Samuel B. Ho

Intestinal Goblet Cells and Mucins in Health and Disease: Recent Insights and Progress

The mucus layer coating the gastrointestinal tract is the front line of innate host defense, largely because of the secretory products of intestinal goblet cells. Goblet cells synthesize secretory mucin glycoproteins (MUC2) and bioactive molecules such as epithelial membrane-bound mucins (MUC1, MUC3, MUC17), trefoil factor peptides (TFF), resistin-like molecule β (RELMβ), and Fc-γ binding protein (Fcgbp). The MUC2 mucin protein forms trimers by disulfide bonding in cysteine-rich amino terminal von Willebrand factor (vWF) domains, coupled with crosslinking provided by TFF and Fcgbp proteins with MUC2 vWF domains, resulting in a highly viscous extracellular layer. Colonization by commensal intestinal microbiota is limited to an outer “loose” mucus layer, and interacts with the diverse oligosaccharides of mucin glycoproteins, whereas an “inner” adherent mucus layer is largely devoid of bacteria. Defective mucus layers resulting from lack of MUC2 mucin, mutated Muc2 mucin vWF domains, or from deletion of core mucin glycosyltransferase enzymes in mice result in increased bacterial adhesion to the surface epithelium, increased intestinal permeability, and enhanced susceptibility to colitis caused by dextran sodium sulfate. Changes in mucin gene expression and mucin glycan structures occur in cancers of the intestine, contributing to diverse biologic properties involved in the development and progression of cancer. Further research is needed on identification and functional significance of various components of mucus layers and the complex interactions among mucus layers, microbiota, epithelial cells, and the underlying innate and adaptive immunity. Further elucidation of the regulatory mechanisms involved in mucin changes in cancer and inflammation may lead to the development of novel therapeutic approaches.

Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer.

BACKGROUND/AIMS Several studies have reported Northern blot data showing that mucin is expressed in a tissue-specific manner. To determine whether expression is limited to specific cell types within these tissues requires histological analysis. METHODS Both immunocytochemistry and in situ hybridization were used to identify cell types expressing the MUC2 and MUC3 mucins in the human small intestine, colon, and colon carcinoma. RESULTS In the normal small intestine and colon, an antibody recognizing the MUC2 apomucin stained goblet cells. In contrast, an antibody recognizing the MUC3 apomucin stained both goblet and absorptive cells. Consistent with this, in situ hybridization showed MUC2 messenger RNA (mRNA) only in goblet cells and MUC3 mRNA in both goblet and absorptive cells. In several samples of moderately well-differentiated colon cancer, MUC2 and MUC3 showed distinct patterns of expression, but the expression level of each was reduced compared with levels in normal tissue; there was considerable tumor-to-tumor and cell-to-cell variability using both mucin antibodies and complementary DNA probes. CONCLUSIONS Individual mucin genes have distinct patterns of expression within mucin-producing tissues, suggesting that the various mucin gene products play distinct functional roles.

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression

BACKGROUND & AIMS Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunners glands of the duodenum. CONCLUSIONS MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Prospective multicenter study of eligibility for antiviral therapy among 4,084 U.S. veterans with chronic hepatitis C virus infection

BACKGROUND:Many veterans may not be candidates for hepatitis C virus (HCV) treatment due to contraindications to therapy. The aims of this study were to determine the proportion of HCV-infected veterans who were eligible for interferon alfa and ribavirin therapy and to evaluate barriers to HCV treatment.METHODS:We prospectively enrolled 4,084 veterans who were referred for HCV treatment over a 1-yr period at 24 Veterans Affairs (VA) Medical Centers. Treatment candidacy was assessed using standardized criteria and the opinion of the treating clinician.RESULTS:Overall, 32.2% (95% CI, 30.8–33.7%) were candidates for HCV treatment according to standardized criteria, whereas 40.7% (95% CI, 39.2–42.3%) were candidates in the opinion of the treating clinician. Multivariable analysis identified ongoing substance abuse (OR = 17.68; 95% CI, 12.24–25.53), comorbid medical disease (OR = 9.62; 95% CI, 6.85–13.50), psychiatric disease (OR = 9.45; 95% CI, 6.70–13.32), and advanced liver disease (OR = 8.43; 95% CI, 4.42–16.06) as the strongest predictors of not being a treatment candidate. Among patients who were considered treatment candidates, 76.2% (95% CI, 74.0–78.3%) agreed to be treated and multivariable analysis showed that persons ≥50 yr of age (OR = 1.37; 95% CI, 1.07–1.76) and those with >50 lifetime sexual partners (OR = 1.44; 95% CI, 1.08–1.93) were more likely to decline treatment.CONCLUSIONS:The majority of veteran patients are not suitable candidates for HCV treatment because of substance abuse, psychiatric disease, and comorbid medical disease, and many who are candidates decline therapy. Multidisciplinary collaboration is needed to overcome barriers to HCV therapy in this population.

Influence of psychiatric diagnoses on interferon-α treatment for chronic hepatitis C in a veteran population

OBJECTIVE:The prevalence of psychiatric problems and substance abuse is high in the veteran population with hepatitis C. The purpose of this study was to retrospectively analyze the effect of preexisting psychiatric conditions in veteran patients undergoing treatment with interferon α-2b (IFN-α) with respect to adverse events, compliance, and treatment response.METHODS:Thirty-three veterans with chronic hepatitis C were treated with IFN-α (5 million units three times weekly) for 6 months, followed by a tapering dose for an additional 6 months. All patients fulfilled standard criteria for treatment eligibility. Psychiatric diagnoses, adverse events, and virological and biochemical responses to therapy were determined.RESULTS:Nineteen of 33 (58%) patients with hepatitis C had documented psychiatric conditions before starting IFN-α therapy. Of the patients with preexisting psychiatric diagnoses, 13/19 (68%) developed major adverse events requiring intervention or discontinuation of therapy. In contrast, 4/14 (29%) patients without psychiatric diagnoses developed major adverse events (p = 0.024) In the psychiatric group, 6/19 (32%) developed major neuropsychiatric side effects compared with 2/14 patients (14%) in the nonpsychiatric group (p = 0.25). Patients with and without psychiatric diagnoses had equivalent biochemical and virological responses to therapy. Overall, only 2/33 (6%) patients had a sustained virological response.CONCLUSIONS:Veterans with chronic hepatitis C and psychiatric diagnoses experienced a significantly greater number of major adverse events during treatment with IFN-α. Veteran patients with hepatitis C should be carefully screened for psychiatric conditions and may require more intensive monitoring during IFN-α therapy.

Integrated Psychiatric/Medical Care in a Chronic Hepatitis C Clinic: Effect on Antiviral Treatment Evaluation and Outcomes

OBJECTIVES:Psychiatric and substance use disorders are common in hepatitis C patients and represent barriers to antiviral treatment. We evaluated the effect of integrating psychiatric and medical care on evaluation for and initiation of antiviral treatment in a cohort of 184 patients with chronic hepatitis C.METHODS:Integrated care consisted of screening for psychiatric problems with Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), Beck Depression Inventory (BDI), Urine Drug Screen (UDS), and Primary Care Posttraumatic Stress Disorder (PC-PTSD) screens, referral based on specified cutoff scores to an established mental health (MH) provider, to a colocated psychiatric clinical nurse specialist (PCNS), or both. Data were collected retrospectively by chart review.RESULTS:Most patients (149/184, 81.0%) had at least one positive screen, 25.5% had a positive UDS. Among patients with positive screens, 38.3% had established MH providers, 47.0% had no MH provider and were referred to the PCNS, and 15.0% refused any psychiatric referral. Patients receiving integrated care with a colocated PCNS were significantly more likely to complete evaluation for and start antiviral treatment than other patients with positive screens, and at a rate similar to that of patients with negative screens. Patients with positive screens followed by any MH provider had significantly greater adherence to antiviral therapy than patients without positive screens.CONCLUSION:An integrated MH and medical approach was associated with rates of antiviral therapy recommendation and initiation similar to patients without risks for psychiatric or substance use problems. MH care was associated with improved adherence to antiviral therapy. Integrated care offers promise as an approach for addressing psychiatric comorbidity in this traditionally difficult to treat population.

Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non‐repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia. Int. J. Cancer 80:210–218, 1999. Published 1999 Wiley‐Liss, Inc.

Supplementation of Saturated Long-chain Fatty Acids Maintains Intestinal Eubiosis and Reduces Ethanol-induced Liver Injury in Mice

BACKGROUND & AIMS Alcoholic liver disease is a leading cause of mortality. Chronic alcohol consumption is accompanied by intestinal dysbiosis, and development of alcoholic liver disease requires gut-derived bacterial products. However, little is known about how alterations to the microbiome contribute to pathogenesis of alcoholic liver disease. METHODS We used the Tsukamoto-French mouse model, which involves continuous intragastric feeding of isocaloric diet or alcohol for 3 weeks. Bacterial DNA from the cecum was extracted for deep metagenomic sequencing. Targeted metabolomics assessed concentrations of saturated fatty acids in cecal contents. To maintain intestinal metabolic homeostasis, diets of ethanol-fed and control mice were supplemented with saturated long-chain fatty acids (LCFA). Bacterial genes involved in fatty acid biosynthesis, amounts of lactobacilli, and saturated LCFA were measured in fecal samples of nonalcoholic individuals and patients with active alcohol abuse. RESULTS Analyses of intestinal contents from mice revealed alcohol-associated changes to the intestinal metagenome and metabolome, characterized by reduced synthesis of saturated LCFA. Maintaining intestinal levels of saturated fatty acids in mice resulted in eubiosis, stabilized the intestinal gut barrier, and reduced ethanol-induced liver injury. Saturated LCFA are metabolized by commensal Lactobacillus and promote their growth. Proportions of bacterial genes involved in fatty acid biosynthesis were lower in feces from patients with active alcohol abuse than controls. Total levels of LCFA correlated with those of lactobacilli in fecal samples from patients with active alcohol abuse but not in controls. CONCLUSIONS In humans and mice, alcohol causes intestinal dysbiosis, reducing the capacity of the microbiome to synthesize saturated LCFA and the proportion of Lactobacillus species. Dietary approaches to restore levels of saturated fatty acids in the intestine might reduce ethanol-induced liver injury in patients with alcoholic liver disease.

Selective induction of mucin-3 by hypoxia in intestinal epithelia.

Epithelial cells line mucosal surfaces (e.g., lung, intestine) and critically function as a semipermeable barrier to the outside world. Mucosal organs are highly vascular with extensive metabolic demands, and for this reason, are particularly susceptible to diminished blood flow and resultant tissue hypoxia. Here, we pursue the hypothesis that intestinal barrier function is regulated in a protective manner by hypoxia responsive genes. We demonstrate by PCR confirmation of microarray data and by avidin blotting of immunoprecipitated human Mucin 3 (MUC3), that surface MUC3 expression is induced in T84 intestinal epithelial cells following exposure to hypoxia. MUC3 RNA is minimally detectable while surface protein expression is absent under baseline normoxic conditions. There is a robust induction in both the mRNA (first evident by 8 h) and protein expression, first observed and maximally expressed following 24 h hypoxia. This is followed by a subsequent decline in protein expression, which remains well above baseline at 48 h of hypoxia. Further, we demonstrate that this induction of MUC3 protein is associated with a transient increase in the barrier restorative peptide, intestinal trefoil factor (ITF). ITF not only colocalizes with MUC3, by confocal microscopy, to the apical surface of T84 cells following exposure to hypoxia, but is also found, by co‐immunoprecipitation, to be physically associated with MUC3, following 24 h of hypoxia. In exploration of the mechanism of hypoxic regulation of mucin 3 expression, we demonstrated by luciferase assay that the full‐length promoter for mouse Mucin 3 (Muc3) is hypoxia‐responsive with a 5.08 ± 1.76‐fold induction following 24 h of hypoxia. Furthermore, analysis of both the human (MUC3A) and mouse (Muc3) promoters revealed potential HIF‐1 binding sites which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia‐regulating transcription factor HIF‐1α. Taken together, these studies implicate the HIF‐1α mediated hypoxic induced expression of mucin 3 and associated ITF in the maintenance of intestinal barrier function under hypoxic conditions. J. Cell. Biochem. 99: 1616–1627, 2006.

The Carboxyl-terminal Sequence of the Human Secretory Mucin, MUC6 ANALYSIS OF THE PRIMARY AMINO ACID SEQUENCE

The distribution of MUC6 suggests that its primary function is protection of vulnerable epithelial surfaces from damaging effects of constant exposure to a wide range of endogenous caustic or proteolytic agents. A combination of genomic, cDNA. and 3′ rapid amplification of cDNA ends techniques was used to isolate the carboxyl-terminal end of MUC6. The 3′ nontandem repeat region contained 1083 base pairs of coding sequence (361 amino acids) followed by 632 base pairs of 3′-untranslated region. The coding sequence consists of two distinct regions; region 1 contained the initial 270 amino acids (62% Ser-Thr-Pro with no Cys residues), and region 2 contained the COOH-terminal 91 amino acids (22% Ser-Thr-Pro with 12% Cys). Although region 1 had no homology to any sequences in GenBank, region 2 had approximately 25% amino acid homology to the COOH-terminal regions of human mucins MUC2, -5, and -5B and von Willebrand factor. The shortness of region 2 would leave little of the peptide backbone exposed to a potentially hostile environment. Antibody studies suggest that MUC6 in its native form exists as a disulfide-bonded multimer. The conservation of the 11 cysteine positions in region 2 suggests the importance of this short region to mucin polymerization.