0 μg). The results revealed that both ligands compete for the binding with Lsa33 as a decrease of 40% in the binding was already detected with 0.25 μg of laminin (*, P < 0.05) (Figure 7C). These experiments were performed in triplicate and Figure 7 shows one representative data of two independent experiments. Figure 7 Inhibition of L. interrogans attachment to immobilized laminin and PLG by recombinant proteins; The effect of laminin concentration on the binding BMS202 cell line of PLG to Lsa33. (A) Laminin or PLG (1 μg/well) was adsorbed onto microtiter plates followed by incubation with increasing concentrations

of Lsa33 (0 to 10 μg) and in (B) laminin was adsorbed onto microtiter plates followed by incubation with increasing concentrations of Lsa25 (0 to 10 μg). In (A) and (B) the incubations were allowed to proceed for 90 min at 37°C. Live leptospires (100 μl/well of 4 X 107 L. interrogans serovar Copenhageni strain M20 leptospires) were added and incubated for another 90 min at 37°C. The unbound leptospires were washed away, and the quantification of bound leptospires Poziotinib chemical structure was performed indirectly by anti – LipL32 AZD3965 supplier antibodies produced in mice (1: 4,000 dilution) followed by horseradish peroxidase

– conjugated antimouse IgG antibodies. Each point represents the mean absorbance value at 492 nm ± standard deviation of three replicates. Data are representative of two independent experiments (*P < 0.05). (C) The effect of laminin on the binding of PLG (10 μg/ml) to immobilized rLIC11834 (10 μg/ml) was assessed with the addition of increasing concentrations of laminin (0 to 1.0 μg). The detection of rLIC11834-bound PLG was performed by use of specific antibodies anti - PLG. Bars represent the mean absorbance values ± standard deviation of four replicates for each condition and are representative MRIP of two independent experiments. Results of statistically significant interference on the binding in comparison with the control (no addition of laminin) are shown: *P < 0.05. Discussion Complement is a key component of the innate immune

system responsible for protection against pathogenic microorganisms [33]. Factor H is a host fluid – phase regulator of the alternative complement pathway. Pathogenic leptospiral complement – resistant strains were found to bind factor H from human serum and this interaction seems to be associated to their serum resistance [31, 34]. C4b – binding protein is an inhibitor of complement classical pathway system. This protein controls the complement classical pathway by interfering with the formation and regeneration of C3 convertase and acting as a cofactor to the serine proteinase factor I in the proteolytic inactivation of C4b [33, 35]. It has been shown that pathogenic leptospiral strains can obtain C4bp from the host and that this acquisition preserves its cofactor activity [36].