James K. Roche

Transforming Growth Factor β1 Ameliorates Intestinal Epithelial Barrier Disruption by Cryptosporidium parvum In Vitro in the Absence of Mucosal T Lymphocytes

ABSTRACT Exposure to oocysts of the protozoan Cryptosporidium parvum causes 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. parvum oocysts 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. parvum oocysts (≥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. parvum oocysts 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.

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

Abstract Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3–12 days, then were infected with 5 × 107 excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height–crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

Expression of immune sensitization to epithelial cell-associated components in the cotton-top tamarin: A model of chronic ulcerative colitis

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 (less than 0.3%). When the data were reanalyzed by age of the animal, the incidence of ECAC-specific cytotoxicity correlated with age greater than 2 yr (r = 0.86, p less than 0.001). Epithelial cell-associated component-specific serum binding was confirmed by a second methodology (enzyme-linked immunosorbent assay), where the A405 for 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 less than 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.

Chronic Inflammatory Bowel Disease: Absence of Adenovirus DNA as Established by Molecular Hybridization

Because previous evidence indicated that adenovirus is tropic for gut wall cells (particularly terminal ileum) and can become chronically established in mammalian cells as a latent infection, we examined, through the use of sensitive molecular hybridization techniques capable of detecting minute amounts of adenovirus DNA, the hypothesis being that adenovirus is specificallly associated with both Crohns disease and ulcerative colitis. DNA of 21 coded specimens from 17 operated patients (6 with Crohns disease, 4 with ulcerative colitis, 7 controls) was hybridized with each of five [ 32 P]DNA probes, prepared by nick translation of adenovirus DNA with Escherichia coli polymerase I and representative of each major adenovirus subgroup (A through E). While quantities as small as 0.2 copies of purified adenovirus DNA per human cell were detectable in standardization experiments, none of the test specimens was positive, either in Crohns disease or ulcerative colitis, whether histologically normal or abnormal. Potential sources of false-negative results were analyzed and judged to be unlikely. Future investigation in this area may await the development of more sensitive assays for viral DNA or for evidence of new candidate virus systems.

Prostanoids in human colonic mucosa: Effects of inflammation on PGE2 receptor expression

Although the tissue concentration of PGE(2) is heightened 3-fold or more during mucosal inflammation, the cellular targets of prostanoids in human mucosa and the resulting changes in cell physiology have not been fully explored. We used a panel of immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2) to cells of histologically normal and inflamed human colonic mucosa, and then examined prostanoid-induced changes in mucosal lymphocyte function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells; EP(4) alone was expressed on lamina propria mononuclear cells. Dual immunostaining in situ identified the CD3(+) T lymphocyte as a major EP(4) receptor-bearing cell in normal mucosa. Flow cytometry of isolated cells showed that 19.2% of lamina propria mononuclear cells were EP(4)(+), and almost 30% of these were CD3(+). In situ hybridization with digoxygenin-labeled RNA probes largely confirmed this localization. During inflammation, mucosal T lymphocytes showed a significant enhancement in EP(4) immunoreactive receptor protein. Computer-assisted densitometry of single cells demonstrated an increase in fluorescence intensity from 4.8 +/- 1.8 to 8.6 +/- 1.8 (p < 0.04). The effects of PGE(2) included a 35% reduction in T lymphocyte IL-2 secretion. COX 1(+) lamina propria cells nearly doubled in number during inflammation; expressed a T lymphocyte marker; but retained an unchanged quantity of immunoreactive COX 1 protein per cell. The number of newly appeared COX 2(+) lymphocytes remained <50% that of COX 1(+) cells. A major perturbation in the number and distribution of PGE(2) receptors and enzymes for prostanoid synthesis occurs in chronic inflammation of the colon, with consequences for mucosal T lymphocyte function.