Sara E. Marshall

Genotype-phenotype analysis of the Crohn’s disease susceptibility haplotype on chromosome 5q31

Background and aims: Recent molecular data suggest that genetic factors may underlie the disease heterogeneity observed in both ulcerative colitis (UC) and Crohn’s disease (CD). A locus on chromosome 5q has been implicated in susceptibility to CD, and recently refined by linkage disequilibrium mapping to a conserved 250 kb haplotype (5q31). No data regarding the contribution of this locus to clinical phenotype exist. In this case control study, we investigated the contribution of this haplotype to both susceptibility and phenotype of CD and UC. Patients and methods: We studied 330 Caucasian CD and 457 UC patients recruited from a single UK centre. Association with disease susceptibility and phenotype was analysed with haplotypes reconstructed from three single nucleotide polymorphisms chosen to span this susceptibility region. Evidence for possible genetic epistasis between IBD5 and NOD2/CARD15 was sought. Results: Linkage disequilibrium across this region was confirmed, with two haplotypes comprising 88% of all chromosomes. Susceptibility to CD, but not to UC, was associated with homozygosity for a common haplotype, H2 (pc=0.002; relative risk (RR) 2.0). Genotype-phenotype analyses demonstrated that this association was particularly strong in patients with perianal disease (pc=0.0005; RR 1.7), especially in individuals homozygous for this haplotype (pc=0.0005; RR 3.0). Importantly, no association with H2 was found in 186 patients without perianal disease. No evidence of epistasis between IBD5 and NOD2/CARD15 was demonstrated. Conclusions: The IBD5 risk haplotype is associated with CD only. Genotype-phenotype analysis reveals that the strongest association is observed in patients with perianal CD. While the precise gene involved is unclear, these data provide further molecular evidence for a genetic basis of the clinical heterogeneity of CD.

Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy

Complex regulation of T cell functions during pregnancy is required to ensure materno‐fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate L‐arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase‐mediated L‐arginine depletion induces down‐regulation of CD3ζ, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase‐mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous L‐arginine restored CD3ζ chain expression and T cell proliferation. Thus, L‐arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.

Characterization of Inflammatory Bowel Disease With Urinary Metabolic Profiling

OBJECTIVES:Distinguishing between the inflammatory bowel disease (IBD), Crohns disease (CD), and ulcerative colitis (UC) is important for both management and prognostic reasons. Discrimination using noninvasive techniques could be an adjunct to conventional diagnostics. Differences have been shown between the intestinal microbiota of CD and UC patients and controls; the gut bacteria influence specific urinary metabolites that are quantifiable using proton high-resolution nuclear magnetic resonance (NMR) spectroscopy. This study tested the hypothesis that such metabolites differ between IBD and control cohorts, and that using multivariate pattern-recognition analysis, the cohorts could be distinguished by urine NMR spectroscopy.METHODS:NMR spectra were acquired from urine samples of 206 Caucasian subjects (86 CD patients, 60 UC patients, and 60 healthy controls). Longitudinal samples were collected from 75 individuals. NMR resonances specific for metabolites influenced by the gut microbes were studied, including hippurate, formate, and 4-cresol sulfate. Multivariate analysis of all urinary metabolites involved principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA).RESULTS:Hippurate levels were lowest in CD patients and differed significantly between the three cohorts (P<0.0001). Formate levels were higher and 4-cresol sulfate levels lower in CD patients than in UC patients or controls (P=0.0005 and P=0.0002, respectively). PCA revealed clustering of the groups; PLS-DA modeling was able to distinguish the cohorts. These results were independent of medication and diet and were reproducible in the longitudinal cohort.CONCLUSIONS:Specific urinary metabolites related to gut microbial metabolism differ between CD patients, UC patients, and controls. The emerging technique of urinary metabolic profiling with multivariate analysis was able to distinguish these cohorts.

Associations between Human Leukocyte Antigens and Nonresponsiveness to Influenza Vaccine

Influenza remains a major cause of morbidity and mortality, particularly in at-risk groups where vaccination reduces complications of infection but is not universally protective. In order to determine whether human leukocyte antigen (HLA) class II polymorphisms modulate anti-influenza antibody responses to vaccination, a cohort of HLA-typed at-risk donors was investigated. The subjects were recruited from a single urban family practice. Hemagglutination-inhibition (HAI) titers were measured immediately before and 28 days after subunit vaccination. Nonresponsiveness was defined as failure to mount an HAI response to any component of the trivalent influenza vaccine. When the nonresponders and responders with HLA class II were compared, the nonresponder group had more HLA-DRB1*07-positive donors (13/32 vs. 6/41 responders; P=.016, Fishers exact test) and fewer HLA-DQB1*0603-9/14-positive donors (2/32 vs. 14/41 responders; P=.0045). Thus, polymorphisms in HLA class II molecules appear to modulate antibody responses to influenza vaccination.

The impact of recipient cytokine genotype on acute rejection after renal transplantation.

Background. Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. As cytokines are major regulators of the immune system, genetic variation in cytokine production or activity may influence susceptibility to acute rejection. This study sought to determine the impact of recipient cytokine and cytokine receptor polymorphisms on acute rejection after renal transplantation. Methods. A total of 209 cadaveric renal transplant recipients were selected for analysis according to the presence or absence of graft rejection in the first 30 days after transplantation. DNA was genotyped for 22 polymorphisms in 11 cytokine and cytokine receptor genes using the polymerase chain reaction with sequence specific primers. Results were stratified by incidence and severity of rejection, and by HLA-DR mismatching. Results. No association between any polymorphism and the incidence or severity of acute rejection was detected. In particular, no association was seen with tumor necrosis factor or interleukin-10 genotype, either alone or in combination. Conclusions. We have failed to demonstrate any association between recipient cytokine genotype and acute rejection after cadaveric renal transplantation. Although more extensive studies may disprove these findings, it would seem premature to use recipient cytokine genotyping to predict transplant outcome, or to guide immunosuppressive therapy after transplantation.

Variation in immunoregulatory genes determines the clinical phenotype of common variable immunodeficiency

Variation in clinical phenotype is a hallmark of many complex diseases. The cause of this clinical heterogeneity is unknown, but it may be determined by genetic factors distinct from those conferring disease susceptibility. Common variable immunodeficiency (CVID) is a complex disease of unknown aetiology and diverse clinical manifestations. We have developed a unified polymerase chain reaction and sequence-specific primer (PCR-SSP) method to simultaneously genotype multiple polymorphisms under identical conditions, and have used this method to test the hypothesis that the clinical phenotype of CVID is determined by immunoregulatory gene polymorphism. Twenty-three polymorphisms in 13 genes were studied in 163 CVID patients. Vitamin D receptor and IL-6 alleles were associated with immunophenotypic abnormalities characteristic of more severe disease; and tumour necrosis factor and IL-10 alleles conferred susceptibility to the granulomatous form of CVID in an interacting fashion. These findings demonstrate that different clinical features of a disease may have unique pathogenetic abnormalities, determined by multiple interacting genetic factors. The ease of application of this efficient, robust genotyping technique to polymorphisms throughout the genome will make it a powerful tool in the investigation of the genetic basis of phenotypic variability in a wide variety of diseases.

European Respiratory Society guidelines for the management of adult bronchiectasis

Bronchiectasis in adults is a chronic disorder associated with poor quality of life and frequent exacerbations in many patients. There have been no previous international guidelines. The European Respiratory Society guidelines for the management of adult bronchiectasis describe the appropriate investigation and treatment strategies determined by a systematic review of the literature. A multidisciplinary group representing respiratory medicine, microbiology, physiotherapy, thoracic surgery, primary care, methodology and patients considered the most relevant clinical questions (for both clinicians and patients) related to management of bronchiectasis. Nine key clinical questions were generated and a systematic review was conducted to identify published systematic reviews, randomised clinical trials and observational studies that answered these questions. We used the GRADE approach to define the quality of the evidence and the level of recommendations. The resulting guideline addresses the investigation of underlying causes of bronchiectasis, treatment of exacerbations, pathogen eradication, long term antibiotic treatment, anti-inflammatories, mucoactive drugs, bronchodilators, surgical treatment and respiratory physiotherapy. These recommendations can be used to benchmark quality of care for people with bronchiectasis across Europe and to improve outcomes. The publication of the first ERS guidelines for bronchiectasis http://ow.ly/wQSO30dU0nE

DONOR CYTOKINE GENOTYPE INFLUENCES THE DEVELOPMENT OF ACUTE REJECTION AFTER RENAL TRANSPLANTATION

Background. Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. Although rejection is mediated by recipient lymphocytes, both donor and recipient factors contribute to the local environment that influences the nature, severity, and duration of the rejection response. Cytokines are a major determinant of this milieu, and this study sought to explore the impact of donor cytokine and cytokine receptor gene polymorphisms on acute rejection after renal transplantation. Methods. A total of 145 cadaveric renal allograft donors were selected for analysis according to the presence or absence of graft rejection in the first 30 days after transplantation. DNA was genotyped for 20 polymorphisms in 11 cytokine and cytokine receptor genes using the polymerase chain reaction with sequence specific primers. Associations were assessed using contingency table analysis and the &khgr;2 test, using a two-set design. Results. A polymorphism at position −174 of the donor IL-6 gene was associated with the incidence (P =0.0002) and severity (P =0.000007) of recipient acute rejection. This finding was independent of HLA-DR matching. Acute rejection was not influenced by recipient IL-6 genotype, or by donor-recipient matching of IL-6 genotype. Conclusion. This study identifies donor IL-6 genotype as a major genetic risk factor for the development of acute rejection after renal transplantation. This provides evidence that donor-derived cytokines play a major role in determining outcome after transplantation, and will contribute to the development of therapeutic algorithms to predict individuals at particularly high risk of acute rejection.

Human Cell Lymphotropic Virus (HTLV) Type-l-Specific CD8+ Cells: Frequency and Immunodominance Hierarchy

Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We used interferon- gamma enzyme-linked immunospot assays with overlapping peptides spanning the entire HTLV-1 proteome to test whether the HTLV-1-specific CD8(+) T cells differed significantly in frequency or immunodominance hierarchy between patients with HAM/TSP and asymptomatic carriers and whether the frequency correlated with provirus load. Tax was the immunodominant target antigen. There was no significant qualitative or quantitative difference in the HTLV-1-specific CD8(+) T cell response between the 2 groups. Virus-specific CD8(+) T cell frequency alone does not indicate the effectiveness of the cytotoxic T lymphocyte response in controlling provirus load at equilibrium.

HLA Class II Polymorphisms and Susceptibility to Recurrent Respiratory Papillomatosis

ABSTRACT Recurrent respiratory papillomatosis (RRP) is characterised by multiple laryngeal papillomas. Left untreated, the lesions enlarge, spread, and endanger the airway. Medical treatments are unsatisfactory, and repeated surgery remains the mainstay of therapy. RRP is caused by human papillomavirus (HPV) infection. However, since oral HPV infection is common and RRP is rare, other host and/or viral factors may contribute to pathogenesis. In an attempt to identify such factors, we have investigated 60 patients. The patients were HLA class I, II, and tumor necrosis factor TNF typed by sequence-specific primer PCR, and the results compared to those for 554 healthy controls by using Fishers exact test. Peripheral blood mononuclear cell proliferative responses of 25 controls and 10 patients to HPV-11 L1 virus-like particles (VLP) were compared. Short-term VLP-specific T-cell lines were established, and recognition of L1 was analyzed. Finally, the L1 open reading frames of HPV isolates from four patients were sequenced. Susceptibility to RRP was associated with HLA DRB1*0301 (33 of 60 patients versus 136 of 554 controls, P < 0.0001). The three most severely affected patients were homozygous for this allele. A range of T-cell proliferative responses to HPV-11 VLP were observed in DRB1*0301-positive healthy donors which were comparable to those in DRB1*0301-negative controls. Individuals with juvenile-onset RRP also mounted a range of VLP responses, and their magnitude was negatively correlated with the clinical staging score (P = 0.012 by the Spearman rank correlation). DRB1*0301-positive patients who responded to L1 recognized the same epitope as did matched controls and produced similar cytokines. Sequencing of clinical isolates excluded the possibility that nonresponsiveness was the result of mutation(s) in L1.