Polymorphisms in Toll-like receptor 4 (TLR4) are associated with protection against leprosy.

Accumulating evidence suggests that polymorphisms in Toll-like receptors (TLRs) influence the pathogenesis of mycobacterial infections, including leprosy, a disease whose manifestations depend on host immune responses. Polymorphisms in TLR2 are associated with an increased risk of reversal reaction, but not susceptibility to leprosy itself. We examined whether polymorphisms in TLR4 are associated with susceptibility to leprosy in a cohort of 441 Ethiopian leprosy patients and 197 healthy controls. We found that two single nucleotide polymorphisms (SNPs) in TLR4 (896G>A [D299G] and 1196C>T [T399I]) were associated with a protective effect against the disease. The 896GG, GA and AA genotypes were found in 91.7, 7.8 and 0.5% of leprosy cases versus 79.9, 19.1 and 1.0% of controls, respectively (odds ratio [OR] = 0.34, 95% confidence interval [CI] 0.20-0.57, P < 0.001, additive model). Similarly, the 1196CC, CT and TT genotypes were found in 98.1, 1.9 and 0% of leprosy cases versus 91.8, 7.7 and 0.5% of controls, respectively (OR = 0.16, 95% CI 0.06--.40, P < 0.001, dominant model). We found that Mycobacterium leprae stimulation of monocytes partially inhibited their subsequent response to lipopolysaccharide (LPS) stimulation. Our data suggest that TLR4 polymorphisms are associated with susceptibility to leprosy and that this effect may be mediated at the cellular level by the modulation of TLR4 signalling by M. leprae.

Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset.

The efficacy of thalidomide (alpha-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor alpha. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2-mediated T cell proliferation and interferon gamma production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.

Effect of rhuIFN-gamma treatment in multibacillary leprosy patients.

Previous studies have shown that when multibacillary leprosy patients were treated with recombinant human interferon gamma (rhuIFN-gamma) for 6-10 months there was an accelerated reduction in the number of acid-fast bacilli in the skin at the site of injection as well as an accelerated bacillary reduction at distal sites. However, this favorable out-come of IFN-gamma treatment was associated with the development of erythema nodosum leprosum (ENL). The present study was undertaken to investigate whether rhuIFN-gamma-induced bacillary clearance could be disassociated from the induction of ENL. rhuIFN-gamma was administered together with thalidomide and conventional multidrug chemotherapy to newly diagnosed leprosy patients. During treatment with this combination of drugs, the mean reduction in bacterial load was the same as the reduction observed with chemotherapy alone. Moreover, the inclusion of thalidomide in the treatment regimen was associated with a low frequency of ENL episodes. A second group of leprosy patients, who had already completed 2 years of chemotherapy, were treated with rhuIFN-gamma only. In those patients who were skin bacilli negative, ENL did not occur during rhuIFN-gamma treatment. In contrast, in bacilli-positive patients the frequency of ENL during rhuIFN-gamma treatment was higher, as was the occurrence of local erythema and induration. However, rhuIFN-gamma treatment without concomitant chemotherapy did not result in a reduction in the bacterial load in the skin of bacilli-positive patients. These findings, taken together, indicate that rhuIFN-gamma does not, by itself, accelerate bacterial clearance, but requires concomitant chemotherapy to achieve the accelerated reduction in bacillary load. Thalidomide reduces the frequency of IFN-gamma-induced ENL, but also eliminates the IFN-gamma-induced bacillary clearance.

Cellular immune response to Mycobacterium leprae infection in human immunodeficiency virus-infected individuals.

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 response to M. leprae antigens as well as the in vitro lymphoproliferative responses to mycobacterial antigens that are usually seen in patients with tuberculoid leprosy were abrogated in the BT HIV-1+ patients. However, production of gamma interferon in response to the same stimuli was preserved in most of the patients. Analysis of cytokine gene expression showed activation of additional cytokine genes in the unstimulated peripheral blood cells of patients with both leprosy and HIV-1 infections as compared with cells from patients with leprosy alone. These results suggest that granuloma formation in leprosy can be independent of the impaired CD4+ T-cell response of the HIV-1 infection. Furthermore, in HIV-1+ individuals with M. leprae infection, activation of cytokine genes is observed even when the circulating CD4+ T-cell count is significantly reduced.

Thalidomide treatment reduces tumor necrosis factor alpha production and enhances weight gain in patients with pulmonary tuberculosis.

BACKGROUND: The monocyte-derived cytokine, tumor necrosis factor alpha (TNF alpha), is essential for host immunity, but overproduction of this cytokine may have serious pathologic consequences. Excess TNF alpha produced in pulmonary tuberculosis may cause fevers, weakness, night sweats, necrosis, and progressive weight loss. Thalidomide (alpha-N-phthalimidoglutarimide) has recently been shown to suppress TNF alpha production by human monocytes in vitro and to reduce serum TNF alpha in leprosy patients. We have therefore conducted a two-part placebo-controlled pilot study of thalidomide in patients with active tuberculosis to determine its effects on clinical response, immune reactivity, TNF alpha levels, and weight. MATERIALS AND METHODS: 30 male patients with active tuberculosis, either human immunodeficiency virus type 1 positive (HIV-1+) or HIV-1-, received thalidomide or placebo for single or multiple 14 day cycles. Toxicity of the study drug, delayed type hypersensitivity (DTH), cytokine production, and weight gain were evaluated. RESULTS: Thalidomide treatment was well tolerated, without serious adverse events. The drug did not adversely affect the DTH response to purified protein derivative (PPD), total leukocyte, or differential cell counts. TNF alpha production was significantly reduced during thalidomide treatment while interferon-gamma (IFN gamma) production was enhanced. Daily administration of thalidomide resulted in a significant enhancement of weight gain. CONCLUSIONS: The results indicate that thalidomide is well tolerated by patients receiving anti-tuberculosis therapy. Thalidomide treatment reduces TNF alpha production both in vivo and in vitro and is associated with an accelerated weight gain during the study period.

Recombinant cytokines for controlling mycobacterial infections.

Knowing how mycobacteria exploit host cytokines to survive and which cytokines have important roles in host defense against mycobacteria should allow the use of these molecules in the treatment of mycobacterial infections. Both interleukin 2 and interferon gamma have been used to treat patients with leprosy, and granulocyte-macrophage colony-stimulating factor is presently being administered to AIDS patients infected with Mycobacterium avium.

Cytokine regulation of disease progression in leprosy and tuberculosis.

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.

GM-CSF activates regenerative epidermal growth and stimulates keratinocyte proliferation in human skin in vivo.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF), an immunomodulator of hematopoietic cells, has also been shown to stimulate human keratinocyte proliferation in vitro and speed healing of wounds in the skin of lepromatous leprosy patients. In this study we have examined the in vivo effects of recombinant human GM-CSF on epidermal keratinocyte proliferation and on expression of proteins marking regenerative epidermal growth. Skin biopsies from GM-CSF injected cutaneous sites were obtained between 1 and 6 d following administration of 7.5 or 15 micrograms of the growth factor. Activation of keratinocyte proliferation, quantified as the expression of the Ki67+ nuclear antigen, was noted 1 d following GM-CSF administration. A regenerative epidermal phenotype, demonstrated by immunohistochemical staining of cellular proteins involucrin, filaggrin, and keratin 16, was similarly noted as early as 1 d following GM-CSF injection. This phenotype persisted as late as 6 d post-injection. These results suggest that GM-CSF injection into human skin induces keratinocyte proliferation as well as regenerative differentiation of the epidermis. To date no other cytokine has been shown to be mitogenic for human keratinocytes both in vivo and in vitro or to alter keratinocyte differentiation along the "alternate" or regenerative pathway.

The influence of thalidomide on the clinical and immunologic manifestation of erythema nodosum leprosum.

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.

Recent advances in cytokine therapy in leprosy.

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.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site. There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

Prolonged treatment with recombinant interferon gamma induces erythema nodosum leprosum in lepromatous leprosy patients.

10 patients with borderline and lepromatous leprosy were selected for a prolonged trial with recombinant interferon gamma (rIFN-gamma). Patients received 30 micrograms intradermally for six injections over a 9-d period, and then either 100 micrograms intradermally every 1 mo for 10 mo or every 2 wk for 5 mo (total, 1.2 mg). Erythema nodosum leprosum (ENL) was induced in 60% of the patients within 6-7 mo, as compared with an incidence of 15% per year with multiple drug therapy alone. The mean whole-body reduction in bacterial index over the first 6 mo was 0.9 log units. Cutaneous induration at the intradermal injection sites of greater than or equal to 15 mm predicted the development of a subsequent reactional state. Monocytes obtained from patients receiving the lymphokine demonstrated an increased respiratory burst and a 2.5-5.1-fold increase in tumor necrosis factor alpha (TNF-alpha) secretion in response to agonists. Patients in ENL had an even higher release of TNF-alpha from monocytes as well as high levels of TNF-alpha in the plasma (mean, 2,000 pg/ml). Thalidomide therapy was required to treat the systemic manifestations of ENL. Control of toxic symptoms with thalidomide was associated with a 50-80% reduction in agonist-stimulated monocyte TNF-alpha secretion. IFN-gamma enhanced the monocyte release of TNF-alpha by 3-7.5-fold (agonist dependent) when added to patient's cells in vitro, and this could be suppressed by the in vitro addition of 10 micrograms/ml of thalidomide.

Rational immunotherapy with interleukin 2.

Interleukin 2 (IL-2), a T lymphocyte product released upon antigen stimulation, has been used for cancer therapy in high doses for more than five years. More recently, its potential as a stimulant of cell-mediated immunity in infectious diseases, particularly those caused by intracellular microbes, has become appreciated. Drawing on the extensive information available as to the structure, cellular and molecular effects of IL-2, this review focuses on its use in patients with lepromatous leprosy and AIDS in low, physiologic doses. The data indicate that IL-2 is effective in stimulating cell-mediated immunity without systemic toxicity.

Effect of cutaneous cell-mediated immune response to rIFN gamma on Mycobacterium leprae viability in the lesions of lepromatous leprosy.

1. Studies were carried out to determine the effect of intra-dermal injections of recombinant human interferon-gamma (rIFN gamma) on the viability of Mycobacterium leprae. Twenty-three untreated and 4 treated multibacillary patients, 12 with lepromatous leprosy (LL) and 15 with borderline lepromatous leprosy (BL), were selected for intradermal administration of rIFN gamma or PPD. Treated patients (LL and BL) had received multi-drug therapy according to the recommendations of the World Health Organization, i.e., rifampicin (600 mg/month), dapsone (100 mg/day) and clofazimine (50 mg/day and 300 mg/month) for 1-4 months. Three daily doses of 10 or 30 micrograms rIFN gamma induced local induration and mononuclear leucocyte accumulation. Bacteria isolated from a punch biopsy of the site 21 days after lymphokine administration were injected into mouse foot pads and evaluated for viability and growth. 2. The local response to rIFN gamma (specific activity 2 x 10(7) units/mg protein) induced a delay or total inhibition of M. leprae growth in the mouse foot pad, indicating that the cellular response to the antigen reduced local M. leprae viability. The extent of reduction in viability depended on the dose of rIFN gamma injected and the extent of local induration induced by the lymphokine. With a vigorous cell-mediated immune response growth was fully inhibited. 3. A similar but less extensive effect on M. leprae viability was observed in response to the local injection of 5 units in 0.1 ml of purified protein derivative of tuberculin (PPD).

Effect of cutaneous cell-mediated immune response to rIFNy on mycobacterium leprae viability in the lesions of lepromatous leprosy / Efeito da célula cutanea - resposta imune intermediária para rIFNy sobre viabilidade de Mycobacterium leprae nas lesöes de lepra lepromatosa

Studies were caried out to determine the effect of intra-dermal injections of recombinant human interferon-gamma (rIFNy) on the viability of Mycobacterium leprae. Twenty-three untreated and 4 treated multibacillary patients, 12 with lepromatous leprosy (LL) and 15 with bordeline lepromatous leprosy (BL), were selected for intradermal administration of rIFNy or PPD. Treated patients (LL and BL) had received multi-drug therapy according to the recommendations of the World Health Organization, i. e., rifampicxin (600 mg/month), dapsone (100 mg/day) and clofazimine (50 mg/day and 300 mg/month) for 1-4 months. Three daily doses of 10 or 30 ug rIFNy induced local induration and mononuclear leucocyte accumulation. Bacteria isolated from a punch biopsy of the site 21 days after lymphokine administration were injected into mouse foot pads and evaluated for viability and growth. The local response to rIFN (specific activity 2 x 10 7 units/mg protein) induced a delay or total inhibition of M. leprae growth in the mouse foot pad, ,indicating that the cellular response to the antigen reduced local M. leprae viability. The extent of reduction in viability depended on the dose of rIFNy injected and the extent of local induration induced by the lymphokine. With a vigorous cell-mediated immune response growth was fully inhibited. A similar but less extensive effect on M. leprae viability was observed in resapo0nse to the local injection of 5 units in 0.1 ml of purified protein derivative of tuberculin (PPD)

Mycobacterium leprae-induced interferon-gamma production by household contacts of leprosy patients: association with the development of active disease.

Identification of individuals at risk for developing leprosy and their early diagnosis are central to effective disease control. Lack of immunologic response to Mycobacterium leprae among persons exposed to the infectious agent may be predictive of susceptibility. M. leprae-induced interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells was used as a measure of immune responsiveness. Household contacts of multibacillary patients likely to be at risk of developing active disease were identified, and a preliminary analysis after 2 years of follow-up is presented. A persistent in vitro negative response to M. leprae was present in 34.6% of the contacts, and a decrease in IFN-gamma production was noted in 52.5%. Five contacts (6.41%) developed leprosy during follow-up and, as predicted, belonged to the group of individuals who were negative or showed reduced levels of IFN-gamma in response to the antigen.