Biomedical subjects

Doug Redelman

Publications and source records attributed to Doug Redelman.

6 recordsLinked to original sources

PubMed2004

Microparticulate beta-glucan upregulates the expression of B7.1, B7.2, B7-H1, but not B7-DC on cultured murine peritoneal macrophages.

Beta-1,3-(D)-glucan from a variety of biological sources has been shown to enhance both humoral and cellular immune responses to a variety of antigens, infectious agents, and tumors. Nevertheless, its mode of action has not been fully defined. We sought to determine whether a 1-2 microm diameter microparticulate form of beta-glucan (MG) from the yeast Saccharomyces cerevisiae could regulate expression of B7 family glycoproteins on resident peritoneal macrophages from BALB/c mice. We discovered that MG uregulated B7.2 mRNA expression and enhanced the surface membrane expression of B7.2 glycoprotein. Although B7.1 mRNA was not upregulated above constitutive levels, MG treatment enhanced B7.1 glycoprotein expression on the macrophages, albeit to a lesser extent than B7.2. At the same time, the gene and cell surface expression of B7-H1, a putative negative regulator of T cell activity, was also upregulated by MG. The expression of B7-DC, another B7 family molecule with negative regulatory activity, was not affected by incubation with MG. This study has demonstrated that a microparticulate form of beta-glucan can enhance B7 co-stimulatory molecule expression on macrophages, thereby enabling these antigen-presenting cells to deliver the second signal to T-lymphocytes that express CD28. In addition, because MG also induces the expression of B7-H1, it may enable macrophages to provide a concomitant downregulatory signal to T-lymphocytes expressing PD-1 or related receptors.

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PubMed2004

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PubMed2004

Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers, and mediators of neurotransmission in the gastrointestinal tract.

BACKGROUND: Interstitial cells of Cajal (ICCs) are mesenchymal cells that play critical roles in gastrointestinal motility as electrical pacemakers and mediators of neuromuscular neurotransmission. Although depletions of ICCs have been implicated in several gastrointestinal motor disorders, quantification of these cells has been difficult due to their varied morphology, regionally changing network density, and overall scarcity. Our goal was to evaluate flow cytometry (FCM) for the enumeration of ICCs. METHODS: We identified murine ICCs in live gastrointestinal muscles or primary cell cultures grown in the presence or absence of stem cell factor (SCF)-expressing STO fibroblasts with fluorescent Kit (CD117) antibodies. Because this technique also labels resident macrophages nonspecifically, we identified the latter with additional fluorescent antibodies. Dispersed cells were analyzed by FCM. RESULTS: ICCs represented 1.63 +/- 0.17% of the total cell count in the distal stomach (n = 18 mice) and 5.85 +/- 0.84% in the proximal colon and 6.28 +/- 0.61% in the distal colon (n = 3 mice). In fundic muscles of W/WV mice (n = 5) that virtually lack ICCs, very few Kit+ cells were detected. FCM identified approximately 2.6- to 7.3-fold more Kit+ ICCs in small intestinal cell cultures grown on STO fibroblasts expressing membrane-bound SCF (n = 6) than in cultures stimulated with soluble SCF (n = 6). CONCLUSIONS: FCM is a sensitive and specific method for the unbiased quantification of ICCs.

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PubMed2003

Immunomagnetic enrichment of interstitial cells of Cajal.

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.

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PubMed2003

Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000-40,000 cells and approximately 60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched approximately 6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested approximately 2,000-4,000 cells from single gastric corpus-antrum muscles and detected an approximately 30-fold increase in c-kit mRNA, no enrichment of mast cells, and an approximately 4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses.

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PubMed2003

Influence of pH treatments on survival of Escherichia coli O157:H7 in continuous cultures of rumen contents.

The pH (i.e., 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, and 7.25) effect on Escherichia coli O157:H7 in an artificial rumen model was investigated. Eight fermenters were inoculated with bovine rumen fluid and were supplied with a diet (75 g of dry matter daily in 12 equal portions [every 2 hr]) containing similar forage-to-concentrate ratio. After an adaptation period (i.e., 3 days for adjusting the rumen fluid [pH 6.2] microbial population to the test pH and 4 days for adjustment to the diet at the test pH), each fermenter was inoculated with 10(9) cells of E. coli O157:H7. Samples were collected hourly for 12 hr and every 2 hr for an additional 12 hr and were analyzed by flow cytometer. E. coli O157:H7 could not be quantified after 24 hr, and detection was only possible after enrichment. Because the pathogen could not be detected 5 days postinoculation (i.e., Day 13), the fermenters were reinoculated with E. coli O157:H7 on Days 17 and 22. E. coli O157:H7 numbers decreased from 10(6) to 10(4)/ml of fermenter contents in a quadratic (P < 0.05) fashion over the 24-hr sampling period, and the rate of reduction was slower (P < 0.05) for pH 7.0 than for other pH treatments. Results suggested that E. coli O157:H7 population were decreased by competitive exclusion and were not affected by culture pH.

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