Placental protein 13 (PP-13): effects on cultured trophoblasts, and its detection in human body fluids in normal and pathological pregnancies.

Placental tissue protein 13 (PP-13), one of the 56 known placental proteins identified till today, was purified from placentas obtained from women at delivery, and used to evoke antibodies against it. The purified PP-13 was lysed to peptides, which were sequenced, leading to the full-length cDNA sequencing and its expression in Escherichia coli. Sequence analysis in databases showed homology to the galectin family. Of the various antibody preparations developed, a pair of monoclonal antibodies (MAbs) coupled to the recombinant PP-13 (PP-13-R) was used for the immunodetection of PP-13 in pregnant women's serum with the solid-phase ELISA format. With a dynamic range of 25-500 pg/mL with no background in non-pregnant women's serum and men's serum, the ELISA test was suitable for the detection of PP-13 in the 1st, 2nd, and 3rd trimesters. PP-13 levels slowly increase during pregnancy. In the 1st trimester, lower than normal PP-13 levels were found in fetal growth restriction (IUGR), preeclampsia (PE), and particularly in early PE (<34 weeks of gestation). In the 2nd and 3rd trimesters, higher than normal concentrations were found in PE, IUGR and in preterm delivery (PTD). Application of PP-13 to cultured trophoblasts elicited depolarization carried by calcium ions, followed by liberation of linoleic and arachidonic acids from the trophoblast membrane, and a subsequent elevation of prostacyclin and thromboxane. These effects were negligible when PP-13 derived from the placentas of patients with IUGR, PE or PTD was used. The results are discussed in view of the potential utilization of PP-13 for early serum screening to assess the risk to develop placental insufficiency, coupled to a differential analysis of the various pathologies by analyzing cultured trophoblasts.

Humoral immune response to immunocomplexed HIV envelope glycoprotein 120.

To further our understanding of the nature of HIV-1 immunogenicity, we injected mice with the virus envelope protein gp120 in different configurations: free, complexed with its receptor CD4, and as an immunocomplex with a monoclonal antibody directed against the V3 loop of the protein. Analyses of the polyclonal sera, as well as of monoclonal antibodies produced in each case, allowed us to conclude that the quality of the humoral immune response depended on the complexation state of the antigen. For the free gp120 and gp120-CD4 complex the responses were directed mainly toward conformational epitopes. However, gp120 immunocomplexed with anti-V3 loop Mab produced, in addition, numerous MAbs directed toward linear epitopes. Epitopes were mapped using immunoblots of gp120 cleaved with S. aureus V8 protease and a combinatorial epitope phage-display library. It was found that some of the linear epitopes had been previously identified as T cell epitopes. These results suggest that the immunocomplexed gp120 may be particularly well taken up by antigen-presenting cells, leading to the processing of the gp120 and the efficient presentation of T cell epitopes. Thus immunocomplexation should afford a means for enhancing the immunogenicity of gp120 and improving its presentation.

Binding of HIV-1 gp120 to an anti-V3 loop antibody reveals novel antigen-induced epitopes.

Antigen association to its corresponding binding site in the immunoglobulin molecule can elicit conformational rearrangements, generating novel epitopes termed metatopes. Such metatopes were characterized for the immunocomplex between the AIDS virus envelope protein, gp120, and M77, a mAb directed against the V3 loop. Five novel mAbs were described (GV1, GV3, GV7, GV8, and GV12). These mAbs were found to bind epitopes harbored in the M77 Fab fragment. Binding to the epitopes was shown to require the complexation of Fab with its antigen. The degree of this antigen requirement was found to be variable for the different mAbs and also for the state of IgG fragmentation. Binding of GV12 to its antigen increased the affinity of M77 for gp120. Moreover, in the presence of GV12, M77 acquired extended cross-reactivity for a second gp120 variant, namely BaL. These results could indicate a novel approach towards improving the performance of anti-HIV antibodies.

Degradation of native human hemoglobin following hemolysis by Prevotella loescheii.

Prevotella loescheii PK1295 can grow on native hemoglobin as a source of heme. Supernatants of P. loescheii cultures hemolysed human erythrocytes and degraded native hemoglobin. These combined activities may provide heme (or iron) for the growth of P. loescheii and other dental plaque bacteria.