Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease

Crohn's disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn's disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Worldwide occurrence of feline hemoplasma infections in wild felid species

While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroy's cat, margay, and ocelot. "Candidatus Mycoplasma haemominutum" was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas "Candidatus Mycoplasma turicensis" was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

Keratinocyte transcriptional regulation of the human c-Myc promoter occurs via a novel Lef/Tcf binding element distinct from neoplastic cells

The proto-oncogene c-Myc is involved in early neoplastic transformations. Two consensus Lef/Tcf binding elements (TBE) were found to be prerequisite for transcriptional transactivation by the armadillo proteins beta-catenin and plakoglobin (PG) together with Tcf4 in human neoplastic cells. In epidermal keratinocytes, c-Myc was reported to be repressed by Lef-1 and PG. Using reporter gene assays, here we demonstrate that deletion of the two consensus TBE fails to abrogate transcriptional regulation by Lef-1/PG in wildtype and beta-catenin-/- keratinocytes, while it reduces transcription in pre-neoplastic PG-/- keratinocytes. We identified a TBE sequence variant downstream of the major transcriptional initiation site that binds Lef-1 in vitro and in vivo, and its mutation compromised transcriptional regulation by Lef-1/PG. Collectively, this study demonstrates that the two consensus TBE's reported in neoplastic cells are dispensable for c-Myc regulation in normal keratinocytes, which instead use a novel TBE sequence variant. This unprecedented finding may have important implications for armadillo target genes involved in carcinogenesis.

Correlation of atrophy and fatty infiltration on strength and integrity of rotator cuff repairs: a study in thirteen patients

In 13 patients, the development of supraspinatus muscle atrophy and fatty infiltration after rotator cuff tendon repair was quantified prospectively via magnetic resonance imaging. Intraoperative electrical nerve stimulation at repair showed that the maximal supraspinatus tension (up to 200 N) strongly correlated with the anatomic cross-sectional muscle area and with muscle fatty infiltration (ranging from 12 N/cm(2) in Goutallier stage 3 to 42 N/cm(2) in Goutallier stage 0). Within 1 year after successful tendon repair (n = 8), fatty infiltration did not recover, and atrophy improved partially at best; however, if the repair failed (n = 5), atrophy and fatty infiltration progressed significantly. The ability of the rotator cuff muscles to develop tension not only correlates with their atrophy but also closely correlates with their degree of fatty infiltration. With current repair techniques, atrophy and fatty infiltration appear to be irreversible, despite successful tendon repair. Unexpectedly, not only weak but also very strong muscles are at risk for repair failure.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance

Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.