Dorsey Bass

Expression of mucosal homing receptor alpha4beta7 by circulating CD4+ cells with memory for intestinal rotavirus.

The integrin alpha4beta7 mediates lymphocyte binding to mucosal addressin cell adhesion molecule-1, and its expression defines lymphocytes capable of trafficking through the intestines and the intestinal lymphoid tissues. We examined the ability of discrete alpha4beta7(hi) and alpha4beta7- subsets of circulating memory phenotype (CD45RA-) CD4+ T cells to proliferate in response to rotavirus, a ubiquitous intestinal pathogen. alpha4beta7(hi) memory (CD45RA-) CD4+ T cells displayed much greater reactivity to rotavirus than alpha4beta7- memory or naive (CD45RA+) CD4+ T cells. In contrast, alpha4beta7- memory cells were the predominant population responsive to mumps antigen after intramuscular vaccination. Our results are consistent with the conclusion that natural rotavirus infection, an enteric pathogen, results in a specific circulating memory CD4+ response that is largely limited to the gut-homing alpha4beta7+ subpopulation. This phenotype is not shared with memory cells elicited by intramuscular immunization (shown here) or by skin contact allergens. The results support the hypothesis that gut trafficking memory CD4+ T cells comprise cellular memory for intestinal antigens and suggest that regulated expression of alpha4beta7 helps target and segregate intestinal versus systemic immune response.

Inflammatory bowel disease-attributable costs and cost-effective strategies in the United States: A review

&NA; The United States spends more for healthcare than any other country in the world. With the rising prevalence of both Crohns disease and ulcerative colitis, inflammatory bowel disease (IBD) represents the leading chronic gastrointestinal disease with increasing healthcare expenditures in the US. IBD costs have shifted from inpatient to outpatient care since the introduction of biologic therapies as the standard of care. Gastroenterologists need to be aware of the national cost burden of IBD and clinical practices that optimize cost‐efficiency. This investigation offers a systematic review of the economics of IBD and evidence‐based strategies for cost‐effective management. (Inflamm Bowel Dis 2010;)

Wernicke Encephalopathy and Beriberi During Total Parenteral Nutrition Attributable to Multivitamin Infusion Shortage

Objective. Wernicke encephalopathy (WE) is an acute neurologic disorder characterized by a triad of ophthalmoplegia, ataxia, and mental confusion. WE is attributable to thiamine (vitamin B1) deficiency. Beriberi is the systemic counterpart of thiamine deficiency and often manifests in cardiovascular collapse. WE is usually associated with alcoholism and malnutrition. It has also been seen in people with gastrointestinal diseases with malabsorption. Patients who have received total parenteral nutrition (TPN) without proper replacement of thiamine have also developed WE. Since November 1996, there has been a shortage of multivitamin infusion (MVI). Many patients who were on chronic TPN with MVI ceased to receive the MVI and were converted to an oral form of the multivitamin. As a result, there have been several reports of children and adults on TPN who have developed WE as a result of thiamine deficiency. With this case report, we bring to attention the association of the MVI shortage and WE. Early diagnosis of WE is important, because if it is treated with thiamine in the acute stages, the neurologic and cardiovascular abnormalities can be reversed. Case Report. We report a 20-year-old female patient with Crohns disease who developed WE as a result of thiamine deficiency. She had Crohns disease since age 9 years and was on chronic TPN. Two months before admission, MVI was discontinued in the TPN because of the shortage of its supply. An oral multivitamin tablet was substituted instead. She was admitted to the hospital for persistent vomiting. In the hospital, she continued to receive TPN without MVI, but continued taking an oral multivitamin preparation. Two weeks after admission, she developed signs of WE including diplopia, ophthalmoplegia, nystagmus, and memory disturbance. She also developed hypotension that was thought to be caused by beriberi. She was treated with 50 mg of intravenous thiamine. Within hours of the intravenous thiamine, her hypotension resolved. The day after the infusion, she no longer complained of diplopia, and her ophthalmoplegia had improved dramatically. Magnetic resonance imaging showed several areas of abnormally high signal on T2-weighted images in the brainstem, thalamus, and mamillary bodies. The topographic distribution of these changes was typical of WE. After 2 months, her mental status and neurologic status had recovered completely. Conclusion. WE and thiamine deficiency should be considered in all patients with malabsorption, malnutrition, and malignancies. WE from thiamine deficiency can occur as a result of cessation of MVI in the TPN infusion. Even if an oral multivitamin preparation is given instead of MVI, patients with malabsorption may not absorb thiamine adequately. Prompt diagnosis of WE is important because it is potentially fatal and readily treatable with thiamine supplementation. Early recognition of WE may be more difficult in children, because the classic triad of symptoms may not develop fully. Magnetic resonance imaging may be useful in these cases to confirm the diagnosis of WE. Because the shortage of MVI is expected to be a long-term, there are likely to be more cases of WE in the pediatric population of TPN-dependent children. Because there is no shortage of intravenous thiamine, it should be administered with TPN even if MVI is not available.

Proteolytic processing of the astrovirus capsid.

ABSTRACT To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35S]methionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (105-fold increase) after trypsin but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.

Studies of the Role for NSP4 in the Pathogenesis of Homologous Murine Rotavirus Diarrhea

A rotavirus (RV) nonstructural protein, NSP4, has recently been proposed to function as an enterotoxin in the pathogenesis of RV diarrhea. The role of NSP4 in the pathogenesis of RV diarrhea was examined by infecting cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice with virulent murine RV and by comparing deduced amino acid sequences of RV gene 10 encoding NSP4 from three distinct sets of virulent and tissue culture-adapted avirulent variant RVs. Homozygous CFTR (CFTR-/-) mice, which do not respond to any known intestinal secretagogues, experienced diarrhea comparable to that in normal CFTR+/+ littermates after RV challenge. Comparison of amino acid sequences of NSP4 from virulent and attenuated pairs of RVs failed to show consistent or significant changes. Together, these data suggest that enterotoxigenic properties of RV NSP4 are not critical in the pathogenesis of murine RV diarrhea and that attenuation of murine RVs is not usually mediated by mutations in the gene encoding NSP4.

Astrovirus, adenovirus, and rotavirus in hospitalized children: Prevalence and association with gastroenteritis

Background Agents of viral gastroenteritis such as astrovirus, rotavirus, and adenovirus are common pediatric pathogens accounting for many physician visits, hospital admissions, and nosocomial infections. Previous hospital-based prevalence studies have examined mainly symptomatic children. Purpose To evaluate the prevalence of astrovirus, rotavirus, and adenovirus infections among hospitalized children less than 6 years of age, regardless of symptoms, and determine association with gastroenteritis. Methods From September 1998 to June 2000, stool specimens were collected twice weekly from children less than five years of age admitted to two wards in a tertiary-care childrens hospital. A total of 480 samples were obtained from 309 hospitalizations. Stools were examined using antibody-based ELISA for astrovirus, rotavirus, and adenovirus. Clinical data was abstracted from patient records. Results Twenty one percent of the children had gastroenteritis symptoms at some point during their hospitalizations (43% were hospital acquired). Astrovirus was detected in 5.2% of all children compared to 6.8% with rotavirus and 0.8% with adenovirus serotypes 40 or 41. Nosocomial acquisition was common. Seventy five percent of astrovirus infections and 90% rotavirus infections were symptomatic. Astrovirus infections were significantly more likely to occur in younger infants and in children with compromised immunity. Rotavirus infections were significantly more likely to cause dehydration. In a three-year passive surveillance of gastroenteritis at the hospital, astrovirus and rotavirus infections peaked simultaneously in winter months. Conclusions Rotavirus and astrovirus are common symptomatic infections on pediatric wards and contribute greatly to inpatient morbidity. Adenoviruses played a limited role in gastroenteritis in hospitalized children in this study.

Maturation and Trafficking Markers on Rotavirus-Specific B Cells during Acute Infection and Convalescence in Children

ABSTRACT We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD− B cells are predominantly large, CD38high, CD27high, CD138+/−, CCR6−, α4β7+, CCR9+, CCR10+, cutaneous lymphocyte antigen-negative (CLA−), L-selectinint/−, and sIgM+, sIgG−, sIgA+/− lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38int/−, CD27int/−, CCR6+, α4β7+/−, CCR9+/− and CCR10−, most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.

Identification and partial characterization of a rhesus rotavirus binding glycoprotein on murine enterocytes

In order to assess the possibility that rotavirus binds to a specific cellular receptor on enterocytes, we have used a viral overlay protein blot assay to study viral binding to murine intestinal brush border membranes (BBM). Infectious double-shelled particles of rhesus rotavirus bound specifically to two approximately 300- and 330-kDa glycoproteins from BBM prepared from suckling mice. Significantly less rotavirus binding was observed when adult BBM were examined. Rats have never been shown to harbor natural group A rotavirus infection and correspondingly, rat BBM showed no rotavirus binding activity. In suckling mice, rotavirus was found to bind to villus tip membranes to a much greater extent than to crypt preparations. Rotavirus binding activity was abolished by treatment of membrane preparations with protease. Analysis by glycolytic digestion of BBM with N- and O-glyconases revealed evidence for both N- and O-linked glycosylation of the rotavirus binding protein. Also neuraminidase digestion showed that O-linked sialic acid residues were required for virus binding. Monoclonal antibodies which immunoprecipitate the 300-kDa viral binding glycoprotein react with the apical surface of suckling but not adult enterocytes by Western blot. Baculovirus-expressed vp4, the rotavirus outer capsid spike protein, bound to the 300- and 330-kDa proteins and competed with rotavirus particles for binding sites. The ability of rotavirus to bind via vp4 to large BBM glycoproteins correlates with in vivo rotavirus cell tropism and host range restriction. Specific host cell receptor expression may be important in rotavirus pathogenesis.

Interferon Gamma and Interleukin 1, But Not Interferon Alfa, Inhibit Rotavirus Entry Into Human Intestinal Cell Lines

Abstract BACKGROUND & AIMS: Rotavirus, an important agent of gastroenteritis in children, causes diarrhea by infecting differentiated villus enterocytes in the small intestine. The aim of this study was to determine whether cytokines that can be expressed by mucosal cells have an effect on the rotavirus susceptibility of cultured human enterocytes. METHODS: Caco-2 and HT-29 cells were pretreated with various cytokines before challenge with rotavirus. RESULTS: Interleukin (IL)-1, interferon (IFN)-alpha, and IFN-gamma pretreatment led to a dose-dependent resistance to rotavirus infection. Maximum effects occurred after 72 hours of pretreatment, whereas no detectable inhibition occurred with <12 hours of pretreatment. Liposomal transfection of single-shelled and double-shelled rotavirus particles bypassed the block to rotavirus replication in IFN-gamma- and IL-1- treated but not IFN-alpha-treated cells. Binding studies with purified, metabolically labeled rotavirus showed no significant difference among IFN-gamma- and IFN-alpha-treated and control Caco-2 cells. Viral entry into Caco-2 cells was significantly inhibited by IFN-gamma and IL-1 but not IFN-alpha. CONCLUSIONS: IFN-alpha and IFN-gamma induce rotavirus resistance by different mechanisms, suggesting that cytokines play a role in host defense against viral agents by changing the phenotype of intestinal epithelial cells. (Gastroenterology 1997 Jul;113(1):81-9)

Lack of a Role for Type I and Type II Interferons in the Resolution of Rotavirus-Induced Diarrhea and Infection in Mice

Rotavirus infects the intestinal epithelium of most mammalian species and causes diarrhea in infants. Previously, we have shown that both type I and II human interferons (IFNs) have potent and mechanistically discreet antiviral effects in vitro against rotavirus. We have also shown that adult IFN-gamma knockout (-/-) mice have no alteration in clearance of primary rotavirus infection. In the present studies, we wished to determine the importance of both IFN types in modulation of degree and duration of disease and infection in mice. Immunocompetent suckling mice were treated orally (5,000 IU) or parenterally (500 IU) with type I and II murine IFNs before and after challenge with virulent murine rotavirus. Treated animals developed diarrhea indistinguishable from that observed in untreated control mice. In other experiments, type I IFN receptor -/- suckling mice and IFN-gamma-/- suckling mice developed diarrhea of similar characteristics and duration and had comparable quantities of viral antigen in their intestines as did immunocompetent mice. Furthermore, type I IFN receptor -/- adult mice infected with rotavirus shed equivalent quantities of viral antigen and with similar kinetics as the control mice. Thus, IFNs do not seem to be major inhibitors of rotavirus diarrhea or replication in mice.