Forming a Complex with MHC Class I Molecules Interferes with Mouse CD1d Functional Expression

CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of) mouse CD1d on the surface of cells. Low pH (3.0) acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.     

Lipid protein interactions: the assembly of CD1d1 with cellular phospholipids occurs in the endoplasmic reticulum.

CD1d1 is a member of a family of lipid Ag-presenting molecules. The cellular ligands associated with CD1d1 were isolated and characterized by biochemical means as an approach to elucidate the mechanism by which CD1 molecules assemble in vivo. Natural ligands of mouse CD1d1 included cellular phosphatidylinositol and phosphatidylinositol-glycans that are synthesized in the endoplasmic reticulum. Further biochemical data revealed that the two CD1d1 mutants, one defective in recycling from-and-to the plasma membrane and the other in efficiently negotiating the secretory pathway, associated with phosphatidylinositol. Thus phosphatidylinositol associated with CD1d1 in the early secretory pathway. Phosphatidylinositol also associated with CD1d1 in Pig-A-deficient cells that are defective in the first glycosylation step of glycosylphosphatidylinositol biosynthesis. Moreover, cellular phosphatidylinositol-glycans are not Valpha14Jalpha15 natural T cell Ags. Therefore, we predict that cellular lipids occlude the hydrophobic Ag-binding groove of CD1 during assembly until they are exchanged for a glycolipid Ag(s) within the recycling compartment for display on the plasma membrane. In this manner, cellular lipids might play a chaperone-like role in the assembly of CD1d1 in vivo, akin to the function of invariant chain in MHC class II assembly.     

Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Background

Host genetics has been shown to play a role in porcine reproductive and respiratory syndrome (PRRS), which is the most economically important disease in the swine industry. A region on Sus scrofa chromosome (SSC) 4 has been previously reported to have a strong association with serum viremia and weight gain in pigs experimentally infected with the PRRS virus (PRRSV). The objective here was to identify haplotypes associated with the favorable phenotype, investigate additional genomic regions associated with host response to PRRSV, and to determine the predictive ability of genomic estimated breeding values (GEBV) based on the SSC4 region and based on the rest of the genome. Phenotypic data and 60 K SNP genotypes from eight trials of ~200 pigs from different commercial crosses were used to address these objectives.

Results

Across the eight trials, heritability estimates were 0.44 and 0.29 for viral load (VL, area under the curve of log-transformed serum viremia from 0 to 21 days post infection) and weight gain to 42 days post infection (WG), respectively. Genomic regions associated with VL were identified on chromosomes 4, X, and 1. Genomic regions associated with WG were identified on chromosomes 4, 5, and 7. Apart from the SSC4 region, the regions associated with these two traits each explained less than 3% of the genetic variance. Due to the strong linkage disequilibrium in the SSC4 region, only 19 unique haplotypes were identified across all populations, of which four were associated with the favorable phenotype. Through cross-validation, accuracies of EBV based on the SSC4 region were high (0.55), while the rest of the genome had little predictive ability across populations (0.09).

Conclusions

Traits associated with response to PRRSV infection in growing pigs are largely controlled by genomic regions with relatively small effects, with the exception of SSC4. Accuracies of EBV based on the SSC4 region were high compared to the rest of the genome. These results show that selection for the SSC4 region could potentially reduce the effects of PRRS in growing pigs, ultimately reducing the economic impact of this disease.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

Regulation of the actin cytoskeleton by rho kinase controls antigen presentation by CD1d

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.     

Antibacterial activity against β- lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: fractional Inhibitory Concentration Index (FICI) determination

Background

The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.

Method

The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.

Results

We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.

Conclusion

The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.