Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India.

BACKGROUND: Post Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required. OBJECTIVE: Previously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis. METHODS/PRINCIPAL FINDING: In the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015-16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility. CONCLUSION: These results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.

Association of a new FCN3 haplotype with high ficolin-3 levels in leprosy.

Leprosy is a chronic inflammatory disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nervous system, leading to a high disability rate and social stigma. Previous studies have shown a contribution of genes encoding products of the lectin pathway of complement in the modulation of the susceptibility to leprosy; however, the ficolin-3/FCN3 gene impact on leprosy is currently unknown. The aim of the present study was to investigate if FCN3 polymorphisms (rs532781899: g.1637delC, rs28362807: g.3524_3532insTATTTGGCC and rs4494157: g.4473C>A) and ficolin-3 serum levels play a role in the susceptibility to leprosy. We genotyped up to 190 leprosy patients (being 114 (60%) lepromatous), and up to 245 controls with sequence-specific PCR. We also measured protein levels using ELISA in 61 leprosy and 73 controls. FCN3 polymorphisms were not associated with disease, but ficolin-3 levels were higher in patients with FCN3 *2B1 (CinsA) haplotype (p = 0.032). Median concentration of ficolin-3 was higher in leprosy per se (26034 ng/mL, p = 0.005) and lepromatous patients (28295 ng/mL, p = 0.016) than controls (18231 ng/mL). In addition, high ficolin-3 levels (>33362 ng/mL) were more common in leprosy per se (34.4%) and in lepromatous patients (35.5%) than controls (19.2%; p = 0.045 and p = 0.047, respectively). Our results lead us to suggest that polymorphisms in the FCN3 gene cooperate to increase ficolin-3 concentration and that it might contribute to leprosy susceptibility by favoring M. leprae infection.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis.

Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR = 75, P < 0.05) and an association of IgG-containing ICs with VL (OR = 621, P = 0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P < 0.001). The assay further established a significant (P < 0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay.

Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 µM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 µM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells.

A modified synthesis and serological evaluation of neoglycoproteins containing the natural disaccharide of PGL-I from Mycobacterium leprae.

In order to generate substantial amounts of neoglycoconjugate needed for commercialization of diagnostic kits and high-throughput detection of leprosy, we developed a facile and high-yield synthesis of the corresponding disaccharide. Herein, the non-reducing disaccharide segment of phenolic glycolipid I from Mycobacterium leprae, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1-->4)-O-2,3-di-O-methyl-alpha-L-rhamnopyranose was synthesized by an improved procedure. The disaccharide was efficiently conjugated to bovine/human serum albumin, via acyl-azide intermediate, to form natural disaccharide-BSA/HSA neoglycoproteins that showed a high activity in serodiagnosis of leprosy. The disaccharide incorporated into the proteins was accurately measured by MALDI-TOF mass spectrometry. The serological activities of the neoglycoproteins against pooled human lepromatous leprosy sera were measured by ELISA and they were detectable at picogram amounts.

[The SLAM-associated protein (SAP) regulates IFN-gamma expression in leprosy]. / La proteína asociada a SLAM (SAP) regula la expresión de IFN-gamma en lepra.

The SLAM-associated protein (SAP) regulates IFN-gamma expression in leprosy. Tuberculoid leprosy patients locally produce Th1 cytokines, while lepromatous patients produce Th2 cytokines. Signaling lymphocytic activation molecule (SLAM) and the SLAM-associated protein (SAP) participate in the differentiation process that leads to the production of specific patterns of cytokines by activated T cells. To investigate the SLAM/SAP pathway in M. leprae infection, we determined the expression of SAP, IFN-gamma and SLAM RNA messenger in leprosy patients. We found a direct correlation of SLAM expression with IFN-gamma expression, whereas the expression of SAP was inversely correlated with the expression of both SLAM and IFN-gamma. Therefore, our data indicate that SAP might interfere with Th1 cytokine responses while SLAM expression may contribute to Th1 responses in leprosy. This study further suggests that the SLAM/SAP pathway might be a focal point for therapeutic modulation of T cell cytokine responses in diseases characterized by dysfunctional Th2 responses.

An adapted ELISA method for differentiating pathogenic from nonpathogenic aPL by a beta 2 glycoprotein I dependency anticardiolipin assay.

With the currently available commercial kits, as well as homemade assays for detecting anticardiolipin antibodies (aCL), it is not possible to discriminate nonpathogenic, beta 2 glycoprotein (GPI)-independent, infection-related antibodies from those of patients with the true autoimmune thrombotic syndrome, known as antiphospholipid syndrome (APS). We devised an assay that is able to differentiate these two types of antibodies by determining the beta 2 GPI requirements to bind in a cardiolipin ELISA. Beta 2 GPI was purified by perchloric acid precipitation, and fixed amounts were used in the dilution solutions of the tested samples that were also tested with no source of beta 2 GPI. The ELISA plates were coated with cardiolipin, as usual, and blocked with a chicken ovalbumin solution. The serum samples had to be highly diluted in order not to have beta 2 GPI from the patient serum. The reaction was detected with alkaline phosphate tablets and developed with pNp in diethanolamine buffer. The adapted ELISA aCL assay described here was able to discriminate infectious [syphilis, hepatitis C virus (HCV), dengue fever, human immunodeficiency virus (HIV) and leprosy] and autoimmune [primary APS and systemic lupus erythematosus (SLE) related APS]. Further testing should be performed to demonstrate that this method consistently differentiates pathogenic antibodies that bind in an aCL ELISA only in the presence of beta 2 GPI.

Activation of signaling lymphocytic activation molecule triggers a signaling cascade that enhances Th1 responses in human intracellular infection.

T cell production of IFN-gamma contributes to host defense against infection by intracellular pathogens, including mycobacteria. Lepromatous leprosy, the disseminated form of infection caused by Mycobacterium leprae, is characterized by loss of cellular response against the pathogen and diminished Th1 cytokine production. Relieving bacterial burden in Ag-unresponsive patients might be achieved through alternative receptors that stimulate IFN-gamma production. We have previously shown that ligation of signaling lymphocytic activation molecule (SLAM) enhances IFN-gamma in mycobacterial infection; therefore, we investigated molecular pathways leading from SLAM activation to IFN-gamma production in human leprosy. The expression of the SLAM-associated protein (an inhibitory factor for IFN-gamma induction) on M. leprae-stimulated cells from leprosy patients was inversely correlated to IFN-gamma production. However, SLAM ligation or exposure of cells from lepromatous patients to a proinflammatory microenvironment down-regulated SLAM-associated protein expression. Moreover, SLAM activation induced a sequence of signaling proteins, including activation of the NF-kappaB complex, phosphorylation of Stat1, and induction of T-bet expression, resulting in the promotion of IFN-gamma production, a pathway that remains quiescent in response to Ag in lepromatous patients. Therefore, our findings reveal a cascade of molecular events during signaling through SLAM in leprosy that cooperate to induce IFN-gamma production and strongly suggest that SLAM might be a focal point for therapeutic modulation of T cell cytokine responses in diseases characterized by dysfunctional Th2 responses.

La proteina asociada a SLAM (SAP) regula la expresion de IFN-gamma en lepra / The SLAM-associated protein (SAP) regulates IFN-gamma expression in leprosy

La inmunidad protectora contra Mycobacterium leprae requiere IFN-gamma. Los pacientes con lepra tuberculoide producen localmente citoquinas Th1, mientras que los pacientes lepromatosos producen citoquinas Th2. La molécula linfocitaria activadora de señales (SLAM) y la proteína aociada a SLAM (SAP) participan en la diferenciación celular que conduce a producción de patrones específicos de citoquinas. A fin de investigar la vía SLAM/SAP en la infección por M. leprae, determinamos expresión de ARN mensajero (ARNm) de SAP, IFN-gamma y SLAM en pacientes con lepra. Obeservamos que la expresión de SLAM correlacionó en forma directa con la expresión de IFN-gamma, mientras que la expresión de SAP interferiría con las respuetas de citoquinas Th1 mientras que SLAM contribuiría con la respuesta Th1 en lepra, señalando a la vía SLAM/SAP como potencial blanco modulador de citoquinas en enfermedades con respuestas Th2 disfuncionales.

La proteina asociada a SLAM (SAP) regula la expresion de IFN-gamma en lepra / The SLAM-associated protein (SAP) regulates IFN-gamma expression in leprosy

La inmunidad protectora contra Mycobacterium leprae requiere IFN-gamma. Los pacientes con lepra tuberculoide producen localmente citoquinas Th1, mientras que los pacientes lepromatosos producen citoquinas Th2. La molécula linfocitaria activadora de señales (SLAM) y la proteína aociada a SLAM (SAP) participan en la diferenciación celular que conduce a producción de patrones específicos de citoquinas. A fin de investigar la vía SLAM/SAP en la infección por M. leprae, determinamos expresión de ARN mensajero (ARNm) de SAP, IFN-gamma y SLAM en pacientes con lepra. Obeservamos que la expresión de SLAM correlacionó en forma directa con la expresión de IFN-gamma, mientras que la expresión de SAP interferiría con las respuetas de citoquinas Th1 mientras que SLAM contribuiría con la respuesta Th1 en lepra, señalando a la vía SLAM/SAP como potencial blanco modulador de citoquinas en enfermedades con respuestas Th2 disfuncionales. (AU)

The origin of dystrophin-glycoprotein complex(DGC)-related muscular dystrophies: the need for protection against an ancestral pathogen?

Because of its crucial role during the early stages of morphogenesis, no genetic defects associated to dystroglycan have been reported so far. Dystroglycan is an important member of the dystrophin-glycoprotein complex (DGC) and in several muscular dystrophies, depending on abnormalities of proteins belonging to or associated with the DGC, it is frequently observed a significant reduction of dystroglycan levels at the sarcolemma. Recently, it has been demonstrated that dystroglycan acts as a receptor for pathogens such as M. leprae and arenaviruses. It is well-known that mutated alleles causing diseases can be selected in order to confer an additional genetic advantage. Herein it is discussed the possibility that mutations leading to a certain number of muscular dystrophies might have been originally selected to indirectly gain a specific advantage: the absence or the lower levels of dystroglycan could have greatly reduced the risk of some ancestral lethal infections specifically directed against muscles.

Anticardiolipin, anti-beta(2)-glycoprotein I and antiprothrombin antibodies in black South African patients with infectious disease.

OBJECTIVES: To investigate IgG, IgM, and IgA, antiphospholipid antibodies (aPL), against cardiolipin (aCL), beta(2)-glycoprotein I (anti-beta(2)GPI), and prothrombin (anti-PT), in black South African patients with infectious disease. Unlike patients with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS), raised levels of aPL in infectious diseases are not usually associated with thrombotic complications. PATIENTS AND METHODS: Serum samples from 272 patients with a variety of infectious diseases (100 HIV positive, 112 leprosy, 25 syphilis, 25 malaria, and 10 HCV patients) were studied and compared with autoantibody levels in 100 normal controls. All three aPL were measured using commercial enzyme linked immunosorbent assay (ELISA) kits. RESULTS: Raised levels of all three aPL were found in all patient groups studied: aCL in 7%, anti-beta(2)GPI in 6%, and aPT in 43% of 100 HIV patients, in 29%, 89%, and 21% of 112 patients with leprosy, in 8%, 8%, and 28% of 25 patients with syphilis, in 12%, 8%, and 28% of 25 patients with malaria, and in 20%, 30%, and 30% of 10 HCV patients studied, respectively. CONCLUSIONS: The prevalence of aCL and anti-beta(2)GPI in black South African HIV positive patients, or those with syphilis, malaria, or hepatitis C virus is lower than reported for mixed race or white populations. aPT were the most prevalent aPL detected in these patient groups, except in patients with leprosy, for whom anti-beta(2)GPI was the most prevalent, and where the spectrum of aPL was similar to that seen in patients with SLE and APS.

Anticardiolipin, anti-beta(2)-glycoproteiun I and antiprothrombin antibodies in black South African patients with infectious disease

OBJECTIVES: To investigate IgG, IgM, and IgA, antiphospholipid antibodies (aPL), against cardiolipin (aCL), beta(2)-glycoprotein I (anti-beta(2)GPI), and prothrombin (anti-PT), in black South African patients with infectious disease. Unlike patients with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS), raised levels of aPL in infectious diseases are not usually associated with thrombotic complications. PATIENTS AND METHODS: Serum samples from 272 patients with a variety of infectious diseases (100 HIV positive, 112 leprosy, 25 syphilis, 25 malaria, and 10 HCV patients) were studied and compared with autoantibody levels in 100 normal controls. All three aPL were measured using commercial enzyme linked immunosorbent assay (ELISA) kits. RESULTS: Raised levels of all three aPL were found in all patient groups studied: aCL in 7%, anti-beta(2)GPI in 6%, and aPT in 43% of 100 HIV patients, in 29%, 89%, and 21% of 112 patients with leprosy, in 8%, 8%, and 28% of 25 patients with syphilis, in 12%, 8%, and 28% of 25 patients with malaria, and in 20%, 30%, and 30% of 10 HCV patients studied, respectively. CONCLUSIONS: The prevalence of aCL and anti-beta(2)GPI in black South African HIV positive patients, or those with syphilis, malaria, or hepatitis C virus is lower than reported for mixed race or white populations. aPT were the most prevalent aPL detected in these patient groups, except in patients with leprosy, for whom anti-beta(2)GPI was the most prevalent, and where the spectrum of aPL was similar to that seen in patients with SLE and APS.

Markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with antiphospholipid antibodies.

OBJECTIVE: To evaluate plasma levels of markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with or without antiphospholipid antibodies (aPL) and to compare them to those found in patients with antiphospholipid syndrome (APS). METHODS: 42 patients with leprosy (35 lepromatous and 7 borderline): 29 aPL(+) and 13 aPL(-), as well as 26 healthy subjects as normal controls (NC) and 79 control aPL patients without leprosy (59 with and 20 without APS) were included in the study. Plasma soluble P and E selectin (sPsel and sEsel), and VCAM-1 (sVCAM-1), prothrombin F1 + 2 fragment (F1 + 2), thrombin-antithrombin complexes (TAT) and D dimer (DD) were measured by ELISA. The protein C pathway was assessed by the ProC global test. RESULTS: Leprosy patients with aPL presented increased median levels of sPsel [ng/ml (82.0 vs 36.0, p < 0.001)] and sVCAM-1 [ng/ml (495 vs 335, p < 0.001)] compared to NC, as observed in control aPL patients without leprosy. Levels of sPsel in aPL(+) patients with leprosy were significantly higher than in aPL(-) ones (52.5 ng/ml), p = 0.005. However, plasma markers of thrombin generation were increased in control aPL patients without leprosy but not in those with leprosy. ProcC global test was abnormal in 24.1% of leprosy patients with aPL compared to 4.4% of NC (p < 0.024), and to 57.2% of control patients with aPL without leprosy (p = 0.005). CONCLUSIONS: We demonstrated that although patients with leprosy present a high prevalence of aPL, and platelet and endothelial cell activation in vivo to the same extent than patients with APS, they do not show a procoagulant state.

Distinguishing features of anti-beta2 glycoprotein I antibodies between patients with leprosy and the antiphospholipid syndrome.

Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.

Markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with antiphospholipid antibodies

OBJECTIVE: To evaluate plasma levels of markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with or without antiphospholipid antibodies (aPL) and to compare them to those found in patients with antiphospholipid syndrome (APS). METHODS: 42 patients with leprosy (35 lepromatous and 7 borderline): 29 aPL(+) and 13 aPL(-), as well as 26 healthy subjects as normal controls (NC) and 79 control aPL patients without leprosy (59 with and 20 without APS) were included in the study. Plasma soluble P and E selectin (sPsel and sEsel), and VCAM-1 (sVCAM-1), prothrombin F1 + 2 fragment (F1 + 2), thrombin-antithrombin complexes (TAT) and D dimer (DD) were measured by ELISA. The protein C pathway was assessed by the ProC global test. RESULTS: Leprosy patients with aPL presented increased median levels of sPsel [ng/ml (82.0 vs 36.0, p smaller 0.001)] and sVCAM-1 [ng/ml (495 vs 335, p smaller 0.001)] compared to NC, as observed in control aPL patients without leprosy. Levels of sPsel in aPL(+) patients with leprosy were significantly higher than in aPL(-) ones (52.5 ng/ml), p = 0.005. However, plasma markers of thrombin generation were increased in control aPL patients without leprosy but not in those with leprosy. ProcC global test was abnormal in 24.1 per cent of leprosy patients with aPL compared to 4.4 per cent of NC (p smaller 0.024), and to 57.2 per cent of control patients with aPL without leprosy (p = 0.005). CONCLUSIONS: We demonstrated that although patients with leprosy present a high prevalence of aPL, and platelet and endothelial cell activation in vivo to the same extent than patients with APS, they do not show a procoagulant state.

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.

High prevalence of antiphospholipid antibodies in leprosy: evaluation of antigen reactivity.

Antiphospholipid antibodies (aPL) have been reported not only in autoimmune disorders but also in various infectious diseases. Accumulating evidence indicates that beta2 glycoprotein I (beta2GPI) and prothrombin are the main proteins to which autoimmune aPL bind. The aim of this study was to evaluate the prevalence of different aPL in patients with leprosy. We included 51 outpatients (42 lepromatous and 9 borderline leprosy) without any clinical feature of the antiphospholipid syndrome (APS). 35 had lupus anticoagulant and 31 had anticardiolipin antibodies (aCL). Anti-beta2GPI antibodies were highly positive in 29/51 and anti- prothrombin antibodies (anti-II) were detected in 23/51. Almost all aCL and anti-beta2GPI were of IgM isotype, while IgG isotype was more frequent among anti-II. No statistical difference was found when aPL were evaluated in patients grouped according to their bacteriological status. Furthermore, patients under treatment (n=33) had a similar frequency of positive aPL compared to patients in vigilance (n=14). Assessing the specificity of antibody binding to CL and beta2GPI in ELISA by means of inhibition studies with cardiolipin-beta2GPI liposomes, leprosy and APS sera showed a similar behaviour. Comparable results were also found in both groups of patients when inhibition experiments with lysate of Mycobacterium leprae were carried out. In summary, leprosy-related aPL resemble those found in patients with APS but the immunoglobulin isotype is different, with IgM much more prevalent in leprosy patients.