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Enteroaggregative Escherichia coli(EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. colistrain associated with human disease. A variety of bacterial characteristics—growth in vitro,biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells—were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism’s ability to form biofilm (P< 0.001), adhere to IEC-6 epithelial cells (P< 0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P< 0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (>3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P< 0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (1010CFU/mouse) significantly abrogated growth shortfalls (by >90%; P< 0.01); furthermore, stool shedding was reduced (days 9–11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium’s effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition.

Neutrophilia is a characteristic of hemolytic uremic syndrome caused by Shiga toxin (Stx2)-producing Escherichia coli. However, the role of neutrophils in the toxin-induced renal injury occurring in enterohemorrhagic E. coliinfection remains undefined. We report the trafficking of neutrophils to the kidney of C57BL/6 mice throughout a 72-hour time course after challenge with purified E. coliStx2 and lipopolysaccharide (LPS). Increased neutrophils were observed in the renal cortex, particularly within the glomeruli where a more than fourfold increase in neutrophils was noted within 2 hours after challenge. Using microarray analysis, an increased number of transcripts for chemoattractants CXCL1/KC (69-fold at 2 hours) and CXCL2/MIP-2 (29-fold at 2 hours) were detected. Ribonuclease protection assays, Northern blotting, enzyme-linked immunosorbent assay, and immunohistochemistry confirmed microarray results, showing that both chemokines were expressed only on the immediate periglomerular epithelium and that these events coincided with neutrophil invasion of glomeruli. Co-administration of Stx2 with LPS enhanced and prolonged the KC and MIP-2 host response (RNA and protein) induced by LPS alone. Immunoneutralization in vivoof CXCL1/KC and CXCL2/MIP-2 abrogated neutrophil migration into glomeruli by 85%. These data define the molecular basis for neutrophil migration into the kidney after exposure to virulence factors of Shiga toxin-producing E. coliO157:H7.

Gastric carcinoma is one of the leading causes of cancer-related death worldwide, but the mechanisms underlying its development and progression still remain largely uncharacterized. As loss of trefoil factor 1 (TFF1) expression leads to neoplastic growth in the antropyloric mucosa of mice, the authors sought to comprehensively study the human TFF1 gene in primary gastric carcinomas. The authors studied the human TFF1 gene in primary gastric carcinomas and normal gastric mucosa at the DNA, RNA, and protein levels through DNA sequencing, quantitative real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry. Strikingly, TFF1 mRNA expression was significantly decreased in all 37 gastric carcinomas studied compared with normal gastric mucosa. Furthermore, six tumor/normal pairs with available histologic samples demonstrated a marked decrease in protein expression in tumor samples. Screening of the entire TFF1 coding region in a panel of 42 human gastric tumors did not reveal any somatic mutations, although a few rare germline sequence variants were identified. These findings demonstrated a significant decrease in the TFF1 transcript in the majority of human gastric carcinomas along with a corresponding reduction in protein expression, both of which occurred in the absence of gene mutation. Dysregulation of TFF1 expression at the transcript level was a critical event in the development of most gastric carcinomas. Cancer 2003. © 2003 American Cancer Society.

The biology of prostanoids in the normal human colon is only beginning to be understood. We used in situ and in vitro techniques to define the lineage, number, per cell enzyme content, and epithelial functional effect of prostaglandin-generating cells, identified by the presence of cyclooxygenase 1 (COX 1). Immunohistochemical results were quantitated densitometrically, and cell surface staining in situ was verified by flow cytometry of isolated cells and by Western blotting. Three populations of COX 1⁺ mucosal cells were identified, based on their morphology and local distribution in human mucosa; these were in the intra-epithelial, crypt apical, and lamina propria regions, with each containing a similar amount of COX 1 protein on a per cell basis. The most numerous were COX 1⁺ mononuclear cells in the lamina propria, identified as CD3⁺ T lymphocytes, both in situ and ex vivo. In toto, 21% of lamina propria mononuclear cells were COX 1⁺, and over 50% of these cells were CD3⁺ T cells. Findings were similar in the colon with mild-moderate inflammation due to ulcerative colitis. Using established surface markers, intra-epithelial and crypt apical COX 1⁺ cells were non-lymphoid CD45⁺ leukocytes; neither IgA (B-lymphocytes) nor α-smooth muscle actin (myelofibroblasts) was co-expressed on these COX 1⁺ cells. Examining the effect of a major product of COX 1 in an in vitro system of human colonic epithelial monolayers, prostaglandin E2 (PGE2) in low concentration (10⁻⁶ M) enhanced epithelial barrier function and partially protected epithelia from the barrier-disruptive consequences of a pro-inflammatory cytokine, IFN-γ. We conclude that the human colon contains three tiers of cell types for local synthesis of prostanoids, distinguishable by their location, morphology, and cell lineage. Further, maintenance of the barrier function of colonic epithelium may be added to other cell functions in mucosa regulated, in part, by prostanoids. © 2002 Wiley-Liss, Inc.

ABSTRACTExposure to oocysts of the protozoan Cryptosporidium parvumcauses intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor β1 (TGF-β1)—a cytokine synthesized and released by cells in the intestine—to preserve the barrier function of human colonic epithelia when challenged with C. parvumoocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-β1 were localized by confocal microscopy, and TGF-β1-induced protein kinase C activation was detected intracellularly by translocation of its α isozyme. TGF-β1 alone enhanced intestinal epithelial barrier function, while exposure to C. parvumoocysts (≥105/monolayer) markedly reduced barrier function to ≤40% of that of the control. When epithelial monolayers were pretreated with TGF-β1 at 5.0 ng/ml, the barrier-disrupting effect ofC. parvumoocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-β1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-β1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-β1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-β1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa α isozyme within 30 s of epithelial exposure to TGF-β1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-β1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-β1 when human colonic epithelium is exposed to an important human pathogen, C. parvum.

Freshly isolated human mucosal T lymphocytes in vitro can markedly diminish an important property of intestinal epithelium—its barrier function. On the other hand, cytokines and their cellular receptors, which maintain homeostasis of epithelia, limit epithelial permeability, and preserve barrier function, are not well characterized. Using a described human colonic epithelial cell monolayer system, we found that transforming growth factor-β1 (TGF-β1) preserved 75% or more of epithelial barrier function, quantitated electrophysiologically, even in the presence of cytokines generated by a high density of barrier-disruptive mucosa-derived mononuclear cells. In opposing the TGF-β1 effect, cytokines able to reduce barrier function were spontaneously secreted by mucosal T cells and were increased in their barrier effect after T-lymphocyte activation. Further, neutralization of individual cytokines with specific monoclonal antibodies abrogated the lymphocyte-induced reduction in epithelial barrier function, and identified interferon gamma (IFN-γ), interleukin (IL)-4, and IL-10, but not IL-6, as the primary cytokines whose barrier effects were curtailed by TGF-β1. Receptors (RI and RII) for TGF-β1 were found to be localized primarily to the apical and basal membranes of surface epithelium in colonic crypts. These findings provide the scientific basis for new strategies to pharmacologically enhance the barrier function of epithelia in mucosal organs regularly exposed to environmental antigens and to T-lymphocyte products. J. Cell. Physiol. 181:55–66, 1999. © 1999 Wiley-Liss, Inc.

Summary Although serological reactivity to human papillomavirus type 16 (HPV-16) proteins has been demonstrated in patients with invasive cervical carcinoma, the degree of seroreactivity to these proteins in women with preinvasive disease and its relationship to the HPV type associated with the disease are unclear. We obtained sera from 27 women undergoing cone biopsy for cervical precursor lesions and 22 controls and analyzed seroreactivity by Western blot to fusion proteins containing portions of the HPV-16 E4, L1 and L2 open-reading frames (ORFs). Positives were analyzed by scanning densitometry and intensity values for each case plotted relative to controls. Cervical biopsy specimens from patients were analyzed for HPV-16 nucleic acids by DNA · DNA in situ hybridization. Mean intensity values for seroreactivity to the pATH-E4 protein approached significance (P = 0.058) and a significantly higher proportion of cases vs controls registered values over 4.0 for pATH-E4 (26% vs 4.5%;P = 0.04) and pATH-L2 (48% vs 18%;P = 0.03) proteins. A significantly higher mean intensity value for E4 was observed for cases containing HPV-16 DNA vs HPV-16 negative cases or controls. Thus, seroreactivity to HPV-16-derived proteins may be more common in women with preinvasive cervical disease, and for some protein targets (E4) may indicate a relatively type-specific response.

Summary Studies of cervical secretions as well as cells composing the endocervix have provided evidence for a functional and potentially important immunological system in the mucosa of that organ. The availability of the tools of cell biology as well as three agents that may be used as probes to infect cervical mucosa experimentally has made possible a detailed approach to define the structural and functional characteristics of local cervical immunity. A long-term goal of these studies is to determine how the cervical immune response may be regulated to reduce local viral replication and virus-associated diseases. With Langerhans cells for antigen presentation, cervical immune responses generally remain detectable for more than 30 days, are predominantly of the IgA isotype, can be influenced by estrogen or progesterone, and are best elicited by local rather than systemic exposure to antigen. Cervical immune responses to the human papillomaviruses (HPV) are of particular importance in this regard because this virus is associated with cervical neoplasia. While responses in serum to HPV-16 proteins L1, E4, and E7 has been found in up to 78% of persons with HPV-associated cervical neoplasms, data showing that a local response of comparable frequency consistently occurs have yet to be confirmed. The current status of local HPV-16-specific immunoglobulin as a potentially useful indicator of HPV-16-related infection or pre-cancer is controversial, and is confounded by several potentially important factors, including patient age, estrogen/progesterone level, smoking status, and sample admixture with serum immunoglobulin.

Near-total pancreatectomy in a neonate presenting with persistent hypoglycemia offered an unusual opportunity to grow preparations enriched in β-cells. Morphology, chromosomal analysis, and immunohistochemistry were used to characterize a subculture (Nesi B) that remained stable through passage 11. Insulin secretion of Nesi B was constitutive at 38–74 nU/μl of medium/ 24 h, increasing modestly in the presence of isobutylmethylxanthine, an inhibitor of cyclic AMP phosphodiesterase. Endocrine cells, exclusive of those in islet regions, were widely distributed throughout the original tissue section, and immunostaining of the Nesi B subculture demonstrated well-differentiated heavily granulated insulin-positive cells, each with a normal modal number of chromosomes (46). Nesi B cell-associated macromolecules were isolated in 3 × 10−3ethylenedinitrilotetraacetate/phosphate-buffered saline and were found to be reactive with a heterologous immune serum elicited to a cloned rat pancreatic β-cell line (RIN-SF). Western (immuno) blotting showed this immunoreactivity to reside primarily in a 95-kDa fraction of Nesi B-derived components. These results indicate that human nesidioblasts can be cultured for a sufficient number of passages to allow isolation and immunochemical characterization of pancreatic β-cell macromolecules shared between rat and human and that may serve as organ-specific antigens for inflammatory disorders of the pancreas.

Chronic colitis is present in up to 50% of adult cotton-top tamarins, but the etiology is unknown. To explore one putative immunopathogenic pathway for the chronic colitis, we determined whether immune sensitization to macromolecules associated with mucosal epithelium of intestine (designated ECAC) had occurred in this primate species. Specifically, we sought to define (a) whether antigenic determinants associated with ECAC are present on tamarin gut epithelium in vivo; (b) if immunoglobulin, capable of binding ECAC, is detectable in tamarin serum; (c) whether the presence of ECAC-specific immunoglobulin is positively correlated with age of the animal or the severity of the colitis, or both; and (d) the number of glycoprotein fractions composing ECAC (denoted as P1 through P4) that are reactive with tamarin immunoglobulin. Expression of ECAC was found by immunofluorescence using characterized oligo-specific or monoclonal antibody: tamarin intestinal epithelium—but not lamina propria, muscularis mucosae, subserosa, or glycocalyx—demonstrated determinants of the ECAC antigen system. Furthermore, coded sera from 10 tamarins with biopsy-proven inflammation involving colonic intestinal mucosa and in which disease activity was moderate to severe showed ECAC-specific cytotoxicity of 3.6% ± 1.6%. Test values for 9 of 10 of those animals were above the upper limit of normal for the assay (2.1%), and exceeded the level of lysis found with serum from histologically normal tamarins less than 2 yr old (<0.3%). When the data were reanalyzed by age of the animal, the incidence of ECAC-specific cytotoxicity correlated with age >2 yr (r = 0.86, p < 0.001). Epithelial cell-associated component-specific serum binding was confirmed by a second methodology (enzyme-linked immunosorbent assay), where the A405for tamarins with lesions of moderate-severe grade (0.35 ± 0.26) clearly exceeded that for young tamarins who were histologically normal (0.02 ± 0.039) (p < 0.05). Most of the reactivity was directed toward the P1 fraction of ECAC. Thus, immune sensitization to a fraction of macromolecules associated with colonic epithelium has been found in the cotton-top tamarin, analogous to findings in humans with chronic inflammatory bowel disease.

Recent data would limit indications for serum CEA measurement primarily to follow-up of resected colonic malignancy, yet physician attitude and usage patterns may lag far behind current findings. This discrepancy was investigated at our institution, where more than 1100 CEAs costing $71,000 are ordered each year. Of 45 physicians (all MDs ordering a CEA test during a preselected month), over 50% believed the test to be worthwhile in initial detection of colonic cancer, and 69% thought an elevated CEA to be an adequate reason to begin an aggressive workup to rule out cancer of the colon in a nonsmoking, previously healthy patient. Impressions of cost were =$30 (50% of true cost) in nearly half of MDs and =20% of true cost in a tenth of MDs. Analysis of the medical record revealed that indications of questionable validity (initial detection of cancer together with follow-up of noncolonic malignancy) accounted for the majority of requested CEAs and included the attempted detection or monitoring of 12 different tumor types in addition to its use as a “general cancer screen.” Patient benefit was realized in none in a random sample of 106 cases (ß=0.11, power =0.89 for an assumed benefit of 2%), while management was altered in only one patient as a direct result of the CEA value. It is important that we continue to inform and educate our colleagues about relatively expensive tests that have only limited and specific application.

We report the case of an 18-yr-old man with quiescent ulcerative colitis complicated by concomitant primary sclerosing cholangitis and rapidly progressive glomerulonephritis. Findings on immunofluorescence microscopy and electron microscopy suggested that glomerular injury occurred secondary to the deposition of circulating immune complexes. Renal disease responded to treatment with corticosteroids. A review of the literature found similar cases of glomerulonephritis and inflammatory bowel disease, but no previous association with sclerosing cholangitis has been recognized. The pertinent clinical, immunological, and pathophysiological aspects of this association are reviewed.

A prior report indicated that sera together with peripheral blood mononuclear cells from patients with Crohn's disease were reactive with epithelial cell-associated components derived fromsmall bowel(ECAC-SB). In the present study, we sought to determine (1) whether similar components (designated ECAC-C) from everted, inflated loops of murinecoloncould be purified to homogeneity in aqueous soluble form and physiochemically characterized; (2) if sera and/or peripheral mononuclear cells from patients with a chronic idiopathic inflammatory disorder of intestinal mucosa were specifically reactive with ECAC-C; (3) whether immunoglobulin was the factor conferring specificity to the anti-ECAC-C response; and (4) if this immunoglobulin was actively synthesized by human lamina propria B lymphocytes isolated from disease-involved intestinal mucosa. Preparative polyacrylamide gel electrophoresis followed by elution of specific bands resulted in the isolation of three major proteins in homogeneous form. Each was a distinctive macromolecule by molecular weight (39,000, 63,000, 148,000), carbohydrate/protein content, and serologically detectable determinants as assessed by quantitative hemagglutination inhibition. By a51Cr release microcytotoxicity assay, ECAC-C-labeled indicator cells were specifically lysed by sera and peripheral blood mononuclear cells from patients with ulcerative colitis (8.0±6.8%) and Crohn's disease (13.2±4.7%), compared with age-/sexmatched controls (0.6±0.9 and 0.2±0.4%, respectively). That the factor conferring ECAC-C specificity was immunoglobulin was demonstrated by (a) the retention of ECAC-C-specific reactivity of patient sera in the presence ofnormalperipheral blood mononuclear cells, (b) the ability of patient sera (without cells) to effect complement-mediated lysis against erythrocytes labeled with ECAC-C (8.0±6.7%) but not with control kidney antigen (0.7±0.7%), and (c) the fact that purified immunoglobulin from patient sera could substitute for the latter in an ECAC-C-specific antibody-dependent cellular cytotoxicity assay. Unique aspects of this ECAC-C reactivity included its absence from sera in other disorders with a known or presumed autoimmune basis (systemic lupus erythematosus, chronic active hepatitis) and the lack of simultaneous reactivity directed toward control antigens, isolated from kidney in a manner analogous to that used for ECAC-C. The importance of ECAC-C-specific immune responses at the mucosal level was also examined using mononuclear cells isolated from disease and control intestinal lamina propria (LPMC). In contrast to sera, supernatants of cultured LPMC from disease-involved gut segments did not contain ECAC-C-specific antibody at levels detectable by our assay system, even though, by radioimmunoassay, 7–89 µg/ml of each immunoglobulin class was newly synthesized. Contrary to its enhancing effect upon peripheral blood mononuclear cells, patient sera did not increase the anti-ECAC-C cytotoxic response of freshly isolated patient mucosal lymphocytes. These findings suggest that colon epithelium-specific immunoglobulin is present in sera of patients with chronic mucosal inflammation and that it may be a useful marker to study autosensitization occurring at epithelial surfaces.

Data from 88 percent of institutions in the United States with facilities for performing open heart surgery were analyzed to determine the current pattern of distribution of these facilities and the extent of their use in the 5 years from 1967 to 1971. Although facilities for open heart surgery were distributed roughly according to population density, the patient case load per hospital varied greatly from institution to institution, ranging in 1971 from 1,616 cases in one hospital to 4 cases in another. A 250 percent increase in the utilization of these facilities occurred between 1967, 2 years before the general introduction of the aortocoronary bypass operation, and 1971. In turn, the percentage of hospitals performing 25 or fewer open heart procedures annually decreased from 52 percent in 1967 to 14 percent in 1971. In 1971 an estimated $249 million in professional fees and hospital costs was incurred by open heart operations alone. A 5 year projection based upon this rate of growth shows that significant improvement in utilization of existing facilities and skilled personnel will occur only if the acquisition of facilities for open heart surgery by additional hospitals is preceded by careful studies documenting need and potential for optimal usage.

Data from 88% of United States institutions that perform cardiopulmonary bypass surgery were analyzed to determine the type, time of storage, anticoagulant, and amount of blood used. The frequency with which cardiopulmonary bypass procedures are performed has more than doubled in the last five years, demanding an ever increasing proportion of the national blood resource. An average of nearly eight units of blood were used per case in 1971. Problems in procurement continue, as a marked number of surgical teams still require fresh, heparinized blood in relatively large amounts. Since the increasing use of saphenous vein bypass surgery may create a prodigious demand upon the blood resource of this country, new policies and practices are needed to reduce the amount of exogenous whole blood used per patient.

A monolayer of mature polarized colonic epithelial cells (T84) able to generate and maintain a barrier to macromolecular flow was used to study pathophysiologic events that occur on microvillus cell exposure to Cryptosporidium parvum. By 24–48 h, several life cycle forms were seen in parasitophorous vacuoles near the apical cell surface, along with a time- and oocyst dose-dependent reduction in epithelial barrier function. As few as 105 organisms constituted a successful infecting dose, and heat inactivation of organisms markedly reduced the monolayer barrier alteration. Horseradish peroxidase flux studies demonstrated a substantial increase in macromolecular permeability of the monolayer, and lactate dehydrogenase determinations indicated modest injury of the T84 epithelial cells on exposure to oocysts. Thus, disruption of the epithelial cell barrier, not just opening of trans cellular channels for ion flow as reported previously, is responsible for the effects of C. parvum oocysts on intestinal epithelium.