Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles

AbstractEndothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1β, tumor necrosis factor-α, interferon-β, interferon-γ, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low virulent and moderate virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE- was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE- vaccine virus was re-adapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE-/P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) when compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading, and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs

Development and evaluation of indirect enzyme-linked immunosorbent assay for a screening test to detect antibodies against classical swine fever virus

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for a screening test to detect antibodies against classical swine fever virus (CSFV). Viral glycoproteins, which were purified from swine kidney cells infected with CSFV ALD/A76 strain by the immunoaffinity purification using monoclonal antibody against E2 protein, were adsorbed on a microtiter plate as the antigen for the antibody detection. Each antibody titer of serum sample was expressed as a sample per positive value calculated with optical absorbance of each sample and that of a positive control. The advantage of this ELISA is its higher sensitivity: most sera containing more than 4 neutralization titers were determined to be positive. This ELISA is unable to discriminate between antibodies against CSFV and those against other ruminant pestiviruses, therefore positive sera in this ELISA should be evaluated by a cross-neutralization test using CSFV, bovine viral diarrhea virus, and border disease virus. Taken together, the indirect ELISA developed in this study is useful screening tool to detect antibodies against CSFV for the large-scale monitoring of classical swine fever

Laz İsmail, Nazım abimi öldürmek istedi

Taha Toros Arşivi, Dosya Adı: Nazım Hikmetİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Virological and immunological analyses in macaque R09-009 following CD8⁺ cell depletion.

<p>(A) Changes in peripheral CD8⁺ T-cell counts after the initial anti-CD8 antibody administration. Group N macaque, R09-009, was administered anti-CD8 antibody at week 108 post-infection and on days 3, 7, and 10 after the first administration. (B) Changes in plasma viral loads. (C) Changes in CD8⁺ T-cell responses specific for SIV Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env. (D) CD8⁺ T-cell responses specific for SIV Gag_206–216, Gag_241–249, Gag_367–381, Vif_114–124, Nef_9–19, Nef_89–97, and Nef_193–203 epitopes at week 113 post-infection. (E) Dominant non-synonymous mutations in plasma viral cDNA regions encoding Gag, Vif, and Nef epitopes. Viral <i>gag</i>, <i>vif</i>, and <i>nef</i> cDNA fragments were amplified from plasma RNA obtained at weeks 110 and 118 post-infection. Amino acid substitutions around SIV Gag_206–216, Gag_241–249, Gag_367–381, Vif_114–124, Nef_9–19, Nef_89–97, and Nef_193–203 epitopes are shown.</p

Plasma viral loads determined by using fivefold-concentrated plasma^a

<p>^aAfter centrifugation of 1 ml of plasma at 25,000 x <i>g</i> for 2 hours, 0.8 ml of its supernatant was discarded for fivefold concentration of plasma. The remaining 0.2 ml was subjected to RNA extraction for quantitation of viral loads (VL) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#ppat.1005247.ref025" target="_blank">25</a>]. Plasma samples of R06-037 and R07-006 at 2 years post-infection (pi) were unavailable (ND, not determined).</p><p>Plasma viral loads determined by using fivefold-concentrated plasma<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#t002fn001" target="_blank">^a</a></p

SIV antigen-specific CD8⁺ T-cell responses in SIV controllers.

<p>(A) Frequencies of CD8⁺ T cells specific for Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env in Group M (upper panels) and Group N (lower) at 4 months (4M), 1 year (1Y), and 2 years (2Y) post-infection. Responses were measured by detection of antigen-specific IFN-γ induction using panels of overlapping peptides spanning the entire SIVmac239 Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env amino acid sequences, respectively. (B) Comparisons of CD8⁺ T-cell frequencies specific for SIV antigens other than Gag and Nef at 4M, 1Y, and 2Y between Groups M and N. Group M had significantly higher frequencies of SIV non-Gag/Nef antigen-specific CD8⁺ T cells at 4M (p = 0.0095 by Mann-Whitney U-test) and 1Y (p = 0.0095). (C) Comparisons of the numbers of CD8⁺ T cell-targeted SIV antigens other than Gag and Nef at 4M, 1Y, and 2Y between Groups M and N. The numbers were significantly higher in Group M at 4M (p = 0.0095 by Mann-Whitney U-test) and 1Y (p = 0.0095).</p