Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80°C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Evaluation of diagnostic tests for human leptospirosis

The IgM-PK-ELISA, an enzyme-linked immunosorbent assay for immunoglobulin M employing a proteinase K-treated antigen, and the "Leptoteste-S" macroagglutination test were evaluated for use in a rapid serodiagnosis of human leptospirosis. The microscopic agglutination test (MAT) was used as reference. The three serological tests were applied to serum samples from patients with leptospirosis (N=89), typhoid fever (N=8), malaria (N=19), syphilis (N=20), hepatitis (N=16) and from clinically healthy donors (N=92). The overall results of the IgM-PK-ELISA and the "Leptoteste-S"are comparable to those of the MAT. However, both tests differed statistically from MAT in terms of the positivity of the acute-phase sera, with approximately 38 per cent of the patients with leptospirosis being identified earlier than when MAT was used. The IgM-PK-ELISA, with 89,9 per cent sensitivity and 97,4 per cent specificity, could be the test of choice for those laboratories which are equipped to perform ELISA. The "Leptoteste-S", with 89.9 per cent sensitivity and 94.8 per cent specificity, seems to be easier to perform and the most accessible to peripheral laboratories for rapid screening of human sera. Both techniques present the important characteristic of detecting early antibodies against leptospires, thus providing a diagnosis during the early stages of the disease.

Search for agglutinating antibodies to Leptospira and Leptospira and Leptonema in horses, Sao Paulo, Brazil

Soros de 922 equinos aparentemente sadios, mantidos na Fazenda do Instituto Butantan (Säo Roque, SP) para produçäo de soros hiperimunes, foram analisados quanto à presença de anticorpos para sorovares de Leptospira interrogans e para Leptonema illini, através da reaçäo de aglutinaçäo microscópica (MA). Entre os 807 (87,5 por cento) animais positivos, 659 (81,7 por cento) reagiram com mais de um sorovar, com títulos entre 1:100 e 1:6.400, havendo predomínio de títulos baixos (<= 1:400), 84 por cento dos soros positivos reagiram com representantes do sorogrupo Icterohaemorrhagiae e 79,2 por cento com Leptonema illini. Dos 23 sorovares utilizados, apenas o tarassovi näo reagiu