Large-scale sequencing identifies multiple genes and rare variants associated with Crohn's disease susceptibility.

Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation.

Significant [C3a] increase in free flaps after prolonged ischemia.

BACKGROUND: Free tissue transfer (FTT) represents a clinical model to measure ischemia-reperfusion injury (IRI). This study was conducted to detect substances relevant for IRI after FTT. METHODS: Eighteen patients underwent lower leg reconstruction with free myocutaneous latissimus dorsi muscles and were monitored clinically and by microdialysis technique. Patients were retrospectively classified as group A (n = 12) (no prolonged IRI) or group B (n = 6) (prolonged IRI). One catheter was placed into the flap and one into the reference tissue. Samples were collected during ischemia and in 90 min steps after reperfusion. Biochemical substances (glucose, pyruvate, lactate, and glycerol) and immunological substances (interleukin 8 [IL-8], complement 3a [C3a], and regulated on activation normal T cell expressed and secreted [RANTES]) were then analyzed. RESULTS: All free myocutaneous latissimus dorsi flaps healed primarily. Minor complications included revisions of the microvascular anastomoses due to hematoma or thrombus formation and increased total flap ischemia time in group B significantly when compared to group A (P < 0.001). No significant differences of biochemical substance concentrations were detected during reperfusion in target and control tissue of both groups. IL-8 and C3a were at detectable levels, whereas the results for RANTES were inconsistent. Either for group A and group B, we found higher concentrations of C3a in target tissue compared with control tissue. Furthermore, during the first 90 min of reperfusion, we found a highly significant increase of C3a (P < 0.001) in the target tissue of patients with increased ischemia time. CONCLUSIONS: Given our results, C3a is a highly sensitive early indicator of ischemia-reperfusion damage. Our results give further insight into development of IRI after complicated FTT.

The variable number of tandem repeat polymorphism in the P-selectin glycoprotein ligand-1 gene is not associated with coronary heart disease.

Genes involved in inflammatory processes are candidates for predisposition to prothrombotic syndromes. The variable number of tandem repeat (VNTR) polymorphism in the P-selectin glycoprotein ligand (PSGL)-1 gene has been associated with ischemic cerebrovascular disease but not with coronary heart disease (CHD). We assessed the effect of the VNTR polymorphism on CHD in two independent case/control studies. In the first study 281 CHD patients and 397 healthy blood donors were genotyped for the VNTR alleles in PSGL-1. The prevalence of homozygous carriers of the PSGL-1 VNTR allele with 15 repeat units was significantly higher in the CHD patients (5.3% vs. 1.5%) than in controls, suggesting an effect of this marker in CHD. To validate the findings genotyping was performed in a second study including 2,578 CHD patients, 731 patients without CHD, and 1084 healthy blood donors. The larger case control study had a power of 99.9% to detect the initially observed difference but failed to confirm the putative role of PSGL-1 VNTR polymorphism in CHD. Frequencies of the PSGL-1 VNTR 15 repeats for homozygous carriers were 2.2% in healthy blood donors, 2.3% in patients without CHD and 2.7%, in CHD cases, respectively. These results demonstrate that the PSGL-1 VNTR polymorphism is not a genetic risk factor for CHD. Adequately powered studies are prerequisites to obtain reliable results about genotype-phenotype relationships of new candidate genes in complex diseases.

High prevalence of cytomegalovirus DNA in plasma samples of blood donors in connection with seroconversion.

BACKGROUND: Human cytomegalovirus (CMV) is considered to latently infect blood cells. Transfusion-transmitted infection (TT-CMV) of immunocompromised patients occurs despite the use of CMV-seronegative or leukoreduced units. STUDY DESIGN AND METHODS: The prevalence of CMV DNA in plasma was investigated in 82 blood donors who had previously been seronegative for CMV and showed anti-CMV immunoglobulin G for the first time, 598 blood donors who were seropositive for at least 1 year, and 150 seronegative blood donors. In a second part of the study, the overall prevalence of CMV DNA in blood donations was assessed based on 31,745 donations. RESULTS: CMV DNA was repeatedly detected in plasma samples of 44 percent of newly seropositive donors (12%-62%, depending on the interval to the last seronegative donation). All steadily seropositive or seronegative donors were negative for the presence of CMV DNA. Detection of CMV DNA in connection with seroconversion was accompanied by significantly increased neopterin, increased alanine aminotransferase, and reduced white blood cell counts, but the sensitivity of these surrogate markers was only 71 percent. The overall prevalence of CMV DNA in blood products due to primary CMV infection of donors was at least 0.13 percent. CONCLUSION: Viremia of newly seropositive donors may be an important reason for the residual risk of TT-CMV despite leukoreduction. Furthermore, transfusion of WBC-reduced blood components from seronegative donors could imply a greater risk of TT-CMV than transfusion of WBC-reduced blood from donors who have been seropositive for at least 1 year, because window-phase donations but no reactivation could be detected in this study.

Elevated levels of endogenous apoptotic DNA and IFN-alpha in complement C4-deficient mice: implications for induction of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE), an autoimmune disease characterized by chronic nephritis, arthritis and dermatitis, and the presence of antinuclear autoantibodies, is associated with complement factor deficiencies in the classical activation pathway. In addition, IFN-alpha seems to be a key cytokine in SLE as an activated IFN-alpha system is regularly observed in patients with SLE. Here, we demonstrate that in lupus-susceptible, complement C4-deficient mice the lack of complement results in elevated intravascular levels of apoptotic DNA. The apoptotic DNA is targeted to the splenic marginal zone where it accumulates and induces IFN-alpha. As such, we present here a unifying hypothesis for the induction of SLE that incorporates the role of complement deficiency and elevated levels of IFN-alpha.

The association between systemic lupus erythematosus and deficiencies of the complement system.

Systemic lupus erythematosus (SLE), an autoimmune disease characterized by chronic nephritis, arthritis or dermatitis and the presence of antinuclear autoantibodies is associated with deficiencies of complement factors of the classical activation pathway. Results accumulated over the past few years with the use of complement gene deficient mice made it possible to update the conventional hypothesis for this association in regard to the etiology of the disease, whereby the early events leading to induction of autoimmunity can be explained by various functions of complement. As a conclusion, a new model for the etiology of the SLE based on the reduced elimination of apoptotic cells, the increased uptake of IgM containing immune complexes into the spleen and the CD21/CD35 dependent B cell toleration in the periphery demonstrates the importance of complement in the prevention of autoimmunity whereas the inflammatory reactions occurring in later stages of the disease are relatively independent from complement. The results obtained with complement deficient mice contribute to a better understanding of tolerance-inducing mechanisms and offers the option to develop new therapeutic procedures for autoimmune diseases.

Antigen localization within the splenic marginal zone restores humoral immune response and IgG class switch in complement C4-deficient mice.

Defects of early complement components (C1, C4 and C2) are associated with the development of systemic lupus erythematosus (SLE). The availability of complement knockout mice has increased our knowledge on the role of complement in the regulation of adaptive immunity. An impaired removal of apoptotic bodies, a disturbed clearance of IgG immune complexes (ICs) and an insufficient B-cell regulation via complement receptors CD21/CD35 have been discussed as explanations for the induction of autoimmunity; however, a unifying hypothesis for the loss of B-cell tolerance in the absence of C1 or C4 is still lacking. Using IgM-containing ICs, we observed a significant accumulation of antigen within the splenic marginal zone (MZ) of C4-deficient mice but not in C3-deficient or complement receptors CD21/CD35-deficient mice. The targeting of antigen toward the MZ restored adaptive immunity (antibody response and class switch) in C4-deficient animals. A new explanation for the association of SLE and complement C4 deficiency would be that in the absence of C4, natural antibodies (IgM type) localize more self-antigen toward the MZ so that the auto-antibody response is increased and autoimmune disease ensues. As such, an inadequate localization of self-antigens might be responsible for the annulment of peripheral B-cell tolerance in the absence of C4.

Frequency and load of hepatitis B virus DNA in first-time blood donors with antibodies to hepatitis B core antigen.

The objective of this study was to determine the frequency and load of hepatitis B virus (HBV) DNA in anti-HBc-positive first-time blood donors; it was designed to contribute to determining whether anti-HBc screening of blood donations might reduce the residual risk of posttransfusion HBV infection. A total of 14 251 first-time blood donors were tested for anti-HBc using a microparticle enzyme immunoassay; positive results were confirmed by a second enzyme-linked immunosorbent assay (ELISA). For the detection of HBV DNA from plasma samples, we developed a novel and highly sensitive real-time polymerase chain reaction (PCR) assay. The 95% detection limit of the method amounted to 27.8 IU/mL, consistent with the World Health Organization (WHO) international standard for HBV DNA. A total of 216 blood donors (1.52%) tested anti-HBc-positive in both tests, and 205 of them (16 HBsAg(+), 189 HBsAg(-)) were tested for HBV DNA. In 14 (87.5%) of the HBsAg-positive blood donors, HBV DNA was repeatedly detected, and in 3 (1.59%) of the HBsAg-negative donors, HBV DNA was also found repeatedly. In the 3 HBV DNA-positive, HBsAg-negative cases, anti-HBe and anti-HBs (> 100 IU/L) were also detectable. HBV DNA in HBsAg-negative as well as HBsAg-positive samples was seen at a low level. Thus, HBV DNA is sometimes found in HBsAg-negative, anti-HBc-positive, and anti-HBs-positive donors. Retrospective studies on regular blood donors and recipients are necessary to determine the infection rate due to those donations. Routine anti-HBc screening of blood donations could probably prevent some transfusion-transmitted HBV infections.