The environmental mycobacterium, M. vaccae has been used in mouse models to support the contempor... more The environmental mycobacterium, M. vaccae has been used in mouse models to support the contemporary hygiene hypothesis that non-pathogenic microorganisms reduce allergy associated T helper (Th)2 responses and inflammatory diseases by augmenting regulatory T cells. However, data for human models and possible mechanisms are limited. We tested the effect of innate immune interactions between human DC and M. vaccae on DC-dependent T cell responses. M. vaccae activation of DC via Toll like receptor (TLR)2 was compared to a specific TLR2 ligand (Pam 3 CSK4) and alternative stimulation with a TLR4 ligand (LPS). M. vaccae induced DC dependent inhibition of Th2 responses, in contrast to Pam 3 CSK4, which had the opposite effect and LPS, which had no polarizing effect. DC maturation, gene expression and cytokine production, in response to each stimulus did not correlate with the specific functional effects. Comparable DC transcriptional responses to M. vaccae and Pam 3 CSK4 suggested that TLR2 mediated transcriptional regulation was not sufficient for inhibition of Th2 responses. Transcription factor enrichment analysis and assessment of signaling events, implicated a role for selective early activation of the CREB pathway by M. vaccae. Further study of the CREB pathway may provide novel insight into the molecular mechanisms of DC-dependent T cell polarization.
The hygiene hypothesis hit the headlines in 1989 when David Strachan observed that hay fever is l... more The hygiene hypothesis hit the headlines in 1989 when David Strachan observed that hay fever is less common in children with older siblings, suggesting that multiple childhood cross-infections from dirty older brothers might protect from allergic disorders. The implication was that elimination of these infections by modern hygiene might be responsible for the increased prevalence of allergies in developed countries. However the notion that mankind’s changing lifestyle is contributing to the increased incidences of various chronic inflammatory disorders was not new. In 1873 Blackley had observed that farmers rarely had hay fever, and in 1966 Leibowitz and colleagues noted that, in Israel, the incidence of multiple sclerosis correlated with the level of sanitation. Similarly, inflammatory bowel disease, which was almost non-existent before the 20th Century, was already known to be increasing in Northern, urban, educated white-collar populations. Meanwhile animal models studied in the 1970’s had shown that different manipulations of the microbial flora of the gut could either increase or decrease susceptibility to autoimmune arthritis.
The findings in Strachan’s 1989 paper were initially considered narrowly in relation to allergic disorders, and this led research in two unhelpful directions. First, there was emphasis on the notion that protection was provided by the burden of recognizable symptomatic childhood infections: subsequent epidemiological surveys have consistently failed to support this hypothesis. Secondly it was thought that the role of these infections might be to drive Th1 cells able to inhibit development of the Th2 cells that mediate allergic disorders: this view ignored the simultaneous increases in Th1- and Th17-mediated disorders, and the increasing evidence for the importance of helminths and of orofecally transmitted organisms. The latter were often subclinical, so had been identified by antibody studies rather than by documenting manifest clinical infections.
Therefore by the late 1990’s several authors were suggesting an inclusive hypothesis that could account for the simultaneous rises in allergies, autoimmunity and inflammatory bowel diseases in rich developed countries, while at the same time incorporating all the different epidemiological clues, in addition to the protective effects of older siblings. These clues included orofecal transmission, living on a farm, economic deprivation, keeping a dog, and having helminth infections.
This reworking of the hygiene hypothesis has three components. First, it suggests that the increases in chronic inflammatory disorders are partly due to an imbalance between effector cells and regulatory cells, rather than an imbalance between Th1 and Th2. Secondly the new synthesis suggests that the most relevant organisms will turn out to be those with very long associations with the mammalian immune system, often as commensals, environmental “pseudocommensals”, subclinical infections or asymptomatic carrier states. (This long association is necessary for the development of evolved dependence and has led us to use the term “Old Friends” hypothesis). Thirdly, it suggests that the most relevant lifestyle changes are those that deprive us of contact with these particular organisms (less contact with animals, soil, and faeces), rather than the details of modern hygiene technology.
This book seeks to summarise the mass of data from epidemiological studies, laboratory experiments, animal models and clinical trials that lead to this view of the hygiene hypothesis. It also seeks to establish the broader significance of the hypothesis, and its boundaries. We first present a Darwinian view of the role of microorganisms in setting up the correct balances within the immune system. Then we consider the history of human interactions with microorganisms in order to analyse how microbial exposures have changed from Paleolithic to modern times. This is followed by chapters on the mechanisms involved in the priming of immunoregulatory circuits by microorganisms, including sections devoted specifically to gut microbiota and skin flora. There are then chapters considering the evidence for and against the importance of the hygiene hypothesis as a mechanism contributing to increases in allergies, multiple sclerosis, inflammatory bowel disease, Type 1 diabetes, depression, atherosclerosis, cancer and neurodegenerative disorders. Finally, there is an important chapter describing other aspects of the modern western lifestyle that might provide alternatives to the hygiene hypothesis, or might be interacting with, and exacerbating, the effects of our changing microbial environment.
This book is therefore unusually interdisciplinary, with implications for those working in essentially all areas of medicine. The year 2009 is the 150th anniversary of the publication of On the Origin of Species (24 November 1859) and the 200th anniversary of Darwin's birth (12 February 1809). We hope that this book will provoke debate and research on the hygiene hypothesis, which should now be seen as a crucial and expanding aspect of Darwinian Medicine.
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 1997
Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B ce... more Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors. Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis. The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2). The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides. beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines. The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine. In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylat...
International journal of leprosy and other mycobacterial diseases : official organ of the International Leprosy Association, 1989
The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-... more The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-test positivity to mycobacterial antigens in children living in the valley, and to assess the effect on this of a series of vaccines against mycobacterial disease. Set up in 1978, 1707 tuberculin-negative children without scars of previous BCG vaccination were vaccinated with BCG Glaxo alone (vaccine A) or with the addition of a suspension of killed Mycobacterium vaccae (vaccine B). One hundred children were vaccinated with BCG Glaxo plus a suspension of M. leprae (vaccine C). Eight to 10 years later about half of the children were found for follow up. At this time further children were skin tested, and the results obtained were related to whether or not they had scars of vaccination with BCG Pasteur (Teheran) given by the local health authorities. Between setting up the study and the first follow up, cases of leprosy or tuberculosis had occurred in some of the villages, although not amo...
The concentration of cortisol in a tissue is regulated by a reversible enzyme &am... more The concentration of cortisol in a tissue is regulated by a reversible enzyme 'shuttle' that can deactivate cortisol by converting it to cortisone, or activate cortisone by converting it to cortisol. The activity of this shuttle, and the direction in which it operates, is regulated by numerous factors including cytokines. This results in large swings in the effective cortisol concentration in sites of inflammation at different phases of an inflammatory response. Thus changes in local cortisol concentration can be largely independent of circulating cortisol levels. The relevant shuttle enzymes are present in skin, blood vessels and nervous tissue, and inhibition of the enzymes in skin enhances the local anti-inflammatory effect of cortisol. We therefore suggest that changes in the activity or direction of action of the shuttle in leprosy lesions may predispose to reactions, requiring exogenous steroid supplements to regain control of the inflammation.
Tuberculosis is characterised by fever, weight loss and necrosis in both lesions and tuberculin s... more Tuberculosis is characterised by fever, weight loss and necrosis in both lesions and tuberculin skin test sites (Koch phenomenon), although the antigens of Mycobacterium tuberculosis are not directly toxic to the tissues. The tissue damage appears to be due to several interacting factors. First, M. tuberculosis induces an immunoregulatory disorder of which a raised percentage of agalactosyl IgG is a marker. This is seen also in rheumatoid arthritis and Crohn's disease and is associated with tissue-damaging inflammation. Subsequently, several properties of M. tuberculosis exacerbate this disorder by triggering cytokine release, and rendering tissues sensitive to the toxicity of tumor necrosis factor (TNF). Moreover, M. tuberculosis, but not bacillus Calmette-Guérin or several Mycobacterium avium strains, produces a factor which increases the toxicity of TNF for individual cells. Thus, M. tuberculosis may distort the normal protective role of TNF so that this cytokine becomes toxic to the host. The immunoregulatory disorder associated with agalactosyl IgG appears to be susceptible to immunotherapy, so novel types of treatment for the immunopathological component of tuberculosis are being explored.
The purpose of this study was to identify Mycobacterium scrofulaceum reliably and rapidly and inv... more The purpose of this study was to identify Mycobacterium scrofulaceum reliably and rapidly and investigate diversity within the species. Fifty-four cultures were identified as Myco. scrofulaceum by preliminary cultural and biochemical tests, thin-layer chromatography and double diffusion. These strains were examined by PCR based on the 65 kDa heat stress protein gene, followed by restriction enzyme analysis of the product with BstEII and HaeIII. This produced seven groups, most with fewer fragments than had been reported previously. The technique was a rapid and reliable method for studying variation within Myco. scrofulaceum but alone, was unable to discriminate between some of these variants and other genetically similar species. When PCR-RFLP results were combined with biochemical tests, the major groups appeared to relate to different disease situations and thus, may have some epidemiological value.
There is much to be gained from examining human diseases within the expanding framework of Darwin... more There is much to be gained from examining human diseases within the expanding framework of Darwinian medicine. This is particularly true of those conditions that change in frequency as populations develop from the human "environment of evolutionary adaptedness" to the living conditions of the rich industrialized countries. This development entails major changes in lifestyle, leading to reductions in contact with environmental microorganisms and helminths that have evolved a physiologic role as drivers of immunoregulatory circuits. It is suggested that a deficit in immunoregulation in rich countries is contributing not only to increases in the incidence of allergic disorders but also to increases in other chronic inflammatory conditions that are exacerbated by a failure to terminate inappropriate inflammatory reponses. These include autoimmunity, neuroinflammatory disorders, atherosclerosis, depression associated with raised inflammatory cytokines, and some cancers.
We describe the use of 5-hydroxytryptamine (5HT) as an adjuvant in the induction of the delayed-t... more We describe the use of 5-hydroxytryptamine (5HT) as an adjuvant in the induction of the delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). Based upon our previous studies with antigen-pulsed macrophages (M phi), we have shown that both the Day 2 early (2 hr) reaction and the Day 3 (24 hr) reaction are augmented if 5HT is incorporated into the priming injection. Furthermore, we have confirmed that in contrast to M phi, antigen-pulsed dendritic cells (DC) fail to prime the early (2 hr) component of DTH. However, DC do prime the early response if injected along with 5HT. A peripheral 5HT antagonist, ICS 205-930, inhibits both the M phi-mediated and the 5HT/DC-primed reactions. These findings support the hypothesis that DTH reactions require a cascade of both inflammatory and immunological signals, and that in mice vascular permeability mediated via 5HT is important in the early phase of the reaction.
Peripheral blood mononuclear cells from 97 predominantly lepromatous leprosy patients and 11 cont... more Peripheral blood mononuclear cells from 97 predominantly lepromatous leprosy patients and 11 control subjects were tested in a lymphoproliferative assay for response to Mycobacterium leprae (whole and sonicated), and sonicated M. vaccae, M. tuberculosis, and M. scrofulaceum, in the presence and absence of three types of interleukin 2 (IL-2) (crude, purified, and recombinant). IL-2 enhanced the response to sonicated M. tuberculosis and M. leprae organisms more often in patients than in control subjects, but not significantly so and only in a minority of patients. This effect was significantly more common (though still only found in a minority of 46%) using M. leprae organisms as antigen, than when using sonicates of M. leprae (19%) or M. vaccae (19%). However it was nearly as frequent using sonicated M. tuberculosis, or M. scrofulaceum. Thus in only nine patients was the effect specific to M. leprae. Enhancement by IL-2 could not be related to the type of IL-2 used, the dose of antig...
The lymph nodes of mice overloaded with mycobacterial products, either by the injection of whole ... more The lymph nodes of mice overloaded with mycobacterial products, either by the injection of whole or ultrasonicated organisms, or as a consequence of severe infection with Mycobacterium ulcerans, contain phagocytic cells which cause spontaneous transformation of the lymph node cells in a low volume, high cell density culture system. This spontaneous mitosis is unaffected by trypsinization but is inhibited by specific antigen and by PHA, and eliminated by treatment with carbonyl iron. Replacement of the macrophages removed with carbonyl iron by a critical number of peritoneal cells, restores the spontaneous transformation. Normal lymph node, thymus or peritoneal lymphocytes will also undergo mitosis if small numbers of peritoneal cells are added to them. This phenomenon therefore appears not to be antigen-dependent, but is probably due to a mediator released from macrophages. The possible role of this phenomenon in the pathogenesis of mycobacterial disease and the 'overloading'...
The environmental mycobacterium, M. vaccae has been used in mouse models to support the contempor... more The environmental mycobacterium, M. vaccae has been used in mouse models to support the contemporary hygiene hypothesis that non-pathogenic microorganisms reduce allergy associated T helper (Th)2 responses and inflammatory diseases by augmenting regulatory T cells. However, data for human models and possible mechanisms are limited. We tested the effect of innate immune interactions between human DC and M. vaccae on DC-dependent T cell responses. M. vaccae activation of DC via Toll like receptor (TLR)2 was compared to a specific TLR2 ligand (Pam 3 CSK4) and alternative stimulation with a TLR4 ligand (LPS). M. vaccae induced DC dependent inhibition of Th2 responses, in contrast to Pam 3 CSK4, which had the opposite effect and LPS, which had no polarizing effect. DC maturation, gene expression and cytokine production, in response to each stimulus did not correlate with the specific functional effects. Comparable DC transcriptional responses to M. vaccae and Pam 3 CSK4 suggested that TLR2 mediated transcriptional regulation was not sufficient for inhibition of Th2 responses. Transcription factor enrichment analysis and assessment of signaling events, implicated a role for selective early activation of the CREB pathway by M. vaccae. Further study of the CREB pathway may provide novel insight into the molecular mechanisms of DC-dependent T cell polarization.
The hygiene hypothesis hit the headlines in 1989 when David Strachan observed that hay fever is l... more The hygiene hypothesis hit the headlines in 1989 when David Strachan observed that hay fever is less common in children with older siblings, suggesting that multiple childhood cross-infections from dirty older brothers might protect from allergic disorders. The implication was that elimination of these infections by modern hygiene might be responsible for the increased prevalence of allergies in developed countries. However the notion that mankind’s changing lifestyle is contributing to the increased incidences of various chronic inflammatory disorders was not new. In 1873 Blackley had observed that farmers rarely had hay fever, and in 1966 Leibowitz and colleagues noted that, in Israel, the incidence of multiple sclerosis correlated with the level of sanitation. Similarly, inflammatory bowel disease, which was almost non-existent before the 20th Century, was already known to be increasing in Northern, urban, educated white-collar populations. Meanwhile animal models studied in the 1970’s had shown that different manipulations of the microbial flora of the gut could either increase or decrease susceptibility to autoimmune arthritis.
The findings in Strachan’s 1989 paper were initially considered narrowly in relation to allergic disorders, and this led research in two unhelpful directions. First, there was emphasis on the notion that protection was provided by the burden of recognizable symptomatic childhood infections: subsequent epidemiological surveys have consistently failed to support this hypothesis. Secondly it was thought that the role of these infections might be to drive Th1 cells able to inhibit development of the Th2 cells that mediate allergic disorders: this view ignored the simultaneous increases in Th1- and Th17-mediated disorders, and the increasing evidence for the importance of helminths and of orofecally transmitted organisms. The latter were often subclinical, so had been identified by antibody studies rather than by documenting manifest clinical infections.
Therefore by the late 1990’s several authors were suggesting an inclusive hypothesis that could account for the simultaneous rises in allergies, autoimmunity and inflammatory bowel diseases in rich developed countries, while at the same time incorporating all the different epidemiological clues, in addition to the protective effects of older siblings. These clues included orofecal transmission, living on a farm, economic deprivation, keeping a dog, and having helminth infections.
This reworking of the hygiene hypothesis has three components. First, it suggests that the increases in chronic inflammatory disorders are partly due to an imbalance between effector cells and regulatory cells, rather than an imbalance between Th1 and Th2. Secondly the new synthesis suggests that the most relevant organisms will turn out to be those with very long associations with the mammalian immune system, often as commensals, environmental “pseudocommensals”, subclinical infections or asymptomatic carrier states. (This long association is necessary for the development of evolved dependence and has led us to use the term “Old Friends” hypothesis). Thirdly, it suggests that the most relevant lifestyle changes are those that deprive us of contact with these particular organisms (less contact with animals, soil, and faeces), rather than the details of modern hygiene technology.
This book seeks to summarise the mass of data from epidemiological studies, laboratory experiments, animal models and clinical trials that lead to this view of the hygiene hypothesis. It also seeks to establish the broader significance of the hypothesis, and its boundaries. We first present a Darwinian view of the role of microorganisms in setting up the correct balances within the immune system. Then we consider the history of human interactions with microorganisms in order to analyse how microbial exposures have changed from Paleolithic to modern times. This is followed by chapters on the mechanisms involved in the priming of immunoregulatory circuits by microorganisms, including sections devoted specifically to gut microbiota and skin flora. There are then chapters considering the evidence for and against the importance of the hygiene hypothesis as a mechanism contributing to increases in allergies, multiple sclerosis, inflammatory bowel disease, Type 1 diabetes, depression, atherosclerosis, cancer and neurodegenerative disorders. Finally, there is an important chapter describing other aspects of the modern western lifestyle that might provide alternatives to the hygiene hypothesis, or might be interacting with, and exacerbating, the effects of our changing microbial environment.
This book is therefore unusually interdisciplinary, with implications for those working in essentially all areas of medicine. The year 2009 is the 150th anniversary of the publication of On the Origin of Species (24 November 1859) and the 200th anniversary of Darwin's birth (12 February 1809). We hope that this book will provoke debate and research on the hygiene hypothesis, which should now be seen as a crucial and expanding aspect of Darwinian Medicine.
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 1997
Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B ce... more Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors. Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis. The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2). The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides. beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines. The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine. In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylat...
International journal of leprosy and other mycobacterial diseases : official organ of the International Leprosy Association, 1989
The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-... more The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-test positivity to mycobacterial antigens in children living in the valley, and to assess the effect on this of a series of vaccines against mycobacterial disease. Set up in 1978, 1707 tuberculin-negative children without scars of previous BCG vaccination were vaccinated with BCG Glaxo alone (vaccine A) or with the addition of a suspension of killed Mycobacterium vaccae (vaccine B). One hundred children were vaccinated with BCG Glaxo plus a suspension of M. leprae (vaccine C). Eight to 10 years later about half of the children were found for follow up. At this time further children were skin tested, and the results obtained were related to whether or not they had scars of vaccination with BCG Pasteur (Teheran) given by the local health authorities. Between setting up the study and the first follow up, cases of leprosy or tuberculosis had occurred in some of the villages, although not amo...
The concentration of cortisol in a tissue is regulated by a reversible enzyme &am... more The concentration of cortisol in a tissue is regulated by a reversible enzyme 'shuttle' that can deactivate cortisol by converting it to cortisone, or activate cortisone by converting it to cortisol. The activity of this shuttle, and the direction in which it operates, is regulated by numerous factors including cytokines. This results in large swings in the effective cortisol concentration in sites of inflammation at different phases of an inflammatory response. Thus changes in local cortisol concentration can be largely independent of circulating cortisol levels. The relevant shuttle enzymes are present in skin, blood vessels and nervous tissue, and inhibition of the enzymes in skin enhances the local anti-inflammatory effect of cortisol. We therefore suggest that changes in the activity or direction of action of the shuttle in leprosy lesions may predispose to reactions, requiring exogenous steroid supplements to regain control of the inflammation.
Tuberculosis is characterised by fever, weight loss and necrosis in both lesions and tuberculin s... more Tuberculosis is characterised by fever, weight loss and necrosis in both lesions and tuberculin skin test sites (Koch phenomenon), although the antigens of Mycobacterium tuberculosis are not directly toxic to the tissues. The tissue damage appears to be due to several interacting factors. First, M. tuberculosis induces an immunoregulatory disorder of which a raised percentage of agalactosyl IgG is a marker. This is seen also in rheumatoid arthritis and Crohn's disease and is associated with tissue-damaging inflammation. Subsequently, several properties of M. tuberculosis exacerbate this disorder by triggering cytokine release, and rendering tissues sensitive to the toxicity of tumor necrosis factor (TNF). Moreover, M. tuberculosis, but not bacillus Calmette-Guérin or several Mycobacterium avium strains, produces a factor which increases the toxicity of TNF for individual cells. Thus, M. tuberculosis may distort the normal protective role of TNF so that this cytokine becomes toxic to the host. The immunoregulatory disorder associated with agalactosyl IgG appears to be susceptible to immunotherapy, so novel types of treatment for the immunopathological component of tuberculosis are being explored.
The purpose of this study was to identify Mycobacterium scrofulaceum reliably and rapidly and inv... more The purpose of this study was to identify Mycobacterium scrofulaceum reliably and rapidly and investigate diversity within the species. Fifty-four cultures were identified as Myco. scrofulaceum by preliminary cultural and biochemical tests, thin-layer chromatography and double diffusion. These strains were examined by PCR based on the 65 kDa heat stress protein gene, followed by restriction enzyme analysis of the product with BstEII and HaeIII. This produced seven groups, most with fewer fragments than had been reported previously. The technique was a rapid and reliable method for studying variation within Myco. scrofulaceum but alone, was unable to discriminate between some of these variants and other genetically similar species. When PCR-RFLP results were combined with biochemical tests, the major groups appeared to relate to different disease situations and thus, may have some epidemiological value.
There is much to be gained from examining human diseases within the expanding framework of Darwin... more There is much to be gained from examining human diseases within the expanding framework of Darwinian medicine. This is particularly true of those conditions that change in frequency as populations develop from the human "environment of evolutionary adaptedness" to the living conditions of the rich industrialized countries. This development entails major changes in lifestyle, leading to reductions in contact with environmental microorganisms and helminths that have evolved a physiologic role as drivers of immunoregulatory circuits. It is suggested that a deficit in immunoregulation in rich countries is contributing not only to increases in the incidence of allergic disorders but also to increases in other chronic inflammatory conditions that are exacerbated by a failure to terminate inappropriate inflammatory reponses. These include autoimmunity, neuroinflammatory disorders, atherosclerosis, depression associated with raised inflammatory cytokines, and some cancers.
We describe the use of 5-hydroxytryptamine (5HT) as an adjuvant in the induction of the delayed-t... more We describe the use of 5-hydroxytryptamine (5HT) as an adjuvant in the induction of the delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). Based upon our previous studies with antigen-pulsed macrophages (M phi), we have shown that both the Day 2 early (2 hr) reaction and the Day 3 (24 hr) reaction are augmented if 5HT is incorporated into the priming injection. Furthermore, we have confirmed that in contrast to M phi, antigen-pulsed dendritic cells (DC) fail to prime the early (2 hr) component of DTH. However, DC do prime the early response if injected along with 5HT. A peripheral 5HT antagonist, ICS 205-930, inhibits both the M phi-mediated and the 5HT/DC-primed reactions. These findings support the hypothesis that DTH reactions require a cascade of both inflammatory and immunological signals, and that in mice vascular permeability mediated via 5HT is important in the early phase of the reaction.
Peripheral blood mononuclear cells from 97 predominantly lepromatous leprosy patients and 11 cont... more Peripheral blood mononuclear cells from 97 predominantly lepromatous leprosy patients and 11 control subjects were tested in a lymphoproliferative assay for response to Mycobacterium leprae (whole and sonicated), and sonicated M. vaccae, M. tuberculosis, and M. scrofulaceum, in the presence and absence of three types of interleukin 2 (IL-2) (crude, purified, and recombinant). IL-2 enhanced the response to sonicated M. tuberculosis and M. leprae organisms more often in patients than in control subjects, but not significantly so and only in a minority of patients. This effect was significantly more common (though still only found in a minority of 46%) using M. leprae organisms as antigen, than when using sonicates of M. leprae (19%) or M. vaccae (19%). However it was nearly as frequent using sonicated M. tuberculosis, or M. scrofulaceum. Thus in only nine patients was the effect specific to M. leprae. Enhancement by IL-2 could not be related to the type of IL-2 used, the dose of antig...
The lymph nodes of mice overloaded with mycobacterial products, either by the injection of whole ... more The lymph nodes of mice overloaded with mycobacterial products, either by the injection of whole or ultrasonicated organisms, or as a consequence of severe infection with Mycobacterium ulcerans, contain phagocytic cells which cause spontaneous transformation of the lymph node cells in a low volume, high cell density culture system. This spontaneous mitosis is unaffected by trypsinization but is inhibited by specific antigen and by PHA, and eliminated by treatment with carbonyl iron. Replacement of the macrophages removed with carbonyl iron by a critical number of peritoneal cells, restores the spontaneous transformation. Normal lymph node, thymus or peritoneal lymphocytes will also undergo mitosis if small numbers of peritoneal cells are added to them. This phenomenon therefore appears not to be antigen-dependent, but is probably due to a mediator released from macrophages. The possible role of this phenomenon in the pathogenesis of mycobacterial disease and the 'overloading'...
Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in lar... more Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.
We previously reported DR2 and DR7-associated regulation of antibody binding to mycobacteria in r... more We previously reported DR2 and DR7-associated regulation of antibody binding to mycobacteria in rheumatoid arthritis sera (RA), but not in tuberculosis (Tb). An extensive analysis of antibody to mycobacteria in matched normal sera, in relation to both HLA class I and class II has revealed no class II correlations, confirming that the original findings were due to RA. There was however a very strong association between IgM binding to M. tuberculosis and Cw1 (P = 0.0004). RA patients have strikingly raised levels of IgG (but not of IgA or IgM) binding to the 65 kD heat shock protein of M. tuberculosis, recently implicated in the pathogenesis of adjuvant arthritis in the rat. Remarkably, levels in RA were significantly higher even than in tuberculosis. Levels of this antibody showed no HLA associations. Thus the 65 kD antigen does not account for the DR7, and DR2 associations of IgM and IgA binding to mycobacteria reported previously, but does suggest a role for cross-reactive autoimmu...
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Books by Graham A W Rook
The findings in Strachan’s 1989 paper were initially considered narrowly in relation to allergic disorders, and this led research in two unhelpful directions. First, there was emphasis on the notion that protection was provided by the burden of recognizable symptomatic childhood infections: subsequent epidemiological surveys have consistently failed to support this hypothesis. Secondly it was thought that the role of these infections might be to drive Th1 cells able to inhibit development of the Th2 cells that mediate allergic disorders: this view ignored the simultaneous increases in Th1- and Th17-mediated disorders, and the increasing evidence for the importance of helminths and of orofecally transmitted organisms. The latter were often subclinical, so had been identified by antibody studies rather than by documenting manifest clinical infections.
Therefore by the late 1990’s several authors were suggesting an inclusive hypothesis that could account for the simultaneous rises in allergies, autoimmunity and inflammatory bowel diseases in rich developed countries, while at the same time incorporating all the different epidemiological clues, in addition to the protective effects of older siblings. These clues included orofecal transmission, living on a farm, economic deprivation, keeping a dog, and having helminth infections.
This reworking of the hygiene hypothesis has three components. First, it suggests that the increases in chronic inflammatory disorders are partly due to an imbalance between effector cells and regulatory cells, rather than an imbalance between Th1 and Th2. Secondly the new synthesis suggests that the most relevant organisms will turn out to be those with very long associations with the mammalian immune system, often as commensals, environmental “pseudocommensals”, subclinical infections or asymptomatic carrier states. (This long association is necessary for the development of evolved dependence and has led us to use the term “Old Friends” hypothesis). Thirdly, it suggests that the most relevant lifestyle changes are those that deprive us of contact with these particular organisms (less contact with animals, soil, and faeces), rather than the details of modern hygiene technology.
This book seeks to summarise the mass of data from epidemiological studies, laboratory experiments, animal models and clinical trials that lead to this view of the hygiene hypothesis. It also seeks to establish the broader significance of the hypothesis, and its boundaries. We first present a Darwinian view of the role of microorganisms in setting up the correct balances within the immune system. Then we consider the history of human interactions with microorganisms in order to analyse how microbial exposures have changed from Paleolithic to modern times. This is followed by chapters on the mechanisms involved in the priming of immunoregulatory circuits by microorganisms, including sections devoted specifically to gut microbiota and skin flora. There are then chapters considering the evidence for and against the importance of the hygiene hypothesis as a mechanism contributing to increases in allergies, multiple sclerosis, inflammatory bowel disease, Type 1 diabetes, depression, atherosclerosis, cancer and neurodegenerative disorders. Finally, there is an important chapter describing other aspects of the modern western lifestyle that might provide alternatives to the hygiene hypothesis, or might be interacting with, and exacerbating, the effects of our changing microbial environment.
This book is therefore unusually interdisciplinary, with implications for those working in essentially all areas of medicine. The year 2009 is the 150th anniversary of the publication of On the Origin of Species (24 November 1859) and the 200th anniversary of Darwin's birth (12 February 1809). We hope that this book will provoke debate and research on the hygiene hypothesis, which should now be seen as a crucial and expanding aspect of Darwinian Medicine.
Papers by Graham A W Rook
The findings in Strachan’s 1989 paper were initially considered narrowly in relation to allergic disorders, and this led research in two unhelpful directions. First, there was emphasis on the notion that protection was provided by the burden of recognizable symptomatic childhood infections: subsequent epidemiological surveys have consistently failed to support this hypothesis. Secondly it was thought that the role of these infections might be to drive Th1 cells able to inhibit development of the Th2 cells that mediate allergic disorders: this view ignored the simultaneous increases in Th1- and Th17-mediated disorders, and the increasing evidence for the importance of helminths and of orofecally transmitted organisms. The latter were often subclinical, so had been identified by antibody studies rather than by documenting manifest clinical infections.
Therefore by the late 1990’s several authors were suggesting an inclusive hypothesis that could account for the simultaneous rises in allergies, autoimmunity and inflammatory bowel diseases in rich developed countries, while at the same time incorporating all the different epidemiological clues, in addition to the protective effects of older siblings. These clues included orofecal transmission, living on a farm, economic deprivation, keeping a dog, and having helminth infections.
This reworking of the hygiene hypothesis has three components. First, it suggests that the increases in chronic inflammatory disorders are partly due to an imbalance between effector cells and regulatory cells, rather than an imbalance between Th1 and Th2. Secondly the new synthesis suggests that the most relevant organisms will turn out to be those with very long associations with the mammalian immune system, often as commensals, environmental “pseudocommensals”, subclinical infections or asymptomatic carrier states. (This long association is necessary for the development of evolved dependence and has led us to use the term “Old Friends” hypothesis). Thirdly, it suggests that the most relevant lifestyle changes are those that deprive us of contact with these particular organisms (less contact with animals, soil, and faeces), rather than the details of modern hygiene technology.
This book seeks to summarise the mass of data from epidemiological studies, laboratory experiments, animal models and clinical trials that lead to this view of the hygiene hypothesis. It also seeks to establish the broader significance of the hypothesis, and its boundaries. We first present a Darwinian view of the role of microorganisms in setting up the correct balances within the immune system. Then we consider the history of human interactions with microorganisms in order to analyse how microbial exposures have changed from Paleolithic to modern times. This is followed by chapters on the mechanisms involved in the priming of immunoregulatory circuits by microorganisms, including sections devoted specifically to gut microbiota and skin flora. There are then chapters considering the evidence for and against the importance of the hygiene hypothesis as a mechanism contributing to increases in allergies, multiple sclerosis, inflammatory bowel disease, Type 1 diabetes, depression, atherosclerosis, cancer and neurodegenerative disorders. Finally, there is an important chapter describing other aspects of the modern western lifestyle that might provide alternatives to the hygiene hypothesis, or might be interacting with, and exacerbating, the effects of our changing microbial environment.
This book is therefore unusually interdisciplinary, with implications for those working in essentially all areas of medicine. The year 2009 is the 150th anniversary of the publication of On the Origin of Species (24 November 1859) and the 200th anniversary of Darwin's birth (12 February 1809). We hope that this book will provoke debate and research on the hygiene hypothesis, which should now be seen as a crucial and expanding aspect of Darwinian Medicine.