Different immunosuppressive mechanisms in multi-drug-resistant tuberculosis and non-tuberculous mycobacteria patients.

Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4(+) CD25(+-) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(+) CD127(-) regulatory T (T(reg) ) cells when compared to NR-TB. Increased numbers of T(reg) cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-ß was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4(+) /interferon (IFN)-γ(+) T cells and enhanced CD4(+) CD25(+) FoxP3(+) , CD4(+) CD25(+) CD127(-) and CD4(+) CD25(+) IL-10(+) T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4(+) CD25(+) FoxP3(+) frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4(+) CD25(+) T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T(reg) /T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.

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.

Thalidomide selectively inhibits tumor necrosis factor alpha production by stimulated human monocytes.

Thalidomide selectively inhibits the production of human monocyte tumor necrosis factor alpha (TNF-alpha) when these cells are triggered with lipopolysaccharide and other agonists in culture. 40% inhibition occurs at the clinically achievable dose of the drug of 1 micrograms/ml. In contrast, the amount of total protein and individual proteins labeled with [35S]methionine and expressed on SDS-PAGE are not influenced. The amounts of interleukin 1 beta (IL-1 beta), IL-6, and granulocyte/macrophage colony-stimulating factor produced by monocytes remain unaltered. The selectivity of this drug may be useful in determining the role of TNF-alpha in vivo and modulating its toxic effects in a clinical setting.

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.

The cutaneous infiltrates of leprosy. A transmission electron microscopy study.

The dermal lesions of 18 patients with leprosy have been examined by transmission electron microscopy. The patients exhibited a spectrum of disease from polar lepromatous to polar tuberculoid with intermediate stages in various states of therapy and relapse. The nature and quantities of inflammatory cells and bacteria have been determined by electron microscopy to supplement previous light and fluorescence microscopy studies. Lepromatous leprosy was characterized by many parasitized foam cells containing large, multibacillary vacuoles with intact, osmiophilic Mycobacterium leprae: Bacteria were embedded in an electron-lucent matrix. No extracellular bacteria were evident. Only small numbers of scattered lymphocytes were found. As one approached the borderline state, smaller numbers of bacilli were present as singlets and doublets in small vacuoles of macrophages. The more reactive forms showed increasing bacillary fragmentation, larger numbers of lymphoid cells, and an occasional epithelioid cell. At the tuberculoid end of the spectrum, clear evidence of an exuberant lymphocyte response was evident. Large numbers of T cells with extremely long and complex filipodia were closely associated with epithelioid and multinucleated giant cells. Many of the mononuclear phagocytes appeared nonviable, and areas of necrosis were evident. Bacillary remnants were scarce and the cytoplasm of the epithelioid cells contained occasional dense bodies and many stacks of endoplasmic reticulum and mitochondria. These results suggest that Leu 3a/OKT4 helper cells may be capable of driving the effector function of mononuclear phagocytes. This would lead to a significant microbicidal effect on M. leprae, perhaps through the production of toxic oxygen intermediates.

Defective gamma interferon production in leprosy. Reversal with antigen and interleukin 2.

Antigen and mitogen-induced gamma interferon (gamma-IFN) production was studied in peripheral blood mononuclear cells from 34 leprosy patients. 17 of 18 lepromatous leprosy and borderline lepromatous patients (LL and BL) failed to release gamma-IFN in response to specific antigen (Mycobacterium leprae) and displayed reduced responses to mitogen (concanavalin A) stimulation. In contrast, cells from six tuberculoid and borderline tuberculoid patients (TT and BT) produced considerable levels of gamma-IFN under the same experimental conditions. Normal controls failed to respond to M. leprae and most displayed good responses to concanavalin A. Mid-borderline patients (BB) showed intermediate levels of gamma-IFN release. gamma-IFN release by lepromatous patients could be partially restored with purified interleukin 2 and M. leprae antigen but not with interleukin 2 alone.

IFNG +874 T>A single nucleotide polymorphism is associated with leprosy among Brazilians.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, a low virulence mycobacterium, and the outcome of disease is dependent on the host genetics for either susceptibility per se or severity. The IFNG gene codes for interferon-γ (IFN-γ), a cytokine that plays a key role in host defense against intracellular pathogens. Indeed, single nucleotide polymorphisms (SNPs) in IFNG have been evaluated in several genetic epidemiological studies, and the SNP +874T>A, the +874T allele, more specifically, has been associated with protection against infectious diseases, especially tuberculosis. Here, we evaluated the association of the IFNG locus with leprosy enrolling 2,125 Brazilian subjects. First, we conducted a case-control study with subjects recruited from the state of São Paulo, using the +874 T>A (rs2430561), +2109 A>G (rs1861494) and rs2069727 SNPs. Then, a second study including 1,370 individuals from Rio de Janeiro was conducted. Results of the case-control studies have shown a protective effect for +874T carriers (OR(adjusted) = 0.75; p = 0.005 for both studies combined), which was corroborated when these studies were compared with literature data. No association was found between the SNP +874T>A and the quantitative Mitsuda response. Nevertheless, the spontaneous IFN-γ release by peripheral blood mononuclear cells was higher among +874T carriers. The results shown here along with a previously reported meta-analysis of tuberculosis studies indicate that the SNP +874T>A plays a role in resistance to mycobacterial diseases.

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.

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.

An immunohistochemical, clinical and electroneuromyographic correlative study of the neural markers in the neuritic form of leprosy.

The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100% of the neuritic biopsies. NGFr positivity was also reduced in 81.8%, PGP staining in 100% of the affected nerves, S100 positivity in 90.9%, and myelin basic protein immunoreactivity in 90.9%. Hypoesthesia was associated with decreased NGFr (81.8%) and PGP staining (90.9%). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6%) and nerve fiber neurofilament staining (45.4%) by immunohistochemistry and with loss of myelinated fibers (100%) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40% of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.

T cell immune responses to mycobacterial antigens in Brazilian tuberculosis patients and controls.

Production of IFN-gamma guarantees helpful T cell-mediated immunity against Mycobacterium tuberculosis infection. We have evaluated the in vitro immune responses to M. tuberculosis antigens using IFN-gamma production among 43 Brazilian tuberculosis (TB) patients prior to and after specific treatment, and 18 community controls. Peripheral blood mononuclear cells (PBMC) were cultivated in the presence either of purified protein derivative, ferritin, 10 kDa, 38 kDa, MPT59, Ag85A or Ag85B. Also, the two M. tuberculosis and M. bovis heat-shock proteins (hsp) 65 and 70 kDa were compared, and 5 day supernatants were harvested for cytokine detection by ELISA. The results showed that the overall profile of primary PBMC in response to most M. tuberculosis antigens was well correlated, since high IFN-gamma levels were induced by Ag85A, Ag85B, 38 kDa, ferritin and 10 kDa, as well as M. tuberculosis hsp65 in TB patients. In addition, analysis was carried out of the in vitro expression of activation molecules on lymphocytes, as CD25 and CD69 expression assessed in 17 TB patients showed induction on CD4+ T cells by Ag85B. Overall, significantly low responses were found in untreated, in comparison with the treated TB patients. Furthermore, internal community but not healthy control individuals have higher immune responses than do TB patients.

Neutrophils isolated from leprosy patients release TNF-alpha and exhibit accelerated apoptosis in vitro.

This study demonstrated that polymorphonuclear neutrophils (PMN) participate in the acute inflammatory response in leprosy as effector cells. Lepromatous patients present intense infiltrate of neutrophils in reactional (ENL) lesions. Circulating PMN of nonreactional patients, healthy donors, and reactional patients were purified and analyzed in vitro. The study confirmed the short lifespan of these cells in culture with progressive changes characteristic of apoptosis. Apoptosis was greatly accelerated in ENL patients as shown by cellular morphology, later confirmed by qualitative and quantitative analysis of fragmented DNA. It was observed that neutrophils stimulated with lipopolysaccharide, Mycobacterium leprae, and lipoarabinomannan secrete interleukin-8 and tumor necrosis factor alpha (TNF-alpha). Thalidomide, a drug known to inhibit TNF-alpha synthesis on monocytes, also exerted an inhibitory effect on TNF-alpha secretion in neutrophils. These data suggest that PMN can participate in the regulation of the immune response in leprosy and can contribute to the amplification of TNF-alpha production at the site of ENL lesion.

Leprosy neuropathy evaluated by NCS is independent of the patient's infectious state.

INTRODUCTION: Leprosy causes nerve injury, which mimics clinical and neurophysiological conditions, rendering it an excellent model of peripheral neuropathy. METHODS: A retrospective study including 822 nerve conduction studies (NCS) of 509 patients was developed to appraise the electrophysiological pattern of leprosy neuropathy. NCS of motor and sensory nerves performed before, during, and after multidrug therapy (MDT) were analyzed. RESULTS: During the three periods of MDT, while NCS alterations were similar regarding extension, topography, damage severity, and type of lesion, NCS showed that sensory was more frequent (sural nerve) (92-96%) than motor impairment (70-77%) (ulnar nerve). CONCLUSION: Once axonal loss has been installed, nerve function is little affected by inflammatory, immune and/or bacterial events since chronic neuropathy has been established, inevitably leading to the well-known leprosy sequelae occurring at any time before and/or after leprosy diagnosis.

Anti-inflammatory drugs block cytokine mRNA accumulation in the skin and improve the clinical condition of reactional leprosy patients.

The aim of this study was to investigate in what ways in vivo anti-inflammatory treatment affects cytokine mRNA expression in situ in both erythema nodosum leprosum and reversal reaction patients. Serial biopsies were collected from the patients undergoing leprosy reactions before and during pentoxifylline (n = 7) or thalidomide (n = 3) treatment for erythema nodosum leprosum and prednisone (n = 3) for reversal reaction. Clinical evolution of the skin lesion was assessed during the study and semiquantitative reverse transcription-polymerase chain reaction was used to investigate cytokine mRNA expression at the lesion site. Results showed expression of interferon-gamma, interleukin-6, interleukin-10, interleukin-12 p40, and tumor necrosis factor-alpha in all patients tested at the onset of reactional episodes, but interleukin-4 mRNA was rarely detected in the lesions (n = 4). Follow-up analysis showed that, irrespective of the drugs used, tumor necrosis factor-alpha mRNA was diminished in 10 of the 13 patients tested. A concomitant decrease of mRNA accumulation was also observed for interferon-gamma (nine of 11 patients), interleukin-6 (nine of 11), and interleukin-12 p40 (six of eight). An inhibitory effect on interleukin-10 mRNA was likewise seen after thalidomide and pentoxifylline, but not subsequent to prednisone treatment. The data also demonstrated that cytokine mRNA inhibition correlates to the resolution of the inflammatory response in situ (n = 10), whereas the persistence/enhancement of cytokine message expression after treatment was associated with worsening of the skin condition, as seen in three erythema nodosum leprosum patients whose maintenance of local inflammation was accompanied by the appearance/persistence of interleukin-4 gene expression in situ subsequent to anti-inflammatory treatment. In summary, the participation of cytokines in leprosy inflammatory episodes seems to be directly associated with the patients' clinical evolution following therapy for reaction.

Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells.

It has recently been demonstrated that laminin alpha2 chains present on the surface of Schwann cells are involved in the process of attachment of Mycobacterium leprae to these cells. In this study, a protein in the M. leprae cell wall that was found to be capable of binding alpha2-containing laminins (merosin) was isolated and characterized. The M. leprae laminin-binding protein was identified as a 21-kDa histone-like protein (Hlp), a highly conserved cationic protein present in other species of mycobacteria. The gene that encodes this protein was PCR amplified, cloned, and expressed, and the recombinant protein was shown to bind alpha2-laminins. More significantly, when added exogenously, Hlp was able to greatly enhance the attachment of mycobacteria to ST88-14 human Schwann cells. The capacity to bind alpha2-laminins and to enhance mycobacterial adherence to Schwann cells was also found in other cationic proteins such as host-derived histones. Moreover, mutation in the hlp gene was shown not to affect the capacity of mycobacteria to bind to ST88-14 cells, suggesting that alternative adhesins and/or pathways might be used by mycobacteria during the process of adherence to Schwann cells. The potential role of Hlp as a fortuitous virulence factor contributing to the pathogenesis of M. leprae-mediated nerve damage is discussed.

Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy.

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.

Binding of alpha2-laminins by pathogenic and non-pathogenic mycobacteria and adherence to Schwann cells.

The ability of Mycobacterium leprae to specifically bind alpha2-laminins of Schwann cells has been described recently as being an important property of the leprosy bacillus, which could explain the neural tropism of M. leprae. Therefore, the extent of the expression of alpha2-laminin-binding properties among mycobacteria was investigated. In an ELISA-based assay, all three species of Mycobacterium tested (M. tuberculosis, M. chelonae and M. smegmatis) expressed laminin-binding capacity, suggesting that the ability to bind alpha2-laminins is conserved within the genus Mycobacterium. This report also demonstrated that not only M. leprae but all the mycobacterial species tested readily interacted with the ST88-14 cells, a human schwannoma cell line, and that the addition of soluble alpha2-laminins significantly increased their adherence to these cells. These results failed to demonstrate the presence in M. leprae of a unique system based on alpha2-laminins for adherence to Schwann cells.

IgM anti-phenolic glycolipid I and IgG anti-10-kDa heat shock protein antibodies in sera and immune complexes isolated from leprosy patients with or without erythema nodosum leprosum and contacts.

The aim of the present work was to evaluate the levels of anti-PGL-I and anti-10-kDa heat shock protein antibodies in serum and immune complexes isolated from leprosy patients, convivients and controls. Leprosy patients with erythema nodosum leprosum or without it were included and a comparative study was done to investigate intergroup differences. Immune complexes were precipitated from serum by polyethylene glycol 3.5%; antibody levels were measured in sera and in dissociated immune complexes by ELISA. Serum antibody levels were then correlated with immune complex-associated antibody levels. The results showed that the erythema nodosum leprosum group differed from controls, contacts and non-erythema patients in their immune complex levels. IgM anti-PGL-I and IgG anti-10-kDa heat shock protein antibodies were constituents of the immune complexes in patients with erythema nodosum leprosum, who exhibited a significant difference in their immune complex composition compared with controls, contacts and non-erythema patients; while free antibody levels (anti-PGL-I and anti-10-kDa) did not differentiate between erythema and non-erythema patients, the measurement of immune complex-associated antibodies demonstrated a significant difference between the two clinical conditions. Furthermore, the measurement of immune complex-associated anti-PGL-I IgM made it possible to differentiate between contacts and controls. The significance of these results is discussed.

Management of erythema nodosum leprosum by thalidomide: thalidomide analogues inhibit M. leprae-induced TNFalpha production in vitro.

Thalidomide is being successfully used for the treatment of erythema nodosum leprosum (ENL), among other disorders with inflammatory and immunological bases. Although the active molecules responsible for the diverse therapeutic activities of the drug and the sequence of reactions triggered inside the cells remain unclear, it was demonstrated that thalidomide (THAL) inhibits TNFalpha mRNA expression and protein production by stimulated monocytes and activated T lymphocytes. Patients treated with THAL experienced a reduction in serum TNFalpha levels and it diminished cytokine gene expression at the lesion site, with a concomitant abrogation of clinical symptoms. It has been reported that thalidomide as well as some its analogues decrease M. leprae-induced TNFalpha and IL-12 mRNA in vitro. THAL also reduced monocyte apoptosis in the cultures. The present data further support thalidomide's effects on TNFa synthesis and the growing need to search for new specific TNFalpha inhibitors (non-teratogenic compounds) that might be potentially used in clinical disorders such as leprosy.