Palsy of the rear limbs in Mycobacterium lepraemurium-infected mice results from bone damage and not from nerve involvement.

A small but relatively constant proportion (3-5%) of mice chronically infected with Mycobacterium lepraemurium (MLM) develops bilateral paralysis of the rear limbs. The aim of the study was to investigate whether or not the bilateral leg palsy results from nerve involvement. Direct bacterial nerve infection or acute/delayed inflammation might possibly affect the nerves. Therefore, palsied animals were investigated for the presence of: (a) histopathological changes in the leg tissues including nerves, bones and annexes, and (b) serum antibodies to M. lepraemurium and M. leprae lipids, including phenolic glycolipid I from M. leprae. Histopathological study of the palsied legs revealed that the paralysis was not the result of direct involvement of the limb nerves, as neither bacilli nor inflammatory cells were observed in the nerve branches studied. Antibodies to brain lipids and cardiolipin were not detected in the serum of the palsied animals, thus ruling out an immune response to self-lipids as the basis for the paralysis. Although high levels of antibodies to MLM lipids were detected in the serum of palsied animals they were not related to limb paralysis, as the nerves of the palsied legs showed no evidence of inflammatory damage. In fact, nerves showed no evidence of damage. Paralysis resulted from severe damage of the leg bones. Within the bones the bone marrow became replaced by extended bacilli-laden granulomas that frequently eroded the bone wall, altering the normal architecture of the bone and its annexes, namely muscle, tendons and connective tissue. Although this study rules out definitively the infectious or inflammatory damage of nerves in murine leprosy, it opens a new avenue of research into the factors that participate in the involvement or the sparing of nerves in human and murine leprosy, respectively.

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.

Antibodies to diverse lipids in the serum of patients with clinically cured leprosy and tuberculosis.

In this study we looked for the presence of antibodies to cardiolipin, cerebrosides, and whole lipids extracted from M. leprae, M. tuberculosis and M. habana, in the serum of patients with clinically cured lepromatous leprosy (sixteen) or tuberculosis (sixteen), 8 to 12 months after arresting the corresponding multi-drug therapy (MDT). Compared to healthy controls (sixteen), both leprosy and tuberculosis ex-patients had still significant levels of antibodies to the three mycobacterial lipids but no detectable levels of antibodies to cardiolipin or cerebroside lipids. Although leprosy and tuberculosis sera recognized the homologous mycobacterial lipids in a preferential fashion, all of them, on the average, reacted more strongly with the lipids of M. habana. This observation backs up, in a certain way, the proposition of using M. habana as a prospective vaccine for leprosy and tuberculosis.

Anticardiolipin antibodies in patients with infectious diseases.

IgG or IgM anticardiolipin antibodies were present in the sera of 67% of 33 patients with Hansen's disease, in 53% of 30 patients with tuberculosis and in 50% of 16 patients with endocarditis. Despite the high frequency of these antibodies, no patient had a history of thrombosis or abortion. Anti-denatured DNA antibodies were tested in patients with tuberculosis and patients with Hansen's disease. Only in the latter group did we observe a statistically significant association between anticardiolipin and anti-denatured DNA antibodies. Anticardiolipin binding activity, however, could not be inhibited by preincubation of sera with a variable concentration of denatured DNA. These data suggest that: a) Anticardiolipin antibodies in infectious diseases do not necessarily participate in the pathogenesis of thrombotic or obstetric complications; b) Anti-denatured DNA and anticardiolipin antibodies in the population studied do not have a cross-reaction.

Evaluation of anti-cardiolipin antibody and its cross-reactivity in sera of patients with lepromatous leprosy.

Using a sensitive and modified solid-phase radioimmunoassay for detecting anti-cardiolipin antibodies, sera of 45 patients with lepromatous leprosy were examined. Nine of the 45 (20%) showed positive levels of anti-cardiolipin antibodies. Inhibition tests revealed that these antibodies significantly cross-reacted with double-stranded (ds) DNA, but not with single-stranded (ss) DNA or extractable nuclear antigens (ENA). We describe the unique pattern of antibody cross-reactivity with cardiolipin and dsDNA in sera of patients with lepromatous leprosy.

Immune complexes in syphilis sera.

Four hundred and forty-nine syphilis sera, positive in VDRL and FTA-ABS tests, were studied for the presence of circulating immune complexes (CIC) by means of anti-antibody (AA) neutralization test. Ninety-three (20.7%) sera had demonstrable CIC. Subsequently, dispersion of CIC by saline suspension of cardiolipin and by extract of rabbit testicular syphiloma was examined. Of 40 CIC-containing sera tested with cardiolipin, dispersion of CIC was noted in 16 instances; of 32 CIC-containing sera examined with syphiloma extract, dispersion was noted in 16 cases. Significantly, CIC that showed definite dispersion by cardiolipin were not dispersed by syphiloma extract and vice versa. Some studies were also performed on lepromatous leprosy and SLE. None of 27 cIC-containing lepromatous leprosy sera were affected by cardiolipin, but in 2 of 9 CIC-containing SLE sera, cardiolipin dispersed the complexes. Cardiolipin-anti-cardiolipin complexes that neutralized AA were frequently observed when cardiolipin was added to sera containing its corresponding antibodies but not CIC. Similar reactions were noted between the syphiloma extract and anti-treponemal antibodies.

Serological and immunohistological studies on lepromatous leprosy.

Sera of patients with lepromatous leprosy were studied for the presence of a variety of antibodies and immune complexes (IC). The frequencies of heterophile, Hanganutziu-Deicher and Forssman antibodies were 61 and 43%, respectively, which were significantly higher than those in other diseases. The frequency of antibodies to cardiolipin was 89% and the frequency of rheumatoid factor was 34%. Circulating IC were demonstrated in 54% of the patients' sera by Raji-cell test and in 43% by anti-antibody inhibition test. Analyses of immunoglobulin classes of IC revealed that IgG was predominant in IC of patients with lepra reaction (LR) and IgM in patients without LR. Immune deposits were found in and between cells of dermis in skin biopsy specimens of patients with LR.

ELISA tests for antibodies against mycobacterial glycolipids.

ELISA tests with purified mycobacterial glycolipids and bovine heart cardiolipin are described. The possible clinical use of ELISA tests with mycobacterial glycolipids for the diagnosis of tuberculosis and other mycobacterioses is discussed.

Reações de microfloculação com cardiolipina e sitolipina em soros de leprosos / Reactions with cardiolipin and micro flocculating sitolipina in sera of lepers

Os autores examinaram 152 soros de leprosos em forma lepromatosa, empregando reação de microfloculação com cardiolipina e sitolipina. Os resultados foram concordantesm em apenas 65%; tiveram pequena discordância (entre positivo ou negativo para duvidoso) 27% e discordância total (negativo para positivo) em 8%. Com a cardiopilina obtiveram: 27% de reações positivas; 19% de reações duvidosas e 54% de reações negativas; com sitolipina: reações positivas 31,6%, reações duvidosas 18,4% e negativas 50%. Os mesmos soros examinados previamente pela técnica quantitativa de fixação de complemento a cardiolipina, forneceram aproximadamente 5% de reações positivas. Os autores concluem que as reações positivas conhecidas de longa data na forma lepromtosa da lepra, com os ant¡genos de lipoides usados em sôro diagnóstico da s¡filis, também ocorrem com os ant¡genos usados em sôro diagnósticoda s¡filis, também ocorrem com os ant¡genos de fosfolip¡ideos puirificados em uso atualmente. Atribuem a positividade menos a falta de especificidade dos mesmos do que à técnica utilizada nas reações.