Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo

CD8+ T cells are generated in response to Leishmania major (Lm) or Toxoplasma gondii parasitic infections, indicating that exogenously delivered Ag can be processed for presentation by MHC class I molecules. We show that presentation of Lm nucleotidase (NT)-OVA is TAP independent in vivo and in vitro, and is inhibited by chloroquine, but not by proteasome inhibitors. In contrast, the presentation of T. gondii P30-OVA relies on the TAP/proteasome pathway. Presentation of OVA- or rNT-OVA-coated beads also bypassed TAP requirement above a certain Ag threshold. TAP was also dispensable for the presentation of wild-type Lm Ags to primed CD8+ T cells in vitro. Finally, in vivo priming of CD8+ T cells involved in acquired resistance to Lm was not compromised in TAP-deficient mice. Thus, Leishmania Ags appear to be confined to an intraphagosomal processing pathway that requires higher concentrations of Ags, suggesting that these parasites may have evolved strategies to impair the efficient endoplasmic reticulum-based, TAP-dependent cross-presentation pathway to avoid or delay CD8+ T cell priming.     

Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection

Activation of innate immune cells by Trypanosoma cruzi-derived molecules such as GPI anchors and DNA induces proinflammatory cytokine production and host defense mechanisms. In this study, we demonstrate that DNA from T. cruzi stimulates cytokine production by APCs in a TLR9-dependent manner and synergizes with parasite-derived GPI anchor, a TLR2 agonist, in the induction of cytokines by macrophages. Compared with wild-type animals, T. cruzi-infected Tlr9(-/-) mice displayed elevated parasitemia and decreased survival. Strikingly, infected Tlr2(-/-)Tlr9(-/-) mice developed a parasitemia equivalent to animals lacking MyD88, an essential signaling molecule for most TLR, but did not show the acute mortality displayed by MyD88(-/-) animals. The enhanced susceptibility of Tlr9(-/-) and Tlr2(-/-)Tlr9(-/-) mice was associated with decreased in vivo IL-12/IFN-gamma responses. Our results reveal that TLR2 and TLR9 cooperate in the control of parasite replication and that TLR9 has a primary role in the MyD88-dependent induction of IL-12/IFN-gamma synthesis during infection with T. cruzi.     

Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.     

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.     

High glucose increases B1-kinin receptor expression and signaling in endothelial cells

The loss of endothelial function is the initiating factor in the development of diabetic vascular disease. Kinins control endothelial function by the activation of two receptors: the B2 which is constitutively expressed, and the B1 which is highly induced in pathological conditions. In the present study, we observed that the levels of B1-receptor mRNA and protein are induced in endothelial cells incubated in high glucose. An increase in B1-receptor was also observed in the endothelial layer of aortas, from 4-week diabetic rats. When cells were grown in high glucose, the B1 agonist des-Arg9-BK increased nitrite levels, whereas in normal glucose nitrite levels were unchanged. Nitrite increase was blocked by L-NAME and 1400W indicating the participation of the inducible Nitric Oxide Synthase (iNOS). iNOS protein levels were also increased in high glucose. These results demonstrate the participation of the B1 receptor in the signaling pathways mediated by kinins in high glucose.     

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.     

Understanding migraine through the lens of maladaptive stress responses: a model disease of allostatic load

The brain and body respond to potential and actual stressful events by activating hormonal and neural mediators and modifying behaviors to adapt. Such responses help maintain physiological stability ("allostasis"). When behavioral or physiological stressors are frequent and/or severe, allostatic responses can become dysregulated and maladaptive ("allostatic load"). Allostatic load may alter brain networks both functionally and structurally. As a result, the brain's responses to continued/subsequent stressors are abnormal, and behavior and systemic physiology are altered in ways that can, in a vicious cycle, lead to further allostatic load. Migraine patients are continually exposed to such stressors, resulting in changes to central and peripheral physiology and function. Here we review how changes in brain states that occur as a result of repeated migraines may be explained by a maladaptive feedforward allostatic cascade model and how understanding migraine within the context of allostatic load model suggests alternative treatments for this often-debilitating disease.     

Human CD4+ T cell response to human herpesvirus 6

Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4(+) T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4(+) T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4(+) T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.