Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate.

The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

Analysis of germline GLI1 variation implicates hedgehog signalling in the regulation of intestinal inflammatory pathways.

BACKGROUND: Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis. METHODS AND FINDINGS: Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C-->G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09-1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1(+/lacZ) mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1(+/lacZ) mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model. CONCLUSIONS: HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

Characterization of intestinal gene expression profiles in Crohn's disease by genome-wide microarray analysis.

BACKGROUND: Genome-wide microarray expression analysis creates a comprehensive picture of gene expression at the cellular level. The aim of this study was to investigate differential intestinal gene expression in patients with Crohn's disease (CD) and controls with subanalysis of confirmed CD susceptibility genes, associated pathways, and cell lineage. METHODS: In all, 172 biopsies from 53 CD and 31 control subjects were studied. Paired endoscopic biopsies were taken at ileocolonoscopy from five specific anatomical locations including the terminal ileum (TI) for RNA extraction and histology. The 41,058 expression sequence tags were analyzed using the Agilent platform. RESULTS: Analysis of all CD biopsies versus controls showed 259 sequences were upregulated and 87 sequences were downregulated. Upregulated genes in CD included SAA1 (fold change [FC] +7.5, P = 1.47 × 10(-41)) and REGL (FC +7.3, P = 2.3 × 10(-16)), whereas cellular detoxification genes including-SLC14A2 (FC-2.49, P = 0.00002) were downregulated. In the CD TI biopsies diubiquitin (FC+11.3, P < 1 × 10(-45)), MMP3 (FC+7.4, P = 1.3 × 10(-11)), and IRTA1 (FC-11.4, P = 4.7 × 10(-12)) were differentially expressed compared to controls. In the colon SAA1 (FC+6.3, P = 5.3 × 10(-8)) was upregulated and thymic stromal lymphopoietin (TSLP) (FC-2.3, P = 2.7 × 10(-6)) was downregulated comparing noninflamed CD and control biopsies, and the colonic inflammatory CD signature was characterized by downregulation of the organic solute carriers-SLC38A4, SLC26A2, and OST alpha. Of CD susceptibility genes identified by genome-wide association scan IL-23A, JAK2, and STAT3 were upregulated in the CD group, confirming the dysregulation of Th17 signaling. CONCLUSIONS: These data characterize the dysregulation of a series of specific inflammatory pathways highlighting potential pathogenic mechanisms as well as areas for translation to therapeutic targets.

Intestinal epithelial cell up-regulation of LY6 molecules during colitis results in enhanced chemokine secretion.

In the healthy colon, intestinal epithelial cells (IEC) form a physical barrier separating the myriad of gut Ags from the cells of the immune system. Simultaneously, IEC use several mechanisms to actively maintain immunologic tolerance to nonpathogenic Ags, including commensal bacteria. However, during inflammatory bowel disease (IBD), the line of defense provided by IEC is breached, resulting in uncontrolled immune responses. As IEC are a principal mediator of immune responses in the gut, we were interested in discerning the gene expression pattern of IEC during development and progression of IBD. Laser capture microdissection and microarray analysis were combined to identify the LY6 superfamily as strongly up-regulated genes in inflamed IEC of the colon in two models of murine colitis. Surface expression of LY6A and LY6C on IEC is induced by several cytokines present within the colitic gut, including IL-22 and IFN-gamma. Furthermore, cross-linking of LY6C results in production of a number of chemokines which are known to be involved in the immunopathogenesis of IBD. Increased chemokine production was cholesterol dependent, suggesting a role for lipid raft structures in the mechanism. As such, LY6 molecules represent novel targets to down-regulate chemokine expression in the colon and limit subsequent inflammation associated with IBD.

Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy.

B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20(+) mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.