In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission... more
In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discrimina...
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Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis (Mtb), is acquired by the respiratory route. It is exquisitely human adapted and a prototypic intracellular... more
Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis (Mtb), is acquired by the respiratory route. It is exquisitely human adapted and a prototypic intracellular pathogen of macrophages, with alveolar macrophages (AMs) being the primary conduit of infection and disease. The outcome of primary infection is most often a latently infected healthy human host, in whom the bacteria are held in check by the host immune response. Such individuals can develop active TB later in life with impairment in the immune system. In contrast, in a minority of infected individuals, the host immune response fails to control the growth of bacilli, and progressive granulomatous disease develops, facilitating spread of the bacilli via infectious aerosols coughed out into the environment and inhaled by new hosts. The molecular details of the Mtb-macrophage interaction continue to be elucidated. However, it is clear that a number of complex processes are involved at the different stages of infection that may benefit either the bacterium or the host. Macrophages demonstrate tremendous phenotypic heterogeneity and functional plasticity which, depending on the site and stage of infection, facilitate the diverse outcomes. Moreover, host responses vary depending on the specific characteristics of the infecting Mtb strain. In this chapter, we describe a contemporary view of the behavior of AMs and their interaction with various Mtb strains in generating unique immunologic lung-specific responses.
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Research Interests: Immunology, Macrophages, Mice, Sheep, Animals, and 4 morePhagocytosis, Tannins, Erythrocytes, and Phagocytes
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Mononuclear leukocytes are able to destroy tumor cells under a variety of circum- stances. In few such instances is the biochemical basis of cytotoxicity understood. Recently, we demonstrated that activated macrophages, stimulated by... more
Mononuclear leukocytes are able to destroy tumor cells under a variety of circum- stances. In few such instances is the biochemical basis of cytotoxicity understood. Recently, we demonstrated that activated macrophages, stimulated by phorbol myristate acetate (PMA), 1 secreted substantial amounts of hydrogen peroxide (1). The release of this oxidant from macrophages correlated with the extracellular lysis of tumor cells
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Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and... more
Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and occurred with cells from lepromatous and tuberculoid leprosy patients as well as control donors. The purified lipoarabinomannans from M. leprae and Mycobacterium tuberculosis had a similar spectrum of inhibition whereas their deacylated derivatives were without effect. All mycobacterial preparations of either a crude or purified state, which suppressed cellular responses, contained appreciable quantities of bacterial lipopolysaccharide by the Limulus amebocyte assay. Contamination with lipopolysaccharide could account for the extent and nonselectivity of the T-cell suppression. Suppression was also monocyte-dependent and in part due to the release of arachidonate metabolites of the cyclooxygenase pathway.
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The effect of multiple intradermal injections (four to six) of 10 micrograms of interferon gamma on the number of Mycobacterium leprae in the skin of patients with polar lepromatous leprosy and borderline lepromatous leprosy was... more
The effect of multiple intradermal injections (four to six) of 10 micrograms of interferon gamma on the number of Mycobacterium leprae in the skin of patients with polar lepromatous leprosy and borderline lepromatous leprosy was evaluated. To achieve a maximum zone of induration and cell emigration a preparatory dose of the lymphokine was required. A second group of three injections, given 3-4 days after the initial series, resulted in lesser degrees of induration and was more in keeping with a partial local hyporesponsive state. A marked emigration of T cells and monocytes into the dermis resulted from injections of interferon gamma and persisted for greater than 21 days. A preponderance of CD4+ cells in the infiltrate was seen within a few days and CD4/CD8 ratios remained elevated for greater than 5 weeks. The bacillary load of injected sites evaluated 21 days after lymphokine administration was reduced in 14/17 patients by factors ranging from 5- to 1000-fold. This occurred predominantly within diffuse lesions and occurred rarely in nodular sites. Biopsy samples of injected sites taken 6 months later demonstrated progressive 10-fold reductions in bacilli and the continued presence of a granulomatous response.
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Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant... more
Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.
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Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to thalidomide therapy were evaluated. Circulating tumor necrosis factor-alpha (TNF alpha) levels were assayed in serum obtained from... more
Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to thalidomide therapy were evaluated. Circulating tumor necrosis factor-alpha (TNF alpha) levels were assayed in serum obtained from lepromatous leprosy patients at diagnosis, during multidrug therapy, at the onset of ENL episodes, and during treatment with thalidomide. Patients with systemic ENL demonstrated the highest serum TNF alpha levels, which decreased significantly during thalidomide treatment. Serum TNF alpha in nonreactional patients was associated with mild flu-like symptoms and local inflammatory lesions. Serum interferon-gamma (IFN-gamma) was also elevated in patients with high TNF alpha levels. Thalidomide therapy reduced not only serum TNF alpha levels and the clinical symptoms but also the dermal infiltration of polymorphonuclear leukocytes and T cells. The expression of intercellular adhesion molecule 1 and major histocompatibility complex class II antigens on the epidermal keratinocytes was also down-regulated. These results indicate that the thalidomide-induced alleviation of clinical symptoms of ENL was associated with a reduction of TNF alpha levels.
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Lepromatous leprosy is characterized by a selective anergy to Mycobacterium leprae and its antigens. The inadequate immune response and the resulting reduced interferon-gamma (IFN-gamma) production lead to a lack of macrophage activation... more
Lepromatous leprosy is characterized by a selective anergy to Mycobacterium leprae and its antigens. The inadequate immune response and the resulting reduced interferon-gamma (IFN-gamma) production lead to a lack of macrophage activation and unrestricted bacterial growth. Purified protein derivative of tuberculin induced a normal local immune response in many lepromatous leprosy patients. Interleukin-2 induced an accelerated equivalent of an antigen response in the skin. In both, monocytes and T cells were recruited, and changes in keratinocytes, including expression of major histocompatibility complex class II antigens, were induced. Skin macrophages appeared to be activated and bacteria were eliminated. Similar effects were generated by IFN-gamma, a more distal molecule in the immune response. Cytokine treatment induced large amounts of tumor necrosis factor-alpha, which is toxic in this context but can be selectively down-regulated by thalidomide without interfering with other monocyte cytokines necessary for normal immune function.
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Studies in our laboratory have focussed on the role of cytokines in the regulation of the cellular immune response and disease progression in two important mycobacterial infection of man, namely leprosy and tuberculosis. Our studies in... more
Studies in our laboratory have focussed on the role of cytokines in the regulation of the cellular immune response and disease progression in two important mycobacterial infection of man, namely leprosy and tuberculosis. Our studies in leprosy have involved the use of key regulatory cytokines such as IFN-gamma in the modulation of the cellular response of infected patients. We have investigated the effect of intradermal administration of low dose IFN-gamma on the lesions of anergic lepromatous patients and have reported an accelerated bacillary clearance from the skin. This was associated with the local accumulation of mononuclear cells and killing of infected macrophages. However, IFN-gamma administration also resulted in the induction of erythema nodosum leprosum, a toxic syndrome associated with excess TNF-alpha production. Both the toxic symptoms and the high levels of TNF-alpha production could be inhibited by thalidomide treatment, a drug we have shown reduces the half life of TNF-alpha mRNA. In preliminary clinical trials in tuberculosis patients we have attempted to use thalidomide to reduce TNF-alpha production and toxicities. These results are discussed.
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ABSTRACT
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Thalidomide has been shown to selectively inhibit TNF-alpha production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-alpha has been shown to play a pivotal role in the pathophysiology of endotoxic shock. Using a mouse... more
Thalidomide has been shown to selectively inhibit TNF-alpha production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-alpha has been shown to play a pivotal role in the pathophysiology of endotoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the production of TNF-alpha and other cytokines and on animal survival. After injection of 100-350 micrograms LPS into mice, cytokines including TNF-alpha, IL-6, IL-10, IL-1 beta, GM-CSF and IFN-gamma were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-alpha levels were reduced by 93%, in a dose-dependent manner, and TNF-alpha mRNA expression in the spleens of mice was reduced by 70%. Serum IL-6 levels were also inhibited by 50%. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1 beta, or IFN-gamma. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 micrograms to 300 micrograms in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death.
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ABSTRACT Background: Although leprosy and other mycobacterial infections cause substantial morbidity worldwide, the host factors that regulate disease susceptibility are poorly understood. Mycobacteria are recognized by the innate immune... more
ABSTRACT Background: Although leprosy and other mycobacterial infections cause substantial morbidity worldwide, the host factors that regulate disease susceptibility are poorly understood. Mycobacteria are recognized by the innate immune system via Toll-like receptors (TLRs). We recently identified a common variant in the transmembrane domain of TLR1, T1805G (I602S) that regulates the immune response to triacylated lipopeptide, a synthetic ligand for TLR1. In the present study, we investigated whether TLR1-deficiency is associated with susceptibility to leprosy and reversal reaction, an immune reaction characterized by Th1 T cell responses. Methods & Results: Allele 1805G was associated with markedly impaired TLR1-mediated NF-κκB signaling in response to stimulation with heat-killed extracts of M. leprae and M. tuberculosis in transfected HEK293 cells. To assess the clinical significance of TLR1-deficiency, we genotyped SNP T1805G in a case-control study of 938 Nepalese leprosy patients. T1805G was not associated with susceptibility to leprosy type, although GG individuals showed a trend toward an increased risk of lepromatous vs. tuberculoid leprosy (OR 5.12, 95% CI 0.64-41.17, P=0.087). However, the 1805G allele was associated with protection from reversal reaction (OR 0.51, CI 0.30-0.88, P=0.013). The GG/GT genotypes were also associated with a lower likelihood of reversal reaction when compared to TT individuals (OR 0.55, CI 0.32-0.97, P=0.036). Conclusion: To our knowledge, this is the first report of an association of a TLR1 polymorphism with an infectious disease and the first time that any genetic variant has been associated with a type I reaction in leprosy. Our findings suggest that TLR1-deficiency may directly impact clinical outcomes such as nerve damage and disability.
Background: Although leprosy and other mycobacterial infections cause substantial morbidity worldwide, the host factors that regulate disease susceptibility are poorly understood. Mycobacteria are recognized by the innate immune system... more
Background: Although leprosy and other mycobacterial infections cause substantial morbidity worldwide, the host factors that regulate disease susceptibility are poorly understood. Mycobacteria are recognized by the innate immune system via Toll-like receptors (TLRs). We recently identified a common variant in the transmembrane domain of TLR1, T1805G (I602S) that regulates the immune response to triacylated lipopeptide, a synthetic ligand for TLR1. In the present study, we investigated whether TLR1-deficiency is associated with susceptibility to leprosy and reversal reaction, an immune reaction characterized by Th1 T cell responses. Methods & Results: Allele 1805G was associated with markedly impaired TLR1-mediated NF-κκB signaling in response to stimulation with heat-killed extracts of M. leprae and M. tuberculosis in transfected HEK293 cells. To assess the clinical significance of TLR1-deficiency, we genotyped SNP T1805G in a case-control study of 938 Nepalese leprosy patients. T18...
Angiogenesis is a crucial process in inflammatory reactions as well as in tumor implantation and growth. Tumors with high rates of invasion and recurrence such as gliomas, are specially dependent on neovascularization. This suggests that... more
Angiogenesis is a crucial process in inflammatory reactions as well as in tumor implantation and growth. Tumors with high rates of invasion and recurrence such as gliomas, are specially dependent on neovascularization. This suggests that inhibition of angiogenesis might reduce the growth of these tumors. Thalidomide has been previously shown to inhibit angiogenesis induced by basic fibroblast growth factor in vivo, using the rabbit corneal micropocket assay. Therefore, the effect of thalidomide and a thalidomide analogue (cc-1069) on the proliferation in vitro of endothelial and glioma cells was tested. We observed a decrease in endothelial cell proliferation in cultures treated with thalidomide or the thalidomide analogue cc-1069. The analogue inhibited endothelial cell proliferation more efficiently than thalidomide. The inhibition occurred in association with a marked decrease in the activity of the nuclear factor SP1 and a moderate inhibition of NF-kappaB activation in nuclear e...
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Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the... more
Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic propertie...
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Human peripheral blood monocytes are permissive for the growth of Mycobacterium tuberculosis, but the fate of nonpathogenic Mycobacterium smegmatis in these cells is not known. Since M. smegmatis may be used as a host with which to... more
Human peripheral blood monocytes are permissive for the growth of Mycobacterium tuberculosis, but the fate of nonpathogenic Mycobacterium smegmatis in these cells is not known. Since M. smegmatis may be used as a host with which to express and screen for M. tuberculosis genes needed for survival in monocytes, we determined whether human peripheral blood monocytes could restrict the growth of Mycobacterium smegmatis. Adherent human peripheral blood monocytes were permissive for the growth of M. smegmatis, as measured by ex vivo [3H]uracil uptake. However, human peripheral blood monocytes which were cultured nonadherently in Teflon wells were able to restrict the growth of M. smegmatis while remaining permissive for the growth of M. tuberculosis H37Ra. The loss of viability of M. smegmatis in nonadherent cells was correlated with an increase in nonspacious phagocytic vacuoles. The killing of M. smegmatis was not blocked by NG-monomethyl-L-arginine, suggesting that it was not due to th...
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13 patients with human immunodeficiency virus type 1 infection class II-IV, but without opportunistic infection or neoplasm, received 6 micrograms (3.6 x 10(4) IU) of polyethylene glycol recombinant human interleukin 2 (PEG IL-2)... more
13 patients with human immunodeficiency virus type 1 infection class II-IV, but without opportunistic infection or neoplasm, received 6 micrograms (3.6 x 10(4) IU) of polyethylene glycol recombinant human interleukin 2 (PEG IL-2) intradermally twice a week for 4 mo were then followed for an additional 6 mo. Clinical, immunological, and viral parameters were monitored in the patients, all of whom were taking zidovudine. The cutaneous administration of PEG IL-2 resulted in an indurated zone resembling a delayed-type hypersensitivity response of 26 +/- 1 mm diameter (676 mm2) at 72-96 h after injection throughout the 4 mo of administration. This dose, which was appreciably lower than in most previous trials, was not associated with local or systemic toxicity. No increase in the viral burden of circulating leukocytes or plasma occurred. A number of immunological functions were stimulated by this course of therapy. All patients demonstrated high levels of lymphokine-activated killer cell...
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The immune responses to Mycobacterium leprae and other mycobacterial antigens were studied in 11 leprosy patients with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Three patients manifested borderline lepromatous... more
The immune responses to Mycobacterium leprae and other mycobacterial antigens were studied in 11 leprosy patients with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Three patients manifested borderline lepromatous leprosy, and eight patients had borderline tuberculoid (BT) leprosy. Despite the low CD4+ T-cell count in the peripheral blood, no histologic or phenotypic change in the cellular infiltrate in either the lepromatous or tuberculoid lesions was observed when compared with HIV-1-negative patients. Lepromatous lesions contained heavily parasitized macrophages and few CD8+ T cells. Lesions from the patients with BT leprosy showed extensive CD4+ T-cell infiltration despite a significant reduction in CD4+ T-cell counts in the peripheral blood. No acid-fast bacilli were detected in the tuberculoid lesions. HIV-1 infection did not alter the lack of response in lepromatous leprosy to M. leprae antigens either in vitro or in vivo. In contrast, the skin test respon...
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Thalidomide, a selective inhibitor of tumor necrosis factor alpha (TNF-alpha) synthesis, suppresses the activation of latent human immunodeficiency virus type 1 (HIV-1) in a monocytoid (U1) line. The inhibition is dose dependent and... more
Thalidomide, a selective inhibitor of tumor necrosis factor alpha (TNF-alpha) synthesis, suppresses the activation of latent human immunodeficiency virus type 1 (HIV-1) in a monocytoid (U1) line. The inhibition is dose dependent and occurs after exposure of the cells to recombinant TNF-alpha, phorbol myristate acetate, lipopolysaccharide, and other cytokine combinations. Associated with HIV-1 inhibition is a reduction in agonist-induced TNF-alpha protein and mRNA production. Thalidomide inhibition of virus replication in the phorbol myristate acetate- and recombinant TNF-alpha-stimulated T-cell line ACH-2 is not observed. The presence of thalidomide also inhibits the activation of virus in the peripheral blood mononuclear cells of 16 out of 17 patients with advanced HIV-1 infection and AIDS. These results suggest the use of thalidomide in a clinical setting to inhibit both virus replication and the TNF-alpha-induced systemic toxicity of HIV-1 and opportunistic infections.