Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 µM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.

Fine specificity of Plasmodium vivax Duffy binding protein binding engagement of the Duffy antigen on human erythrocytes.

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax.

Expression, purification, and characterization of the immunological response to a 40-kilodalton Plasmodium vivax tryptophan-rich antigen.

We describe here an approximately 40-kDa Plasmodium vivax tryptophan-rich antigen (PvTRAg40) which contains 321 amino acids and 11.4% tryptophan residues. This protein shows 65% homology (35% identity) with the previously described PvTRAg, besides sharing 23 of 27 positionally conserved tryptophan residues and similar genomic organization. The nucleotide sequence of the entire tryptophan-rich domain of PvTRAg40 was identical among 35 P. vivax clinical isolates. The protein is expressed by ring, trophozoite, and schizont stages of the parasite. The cDNA covering exon 2 of PvTRAg40 was cloned and expressed in the pPROEXHTa vector, and recombinant protein was purified. A high humoral immune response (90.7% seropositivity; n = 43) against this recombinant protein was detected in humans during the course of natural P. vivax infection. Eighty percent of the total of 20 P. vivax-exposed individuals exhibited lymphoproliferative responses against this antigen. The T cells of these individuals produced larger amounts of interleukin-12 (IL-12), IL-4, and IL-10 than gamma interferon and tumor necrosis factor alpha cytokines in response to the recombinant protein. Production of Th2-biased cytokines, conserved T- and B-cell epitopes, and an enhanced humoral immune response indicate that PvTRAg40 could possibly induce antibody-mediated immune protection against infection.

Plasmid DNA acquires immunogenicity on exposure to singlet oxygen.

In the present study, the effect of singlet oxygen (1O2) (generated by ultraviolet (UV) irradiation of methylene blue) on plasmid DNA has been analyzed by UV spectroscopy, fluorescence spectroscopy, and S1 nuclease digestibility. Both native and 1O2-modified plasmid DNA were treated with a number of restriction enzymes to map out the sites damaged by 1O2. It was also observed that, on exposure to 1O2, native plasmid DNA that is non-immunogenic acquired the ability to elicit an immune response in experimental animals. However, the induced antibodies exhibited appreciable cross reactivity with various polynucleotides and nucleic acids. The data indicate that the antibodies, though cross-reactive, preferentially bind 1O2-modified epitopes on plasmid DNA. Gel retardation assay further substantiated the enhanced recognition of 1O2-modified plasmid DNA over the native form. The antibodies developed were then subjected to competition ELISA with sera from various diseases such as systemic lupus erythematosus, rheumatoid arthritis, and cancer. These results suggest that upon exposure of DNA to 1O2, neo-epitopes are generated, which may be one of the factors for the induction of circulating autoantibodies in the three diseases.