymes called granzymes, and
chemokines. Granzymes pass through the pores and
Whether activated by T-dependent or T-independent activate the enzymes that lead to apoptosis of the
antigens, a B cell gives rise to a clone of plasma cells. infected cell by means of destruction of its structural
⇒ Each of these effector cells secretes up to 2000 cytoskeleton proteins and by chromosomal degradation.
antibodies per second into the body fluids for As a result, the cell breaks into fragments that are
its 4- to 5-day lifespan. subsequently removed by phagocytes. Perforins can also
⇒ The specific antibodies help eliminate the sometimes result in cell lysis. (When NK cells are
foreign invader from the body. carrying out ADCC, they are sometimes also referred to
as killer cells.)

3. Neutralization of Exotoxins
For an exotoxin to cause harm it must first bind to

receptors on a susceptible host cell. Antitoxin antibodies,
mainly IgG, are made against protein exotoxins. They
combine with the exotoxin molecules before they can
interact with host target cells and thus neutralize the
toxin.
IN THE CELL-MEDIATED RESPONSE,

CYTOTOXIC-T CELLS DEFEND AGAINST
INTRA-CELLULAR PATHOGENS
⇒ Antigen-activated cytotoxic T cells kills cells

that are infected by pathogens (e.g., viruses,
bacteria).
⇒ Host cells infected by viruses and other
pathogens display antigens complexed with
class I MHC molecules on their surfaces.
⇒ Cytotoxic T cells have specific receptors that
4. Neutralization of Viruses recognize and bind to antigen-class I MHC
markers. (Note that this differs from Helper T
In order for viruses to infect a cell and replicate, they cells which bind to antigen-class II MHC
must first adsorb to receptors on the host cell's plasma complexes.)
membrane. Antibodies made against the epitopes on ⇒ The cytotoxic T receptor can bind to any cell
viral capsids or on viral envelope glycoproteins that in the body displaying the antigen-class I
function in the attachment of the virus to the host cell MHC marker since class I MHC is present on
receptor prevent viral adsorption (see Fig). Neutralizing all nucleated cells.
antibodies are especially important in preventing viral
⇒ When a cytotoxic T cell binds to an infected
reinfection.
cell (target cell), it releases chemicals that
forms a lesion in the infected cell's membrane.
⇒ Cytoplasm escapes through the lesion and
eventually cell lysis occurs.
⇒ Destruction of the host cell not only removes
the site where pathogens can reproduce, but
also exposes the pathogens to circulating
antibodies from the humoral response.
⇒ Cytotoxic cells continue to live after
destroying the infected cell and may kill many
others displaying the same antigen-class I
MHC marker.
6. Agglutination of Microorganisms
Agglutination is mainly a function of antibodies with

multiple reactive Fab sites such as IgM and IgA. The
antibodies link microorganisms together (cause them to
agglutinate) so they can be filtered out of the lymph and
blood and be phagocytosed more effectively by the fixed
macrophages of the mononuclear phagocytic
(reticuloendothelial) system (see Fig)

Helper T lymphocytes function in both humoral and Helper T cells that bind to a class II MHC-antigen
cell-mediated immunity complex on these cells are induced to differentiate into
either of two clones of cells:
Cells that take up antigens, such as B cells and
macrophages alert the immune system, via helper T 1. Activated helper T cells: Secrete chemicals that
cells, of the presence of a foreign antigen. stimulate other lymphocytes (e.g., stimulates
⇒ Such cells engulf foreign material (e.g., differentiation of B cells into antibody-secreting plasma
bacteria) cells and induces cytotoxic T cells to become active
⇒ The foreign protein (e.g., from broken down killers).
bacteria) binds to a class II MHC molecule and
is conveyed to the outside of these cells 2. Memory helper T cells: These cells become
⇒ The class II MHC molecule with bound important when there is a second exposure to the antigen
foreign antigen is recognized by a helper T
cell.
E11. IMMUNITY AND HEALTH AND DISEASE
A. Immunity can be achieved naturally or artificially ⇒ Some antibodies (IgA) are transferred to nursing
infants though breast milk.
Active immunity is the immunity conferred by recovery ⇒ Provides temporary protection to newborns
from an infectious disease. whose immune systems are not fully operational
at birth. - - Persists as long as the antibodies last
⇒ Depends on response by the person's own (a few weeks or months), but it can provide
immune system protection from infections until a baby's own
⇒ May be acquired naturally from an infection to immune system has matured.
the body or artificially by immunization, also
known as vaccination B. The immune system's capacity to distinguish self
⇒ Vaccines may be inactivated bacterial toxins, from non-self limits blood transfusion and tissue
killed microbes, parts of microbes, or viable but transplantation
weakened microbes.
⇒ In all cases the organisms can no longer cause The body's immune system distinguishes between self (the
the disease but can act as antigens and stimulate body's own cells) and nonself (foreign cells). Nonself
an immune response. includes pathogens and cells from other individuals of the
⇒ A person vaccinated against an infectious agent same species.
who encounters the pathogen will show the
same rapid, memory-based secondary response 1. Blood groups and blood transfusion
as someone who has had the disease.
The human ABO blood groups provide a good example of
Passive immunity is immunity which has been transferred nonself recognition. The antigen present on the surface of
from one individual to another by the transfer of the erythrocytes is not antigenic to that person but may be
antibodies. recognized as foreign if placed in the body of another
individual.
⇒ Naturally occurs when IgG antibodies cross the ⇒ Individuals of blood type A have the A antigen
placenta from a pregnant woman to her fetus. and make anti-B antibodies.

⇒ Individuals of blood type B have the B antigen C. Abnormal immune function can lead to disease
and make anti-A antibodies.
⇒ Individuals of blood type AB have the A and B 1. Allergies (Hypersensitivity): Allergy = A
antigen and make no antibodies. hypersensitivity of the body's defense system to an
⇒ Individuals of blood type O have neither the A environmental antigen called an allergen. Some believe
nor B antigen and make anti-A and anti-B these reactions to be evolutionary remnants to infection by
antibodies. parasitic worms due to similarities in the responses. IgE
class antibodies are commonly involved in allergic
Blood group antibodies can cause blood of a different reactions; these antibodies recognize pollen as allergens
antigenic type to agglutinate, resulting in a life-threatening
transfusion reaction. ⇒ IgE antibodies attach by their tails to
⇒ Type O individuals are universal donors since noncirculating mast cells found in connective
their blood has neither antigen. tissues. (Remember mast cells are involved in
⇒ Type AB individuals are universal recipients the inflammatory response.)
since they produce neither antibody A or ⇒ When a pollen grain bridges the gap between
antibody B. two adjacent IgE monomers, the mast cell
⇒ The blood group antibodies are present in the responds with a reaction called degranulation.
body before a transfusion occurs because they ⇒ Degranulation involves the release of histamine
form in response to the body's normal bacterial and other inflammatory agents.
flora that have epitopes very similar to blood ⇒ Histamine causes dilation and increased
group antigens. This phenonomenon is termed permeability of small blood vessels, which
as cross reactivity. results in the common symptoms of an allergy.
⇒ Antihistamines are drugs used to treat allergies
Usually IgM class antibodies do not cross the placenta, since they interfere with the action of histamine.
thus they present no harm to a developing fetus with a ⇒ Anaphylactic shock is a life-threatening reaction
blood type different from the mother. to injected or ingested antigens; it is the most
⇒ The Rh factor is another blood group antigen. serious type of acute allergic response.
Rh factor causes problems when a mother is Rh ⇒ Occurs when mast cell degranulation causes a
negative and her fetus is Rh positive (inherited sudden dilation of peripheral blood vessels and a
from the father). drastic drop in blood pressure.
⇒ When small amounts of fetal blood cross the ⇒ Death may occur in a few minutes.
placenta and come into contact with the mother's ⇒ This hypersensitivity may be associated with
lymphocytes, the mother develops antibodies foods (peanuts, fish) or insect venoms (wasp or
against the Rh factor. bee stings).
⇒ Usually only a problem in the second child since ⇒ Epinephrine may be injected to counteract the
the response will be quick due to sensitization allergic response.
and formation of memory cells during the first
baby's gestation. 2. Autoimmune diseases
⇒ Unlike blood group antibodies, Rh antibodies
are IgG class, which can cross the placenta. Autoimmune disease = An immune system reaction
⇒ The mother's antibodies cross the placenta and against self Examples:
destroy the red blood cells of the Rh positive
fetus. ⇒ Some cases involve immune reactions against
components of the body's own cells, which are
2. Tissue grafts and organ transplantation released by the normal breakdown of skin and
other tissues, especially nucleic
The MHC is a biochemical fingerprint unique to each acids(DNA/RNA) in Systemic Lupus
individual. Erythematous(SLE).
⇒ Rheumatoid arthritis is an autoimmune disease
⇒ Complicates tissue grafts and organ transplants in which inflammation damages cartilage and
since foreign MHC molecules are antigens and bones in joints.
cause cytotoxic T cells to mount a cell-mediated ⇒ Destruction of insulin-producing pancreas cells
response. In the case of bone marrow by an autoimmune reaction appears to cause
transplants, which are used to treat leukemia and insulin-dependent diabetes.
other cancers, the graft rather than the host is the ⇒ In multiple sclerosis, T cells reactive against
source of immune rejection myelin infiltrate the central nervous system and
⇒ The donated bone marrow contains lymphocytes destroy the myelin of neurons.
that will react against the recipient. ⇒ Antibodies produced to repeated streptococcal
⇒ This graft versus host rejection is limited if the infections may react with heart tissues and cause
MHC molecules of the donor and recipient are valve damage in some people.
well matched. ⇒ Other autoimmune diseases are Grave's disease
and Rheumatic fever.
The reactions of the immune system to transfusions, tissue
grafts, and organ transplants are normal reactions of a
healthy immune system, not disorders of the system.


3. Immunodeficiency diseases strains: HIV-1 and HIV-2. HIV infects cells,

including helper T cells.
Immunodeficiency refers to a condition where an ⇒ After entry, HIV RNA is reverse-transcribed and
individual is inherently deficient in either humoral or cell- the product DNA is integrated into the host cell
mediated immune defenses. genome.
Severe combined immunodeficiency (SCID) is a ⇒ In this provirus form, the viral genome directs
congenital disorder in which both the humoral and the production of new virus particles.
cell-mediated immune defenses fail to function. ⇒ HIV is not eliminated from the body by
antibodies for several reasons:
Not all cases of immunodeficiency are inborn conditions. ⇒ The latent provirus is invisible to the immune
⇒ Some cancers, like Hodgkin's disease, damage system.
the lymphatic system and make the individual ⇒ The virus undergoes rapid mutational changes in
susceptible to infection. antigens during replication which eventually
⇒ Some viral infections cause depression of the overwhelms the immune system.
immune system (e.g., AIDS). ⇒ The population of helper T-cells eventually
declines to the point where cell-mediated
Acquired immunodeficiency syndrome (AIDS): immunity collapses. Researchers are not entirely
Acquired immunodeficiency syndrome is a severe sure why this occurs.
immune system disorder caused by infection with the ⇒ Secondary infections characteristic of HIV
human immunodeficiency virus (HIV). Individuals infection develop (Pneumocystis pneumonia and
with AIDS are highly susceptible to opportunistic Kaposi's sarcoma).
diseases, infections, and cancers that take advantage
of a deficient immune system. Mortality rate
approaches 100%. HIV probably evolved from
another virus in central Africa and may have gone
unrecognized for many years. There are two major

E12. IMMUNIZATION AND VACCINES:
Immunization is the means of providing specific ⇒ Live vaccines are used against a number of viral
protection against most common and damaging pathogens. infections (polio (Sabin vaccine), measles, mumps,
Specific immunity can be acquired either by passive or by rubella, chicken pox, hepatitis A, yellow fever, etc.).
active immunization and both modes of immunization can The only example of live bacterial vaccine is one
occur by natural or artificial means. against tuberculosis (Mycobacterium bovis: BCG).
While live vaccines normally produce only self-
Passive Immunity: Immunity can be acquired, without limiting non-clinical infections and subsequent
the immune system being challenged with an antigen. This immunity, they carry a serious risk of causing overt
is done by transfer of serum or gamma-globulins from an disease in immunocompromised individuals.
immune donor to a non-immune individual. Alternatively, ⇒ Killed (heat, chemical or UV irradiation) viral
immune cells from an immunized individual may be used vaccines include those for polio (Salk vaccine),
to transfer immunity. Passive immunity may be acquired influenza, rabies, influenza, rabies, etc. Most
naturally or artificially. bacterial vaccines are killed organisms ( typhoid,
cholera, plague, pertussis, etc.).
Naturally acquired passive immunity: Immunity is ⇒ Other bacterial vaccines utilize their cell wall
transferred from mother to fetus through placental transfer components (haemophilus, pertussis, meningococcus,
of IgG or colostral transfer of IgA. pneumococcus, etc.). Some viral vaccines (hepatitis-
B, rabies, etc.) consist of antigenic proteins cloned
Artificially acquired passive immunity: Immunity is into a suitable vector (e.g., yeast). When the
often artificially transferred by injection with γ-globulins pathogenic mechanism of an agent involves a toxin, a
from other individuals or γ-globulin from an immune modified form of the toxin (toxoid) is used as a
animal. Passive transfer of immunity with immune vaccine (e.g., diphtheria, tetanus, cholera). These
globulins or γ-globulins is practiced in numerous acute subunit vaccines are designed to reduce the toxicity
situations of infections (diphtheria, tetanus, measles, problems. Each type of vaccine has its own
rabies, etc.), poisoning (insects, reptiles, botulism), and as advantages and disadvantages.
a prophylactic measure (hypogammaglobulinemia). In ⇒ Anti-idiotype antibodies are also under trial.
these situations, γ-globulins of human origin are preferable Similarly, DNA vaccines and immunodominant
although specific antibodies raised in other species are peptides (recognized by the MHC molecules) are
effective and used in some cases (poisoning, diphtheria, under investigation, particularly for protection
tetanus, gas gangrene, botulism). While this form of against viral diseases.
immunization has the advantage of providing immediate ⇒ The protective immunity conferred by a vaccine may
protection, heterologous γ-globulins are effective for only be lifelong (measles, mumps, rubella, small pox,
a short duration and often result in pathological tuberculosis, yellow fever, etc.) or may last as little as
complications (serum sickness) and anaphylaxis. six months (cholera).
Active Immunity: This refers to immunity produced by Application of immunological techniques:

the body following exposure to antigens.
1. Monoclonal Antibodies with Virtually Any
Naturally acquired active immunity: Exposure to Desired Specificity Can Be Readily Prepared
different pathogens leads to sub-clinical or clinical
infections which result in a protective immune response 2. Proteins Can Be Detected and Quantitatively
against these pathogens. determined by Using an Enzyme-Linked
Immunosorbent Assay (ELISA).
Artificially acquired active immunity: Immunization
may be achieved by administering live or dead pathogens 3. Western Blotting Permits the Detection of
or their components. Vaccines used for active Proteins Separated by Gel Electrophoresis
immunization consist of live (attenuated) organisms, killed
whole organisms, microbial components or secreted toxins 4. Immune-Fluorescent Markers Make Possible the
(which have been detoxified). Visualization of Proteins in the Cell using
fluorescence microscopy
⇒ The first live vaccine was cowpox virus introduced
by Edward Jenner as a vaccine for smallpox; 5. Gold (heavy metal) tagged antibodies make
however, variolation, innoculation using pus from a Possible the Visualization of Proteins in the Cell
patient with a mild case of smallpox has been in use using electron microscopy
for over a thousand years.

E13. IMMUNITY AGAINST INFECTIOUS PATHOGENS
I. Introduction 1. CR1 and CR3 receptors are able to interact with C3b
and iC3b on the microbial membrane, generated as a
During evolution, an extremely complex system of anti- consequence of complement activation by the alternative
infections defenses has emerged. But at the same time as pathway, a property common to many bacteria (see
vertebrates and mammals developed their defenses, below).
microbes continued evolving as well, and many became 2. Mannose receptors on phagocytic cells may mediate
adept at avoiding the consequences of the anti-infections ingestion of organisms with mannose-rich
defense mechanisms. The interplay between host polysaccharides, such as Candida albicans. Mannose
defenses, microbial virulence, and microbial evasion mediated phagocytosis is amplified by a mannose-
mechanisms determines the outcome of the constant binding protein that promotes phagocytosis through
encounters between humans and pathogenic organisms. complement activation and direct interaction with the
C1q (collectin) receptor on phagocytic cells.
II. Nonspecific Anti-Infectious Defense Mechanisms 3. C-reactive protein binds to certain bacterial
polysaccharides and has very similar effects to the
Nonspecific defense mechanisms play a most important mannosebinding protein, activating complement and
role as a first line of defense, preventing penetration of promoting phagocytosis, both through CR1 and CR3, as
microorganisms beyond the outer exposed surfaces of the well as by other receptors, including the FcγRI and the
body. The following is a brief description of the most C1q receptor, both of which bind this protein.
important nonspecific defense mechanisms.
D. Activation of the Complement System via the
A. Physical and Chemical Barriers, including the Alternative Pathway. A variety of microorganisms
integrity of the epithelial and mucosal surfaces, the flow (bacteria, fungi, viruses, and parasites) can activate
of mucosal secretions in the respiratory tract, the acidity complement by the alternative pathway (see Table 1). In
of the gastric contents, and the secretion of lysozyme in most cases where adequate studies have been carried out,
tears, saliva, and most secretions. The importance of polysaccharidic structures have been proven to be
these barriers is apparent from the prevalence of responsible for complement activation of the alternative
infections when their integrity is compromised. pathway. This activation will lead to phagocytosis,
through the generation of C3b, and to chemotaxis,
B. Inducible Nonspecific Responses, including fever through the release of C3a.
and release of interferons, activated when infectious
agents manage to invade, particularly effective in
preventing viral replication.
C. Phagocytosis. As a microbe penetrates beyond the

skin or mucosal surface, it will encounter cells able to
ingest it. Two types of cells are particularly adept at
nonimmune phagocytosis: tissue macrophages and
granulocytes (particularly neutrophils). This nonimmune
phagocytosis involves a variety of recognition systems
(Fig. 1).
E. Acute Phase Reaction and Leukocyte Chemotaxis.

The initial recognition by phagocytes and the activation
of the complement system by the alternative pathway, by
themselves, may not be sufficient to eradicate the
invading microorganism, but the response is quickly
amplified by a multitude of cytokines released by
macrophages activated as a consequence of phagocytosis,
including IL-1, IL-8, and TNF-α.
1. IL-1 and TNF- α cause an increase in body
temperature, mobilize neutrophils from the bone marrow,
and upregulate the synthesis of a variety of proteins
known as acute phase reactants, including C-reactive
protein and the mannose-binding protein mentioned
above.
2. TNF α and IL-1 up-regulate the expression of cell
adhesion molecules in the endothelial cells of
neighboring endothelial cells, thus promoting adherence
of leukocytes, and increase vascular permeability. Both

factors facilitate the migration of leukocytes off the G. g/D T Lymphocytes are predominantly localized to
vessels, toward the focus of infection. the mucosal epithelia, where they appear to recognize
3. IL-8 has chemotactic properties. Together with other infected epithelial cells by a nonimmunological
chemotactants, such as C5a and bacterial peptides, it mechanisms (i.e., not involving the T-cell receptors),
attracts neutrophils toward the focus of infection. which are subsequently immunization. It was eventually
demonstrated that the cell wall polysaccharides of several
F. Natural Killer (NK) Cells are able to destroy viral strains of enterobacteriaceae and the AB
infected cells as a consequence of the delivery of an oligosaccharides of human erythrocytes are structurally
activating signal in the absence of an inhibitory signal. similar. Thus, crossreactive antibodies to
1. The activating signal is delivered as a consequence of enterobacteriaceae are responsible for the “spontaneous”
the interaction between a recognition molecule (NKR- development of antibodies to human red cell antigens in
P1) on the NK cells and glycoproteins expressed on the chickens.
membrane of the infected cells.
2. The inhibiting signal is usually delivered as a 3. Newborn babies of blood groups A, B, or O do not
consequence of the interaction between a membrane have either anti-A or anti-B isohemagglutinins, but will
protein in the NK cell membrane and MHC-I molecules develop them during the first months of life, as they get
on the membrane of normal cells. Viral infected cells exposed to common bacteria with polysaccharide
often have a reduced expression of MHC-I molecules capsules. However, newborns are tolerant to their own
due either to a down-regulation of cellular protein blood group substance, so they will only make antibodies
synthesis or a specific inhibition of MHC-I transport to against the blood group substance that they do not
the cell membrane, discussed later in this chapter. express. Blood group AB individuals never produce AB
isoagglutinins.
B. Other Mechanisms for the Generation of Natural IV. The Protective Role of Specific Antibodies
Antibodies. Cross-reaction is probably the most
common explanation for the emergence of “natural” A. The Humoral Immune Response. If a pathogen is
antibodies, but other mechanisms, such as the mitogenic not eliminated by nonimmunological means and
effects of Tindependent antigens and the nonspecific continues to replicate, it will eventually spread through
stimulatory effects of lymphokines released by antigen- the blood and lymph, and will usually be trapped by
stimulated T lymphocytes, which could activate B cells macrophages and follicular interdigitating cells in the
responding to other antigens, could explain the rise of lymph nodes and spleen. Those cells are able to
“nonspecific” immunoglobulins that is observed in the internalize antigens interacting with receptors such as the
early stages of the humoral response to many different mannose receptor (see above), process the antigen in the
antigens. It is only a matter of random probability that endosomic compartment, and express MHC-II-associated
some of those “nonspecific” immunoglobulins may play antigen-derived peptides. This creates the ideal
the role of “natural antibody” relative to an unrelated conditions for the onset of an immune response: B
antigen. lymphocytes can interact with membrane-bound antigen
while helper T cells recognize MHC-II with antigen-
C. The Significance of “Natural” Antibodies. derived peptides presented by the same APC. The
“Natural” antibodies may play an important protective antigen-recognizing cells will interact and costimulate
role. Antibodies elicited to E. coli K100 cross-react with each other and, after a time lag necessary for
the polyribophosphate of Haemophilus influenzae and proliferation and differentiation of B cells into antibody-
can protect experimental animals against infection with producing cells, circulating antibody will become
the latter organism. It is logical to assume that such detectable.
cross-immunizations may be rather common and play an 1. A primary immune response will take 5 to 7 days
important protective role against a variety of infectious (some times as long as 2 to 3 weeks) to be detected. The
agents. predominating isotype of the antibodies made early in a
primary immune response is IgM.

2. A secondary immune response has a shorter lag binding to cell receptors or its ability to release nucleic
phase (as short as 3–4 days) and the predominant isotype acid into the cell.
of the antibodies is IgG. 6. Mucosal protection. Secretory antibodies seem to
3. The differentiation between IgM and IgG antibodies is play their protective role by preventing the attachment
a useful index for discriminating between recent and past and penetration of microbial agents through mucosal
infections, particularly in the case of viral infections. surfaces.
B. Antibody-Dependent Anti-Infectious Effector C. Factors Influencing the Effectiveness of an Anti-

Mechanisms. As soon as specific antibodies become Infectious Humoral Response. The effectiveness of the
available, they can protect the organism against infection humoral immune response in eliminating or preventing
by several different mechanisms. the proliferation of an infectious agent or in neutralizing
1. Complement-mediated lysis of a microorganism or a toxin before it causes severe disease depends on
virus-infected cell can result from the activation of the whether or not antibodies can be available in time to
complete sequence of complement. However, as prevent the infection to develop or the toxin to reach its
discussed below, both mammalian cells and most cellular target.
pathogenic microorganisms have developed mechanisms 1. If the relevant antibodies are present in circulation, as
that allow them to resist complement-mediated lysis. a result of vaccination, previous infection, or
2. Opsonization and phagocytosis are, in contrast, of crossreaction between different microorganisms,
extreme physiological significance. Several proteins can protection is most effective, the microorganism or its
opsonize and promote phagocytosis, as discussed above, toxin(s) will be almost immediately neutralized, and the
but IgG antibodies are the most efficient opsonins, infection will remain subclinical.
particularly when complement is activated and C3b joins 2. If preformed antibodies are not available, protection
IgG on the microbial cell membrane. will depend on whether antibody synthesis can take place
a. Opsonins. Because of their role in promoting before the “incubation period” (period of time during
opsonization, IgG antibodies and C3b are considered which the infectious agent is multiplying but has not yet
“opsonins.” Both C3b and IgG alone are efficient reached sufficient mass to cause clinical disease) is over.
opsonins, but their effects are synergistic. a. Some infectious agents, such as the influenza virus,
b. Fc and CR1 receptors, expressed on the membranes have very short incubation periods (about 2 to 3 days)
of all phagocytic cells, mediate phagocytosis and also and, in such cases, not even a secondary immune
deliver activating signals to the cells, inducing release of response can be protective.
cytokines, as mentioned above. b. In most infections, the duration of the incubation
c. Killing through opsonization has been demonstrated period is sufficiently long as to allow a secondary
for bacteria, fungi, and viruses, while phagocytosis of immune response to provide protective antibodies. Thus,
antibody/complement-coated unicellular parasites has not for many infections, particularly the common viral
been clearly demonstrated. diseases of childhood, previous exposure and acquisition
d. The biological significance of phagocytic cells as of memory ensure that antibody will be produced in time
ultimate mediators of the effects of opsonizing antibodies to maintain subsequent exposures, which play the role of
is obvious; the protective effects of antibodies are lost in natural “booster” doses, probably explaining the
patients with severe neutropenia or with severe “immunity for life” associated with them.
functional defects of their phagocytic cells, and those c. The goal of prophylactic immunization may vary from
patients have increased incidence of infections with a case to case. In diseases with very short incubation
variety of opportunistic organisms. periods, it is essential to maintain the levels of
neutralizing antibody in circulation necessary to
3. Antibody-dependent cell-mediated cytotoxicity immediately abort infection. In most other diseases, it
(ADCC). Cells with Fc receptors may be able to may be sufficient to induce immunological memory,
participate in killing reactions that target antibody-coated since once memory has been induced, the immune
cells. IgG1, IgG3, and IgE antibodies and cells with system will be able to respond in time to prevent the
FcγR or FcεR are usually involved. Large granular development of clinical infection.
lymphocytes or monocytes are the most common effector d. Protection by humoral immunity is only possible if the
cells in ADCC, but in the case of parasitic infections, infectious agent is easily available to the antibodies
eosinophils play the principal role in cytotoxic reactions. produced against it. Thus, organisms that grow
Different effector mechanisms are responsible for killing intracellularly are not easy to eliminate by antibodies. In
by different types of cells: large granular lymphocytes addition, as discussed in detail later in this chapter, those
kill through the release of granzymes and signaling for organisms able to change their antigenic make-up during
apoptosis; monocytes kill by releasing oxygen active the course of an infection can persist in spite of a
radicals and nitric oxide; eosinophil killing is mostly vigorous humoral response.
mediated by the release of a “major basic protein,”
which is toxic for parasites. V. The Protective Role of Cell-Mediated Immunity
4. Toxin neutralization. Many bacteria release toxins,
which are often the major virulence factors responsible Many organisms have the ability to grow and replicate
for severe clinical symptoms. Antibodies to these toxins intracellularly, some as an absolute requirement, others
prevent their binding to cellular receptors and promote as an option that allows them to survive after
their elimination by phagocytosis. phagocytosis. Antibodies are largely ineffective against
5. Virus neutralization. Most viruses spread from an those organisms, and T lymphocytes play a major role in
initial focus of infection to a target tissue via the blood their elimination, as reflected by the finding of
stream. Antibodies binding to the circulating virus lymphocytic infiltrates in tissues infected by intracellular
change its external configuration and prevent either its infectious agents, such as viruses. The immune system

has two basic options to eliminate those organisms: to 2. Inflammatory reaction. In concert with the release of
kill the infected cell, or to enhance the infected cell's chemotactic cytokines, the expression of CAMs in
ability to kill intracellular organisms. Either option neighboring microvessels is up-regulated, favoring
requires the persistent activation of the TH1 adherence and migration of monocytes and granulocytes
subpopulation of helper cells. to the extravascular space. Inflammatory cells
accumulate in the area of infections and, as a
A. Lymphocyte-Mediated Cytotoxicity. It can be easily consequence of crossactivation circuits involving
demonstrated that viral-infected cells are lysed as a phagocytes and TH1 lymphocytes, the localized
consequence of their incubation with “immune” macrophages and granulocytes become activated.
lymphocytes, obtained from an animal previously Consequently, a. the enzymatic contents and respiratory
exposed to the same virus. burst of the cells become more intense, making them
1. Recognition of infected cells better suited for killing intracellular organisms.
a. Virus-infected cells that express MHC-I molecules b. Phagocytosis is enhanced.
with associated viral peptides can be easily recognized c. Activated macrophages release higher levels of
by CD8+ cytotoxic T cells. While CD8+ lymphocytes, cytokines, including IL-12, which continues to promote
when strongly stimulated, are able to release IL-2 and the differentiation of TH1 cells, as well as IL-1, IL-6, and
differentiate into effector cytotoxic T cells, usually that TNF-α, which in association with IL-8, play the major
differentiation requires the collaboration of TH1 helper T role in inducing the synthesis of reactive proteins,
cells. The activation of CD4+ TH0 cells and their increasing body temperature, and several other metabolic
differentiation into TH1 helper T cells is likely to take effects characteristic of the inflammatory reaction.
place in lymphoid tissues, since macrophages are often
infected by viruses and virus-derived peptides are likely VI. The Immune Deficiency Syndromes as Models for
to be presented by those cells both in association with the Study of the Importance of Immune Defenses
MHC-I and MHC-II molecules. against Infections in Humans
b. Macrophages are often infected by intracellular
organisms of all kinds, including bacteria, fungi, and Most of our information about the immune system in
protozoa. Thus, recognition by CD4+ and CD8+ T humans has been learned from the study of patients with
lymphocytes is likely to take place, resulting in the immunodeficiency diseases. The most characteristic
differentiation of effector CD8+ cytotoxic T cells. clinical features of immunocompromised patients are the
2. Killing involves recognition of the target cell, release repeated or chronic infections, often caused by
of perforins and esterases, and delivery of apoptosis opportunistic agents. There are some characteristic
signals through the fas-fas ligand interaction. associations between specific types of infections and
generic types of immune deficiency, which provide the
B. Lymphocyte-Mediated Activation of Macrophages best data about the role of the different components of
and Other Inflammatory Cells. In many cases, the cells the immune system.
infected by intracellular organisms are the tissue A. Patients with antibody deficiencies and conserved
macrophages, and the persistence of the infection cell-mediated immunity suffer from repeated and chronic
depends on a delicate balance between a state of relative infections with pyogenic bacteria.
inactivity by the macrophage and mechanisms that allow B. Patients with primary deficiencies of cell-mediated
the infectious agent to escape proteolytic digestion once immunity usually suffer from chronic or recurrent fungal,
inside the cytoplasm (see below). parasitic, and viral infections.
C. Neutrophil deficiencies are usually associated with
1. Macrophage activation bacterial and fungal infections caused by common
a. CD4+ TH1 lymphocytes are activated as a organisms of low virulence, usually kept in check
consequence of their interaction with infected through nonimmune phagocytosis and other inate
macrophages expressing MHC-II-associated peptides and resistance mechanisms.
releasing IL-12. Once activated, TH1 lymphocytes D. Isolated complement component deficiencies are also
release a variety of lymphokines, particularly interferon- associated with bacterial infections, most frequently
g, which activates macrophages, enhancing their ability involving Neisseria gonorrhoeae and N. meningitidis,
to kill intracellular organisms and GM-CSF, which whose elimination appears to require the activation of the
promotes differentiation and release of granulocytes and membrane attack complex.
monocytes from the bone marrow and delivers
costimulatory signals to B cells. VII. Escape from the Immune Response
b. Once the initial activation signals are delivered,
macrophages and lymphocytes enter in a complex cycle Many infectious agents have developed the capacity to
of self and mutual activation involving a variety of avoid the immune response. Several mechanisms are
cytokines. In addition, several of these cytokines may involved.
activate other types of cells and have chemotactic A. Anti-Complementary Activity has been
properties, for which reason they are known as characterized for bacterial capsules and outer proteins of
chemotactic cytokines or chemokines. This group of some bacteria. For example, the M protein of group A
cytokines includes interleukin-8 (IL- 8), RANTES, Streptococcus inactivates complement convertases,
macrophage inflammatory proteins (MIP), preventing activation of the alternative pathway. The
macrophage chemotactic proteins (MCP), and anti-complementary activity of bacterial outer
migration-inhibition factor (MIF). Collectively the components has as a net result a decreased level of
chemokines attract, activate, and retain leukocytes to the opsonization by C3b and other complement fragments.
area where a cell-mediated immune reaction is taking
place.

B. Resistance to Phagocytosis can be evident at bactericidal activity until IgA-specific anti-Neisseria

different levels of the sequence of events associated with meningitidis antibodies were removed. This observation
ingestion and digestion of microorganisms: suggests that the IgA antibodies acted as a “blocking
1. Resistance to ingestion is usually associated with factor,” preventing opsonizing IgG antibodies from
polysaccharide capsules, which repeal and inhibit the binding to the same epitopes.
function of phagocytic cells. Haemophilus, Pasteurella, 3. Antiviral antibodies are not always neutralizing and
Klebsiella, Pseudomonas, and some yeasts (e.g., protective.
Cryptococcus neoformans) have such capsules. a. Retroviruses induce both neutralizing and non-
2. Ability to survive after ingestion is characteristic of neutralizing (blocking) antibodies. Both seem to
the group of bacteria known as facultative intracellular recognize the same epitopes; when non-neutralizing
(Mycobacteria, Brucella, Listeria, and Salmonella), as antibodies predominate, they block the binding of
well as of some fungi and protozoa (Toxoplasma, neutralizing antibodies.
Trypanosoma cruzi, and Leishmania). These b. Hepatitis B virus induces the synthesis of large
microorganisms survive inside phagocytosis by several quantities of soluble antigenic proteins by the infected
different strategies. host cells. These circulating antigens will block the
a. Secretion of molecules that prevent the formation of antiviral antibodies before they reach the infected cells,
phagolysosomes; these bacteria survive inside in a sense, acting as a “deflector shield” that protects the
phagosomes that are relatively devoid of toxic infected tissues from “antibody aggression.”
compounds (Mycobacterium tuberculosis, Legionella
pneumophila, and Toxoplasma gondii). D. Loss and Masking of Antigens have been
b. Synthesis of outer coats that protect the bacteria demonstrated with schistosomula (the larval forms of
against proteolytic enzymes and free toxic radicals (such schistosoma).
as the superoxide radical). 1. In the few hours after hatching, there is a rapid
c. Depression of the response of the infected phagocytic decrease in the surface antigens, as reflected by a
cells to cytokines which usually activate their killing progressive decrease in the binding of purified and
functions, such as interferon-γ. fluorescein-labeled heterologous antischistosomular
d. Exit from the phagosome into the cytoplasm, where antibodies to the parasite membrane. This is observed
the bacteria can live and multiply unharmed even in parasites cultured in serumfree medium.
(Trypanosoma cruzi). 2. Schistosomula recovered from infected animals have
e. The resistance to phagocytosis of several organisms host proteins bound to the outer layers, masking the
seems to involve a combination of these strategies. remaining parasite antigens.
i. Mycobacterium leprae is coated with a phenolic
glycolipid layer that scavenges free radicals and releases E. Antigenic Variation has been characterized in
a compound that inhibits the effects of interferon-γ. In bacteria (Borrelia recurrentis), parasites (trypanosomes,
addition, the release of IL-4 and IL-10 is enhanced, the agents of African sleeping sickness; Giardia
contributing to the down-regulation of TH1 lymphocytes. lamblia), and viruses (human immunodeficiency virus,
ii. Two glycolipids present in the membrane of HIV).
Leishmania parasites inhibit the response of monocytes 1. African trypanosomes have a surface coat constituted
and neutrophils to cytokines and a variety of stimuli; in mainly of a single glycoprotein (variant-specific surface
addition, Leishmania seems to resist the effects of glycoprotein or VSG), for which there are about 103
proteolytic enzymes in the phagolysosome. genes in the chromosome. At any given time, only one of
f. Viruses taken up by phagocytes as antigen-antibody those genes is expressed, the others remain silent. For
complexes often infect the phagocytic cells. The every 106 or 107 trypanosome divisions, a mutation
infection of phagocytic cells is frequent when viruses occurs that replaces the active VSG gene on the
disseminate through the blood stream. expression site by a previously silent VSG gene. The
g. Phagocytic cells are also infected by the HIV virus, previously expressed gene is destroyed, and a new VSG
either as a consequence of ingestion of virus-IgG protein is coded, which is antigenically different. The
antibody complexes or as a consequence of interactions emergence of a new antigenic coat allows the parasite to
with a CD4+ related molecule which is expressed in their multiply unchecked. As antibodies emerge to the newly
membrane. In either case, the infection of the expressed VSG protein, parasitemia will decline, only to
monocyte/macrophage is not cytotoxic, but the virus increase as soon as a new mutation occurs and a different
continues to replicate in small amounts and these cells VSG protein is synthesized.
become permanent sources of reinfection. 2. Giardia lamblia has a similar mechanism of variation
but the rate of surface antigen replacement is even faster
C. Ineffective Immune Responses (once every 103 divisions).
1. Polysaccharide capsules are not only antiphagocytic, 3. Borrelia recurrentis, the agent of relapsing fever
but poorly immunogenic and elicit predominantly IgM carries genes for at least 26 different variable major
and IgG2 antibodies, which are inefficient as opsonins proteins (VMP) which are sequentially activated by
(the FcγR of phagocytic cells recognize preferentially duplicative transposition to an expression site. The
IgG1 and IgG3 antibodies). successive waves of bacteremia and fever correspond to
2. Neisseria meningitidis often induces the synthesis of the emergence of new mutants which, for a while, can
IgA antibodies. In vitro data suggest that IgA can act as a proliferate unchecked until antibodies are formed.
weak opsonin or induce ADCC (monocytes/macrophages 4. HIV exhibits a high degree of antigenic variation,
and other leukocytes express Fcα receptors on their which seems to be the result of errors introduced by the
membranes), but the physiological protective role of IgA reverse transcriptase when synthesizing viral DNA from
antibodies is questionable. Patient sera with high titers of the RNA template. The mutation rate is relatively high
IgA antibodies to N. meningitidis failed to show (one in every 103 progeny particles), and the immune

response selects the mutant strains that present new X. Abnormal Consequences of the Immune Response
configurations in the outer envelope proteins, allowing
the mutant to proliferate unchecked by preexisting A. The Activation of T lymphocytes by Bacterial
neutralizing antibodies. Superantigens
1. A variety of bacterial exotoxins, such as
F. Cell-to-Cell Spread allows infectious agents to staphylococcal enterotoxins-A and -B (SE-A and SE-
propagate without being exposed to specific antibodies or B), staphylococcal toxic shock syndrome toxin-1
phagocytic cells. (TSST-1), exfoliating toxin, and streptococcal exotoxin
1. Listeria monocytogenes, after becoming intracellular, A, as well as other unrelated bacterial proteins (such as
can travel along the cytoskeleton and promote the fusion streptococcal M proteins) have been characterized as
of the membrane of an infected cell with the membrane “superantigens.”
of a neighboring noninfected cell, which is subsequently 2. Superantigens are defined by their ability to stimulate
invaded. T cells without being processed. The stimulation of T
2. Herpes viruses, retroviruses, and paramyxoviruses cells is polyclonal; thus the designation of “superantigen”
cause the fusion of infected cells with noninfected cells is a misnomer, but it has gained popularity and is widely
allowing viral particles to pass from cell to cell. used in the literature.
3. The best studied superantigens are the staphylococcal
G. Integration of Microbial Genomes is a tactic enterotoxins, which are potent polyclonal activators of
exclusive of viruses, particularly DNA viruses and murine and human T lymphocytes, inducing T-cell
retroviruses. The integration of viral genomes in the host proliferation and cytokine release. TSST-1 also appears
genome allows the virus to cause persistent or latent to activate monocytes and is a potent B-cell mitogen,
infections, with minimal replication of the integrated inducing B-cell proliferation and differentiation.
virus and minimal expression of viral proteins in the 4. Mechanism of action. Superantigens bind directly
membranes of infected cells. and simultaneously to the nonpolymorphic area of class
II MHC on professional accessory cells (macrophages
H. Immunosuppressive Effects of Infection. Although and related cells) and to the Vβ chain of the α/β TcR
immunosuppressive effects have been described in (Fig.).
association with bacteria and parasitic infections, the best a. For example, staphylococcal enterotoxins bind
documented examples of infection-associated exclusively to specific subfamilies of Vβ chains that are
immunosuppression are those described in viral expressed only by certain individuals. When expressed,
infections. these Vβ chain regions can be found on 2–20% of a
1. Measles. Patients in the acute phase of measles are positive individual's T cells, and the cross-linking of the
more susceptible to bacterial infections, such as TcR2 and of the APC by the enterotoxin activates all T
pneumonia. Both delayed hypersensitivity responses and cells (both CD4 and CD8+) expressing the specific Vβ
then in vitro lymphocyte proliferation in response to region recognized by the enterotoxin.
mitogens and antigens are significantly depressed during b. The massive T-cell activation induced by
the acute phase of measles and the immediate superantigens results in the release of large amounts of
convalescence period, usually returning to normal after 4 IL-2, interferon-γ, lymphotoxin (TNF-β), and TNF-α.
weeks. Recent investigations suggest that infection of c. Patients infected by bacteria able to release large
monocytes/macrophages with the measles virus is amounts of superantigens (e.g., S. aureus-releasing
associated with a down-regulation of interleukin-12 enterotoxins or TSST-1 and Group A Streptococcus-
synthesis, which can explain the depression of cell- releasing) may develop septic shock as a consequence of
mediated immunity associated with measles. the systemic effects of these cytokines, which include
2. Cytomegalovirus. Mothers and infants infected with fever, endothelial damage, profound hypotension,
cytomegalovirus show depressed responses to CMV disseminated intravascular coagulation, multiorgan
virus, but normal responses to T-cell mitogens, failure, and death.
suggesting that, in some cases, the immunosuppression d. After the initial burst of cytokine release, the
may be antigenspecific, while in measles it is obviously stimulated T cells either undergo apoptosis or become
nonspecific. anergic. This effect could severely disturb the ability of
3. Influenza virus has been found to depress CMI in the immune system to adequately respond to
mice, apparently due to an increase in the suppressor superantigen-releasing baceria.
activity of T lymphocytes.
4. Epstein-Barr virus releases a specific protein that has
extensive sequence homology with interleukin-10. The
biological properties of this viral protein are also
analogous to those of interleukin-10; both are able to
inhibit lymphokine synthesis by T-cell clones.
5. Human immunodeficiency virus. HIV infection is
associated with depletion of CD4+ cells, which are the
primary target of the virus. The depletion of CD4 cells
results mostly from viral replication itself and by the
priming of infected cells for apoptosis, which is triggered
by stimuli that normally would cause T-cell proliferation.
In addition, FcR-dependent phagocytosis is depressed in
HIV-infected monocyte-derived macrophages, further
contributing to the immunological compromise of the
infected patients.

B. Infection as a Consequence of the Uptake of the infection on the immune system, and the possibility
Antigen-Antibody Complexes. that the immune response may be more of a problem than
The immune response, in some cases, facilitates the the infection itself.
access of infectious agents to cells in which they will be
able to proliferate.
1. Macrophages are often infected by intracellular
organisms that are ingested as a consequence of
opsonization.
2. Babesia rodhaini, a bovine intraerythrocytic parasite,
penetrates the host's cells after it has bound complement,
particularly C3. Absorption of C3b-containing
circulating antigen-antibody complexes to the CR1
expressed by red cells allows the parasite to gain access
to the red cell, which becomes its permanent location.
C. Post-Infectious Tissue Damage. Several examples of

deleterious consequences of the immune response have
to do with the predominant role of the immune response
in the pathogenesis of the disease.
1. Immune-complex-induced inflammation. Antigen-

antibody complexes, if formed in large amounts, can
cause disease by being trapped in different capillary
networks and leading to inflammation. The clinical
expression of immune complex-related inflammation
depends on the localization of the trapped complexes:
vasculitis and purpura, when the skin is predominantly
affected, glomerulonephritis if trapping takes place on
the glomerular capillaries, arthritis when the joints are
affected. Viruses are often involved in the formation of
circulating antigen-antibody complexes.
2. Immune destruction of infected cells and tissues.

An immune response directed against an infectious agent
may be the main cause of damage to the infected tissue.
a. In subacute sclerosing panencephalitis, a
degenerative disease of the nervous system associated
with persistent infection with the measles virus, the
response against viral epitopes expressed in infected
neurons is believed to be the primary mechanism of
disease.
b. In some forms of chronic active hepatitis, the
immune response directed against viral epitopes
expressed by infected hepatocytes seems to cause more
tissue damage than the infection itself.
c. Cross-reactions with tissue antigens have been
proposed as the basis for the association of streptococcal
infections with rheumatic carditis and
glomerulonephritis. Antibodies to type 1 streptococcal M
protein crossreact with epitopes of myocardium and
kidney mesangial cells and cause inflammatory changes
in the heart and glomeruli, respectively.
X. Epilogue
The outcome of an infectious process depends on a very

complex set of interactions with the immune system. A
successful pathogen has developed mechanisms that
avoid fast elimination by an immunocompetent host.
These mechanisms allow the infectious agent to
replicate, cause disease, and spread to other individuals
before the immune response is induced. The immune
response, on the other hand, is a powerful weapon that,
once set in motion, may destroy friendly targets. Thus,
the therapeutic strategies in infectious diseases have to
consider all these questions, such as the particular
survival strategy of the infectious agents, the effects of

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UNIT 4: CELL COMMUNICATION AND CELL SIGNALING

A. Host parasite interaction:

Anaerobic diphtheroids occur below the skin surface in

A number of fungi, including Candida, occur on the scalp

Both the nose and mouth can be heavily colonized by

The majority of bacteria here are anaerobes. Common

The mucous membranes of the mouth can have the same

Dental caries is one of the commonest infectious

The surfaces of the teeth and the gingival crevices carry

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 1

Some of the species of the normal flora are positively

The importance of these species for health is sometimes

After treatment with clindamycin, overgrowth by

Ways in which the normal flora prevents colonization by

Gut bacteria also release organic acids, which may have

2 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

The disadvantages of the normal flora lie in the potential

This may happen:

Overgrowth by potentially pathogenic members of the

Under these conditions, the potential pathogens take the

All living animals are used as habitats by other

As the normal flora demonstrates, pathogenesis is not the

All associations in which one species lives in or on the

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 3

4 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

The most obvious disadvantage of parasitism arises from

THE EVOLUTION OF PARASITISM

As so many organisms are parasitic and every group of

Bacterial parasites evolved through accidental contact

In the case of bacteria, it is easy to see how accidental

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 5

6 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

Parasite adaptations to overcome host inflammatory and

We can view the evolution of parasitism and the

Pressure of infection has been a major influence in

Pressure of infection has been a major selective influence

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 7

8 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

In contrast, a transfer host is not necessary for the

Because, by definition, parasitic organisms are dependent

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 9

resistance. Usually the greater the number of organisms

10 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

The determinants of pathogenicity are now discussed in

Viral Spread and Cell Tropism

Mechanisms of viral spread vary, but the most common

Viruses exhibit cell, tissue, and organ specificities. These

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 11

Recovery from Infection: The host will either succumb

12 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

Leaving the Host Pathogenicity Islands

The gene for diphtheria toxin from Corynebacterium

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 13

A2. Cell fusion in both normal and abnormal cells

14 INSTITUTE FOR ADVANCED STUDIES, JODHPUR

Other studies discovered that cell fusion can determine

Another possibility is that tumor cells can acquire the

INSTITUTE FOR ADVANCED STUDIES, JODHPUR 15

B1. CELL SIGNALLING: