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Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that Ho somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.

Background: Cutaneous leishmaniasis (CL) is a serious public health problem in many tropical countries. The infection is caused by a protozoan parasite of Leishmania genus transmitted by Phlebotominae sandflies. In the present study, we constructed a eukaryotic expression vector to produce a fusion protein containing LmSTI1 from Leishmania major (L. major) and PpSP42 from Phlebotomus papatasi (Ph. papatasi). In future studies we will test this construct as a DNA vaccine against zoonotic CL. Methods: The nucleotide sequences encoding the LmSTI1 protein and a fragment encoding 79% of PpSP42 were amplified using L. major and Ph. papatasi genomic DNA, respectively. The amplicons were cloned into the pcDNA3.1(+) eukaryotic expression vector. The recombinant plasmid pcDNA-LmSTI1Pp42 was propagated in Escherichia coli (E. coli) and used to transfect HEK-293T cells. The expressed fusion protein was analyzed by Western blotting using anti-LmSTI1 mouse serum. Results: Sequences encoding LmSTI1 and partial PpSP42 were cloned into pcDNA3.1(+). Production of the recombinant LmSTI1Pp42 fusion protein was confirmed by Western blotting. Conclusions:An LmSTI1Pp42 fusion protein was expressed in HEK-293T cells. This construct may be an effective DNA vaccine against CL. [ABSTRACT FROM AUTHOR]

Objective(s): After decades of containment, pertussis disease, caused by Bordetella pertussis seems to be re-emerging and still remains a major cause of reported vaccine-preventable deaths worldwide. The current licensed whole-cell vaccines display reactogenicity while acellular vaccines are expensive and do not induce Th1-type immune responses that are required for optimum protection against the disease. Thus, there is an urgent need to develop new vaccines and the recombinant technology seems to be the method of choice for this purpose. The present study was an attempt to develop a new, simplified, cost-effective and well-defined vaccine against Bordetella pertussis, with capacity to induce a Th1 response. Materials and Methods: A fusion DNA fragment encoding the N-terminal region of pertussis toxin S1 subunit and filamentous hemagglutinin type 1 immunodominant domain was constructed and the corresponding fusion protein (F1S1) was produced in Escherichia coli. F1S1 in conjunction with imiquimod was administered by subcutaneous (SC) and intranasal (IN) routes to BALB/c mice. Results: This vaccine formulation could elicit high levels of IFN-γ, serum IgG (with higher IgG2a/IgG1 ratio) and lung IgA after the SC and, to a lesser extent, following the IN administration. Conclusion: Our results indicate that the above-mentioned important proteins of B. pertussis could be successfully produced in E. coli as a single fusion protein. Furthermore, this protein could induce proper systemic and mucosal immune responses after administration via SC or IN routes. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model.Results: Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05).Conclusions: Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies showed that P2 receptors are involved in neutrophil migration via stimulation of chemokine release and by facilitating chemoattractant gradient sensing. Here, we have investigated whether these receptors are involved in LPS-induced neutrophil transendothelial migration (TEM) using a Boyden chamber where neutrophils migrated through a layer of lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs). In line with a role of P2 receptors, neutrophil TEM was inhibited by the P2 receptor antagonists suramin and reactive blue 2 (RB-2) acting on the basolateral, but not luminal, HUVECs’ P2 receptors. HUVECs express P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11. The involvement of P2Y4 was unlikely as this receptor is insensitive to suramin while P2Y1, P2Y6 and P2Y11 were excluded with available selective antagonists, leaving P2Y2 as the only candidate. Indeed, the P2Y2 knockdown in HUVECs inhibited neutrophil TEM compared to control HUVECs transfected with scrambled siRNA. Moreover, UTP, a P2Y2 ligand, markedly potentiated LPS-induced TEM. Interestingly, IL-8 and ICAM-1 had a modest effect on neutrophil TEM in this 3h assay which was significantly diminished by the inhibition of Rho kinase in HUVECs with Y27632. In summary, endothelial P2Y2 receptors control the early LPS-induced neutrophil TEM in vitro via Rho kinase activation. [Copyright &y& Elsevier]