Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection.

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.

The role of indoleamine 2, 3-dioxygenase in lepromatous leprosy immunosuppression.

To elucidate further the possible role of the tryptophan, rate-limiting enzyme indoleamine 2, 3-dioxygenase (IDO) in leprosy, the distribution of IDO-positive cells and IDO activity in the skin biopsies and sera of these patients representing the entire spectrum of the disease were studied. An increased number of macrophages/dendritic cells (DC-lineage IDO(+) cells were found in lepromatous (LL) compared to tuberculoid (BT) and reversal reaction (RR) patients. IDO-positive cells showing CD68 and CD86 surface markers predominated in LL lesions, while higher levels of IDO activity were observed in the sera of LL versus BT patients. Tests revealed an increased IDO message in Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) by real-time polymerase chain reaction (PCR) and increased IDO expression in M. leprae-stimulated CD14(+) cells of both healthy controls (HC) and LL patients, as evaluated via flow cytometry. Increased M. leprae-induced IDO-protein synthesis was also confirmed by Western blot. Based on our in vitro studies, it was confirmed that M. leprae up-regulated IDO expression and activity in HC and LL monocytes. Interferon (IFN)-γ synergized with M. leprae in promoting IDO expression and activity in monocytes. IDO expression induced by both IFN-γ and M. leprae was abrogated by 1-methyltryptophan (1-MT). Our data suggest that M. leprae chronic infection activates the suppressive molecule IDO which, in turn, contributes to the specific immunosuppression observed in LL leprosy.

Jarisch-Herxheimer reaction among syphilis patients in Rio de Janeiro, Brazil.

The Jarisch-Herxheimer reaction (JHR) is a syndrome observed after antimicrobial treatment of some infectious diseases. The syndrome has clinical characteristics of an inflammatory reaction to antibiotic treatment. A prospective study of patients with a clinical and laboratory diagnosis of syphilis was conducted at a sexually transmitted diseases clinic in Rio de Janeiro, Brazil. Patients were treated with benzathine penicillin and observed for the JHR. A total of 115 patients were included in this study. Fifty-one patients (44%) had secondary syphilis; 37 (32%), primary; 26 (23%), latent; and one (1%), tertiary syphilis. Ten patients (9%) developed the JHR. All JHRs occurred in patients with secondary and latent syphilis. No patients experienced an allergic reaction to penicillin. The JHR occurred less frequently than in previous studies. It is important that health-care professionals recognize the clinical characteristics of the JHR so that it is not misinterpreted as an allergic reaction to penicillin.

Hansen's disease in a general hospital: uncommon presentations and delay in diagnosis.

BACKGROUND: The question was raised as to why 'obvious' signs of leprosy, Hansen's disease (HD), are often missed by medical doctors working in a HD endemic area. METHODS: This study describes a small sample of patients who were diagnosed with HD during their hospital admission and not before. The discussion is whether the typical early signs and symptoms of HD are just not recognized, or whether unusual presentations confuse the attending physician. RESULTS: A total of 23 HD patients were hospitalized during the study period, of which 6 (26%) were only diagnosed with HD during their admission. All were classified as lepromatous leprosy (LL) with a history of signs and symptoms of HD. In nearly all patients, a suspicion of HD might have been raised earlier if a careful history and dermato-neurological examination had been done. CONCLUSIONS: Multibacillary (MB) HD, especially close to the lepromatous end of the spectrum, may mimic other diseases, and the patient can not be diagnosed without a biopsy or a slit skin smear examination. Clinicians working in a HD endemic area (Rio de Janeiro) do not always include HD in their differential diagnosis, especially when the clinical presentation is unusual. HD should be considered in all patients with skin lesions not responding to treatment, especially when they have neurological deficits, and live or have lived in an HD endemic area. Due to the increase in global travel and immigration, doctors in low endemic areas need to consider HD as a possible diagnosis.

Effectiveness of BCG vaccination among leprosy contacts: a cohort study.

The study assessed the effectiveness of BCG vaccination against leprosy among the contacts of 1161 leprosy patients at the FIOCRUZ Leprosy Outpatient Clinic, RJ, Brazil, from June 1987 to December 2006. Following National Leprosy Program guidelines, the clinic has administered one-to-two doses to all healthy contacts since 1991. Among the 5680 contacts, 304 (5.4%) already had leprosy. Of the 5376 eligible healthy contacts, 3536 were vaccinated, 30 of whom were excluded due to previous or current tuberculosis, or HIV. In 18 years of follow up, 122 (2.15%) incident cases were diagnosed (58 vaccinated and 64 not), 28 occurring in the first year of follow up (21 vaccinated, 16 with no scar). The protection conferred by BCG was 56% and was not substantially affected by previous BCG vaccination (50% with a scar and 59% without). The risk of tuberculoid leprosy during the initial months was high among those vaccinated with no scar. However, it had substantially declined by the first year and in the following years, when the protection rate in this group reached 80%. Since Brazil is endemic for leprosy and the detection rate is not declining satisfactorily, vaccinating all contacts could be an effective means of substantially reducing the incidence of leprosy.

Clinical, immunological and histological aspects of an uncommon type II reaction in patients with lepromatous leprosy.

This study reports three cases of an unusual leprotic reaction characterized by superficial bullous ulcerative cutaneous lesions associated with high fever, malaise and oedema in patients with leprosy. Two patients responded to thalidomide treatment, with regression of the symptoms and skin ulcers. The third patient responded to thalidomide plus prednisone. Analysis of the ulcerated skin lesions showed dermal oedema with mononuclear cell infiltrate enriched for gammadelta-positive T lymphocytes and an increased number of Mycobaterium leprae bacilli within capillary endothelium. In contrast, gammadelta+ cells were decreased in or absent from the blood. Tumour necrosis factor-alpha and interleukin-6 were raised in the serum of the patients at the onset of the reaction. After the episode, cytokine levels and the percentage of gammadelta+ cells in the blood returned to normal. These cases characterize an uncommon leprotic reaction with clinical similarities to type II reaction and may indicate a significant role for gammadelta+ T cells in its pathogenesis.

Increased Langerhans cell accumulation after mycobacterial stimuli.

AIMS: To evaluate the role of Langerhans cells (LCs) in the local activation of leprosy lesions. LCs, acting as tolerance inducers and immune stimuli, are dendritic cells recently implicated in cutaneous homeostasis. The role of LCs in the defence against mycobacterial infection remains poorly understood. METHODS AND RESULTS: The number and distribution of CD1a+ skin cells and HLA-DR and intercellular adhesion molecule (ICAM)-1 expression were analysed in leprosy skin lesions and in delayed-type hypersensitivity (DTH) tests. The results showed a high number of LCs in tuberculin and lepromin tests, in tuberculoid lesions and in the epidermis and dermis during type I and II reactions. In multibacillary lesions, however, the number of LCs was consistently low in comparison with other groups. Increased numbers of LCs were accompanied by marked HLA-DR and ICAM-1 expression, suggesting a strong relationship between these immunological events. CONCLUSIONS: CD1a+ cells are implicated in the local immunological events taking place after mycobacterial stimuli and may account for the local activation of all types of reactional episodes in leprosy.

Long-term culture of multibacillary leprosy macrophages isolated from skin lesions: a new model to study Mycobacterium leprae-human cell interaction.

BACKGROUND: Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell-mediated immunity. In the tuberculoid forms, granuloma-activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood. OBJECTIVES: To establish an ex vivo methodology capable of evaluating the activation pathways, grade and fate of cultured macrophages isolated from MB lesions. METHODS: Skin biopsies from patients with borderline tuberculoid, bordeline lepromatous and lepromatous leprosy (LL) were characterized by immunohistochemistry and transcriptional analysis. To isolate inflammatory cells, a portion of the samples was submitted to enzymatic digestion. These same cells, maintained in culture for a minimum 7-day period, were characterized morphologically and via flow cytometry at different culture time points. Cytokine [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10] mRNA levels were quantified by real-time polymerase chain reaction and protein secretion in the culture supernatants was measured by enzyme-linked immunosorbent assay and the nitric oxide levels by Griess reagent. RESULTS: RNA expression in tuberculoid and MB lesions showed the profile expected of characteristic Th1 and Th2 responses, respectively. The inflammatory cells in all biopsies were successfully isolated. Although the number of cells varied between biopsies, it was highest in LL biopsies. The frequency of isolated CD14+ and CD3+ cells measured by flow cytometry correlated with the percentages of macrophages and lymphocytes in the lesions. Throughout the culture period, CD68+ macrophages showed morphological changes. A progressive increase in cell number and reduction of infected cells were perceptible in the cultures. In contrast to the biopsies, TNF-alpha, IFN-gamma and IL-10 expression in the tuberculoid and MB leprosy cells in 24-h culture and the cytokine levels in the supernatants did not differ significantly. During the culture period, cytokine expression in the MB cells progressively declined, whereas, from days 1 to 7, nitrite levels progressively increased. After day 40, the remaining macrophages were able to ingest fluorescein isothiocyanate-labelled M. leprae. These data need to be confirmed. CONCLUSIONS: This study confirmed the feasibility of obtaining ex vivo macrophages from leprosy lesions and keeping them in long-term culture. This procedure may open new pathways to studying the interaction between M. leprae and human macrophages, which might, in turn, lead to the development of therapeutic tools capable of overcoming the specific anergy found in patients with MB leprosy.

Expression of B7-1 costimulatory molecules in patients with multibacillary leprosy and reactional states.

BACKGROUND: The expression of B7 as a costimulatory molecule on the surface of antigen-presenting cells such as macrophages and on dendritic cells characterizes the efficiency of the cell-mediated immune response. AIMS: Our purpose was to evaluate B7-1 expression in peripheral blood mononuclear cells (PBMCs) immediately after cell isolation ('spontaneous' B7 expression), and in inflammatory cells from cutaneous lesions of patients with multibacillary leprosy (MB-L) without and during the reactional states of erythema nodosum leprosum (ENL) or reversal reaction (RR). METHODS: Peripheral blood samples and skin biopsies of eight patients without (MB-L) and with reactional episodes (ENL and RR) were studied using antibodies against B7-1, CD1b, DR and CD14 in flow-cytometry and immunohistochemistry experiments. RESULTS: The flow-cytometry studies (mean +/- SD% of fluorescent cells) revealed significant B7-1 expression on PBMCs isolated from patients with ENL (8.0 +/- 0.6%) and RR (15.0 +/- 1.4%) compared with that observed for patients with MB-L (0.4 +/- 0.2%). Similar results were observed for cutaneous lesions of these patients by immunohistochemical assays. One patient studied before and during ENL revealed weak B7 expression before the reactional episode (0.3% of cells) compared with the marked level of B7-expressing cells detected during ENL (8.5% fluorescent cells). Interestingly, an even higher B7 expression (15% of cells) was observed in patients with RR. CONCLUSIONS: Our results strongly suggest that B7 expression precedes reactional episodes in MB-L, which could be related to the acquisition of effective immunity to Mycobacterium leprae during reactional episodes in leprosy. We propose B7 expression as a marker of CMI response in reactional episodes in leprosy.

Double-blind trial of the efficacy of pentoxifylline vs thalidomide for the treatment of type II reaction in leprosy.

Type II reaction in leprosy, or erythema nodosum leprosum (ENL), is often characterized by severe clinical symptoms together with nerve function impairment leading to permanent disabilities. Thalidomide has been shown to be a highly effective drug for the treatment of ENL. It is, however, contraindicated for women of childbearing age due to its teratogenicity. On the other hand, pentoxifylline, used to treat hypercoagulable states, is not teratogenic and, like thalidomide, can inhibit the synthesis of tumor necrosis factor-a and other cytokines. In the present randomized double-blind clinical study we compared the effectiveness of orally administered pentoxifylline vs thalidomide in treating type II reaction in 44 patients. Daily doses of 300 mg thalidomide or 1.2 g pentoxifylline were administered for 30 days to multibacillary leprosy patients undergoing type II reaction. Randomly chosen patients were included in the study before, during, and after specific multidrug therapy. Clinical evaluations were performed on the 1st, 7th, 14th, 21st, and 30th days of treatment and laboratory tests were carried out on the 1st and 30th days. As expected, overall, thalidomide proved to be more effective in the treatment of type II leprosy reaction. Nevertheless, continuous treatment with pentoxifylline was effective in relieving the clinical signs of ENL, especially limb edema and systemic symptoms, in 62.5% of the patients.

Double-blind trial of the efficacy of pentoxifylline vs thalidomide for the treatment of type II reaction in leprosy

Type II reaction in leprosy, or erythema nodosum leprosum (ENL), is often characterized by severe clinical symptoms together with nerve function impairment leading to permanent disabilities. Thalidomide has been shown to be a highly effective drug for the treatment of ENL. It is, however, contraindicated for women of childbearing age due to its teratogenicity. On the other hand, pentoxifylline, used to treat hypercoagulable states, is not teratogenic and, like thalidomide, can inhibit the synthesis of tumor necrosis factor-a and other cytokines. In the present randomized double-blind clinical study we compared the effectiveness of orally administered pentoxifylline vs thalidomide in treating type II reaction in 44 patients. Daily doses of 300 mg thalidomide or 1.2 g pentoxifylline were administered for 30 days to multibacillary leprosy patients undergoing type II reaction. Randomly chosen patients were included in the study before, during, and after specific multidrug therapy. Clinical evaluations were performed on the 1st, 7th, 14th, 21st, and 30th days of treatment and laboratory tests were carried out on the 1st and 30th days. As expected, overall, thalidomide proved to be more effective in the treatment of type II leprosy reaction. Nevertheless, continuous treatment with pentoxifylline was effective in relieving the clinical signs of ENL, especially limb edema and systemic symptoms, in 62.5 percent of the patients.

High prevalence of vasomotor reflex impairment in newly diagnosed leprosy patients.

BACKGROUND: Initial nerve damage in leprosy occurs in small myelinated and unmyelinated nerve fibers. Early detection of leprosy in the peripheral nervous system is challenging as extensive nerve damage may take place before clinical signs of leprosy become apparent. PATIENTS AND METHODS: In order to determine the prevalence of, and factors associated with, peripheral autonomic nerve dysfunction in newly diagnosed leprosy patients, 76 Brazilian patients were evaluated prior to treatment. Skin vasomotor reflex was tested by means of laser Doppler velocimetry. Blood perfusion and reflex vasoconstriction following an inspiratory gasp were registered on the second and fifth fingers. RESULTS: Vasomotor reflex was impaired in at least one finger in 33/76 (43%) patients. The fifth fingers were more frequently impaired and suffered more frequent bilateral alterations than the second fingers. Multivariate regression analysis showed that leprosy reaction (adjusted odds ratio = 8.11, 95% confidence interval: 1.4-48.2) was associated with overall impaired vasomotor reflex (average of the four fingers). In addition, palmar erythrocyanosis and an abnormal upper limb sensory score were associated with vasomotor reflex impairment in the second fingers, whereas anti-phenolic glycolipid-I antibodies, ulnar somatic neuropathy and a low finger skin temperature were associated with impairment in the fifth fingers. CONCLUSIONS: A high prevalence of peripheral autonomic dysfunction as measured by laser Doppler velocimetry was observed in newly diagnosed leprosy patients, which is clinically evident late in the disease. Autonomic nerve lesion was more frequent than somatic lesions and was strongly related to the immune-inflammatory reaction against M. leprae.

Interleukin-10 promoter single-nucleotide polymorphisms as markers for disease susceptibility and disease severity in leprosy.

We have determined IL-10 promoter genotypes of five single-nucleotide polymorphisms (SNPs): T-3575A, A-2849G, C-2763A, -A-1082G and C-819T. The haplotype frequencies were defined in healthy subjects compared to leprosy patients, and analyzed for their occurrence in multi- (MB) vs paucibacillary (PB) as severe and mild forms of leprosy, respectively. Haplotypes defined by three SNP positions (-3575, -2849 and -2763) captured significant differences between controls and patients (P=0.04). The haplotype carrying -3575A, -2849G and -2763C was associated with resistance to leprosy and to the development of severe forms of the disease using either a binomial (controls vs cases, P=0.005, OR=0.35, CI=0.13-0.91) or ordinal (controls vs PB vs MB, P=0.006, OR=0.32, CI=0.12-0.83) model. By contrast, the IL-10 haplotype -3575T/-2849A/-2763C was found to be associated with susceptibility to leprosy per se (P=0.027, OR=2.37, CI=1.04-5.39), but not leprosy type. The data suggest that the IL-10 locus contributes to the outcome of leprosy.

Impairments in multibacillary leprosy; a study from Brazil.

This is a retrospective cohort study of 103 multibacillary leprosy patients (18% BB, 48% BL and 34% LL) followed during and after treatment, in a tertiary referral centre with an outpatient clinic in an endemic area in Brazil, for an average period of 65 months since the start of multidrug therapy (24-dose MDT). The objective of the study was to identify the role of overt neuritis (presence of pain in a peripheral nerve trunk, with or without enlargement or neural function damage), in the development of impairments. They were evaluated using the World Health Organization disability grade before treatment, at the end of the treatment, and at the end of the follow-up period. Thirty-four percent of patients presented overt neuritis during MDT, and 45% had overt neuritis episodes during the follow-up period; the most commonly affected nerves were ulnar, fibular and posterior tibial nerves, and the neuritic episodes were carefully treated with steroid therapy and physiotherapy. Impairments were associated with: affected (painful and/or thick) nerves at diagnosis (P < 0.005); delay in diagnosis (P = 0.010); impairments already present at the start of treatment (P = 0.00041 at the end of MDT, and P = 0.000013 at the end of follow-up); occurrence of overt neuritis episodes during MDT (P = 0.0016) or the whole follow-up (P = 0.015). These data draw attention to the importance of early diagnosis and of good neurological examination throughout the follow-up, as well as suggest the importance of neuritis in the induction of impairments in multibacillary leprosy.

Leprosy and AIDS: two cases of increasing inflammatory reactions at the start of highly active antiretroviral therapy.

Reported here are the cases of two HIV-positive patients with skin lesions suggestive of leprosy, based on clinical and pathological analysis, which worsened during the few weeks following initiation of highly active antiretroviral therapy. The lesions improved after a few weeks of multidrug therapy for leprosy. Mycobacterium leprae was confirmed by polymerase chain reaction analysis of blood in case 1 and of a biopsy sample in case 2. Neither Mycobacterium avium complex nucleic acid, which is usually associated with immune restoration syndrome, nor mycobacterial cutaneous manifestations were detected in either case.

Leprosy and AIDS: two cases of increasing inflammatory reactions at the start of highly active antiretroviral therapy

Reported here are the cases of two HIV-positive patients with skin lesions suggestive of leprosy, based on clinical and pathological analysis, which worsened during the few weeks following initiation of highly active antiretroviral therapy. The lesions improved after a few weeks of multidrug therapy for leprosy. Mycobacterium leprae was confirmed by polymerase chain reaction analysis of blood in case 1 and of a biopsy sample in case 2. Neither Mycobacterium avium complex nucleic acid, which is usually associated with immune restoration syndrome, nor mycobacterial cutaneous manifestations were detected in either case

Evaluation of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules and dendritic cells on the immune response in leprosy.

The cell activation depends on T cell antigen receptor binding to antigen plus MHC and costimulation. The binding of CD28, expressed on the T cell surface to B7 (B7-1 or CD80/B7-2 or CD86) present on the antigen--presenting cells (APCs), determines, in several T cell function models, if activation or anergy follows antigenic stimulation. In leprosy, the role of CD80 and CD86 as costimulatory signal in M. leprae-specific cellular immunity has not yet been defined. We investigated the role of B7-CD28 pathway of T cell activation in the in vitro response to M. leprae, following stimulation in the presence of monocytes or dendritic cells (DCs) as APCs. Monocytes were purified, by cold aggregation, from peripheral blood mononuclear leukocytes (PBMC), isolated from leprosy patients. In order to obtain DCs, the monocytes were cultured in the presence of IL-4 and GM-CSF. T cells were purified from PBMC by negative selection with mABs and C'. The phenotype of the cell populations was monitored by FACS. Lymphoproliferative assays were performed with T cells, in the presence of monocytes or DCs. The cells were stimulated by M. leprae in the presence of anti-CD80 antibody (Ab) and/or anti-CD86 antibody (Ab) (Innogenetics). In some experiments Il-10, Il-12 and anti-Il-12 Ab were also added to the culture. We observed a significantly more efficient APC function for DCs when compared to monocytes in T cell in vitro responses to M. leprae. Regardless of the clinical form of Leprosy, the M. leprae-specific immune response was markedly reduced in the presence of anti-CD86 Ab. Il-12 increase the immune response to M. leprae while IL-10 or anti-IL-12 Ab reduce this response when monocytes or DCs were used as APCs.