3(S)-hydroxy-leukotriene B4 is a potent granulocyte chemotaxin in the guinea pig dermis

Inflammation Research -     

Identification of amino acid residues of anthrax protective antigen involved in binding with lethal factor

Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for the translocation of LF to the cytosol. The binding of LF to cell surface receptor-bound PA is a prerequisite for the formation of lethal toxin. It has been hypothesized that hydrophobic residues P184, L187, F202, L203, P205, I207, I210, W226, and F236 of domain 1b of PA play an important role in the binding of PA to LF. These residues are normally buried in the 83-kDA version of PA, PA83, as determined by the crystal structure of PA. However, they become exposed due to the conformational change brought about by the cleavage of PA83 to PA63 by a cell surface protease. Mutation of the above-mentioned residues to alanine resulted in mutant proteins that were able to bind to the cell surface receptors and also to be specifically cleaved by the cellular proteases. All the mutant proteins except the F202A, L203A, P205A, and I207A mutants were able to bind to LF and were also toxic to macrophage cells in combination with LF. It was concluded that residues 202, 203, 205, and 207 of PA are essential for the binding of LF to PA.     

A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.     

Direct detection and identification of Mycobacterium tuberculosis and Mycobacterium bovis in bovine samples by a novel nested PCR assay: correlation with conventional techniques

Mycobacterium tuberculosis and M. bovis infect animals and humans. Their epidemiologies in developed and developing countries differ, owing to differences in the implementation of preventive measures (World Health Organization, 1999). Identification and differentiation of these closely related mycobacterial species would help to determine the source, reservoirs of infection, and disease burden due to diverse mycobacterial pathogens. The utility of the hupB gene (Rv2986c in M.tuberculosis, or Mb3010c in M.bovis) to differentiate M. tuberculosis and M. bovis was evaluated by a PCR-restriction fragment length polymorphism (RFLP) assay with 56 characterized bovine isolates (S. Prabhakar et al., J. Clin. Microbiol. 42:2724-2732, 2004). The degree of concordance between the PCR-RFLP assay and the microbiological characterization was 99.0% (P < 0.001). A nested PCR (N-PCR) assay was developed, replacing the PCR-RFLP assay for direct detection of M. tuberculosis and M. bovis in bovine samples. The N-PCR products of M. tuberculosis and M. bovis corresponded to 116 and 89 bp, respectively. The detection limit of mycobacterial DNA by N-PCR was 50 fg, equivalent to five tubercle bacilli. M. tuberculosis and/or M. bovis was detected in 55.5% (105/189) of the samples by N-PCR, compared to 9.4% (18/189) by culture. The sensitivities of N-PCR and culture were 97.3 and 29.7, respectively, and their specificities were 22.2 and 77.7%, respectively. The percentages of animals or samples identified as infected with M.tuberculosis or M. bovis by N-PCR and culture reflected the clinical categorizations of the cattle (P of <0.05 to <0.01). Mixed infection by N-PCR was detected in 22 animals, whereas by culture mixed infection was detected in 1 animal.     

Immunosuppression due to chronic ochratoxicosis in broiler chicks

Crystalline ochratoxin A (OA) was added to the feed of broiler chicks at 0.5 ppm and 2.0 ppm, and humoral and cell-mediated immunity (CMI) were studied. CMI was assessed by skin sensitivity testing, graft versus host (GVH) reaction and T lymphocyte count. Humoral immunity was examined by measuring the haemagglutinin (HA) response to sheep RBC (SRBC). In addition, total lymphocyte counts, total serum protein, albumin and globulin were determined and the phagocytic activity of splenic macrophages was measured in the nitroblue tetrazolium test (NBT). The weights of lymphoid organs were also recorded at post-mortem examination of the birds. Highly significant reductions in CMI were indicated by diminished skin sensitivity, GVH reactions and T lymphocyte counts. On the other hand, only the overall HA titres differed significantly between the various treatment groups. Total lymphocyte counts, total serum protein, serum albumin and serum globulin were significantly depressed on the 21st day of intoxication. The number of NBT-positive cells was drastically reduced in both the intoxicated groups compared with controls (P less than 0.05) and the weights of thymus, bursa of Fabricius and spleen of intoxicated birds were significantly reduced. The study illustrated the immunosuppressive effects of ochratoxicosis in broiler chicks.