Application of an improved method for the recombinant k 39 enzyme-linked immunosorbent assay to detect visceral leishmaniasis disease and infection in Bangladesh.

Several serology-based immunoassays are used to diagnose visceral leishmaniasis (VL), a chronic protozoan parasitic disease caused by the Leishmania donovani complex. These tests are primarily designed to diagnose the most severe clinical form of VL, known as kala-azar. However, leishmanial infection is frequently asymptomatic and may manifest only as a positive serologic response or positive leishmanin skin test. We modified a previously described enzyme-linked immunosorbent assay (ELISA) that detects patient antibodies reactive with the recombinant Leishmania protein K39 (rK39) to confirm suspected kala-azar and to detect asymptomatic infection in a community study in Bangladesh. With the inclusion of a standard curve on each ELISA plate, the rK39 ELISA was more repeatable (kappa coefficient of agreement=0.970) and more reliable compared to the original method (kappa=0.587, P<0.001). The cutoff point for a positive antibody response was chosen based on the 99th percentile of the ELISA distribution for the negative-control sera. However, we found that sera from all patients with active kala-azar yielded values more than twice the magnitude of this cutoff. Using receiver-operator characteristic curves, we determined a second cutoff value predictive of kala-azar. Using these criteria, the sensitivity and specificity of the modified ELISA for kala-azar were 97.0% and 98.9%, respectively, for sera from our study population. We hypothesize that individuals with antibody levels greater than the 99th percentile of the negative controls but less than the cutoff point for kala-azar have asymptomatic leishmanial infections.

Normal ligand binding and signaling by CD47 (integrin-associated protein) requires a long range disulfide bond between the extracellular and membrane-spanning domains.

CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys(33) and Cys(263). Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPalpha1 binding, and reduced CD47/SIRPalpha1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca(2+) responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with alpha(v)beta(3) integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.