Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Th17 cells and polyfunctional HIV-specific T-cell responses.

Human immunodeficiency virus (HIV) infection leads to severe CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) that persists despite the initiation of highly active antiretroviral therapy (HAART). It is not known whether restoration of gut mucosal CD4+ T cells and their functions is feasible during therapy and how that relates to immune correlates and viral reservoirs. Intestinal biopsies and peripheral blood samples from HIV-infected patients who were either HAART naive or on long-term HAART were evaluated. Our data demonstrated that gut CD4+ T-cell restoration ranged from modest (<50%) to high (>50%), compared with uninfected controls. Despite persistent CD4+ T-cell proviral burden and residual immune activation in GALT during HAART, effective CD4+ T-cell restoration (>50%) was achieved, which was associated with enhanced Th17 CD4+ T-cell accumulation and polyfunctional anti-HIV cellular responses. Our findings suggest that a threshold of>50% CD4+ T-cell restoration may be sufficient for polyfunctional HIV-specific T cells with implications in the evaluation of vaccines and therapeutics.

Analysis of the Foxp3/scurfin gene in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease typified by transmural inflammation affecting any part of the gastrointestinal tract. CD4(+)CD25(+) regulatory T cells (T(reg) cells) play important roles in intestinal homeostasis. Adoptive transfer of regulatory T cells into mice with chronic colitis prevented inflammation and reappearance of normal intestinal architecture. The Foxp3 gene encodes a protein that is a member of the forkhead/winged-helix family of transcriptional regulators and is involved in the regulation of T cell activation. Mutation of Foxp3 gene may lead to development of autoimmune disease and inflammatory bowel disease. We investigated 10 single-nucleotide polymorphisms (SNPs) of the Foxp3 gene in CD patients and assessed allele and genotype frequencies in 93 patients with Crohn's disease (61 female and 32 male), 82 patients with primary biliary cirrhosis (PBC) (all female), and 108 normal controls (51 female and 57 male). We found that 3 SNPs (-6054, -/ATT; -3279, A/C; -924, A/G) in the promoter region and 1 SNP (IVS9+459, T/C) in the intron 9 region existed in CD patients. No significant differences of allele and genotype frequencies of all four SNPs were observed between CD patients and controls (both female and male groups). However, there was a significance difference of the comparison between PBC patients and controls in the IVS9+459 SNP. These data indicate that SNPs of the Foxp3 gene are not significantly associated with CD but are associated with PBC.

The biology of TNF blockade.

Rheumatoid arthritis and Crohn's disease are costly diseases that result in significant long-term patient disability. They are chronic inflammatory diseases that are associated with increased production of Tumor Necrosis Factor (TNF). Blockage of this cytokine with bio-engineered compounds has significantly changed therapy of these diseases and has ushered in the era of biological therapy. The pro-inflammatory role of TNF is mediated by its essential respiratory burst function that is effectively inhibited by anti-TNF therapy. Anti-TNF therapy is effective in approximately two-thirds of patients to whom it is administered, but the effect is temporary. Lack of response to anti-TNF therapy stems from interplay of host-factors including: host cytokine response, disease phenotype, and antibody response to the anti-TNF agents. NOD 2, a defect present in approximately 50% of Crohn's disease patients, bears no relationship to non-response. Additionally, TNF promoter gene polymorphisms and TNF receptor gene heterogeneity play a significant role in non-response and disease course/severity. Adverse effects of anti-TNF therapy include early and delayed hypersensitivity reactions, cell-mediated infections, lupus-like syndrome, demyelinating diseases, and exacerbation of CHF.

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis.

The characterization of differentially expressed genes provides a powerful tool for identifying molecules that may be involved in the pathogenesis of disease. We have used two independent techniques to identify overexpressed transcripts in bile duct cells and in liver from patients with primary biliary cirrhosis (PBC). In the first method, we used suppressive subtractive hybridization to compare mRNA from isolated PBC bile duct epithelial cells (BECs) to normal BECs and identified 71 clones as transcribed at higher levels in PBC-BECs. Amongst these clones, 62/71 had matches in a non-redundant nucleotide database and 9/71 had matches in an EST database. Of the 62 clones, 51/62 include a complexity of genes involved in cell proliferation, signal transduction, transcription regulation, RNA processing, carbohydrate metabolism and hypothetical/unknown proteins; 4/62 were identified as interstitial collagenase and collagenase precursors, 4/62 as ribosomal proteins, 3/62 as mitochondrial DNA. The mitochondrial cDNA sequences included cytochrome c oxidase, Wnt-13, and the pHL gene, a c-myc oncogene containing coxIII sequence. In the second method, we constructed cDNA libraries from three different PBC livers and sequenced a total of 12,324 independent clones. These 12,324 clones underwent virtual subtraction with 2,814,148 independent clones from Incyte LifeSeq libraries. Twenty one sequences were identified as unique to PBC liver. Collectively, these approaches identified a number of genes involved in signalling, RNA processing, mitochondrial function, inflammation, and fibrosis. Interestingly, both Wnt-13 and Notch transcripts are overexpressed in PBC liver. Further studies are needed to focus on the significance of these genes during the natural history of disease.

Microsporidial AIDS cholangiopathy due to Encephalitozoon intestinalis: case report and review.

Microsporidia are increasingly recognized as opportunistic infections in immunodeficient patients, predominantly patients with AIDS. The two microsporidia most commonly associated with disease in AIDS patients are Enterocytozoon bieneusi and Encephalitozoon intestinalis (previously known as Septata intestinalis). The most common clinical presentation of microsporidiosis in AIDS patients is diarrhea, most commonly caused by the Enterocytozoon bieneusi species. Encephalitozoon intestinalis is a recently described species that has been reported to cause disseminated human infection including cholangitis. We report a case of AIDS cholangiopathy that presented with abdominal pain and cholestatic liver tests. Ultrasound examination and ERCP revealed a picture of sclerosing cholangitis. Bile samples obtained at ERCP were negative for microsporidia; stool studies for microsporidia and cryptosporidia were also negative. No organisms were identified on routine light microscopy of the biopsy specimens from the duodenum, ampulla, and bile duct. E. intestinalis spores were demonstrated in the bile duct biopsies, by methylene blue and azure 11 staining and confirmed by electron microscopy. Albendazole therapy was successful in eradicating E. intestinalis with clinical improvement and improvement in CD4 count. However, the cholangiographic picture did not improve and repeat cholangiography revealed progressive bile duct injury. Albendazole therapy was delayed and may have been too late to prevent bile duct damage; the drug had to be approved by the US Food and Drug Administration for compassionate use. This is an unusual case of sclerosing cholangitis caused by an unusual organism and requiring biliary sphincterotomy and stent placement for progressive stricturing despite eradication of the infection.

Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR.

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.

Bacteroides fragilis enterotoxin gene sequences in patients with inflammatory bowel disease.

We identified enterotoxigenic Bacteroides fragilis in stool specimens of patients with inflammatory bowel disease and other gastrointestinal disorders. The organism was detected in 11 (13.2%) of 83 patients with inflammatory bowel disease. Of 57 patients with active disease, 19.3% were toxin positive; none of those with inactive disease had specimens positive for enterotoxigenic Bacteroides fragilis gene sequences.

Are infectious agents involved in primary biliary cirrhosis? A PCR approach.

BACKGROUND/AIMS: A variety of data suggest that microbial infections and, in particular, atypical mycobacteria infections, may either initiate and/or be associated with the pathogenesis of primary biliary cirrhosis. METHODS: To address this hypothesis, use was made of polymerase chain reaction techniques and primers specific for the 16s rRNA gene of Eubacteria, Archaeabacteria, Mycobacteria and Helicobacter to determine if such sequences were detectable in liver tissue specimens from 29 patients with primary biliary cirrhosis. Similar liver tissues from patients with primary sclerosing cholangitis, chronic hepatitis, alcoholic liver disease and otherwise normal donors were analyzed in parallel. Genomic DNA was extracted from each of these liver tissue specimens using sterile techniques to avoid possible laboratory contamination. The DNA was subjected to polymerase chain reaction amplification using bacterial genus specific primers and the amplified products cloned and sequenced. Sequence data were analyzed by searching for homology to existing genes. RESULTS: Sequences from primary biliary cirrhosis and control livers corresponded to those found in a variety of bacteria, but no consensus sequence was found in primary biliary cirrhosis specimens. Neither Archaeabacteria nor Mycobacteria products were detected in liver specimens of patients with primary biliary cirrhosis, and Helicobacter pylori DNA was detected in only one primary biliary cirrhosis patient. CONCLUSIONS: Although bacterial infection, particularly with intracellular organisms, has been suggested to play a role in the initiation of primary biliary cirrhosis, there is no evidence from this study to suggest an ongoing chronic infectious process.

Is there a serological difference between men and women with primary biliary cirrhosis?

OBJECTIVE: Primary biliary cirrhosis (PBC) is an autoimmune disease affecting small intrahepatic bile ducts of the liver, causing destruction of the epithelium that results in eventual fibrosis and scarring. We still lack a complete epidemiological description of this disease, although interesting geographic differences in prevalence have been described. One consistent feature has been the relative scarcity of men with PBC. In fact, published ratios of women to men range from 3:1 to as high as 22:1. Thus far, the only clinical difference reported between men and women with PBC is a putative higher risk of hepatocarcinoma in men. Previous serological studies have shown that about 95% of all patients possess antimitochondrial antibodies to members of the highly conserved 2-oxo-acid dehydrogenase family of proteins, namely pyruvate dehydrogenase complex E2 (PDC-E2), branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2), and 2-oxo glutarate dehydrogenase complex E2 (OGDC-E2). However, there has been no information as to whether there is a difference in serological response between men and women. Using the serological hallmark of antimitochondrial antibodies (AMAs) and taking advantage of the availability of recombinant mitochondrial autoantigens, investigations were performed to determine if there were any serological differences between men and women with PBC. METHODS: Sera were collected from 88 patients with PBC, of whom 46 were men and 42 were women. Using a combination of immunoblotting and enzyme-linked immunoabsorbent assay (ELISA) against beef heart mitochondria (BHM), recombinant PDC-E2, BCOADC-E2, and OGDC-E2, we determined the relative autoantibody reactivities of our study population. RESULTS: Both men and women with PBC produced high titer antimitochondrial antibodies. The frequency of reactivity was similar in both groups and included, in descending order, PDC-E2, E3BP (Protein X), BCOADC-E2, and finally OGDC-E2. More importantly, antigenic specificity was nearly identical regardless of gender. CONCLUSIONS: AMAs are the serological hallmark of PBC in both men and women, and there is no significant difference in reactivity between the two groups of patients.

Sclerosing mesenteritis seen clinically as pancreatic pseudotumor: two cases and a review.

Sclerosing mesenteritis is an uncommon nonneoplastic inflammatory process in the mesentery that is seen as a pseudotumor, usually involving the small bowel mesentery, the mesenteric fat, and less commonly, the mesentery of the large bowel. We report two cases of sclerosing mesenteritis and review the literature on this rare disease. Both patients had pain, profound weight loss, and a mass on computed tomography (CT) scan of the abdomen. The provisional diagnosis was pancreatic neoplasm on the basis of clinical presentation and imaging studies. The diagnosis of sclerosing mesenteritis was established by histologic findings in biopsy material obtained at laparotomy in both cases. Interval histologic studies in one patient who had a high CA 19-9 level, progressive biliary ductal and partial duodenal compression, revealed a transitional histologic pattern from predominant inflammation and fat necrosis to predominant fibrosis. This may explain the varied descriptive terms used in the literature to describe this entity.

Detection of Bacteroides fragilis enterotoxin gene by PCR.

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.

A study of antimitochondrial antibodies in a random population in Estonia.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the spontaneous destruction of the small intrahepatic bile ducts. The hallmark serologic feature of PBC is the presence of high-titer antimitochondrial antibodies (AMA). Both the incidence and prevalence of PBC varies geographically; epidemiological data may provide valuable insight regarding the pathogenic mechanisms and etiology of disease. Thus far, the majority of studies on the occurrence of PBC and AMAs have been derived from autopsy, mortality figures, or hospital admission records. The numbers reported reflect only those patients with clinical disease. To address this issue, an adult population sample representing all age groups in the village of Karksi-Nuia in southern Estonia was selected for a study of AMA incidence. This village has unique features that make it ideal for such a study. First, the village is remote and a substantial number of families have lived in the area for generations. There is also a limited influx of new families into the village, therefore providing a limited genetic repertoire. In this unselected adult population, we examined AMA incidence by both immunoblot and ELISA, using native and recombinant antigens. Of the 1461 people studied, 13 (0.89%) were AMA positive. A similar frequency (0.96%) was found among 104 persons from a neighboring village, who subsequently joined the study. Our study suggests that the presence of AMA in Estonia is in agreement with the reported incidence of less than 1% AMA in a mixed hospital population.

Association of cocaine and methamphetamine use with giant gastroduodenal ulcers.

OBJECTIVES: Giant gastric and duodenal ulcers (>2-3 cm in greatest dimension) are reported to have higher rates of complication and mortality and to be associated with increasing age, renal failure, and use of nonsteroidal antiinflammatory drugs (NSAIDs). This study investigated the outcome and associations of gastric and duodenal ulcers >2.5 cm compared to ulcers of lesser size. METHODS: Records from all patients with gastric and duodenal ulcers >0.5 cm diagnosed by upper endoscopy between January 1994 and September 1995 were studied for evidence of concurrent use of aspirin, NSAIDs, methamphetamine, and cocaine, as well as for transfusion requirements, length of hospital stay, mortality, surgery, rebleeding, Helicobacter pylori infection, and malignancy. RESULTS: A logistic regression analysis of the 220 patients identified revealed that recent methamphetamine and/or cocaine use was significantly predictive of giant ulcer formation (p = 0.0002) with an odds ratio of 9.66. Also significant was younger age (p = 0.026) and aspirin or NSAID use (p = 0.046). H. pylori infection was significant only for giant gastric ulcers (p = 0.031). Ulcer size did not predict mortality, rate of rebleeding, requirement for surgery, transfusion requirements, or length of hospital stay. However, giant gastric ulcers were significantly more likely to be malignant (p = 0.002). CONCLUSIONS: Giant gastric and duodenal ulcers were strongly associated with stimulant abuse. They were also associated with younger age and use of aspirin or NSAIDs. Additionally, giant gastric ulcers were associated with malignancy and H. pylori infection. Ulcer size did not predict rate of complications or outcome.

Treatment of irritable bowel syndrome with Lacteol Fort: a randomized, double-blind, cross-over trial.

OBJECTIVE: To assess the efficacy of Lacteol Fort, an antidiarrheal drug, in patients suffering from the chronic intestinal disease known as irritable bowel syndrome (IBS). DESIGN: The randomized, double-blind, cross-over trial versus placebo was carried out from 1992 to 1994. This trial consisted of administering a 6-wk treatment with a first drug (Lacteol Fort or placebo), followed by a wash-out period of 2 wk, and then the administration of a second drug for a further 6 wk (placebo or Lacteol Fort). Among the 29 patients eligible after recruitment, 18 adults with well documented IBS fulfilled the inclusion criteria. Four patients were dropped for loss of materials used in the study and seven for lack of compliance. The patient's initial state was assessed using a questionnaire relating to six criteria: abdominal pain, bloating or gas, daily number of stools, consistency, mucus content, and general physical state. During the treatment, these criteria were evaluated daily by the patients themselves. RESULTS: All investigated criteria were scored, and then a daily mean index was calculated. The statistical analysis of the daily mean index values showed that the number of patients (nine cases) obtaining better results with Lacteol Fort than with placebo was statistically significant (p = 0.018). CONCLUSIONS: This double-blind, placebo-controlled, cross-over trial demonstrated that Lacteol Fort leads to a statistically significant therapeutic benefit in 50% of patients, when taking into consideration all of the six selected clinical criteria considered representative of IBS.

Analysis of function, specificity and T cell receptor expression of cloned mucosal T cell lines in Crohn's disease.

Monoclonal populations of mucosal T cells were established from the earliest visible lesions in eight patients with well defined Crohn's disease. The FACS phenotype of all the mucosal derived clones to date are TCR alpha/beta+, CD3+, CD4+, and CD45RO+ memory cells. TCR variable region Beta chain analysis revealed predominantly V beta families 1, 2, 5.1, 5.2, 6, 7 and 8, with V beta family analysis supporting antigen expansion in the diseased mucosa. Putative autoreactivity was evaluated by stimulating individual clones with a battery of antigens and determining proliferation and IL-2 production by thymidine incorporation at 72 h. Antigens tested included crude Crohn's diseased (CD) colon and small bowel homogenates, CD brush border preparations, crude CD colon and small bowel mucin, and purified CD small bowel mucin. Controls included clone, APC, tetanus toxoid and either PHA or Staphylococcus enterotoxin B. A total of 200 clones were studied with 29.5% or 59 clones demonstrating proliferation and/or IL-2 production. T cell receptor V beta gene usage evaluated in a small number of reactive clones correlated with the expanded patient families. Seven of the fifteen represented families revealed diverse T cell receptor gene use and no disease overlap.

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.