Fingerstick test quantifying humoral and cellular biomarkers indicative for M. leprae infection.

OBJECTIVES: New user-friendly diagnostic tests for detection of individuals infected by Mycobacterium leprae (M. leprae), the causative pathogen of leprosy, can help guide therapeutic and prophylactic treatment, thus positively contributing to clinical outcome and reduction of transmission. To facilitate point-of-care testing without the presence of phlebotomists, the use of fingerstick blood (FSB) rather than whole blood-derived serum is preferred. This study is a first proof-of-principle validating that previously described rapid serum tests detecting antibodies and cytokines can also be used with FSB. METHODS: Quantitative detection of previously identified biomarkers for leprosy and M. leprae infection, anti-M. leprae PGL-I IgM antibodies (αPGL-I), IP-10 and CRP, was performed with lateral flow (LF) strips utilizing luminescent up-converting reporter particles (UCP) and a portable reader generating unbiased read-outs. Precise amounts of FSB samples were collected using disposable heparinized capillaries. Biomarker levels in paired FSB and serum samples were determined using UCP-LF test strips for leprosy patients and controls in Bangladesh, Brazil, South-Africa and the Netherlands. RESULTS: Correlations between serum and FSB from the same individuals for αPGL-I, CRP and IP-10 were highly significant (p < .0001) even after FSB samples had been frozen. The αPGL-I FSB test was able to correctly identify all multibacillary leprosy patients presenting a good quantitative correlation with the bacterial index. CONCLUSIONS: Reader-assisted, quantitative UCP-LF tests for the detection of humoral and cellular biomarkers for M. leprae infection, are compatible with FSB. This allows near-patient testing for M. leprae infection and immunomonitoring of treatment without highly trained staff. On site availability of test-result concedes immediate initiation of appropriate counselling and treatment. Alternatively, the UCP-LF format allows frozen storage of FSB samples compatible with deferred testing in central laboratories.

DNAhsp65 Vaccine as Therapy against Paracoccidioidomycosis.

The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.

Determinação dos níveis séricos de MBL, dos anticorpos anti-PGL-I e dos alelos HLA de classe I e classe II em pacientes com hanseníase / Determination of MBL and anti-PGL-I serum levels and HLA ClassI and II in patients with leprosy

A hanseníase é uma doença infecto-contagiosa e endêmica, constitui grave problema de saúde pública por causar incapacidade física permanente. A doença caracteriza-se por apresentar um amplo espectro de manifestações clínicas que correspondem a distintos padrões de resposta imunológica diante do mesmo agente etiológico, o Mycobacterium leprae. Métodos sorológicos têm sido desenvolvidos com a finalidade de auxiliar no diagnóstico precoce e estados reacionais, no monitoramento da eficácia da quimioterapia e no estudo da epidemiologia da hanseníase. Este estudo teve como objetivos: avaliar os níveis séricos de Lectina Ligada a Manose (MBL) e dos anticorpos anti-PGL-I em poacientes com hanseníase, em atividades, atendidos no Instituto Lauro de Souza Lima, SP, com ou sem o tratamento de poliquimioterapia e nos estados reacionais tipo 1 e 2; correlacionar os resultados dos nívceis séricos de MBL, e dos anticorpos anti-PGL-I com o índice baciloscópico (IB); avaliar a freqüência dos alelos HLA de classe I e II (locus A, B, DR e DQ) neste grupo de pacientes. Para a avaliação do anticorpo anti-PGL-I e da MBL foram estudados 69 pacientes sendo 21 do gênero feminino e 48 do gênero masculino, com idade variando de 11 a 72 anos. Quanto a classificação clínica, 12 eram tuberculóides (HT), 23 vichovianos (HV) e 34 dimorfos [18 dimorfos-tuberculóides (HDT), 09 dimorfos-dimorfos (HDD) e 07 dimorfos-virchovianos (HDV)]. Entre os pacientes avaliados, 38 apresentaram reações, sendo 20 reação tipo 1 e 18 reação tipo 2. Quanto ao esquema de tratamento, 43 pacientes eram multibacilares (MB) e 26 paucibacilares (PB). Neste estudo foram avaliados marcadores labaratoriais séricos MBL e anti-PGL-I pelo método imunoenzimático (ELISA) e os antígenos de histocompatibilidade pelo método Reverse Line blot. Os resultados dos níveis de anti-PGL-I forma mais altos nos pacientes MB comparados aos PB e mostraram uma forte associação com a carga bacilar. Com relação à MBL, nossos achados sugerem...

Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages.

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

Characterization of langerhans cells in epidermal sheets along the body of Armadillo (Dasypus novemcinctus).

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.

Enhancement of the sensitivity of the whole-blood gamma interferon assay for diagnosis of Mycobacterium bovis infections in cattle.

In this study, we determined if the sensitivity of the currently available in vitro test to detect bovine tuberculosis could be enhanced by adding the following immunomodulators: interleukin-2 (IL-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); antibodies neutralizing IL-10 and transforming growth factor beta (TGF-beta); mono-methyl-l-arginine, which blocks nitric oxide production; and l-methyl-tryptophan, which interferes with the indoleamine dioxygenase pathway. Blood was obtained from uninfected control cattle, experimentally infected cattle, cattle responding positively to the skin test in tuberculosis-free areas (false positives), and cattle naturally infected with Mycobacterium bovis from New Zealand and Great Britain. Gamma interferon (IFN-gamma) responses to bovine purified protein derivative (PPD-b), avian purified protein derivative, and a fusion protein of ESAT-6 and CFP-10 were measured. Mono-methyl-l-arginine, l-methyl-tryptophan, or an antibody neutralizing TGF-beta had minimal impact on IFN-gamma production. IL-2 and GM-CSF promoted IFN-gamma release whether antigen was present or not. In contrast, adding an antibody against IL-10 enhanced only antigen-specific responses. In particular, addition of anti-IL-10 to ESAT-6/CFP-10-stimulated blood cultures enhanced the test sensitivity. Furthermore, whole blood cells from field reactors produced substantial amounts of IL-10 upon stimulation with PPD-b or ESAT-6/CFP-10. Testing "false-positive" cattle from tuberculosis-free areas of New Zealand revealed that addition of anti-IL-10 did not compromise the test specificity. Therefore, the use of ESAT-6/CFP-10 with anti-IL-10 could be useful to detect cattle potentially infected with tuberculosis, which are not detected using current procedures.

Lymphoproliferation and in vitro antibody synthesis in leprosy patients

An in vitro system to assess B-cell function in leprosy patients is described. In vitro lymphoproliferation and antibody synthesis by peripheral blood mononuclear cells (PBMC) in response to pokeweed mitogen (PWM) and Formalin-treated Staphylococcus aureus Cowan I (FSA) from 31 leprosy patients and 13 healthy controls were studied. DNA synthesis was induced by both PWM and FSA in PBMC from all of the leprosy patients and control subjects. Lepromatous leprosy (LL) patients' cells showed higher responses to both PWM and FSA. However, these increases were not statistically significant. The levels of secreted IgM, IgG, or IgA were examined in the 7-day culture supernatants of PBMC cultured with or without PWM or FSA using an enzyme-linked immunosorbent assay. Wide individual variations were observed in in vitro antibody synthesis. IgM secretion in PBMC from normal subjects and various groups of leprosy patients in response to PWM and FSA was comparable. In vitro IgG secretion in response to PWM was the highest in cells from LL patients; it was significantly decreased in cells from tuberculoid leprosy (TT) patients (p less than 0.01). The levels in cells from borderline leprosy (BB) patients were intermediate in response to the same mitogen. Cells from leprosy patients as a group showed a higher spontaneous secretion of IgA in comparison with cells from normal subjects. Overall, the in vitro Ig secretion by PBMC in different patient groups appears to reproduce the spectrum of antibody levels observed in patients in vivo. Thus, the present in vitro culture system may help to delineate the mechanisms of B-cell dysregulation in leprosy

A dot enzyme immunoassay for detection of IgM antibodies against phenolic glycolipid-I in sera from leprosy patients.

A visual dipstick dot enzyme immunoassay (EIA) for diagnosis of leprosy is described. The assay is based on detection of IgM antibodies against phenolic glycolipid (PGL-I) in sera from leprosy patients. The antigen (PGL-I or synthetic disaccharide of PGL-I) was dotted on a nitrocellulose pad stuck on a plastic strip (dipstick). Sera were used at a dilution of 1:200. Peroxidase coupled mouse anti-human IgM monoclonal antibodies were used as the conjugate. A positive test gave a blue dot against a white background. The test was highly specific for leprosy, and was quite sensitive for detection of bacilliferous (BL/LL) leprosy. The antigen dotted and preblocked dipsticks stored at room temperature upto 4 months of observation period, were unable in the assay.

Antispermatozoal antibodies in leprosy with special reference to their morphological patterns.

Sixty two male patients with polar leprosy--38 lepromatous and 24 tuberculoid types were investigated for the presence of antispermatozoal antibodies with special reference to their morphological patterns. Antibodies were detected by three different immunological techniques. Sperm agglutination was found to be the most sensitive. The incidence of antibodies was higher in patients with lepromatous leprosy and was directly proportional to the duration of the disease in both types of leprosy. Morphologically, head-to-head type of agglutination was observed in 50 percent of the patients, mixed in 41.7 percent and tail-to-tail type in 8.3 percent. There was no correlation between the number of ENL attacks and the incidence of anti-bodies. In polar tuberculoid leprosy patients the histological findings of testicular biopsy indicated cell mediated tissue damage occurring in a non-infective form.

Anti-hapten antibodies and autoantibodies in Japanese patients with lepromatous leprosy.

In the sera of patients with lepromatous leprosy anti-DNP antibodies were detected in order to determine the mode of polyclonal B-cell activation. Anti-DNP antibodies were found in 30% of the patients with active lepromatous leprosy and in 8% of those with inactive lepromatous leprosy. The level of anti-DNP antibodies in active patients was significantly higher than the level in inactive patients and control. However, the presence of anti-DNP antibodies was unrelated to the production of circulating immune complexes and antinuclear antibodies. These results suggest that polyclonal B-cell activation might occur but that the B-cell clones stimulated by M. leprae are different from patient to patient.

Suppression of the antibody response to sulfolipids in leprosy patients by 4,4'-diaminodiphenyl sulfone (DDS).

Antibodies to sulfolipids were demonstrated in patients suffering from lepromatous leprosy. The antibody titre was found to decrease gradually on treatment with DDS. This effect was maximum for patients undergoing treatment for more than 1 year.

Absence of antibodies to muramyl dipeptide in patients with tuberculosis or leprosy.

The enzyme-linked immunosorbent assay (ELisa) was used to detect the presence of antibodies to muramyl dipeptide (MDP) in serum of patients with leprosy or tuberculosis. Using a conjugate of MDP-lysine to horse radish peroxidase, no such antibodies could be detected in sera of either patients or controls. Antibodies to a sonicate antigen of Mycobacterium tuberculosis were found in sera of all individuals tested and the binding of these antibodies to the M. tuberculosis antigen could not be inhibited by MDP. On the other hand, binding of MDP to anti-MDP antibodies, raised in rabbits, was largely inhibited by free MDP, slightly inhibited by M. tuberculosis antigen and was not inhibited by the patients' sera.

Serologia en lepra: anticuerpos antilipoideos y antitreponómicos

Se ha practicado la Pálida reacción (la desviación del complemento empleando antígeno treponémico, derivado del Treponema de Reiter) en 20 sueros de enfermos leprosos con R.W. (Antígeno Bordet), positivos, y con Mainicke, Khan y Citocol, también positivas en parte o todas. Dos sueros resultaron positivos a la Pálida reacción, uno fue dudoso, todos los demás negativos. Se concluye que en todos estos sueros hay presencia de anticuerpos antilipoideos; pero en dos solamente hay presencia también de anticuerpos antitreponémico, y un, de modo dudoso.