John P. Hegarty

Resident human hepatitis lymphocytes are phenotypically different from circulating lymphocytes

BACKGROUND/AIMS Murine and human studies have documented the existence of subpopulations of lymphocytes in particular tissues that differ phenotypically and functionally from those in peripheral blood and may mature locally. Since little is known about lymphocyte subpopulations in the normal human liver, we have analysed the surface phenotypes of lymphocytes isolated from liver specimens taken from 15 donors at the time of liver transplantation, and compared these with those of peripheral blood lymphocytes. METHODS Hepatic lymphocytes were prepared by mechanical dissociation and enzymatic digestion of liver tissue. The cells were stained with a panel of monoclonal antibodies (CD3, CD4, CD8, CD19, CD56, gammadeltaTCR, alphabetaTCR, CD8alpha-chain, CD8alphabeta dimer), and analysed by flow cytometry. In situ characterisation of hepatic lymphocytes was by haematoxylin and eosin staining of fixed liver sections and by immunohistochemical staining for common leukocyte antigen and CD3. RESULTS Significant numbers of hepatic T lymphocytes were localised to the portal tracts and parenchyma of normal liver specimens. Flow cytometry revealed that the CD4/CD8 ratio (1:3.5) was consistently reversed compared with that in peripheral blood (2:1). Other lymphocyte populations identified include double positive CD3+CD4+CD8+ cells which accounted for a mean of 5.5% (range 3-11.6%) of hepatic CD3+ cells compared with 1.3% in blood (range 0.7-3.6%; p < 0.007), and double negative CD3+ CD4-8- cells (14.5%; range 2.7-29% compared with 5.0%; range 2.1-10.8%, p < 0.02). Over 15% (range 6.8-34%) of all hepatic CD3+ cells expressed a gammadeltaTCR compared to 2.7% (range 0.9-4.7%) of CD3+ peripheral blood lymphocytes (p < 0.004) and almost 50% of these coexpressed CD8. The CD8 alpha-chain was expressed without the beta-chain (CD8alpha+beta-) by 15.4% (range 4-29.1%) of hepatic T cells, but this phenotype was undetectable among peripheral blood lymphocytes (p < 0.009). Cells expressing both the T cell marker CD3 and the natural killer cell marker CD56 constituted 31.6% (range 14-54%) of all hepatic CD3+ lymphocytes but were rarely present amongst peripheral blood lymphocytes (0-6%; p < 0.0001). CONCLUSIONS These data are the first to describe and quantify unconventional T lymphocyte subpopulations in the normal adult human liver which may have specialised functions in regional immune responses and which may differentiate locally. These findings have important implications for our understanding of hepatic immunoregulation and the pathogenic mechanisms involved in viral and immune-mediated liver disease and allograft rejection.

Distinct MHC class I and II alleles are associated with hepatitis C viral clearance, originating from a single source

The role of cytotoxic T lymphocyte responses, restricted by human leukocyte antigen (HLA) class I alleles, is recognized as highly significant in the successful clearance of hepatitis C virus (HCV). The frequency of class I alleles in females inoculated with HCV genotype 1b from a single source was examined for an association with outcome. Class I typing was performed using polymerase chain reaction sequence‐specific primers in 227 female subjects: 141 had chronic infection and 86 had viral clearance. Statistical analysis included χ2 testing and multiple logistic regression analysis. A*03, B*27, and Cw*01 occurred more frequently in those with viral clearance (39.5%, 14%, and 9.3%, respectively) compared with those with chronic infection (19.1%, 2.1%, and 1.4%, respectively; P ≤ .005). B*08 occurred more often in those with chronic infection compared with viral clearance (39.7% vs. 19.8%; P = .002). In combination with previously reported class II allele associations, over 75% that successfully eliminate HCV carry either A*03, DRB1*0101, or *0401, compared with only 37% of those with chronic infection (P < .0001). The haplotypes A*03‐B*07‐DRB1*15‐DQB1*0602 and A*02‐B*27‐Cw*01‐DRB1*0101‐DQB1*0501 are associated with viral clearance (P = .004 and .01, respectively). By multiple logistic regression analysis, the alleles A*03, B*27, DRB1*0101, *0401, and *15 are associated with viral clearance, and B*27 has the strongest association (odds ratio [OR] 7.99). The haplotype A*01‐B*08‐Cw*07‐DRB1*03011‐DQB1*0201 is associated with chronic infection (P = .002), being independent for DQB1*0201 (OR 0.27). In conclusion, certain class I alleles are associated with outcome in this homogenous cohort. More significantly, either HLA‐A*03, ‐DRB1*0101, or ‐*0401 are carried by an overwhelming majority of those subjects who successfully clear HCV. (HEPATOLOGY 2004;40:108–114.)

Occurrence of Helicobacter pylori in surface water in the United States

The primary mode of transmission of the human pathogen Helicobacter pylori is unresolved. This study examined the possibility that H. pylori is water‐borne. Because methods for the direct culture of H. pylori from water samples remain elusive, a microscopic technique was used for detection of this organism. Actively respiring micro‐organisms binding monoclonal anti‐H. pylori antibody were found in the majority of surface and shallow groundwater samples tested (n = 62), indicating that H. pylori may be present in aquatic environments in the US and supporting a water‐borne route of transmission for this organism. There was no significant correlation between the occurrence of either total coliforms or Escherichia coli in the water and the presence of H. pylori. Our results indicate that routine screening of water supplies for the presence of traditional indicator organisms may fail to protect the consumer from exposure to H. pylori.

Presence of Helicobacter pylori in drinking water is associated with clinical infection.

Helicobacter pylori was detected using molecular methods in untreated well water. The presence of H. pylori in the wells correlated with infection in consumers and with the presence of Escherichia coli, indicating fecal contamination. Consumption of untreated well water should be considered a risk factor for H. pylori infection.

Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease

Lin Z, Hegarty JP, Cappel JA, Yu W, Chen X, Faber P, Wang Y, Kelly AA, Poritz LS, Peterson BZ, Schreiber S, Fan J‐B, Koltun WA. Identification of disease‐associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease.

Effect of Oxidizing Disinfectants (Chlorine, Monochloramine, and Ozone) on Helicobacter pylori

ABSTRACT The susceptibility of Helicobacter pylori to disinfectants was compared to that of Escherichia coli. H. pylori is more resistant than E. coli to chlorine and ozone but not monochloramine. H. pylori may be able to tolerate disinfectants in distribution systems and, therefore, may be transmitted by a waterborne route.

The MHC is a major determinant of viral status, but not fibrotic stage, in individuals infected with hepatitis C

BACKGROUND & AIMS In hepatitis C infection, several studies have examined the role of the major histocompatibility complex (MHC) in determining outcome, with variable results. To clarify the importance of MHC, we examined class II DR and DQ antigens in a homogenous cohort of women exposed to hepatitis C genotype 1b from a single inoculum. METHODS Of 243 participants, 95 had spontaneous viral clearance and 148 are chronically infected. The frequencies of HLA class II DR and DQ antigens were compared between the 2 groups and between liver biopsy findings of 145 chronically infected subjects. RESULTS DRB1*0101 and DQB1*0501 alleles were more frequent in subjects who sustained viral clearance than in chronically infected subjects (32.3% and 36.8% vs. 8.8% and 14.2%, respectively; P = 0.002). DRB1*03011 and DQB1*0201 occurred more frequently in chronically infected subjects than in those who cleared the virus (41.5% and 42.6% vs. 16.7% and 15.8%, respectively; P = 0.001). Both DRB1*03011 and DQB1*0201 were significantly less frequent in those with higher inflammatory scores on liver biopsy. CONCLUSIONS We show that in a homogenous cohort of women infected with the same hepatitis C virus, several HLA antigens are associated with either viral clearance or persistence. This suggests a strong role for host immunogenetic factors in determining outcome in hepatitis C infection.

NOD2/CARD15 Mutations Correlate With Severe Pouchitis After Ileal Pouch-Anal Anastomosis

PURPOSE: Pouchitis and Crohns-like complications can plague patients after IPAA. NOD2 is an intracellular sensor for bacterial cell wall peptidoglycan. NOD2 mutations compromise host response to enteric bacteria and are increased in Crohns disease. We hypothesize that IPAA patients with complications (Crohns disease-like/pouchitis) have a higher rate of NOD2 mutations compared with asymptomatic IPAA patients. METHODS: Patients were retrospectively subclassified into the following groups: 1) IPAA with Crohns-like complications (n = 28, perianal fistula, pouch inlet stricture/upstream small-bowel disease, or biopsies showing granulomata) occurring at least 6 months after ileostomy closure; 2) IPAA with mild pouchitis (n = 33, ≤3 episodes/y for 2 consecutive years); 3) IPAA with severe pouchitis (n = 9, ≥4 episodes/y for 2 consecutive years or need for continuous antibiotics); 4) IPAA without complications or pouchitis (n = 37); 5) patients with Crohns disease with colitis undergoing total proctocolectomy/ileostomy (n = 11); and 6) healthy controls (n = 269). The 3 NOD2 single-nucleotide polymorphism mutations (rs2066844, rs2066845, and rs2066847) previously identified as associated with Crohns disease were genotyped using polymerase chain reaction. Groups were compared by use of &khgr;2 with Yates continuity correction. RESULTS: NOD2 mutations were found in 8.5% of healthy controls. NOD2 mutations were significantly higher in the severe pouchitis group (67%) compared with both asymptomatic IPAA (5.4%, P < .001) and IPAA with Crohns disease-like complications (14.3%, P = .008) groups. CONCLUSIONS: 1) Asymptomatic IPAA patients have a low incidence of NOD2 mutations not significantly different from patients with mild pouchitis or healthy controls. 2) Patients with severe pouchitis had the highest incidence of NOD2 mutations, suggesting that this group may have a compromised host defense mechanism to enteric bacteria. 3) Patients with Crohns-like complications after IPAA have a significantly lower incidence of NOD2 mutations than patients with severe pouchitis, suggesting a different genetic makeup in these 2 patient groups. Preoperative assessment of NOD2 in the equivocal IPAA candidate may predict severe pouchitis and might assist in preoperative surgical decision making.

Identification of Disease-Associated DNA Methylation in B Cells from Crohn’s Disease and Ulcerative Colitis Patients

BackgroundChanges in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay.AimsIn this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn’s disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood.MethodsWe examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals.ResultsUsing this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells.ConclusionsIBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.

Mutations in IRGM are associated with more frequent need for surgery in patients with ileocolonic Crohn's disease.

BACKGROUND: There are no clear criteria for judging the severity of disease in patients with Crohns disease. Yet classification of patients into low- and high-risk severity groups would benefit both medical and surgical management. At the time of this study, approximately 80 single-nucleotide polymorphisms within 55 genes had been associated with IBD. OBJECTIVE: The aim of this study was to identify genetic determinants (single-nucleotide polymorphisms) that could be markers of Crohns disease severity by the use of frequency of ileocolic surgery as a surrogate for disease severity. DESIGN: Sixty-six patients (30 male) with ileocolonic Crohns disease who previously underwent ileocolectomy were retrospectively studied. The severity of Crohns disease was quantified by dividing the total number of ileocolectomy procedures by the time between IBD diagnosis and the patients last clinic visit, the rationale being that more severe disease would be associated with a more frequent need for surgery. Genotyping for the 83 single-nucleotide polymorphisms associated with IBD was done on a customized Illumina Veracode genotyping platform. Three genetic models (general, additive, and dominant) were used to statistically quantify the genetic association of the studied single-nucleotide polymorphisms to the frequency of surgery after adjusting for covariates (age, smoking, family history, disease location, and disease behavior). RESULTS: For the entire group the average number of ileocolectomies per patient was 1.7 (range, 1–5) with an average duration of disease of 14.7 years. Single-nucleotide polymorphism rs4958847 in the IRGM gene (immunity-related GTPase family, M) was the most significant single-nucleotide polymorphism in all 3 models tested (p = 0.007) as being associated with ileocolectomy, and it remained significant even after a Benjamini-Hochberg false-discovery correction for multiple observations. Patients carrying the “at-risk” allele for this single-nucleotide polymorphism (n = 20) had an average of 1 surgery every 6.87 ± 1.33 years in comparison with patients carrying the wild-type genotype (n = 46) who averaged 1 surgery in 11.43 ± 1.21 years (p = 0.007, Mann-Whitney U test). CONCLUSIONS: Single-nucleotide polymorphism rs4958847 in the IRGM gene correlated very significantly with frequency of surgery in patients with ileocolonic Crohns disease. IRGM is a mediator of innate immune responses and is involved in autophagy. The presence of this IRGM SNP may be a marker for disease severity and/or early recurrence after ileocolectomy and may assist in surgical and medical decision making.