Michael J. Rutten

Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells

The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]i were also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]i in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]iwere also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]iin the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.

Helicobacter pylori induces apoptosis in Barrett's-derived esophageal adenocarcinoma cells

Helicobacter pylori may protect against the development of dysplasia in Barrett’s epithelium of patients with gas-troesophageal reflux disease. The aim of this study was to determine whether H. pylori preferentially induces apoptosis in Barrett’s-derived cancer cells compared to normal cells. A Barrett’s-derived adenocarcinoma cell line (OE33) was grown. H. pylori wild-type, isogenic vacA-, cagA-, and picB-/cagE- mutant strains were grown on agar plates. Intact or sonicated bacteria were used to treat normal and OE33 cells for 24 hours, and Hoechst dye binding was performed to measure apoptosis. FAS protein expression was determined by Western immunoblotting. OE33 cells treated with intact H. pylori wild-type strains produced significant (P < 0.05) dose-dependent increases in apoptosis compared to normal esophageal cells. H. pylori wild-type and vacA- isogenic strains were more effective than cagA- and picB-/cage- isogenic strains in inducing apoptosis in OE33 cells. In OE33 cells, H. pylori sonicates produced lower levels of apoptosis than intact bacteria. Wild-type H. pylori strains increased Fas protein expression in OE3 3 cells at 18 hours. H. pylori induced apoptosis at a higher rate in the Barrett’s-derived human esophageal adenocarcinoma cells than in normal esophageal cells. The H. pyloriinduced apoptosis was primarily dependent on intact bacteria and the presence of the cagA and picB/cagE gene products. H. pyloriinduced apoptosis may involve the Fas-caspase cascade.

Bismuth subsalicylate increases intracellular Ca2+, MAP-kinase activity, and cell proliferation in normal human gastric mucous epithelial cells.

Clinical and laboratory studies have shown that bismuth subsalicylate (BSS) is helpful in the healing of gastric ulcers because of the bactericidal effects of bismuth (Bi3+) on H. pylori. Bismuth or BSS has also been reported to possess other nonbactericidal or “gastroprotective” effects in the stomach. It is known in other cell types that the effects of extracellular divalent or trivalent cations (e.g., Ca2+) can activate a plasma membrane-bound calcium-sensing receptor (CaSR). In a previous study, we found the existence of a CaSR which was activated by extracellular Ca2+ and found to increase intracellular Ca2+ [Ca2+]i, MAP-kinase activity, and gastric epithelial cell proliferation. In the present study, we were interested in determining whether the effects of the trivalent cation Bi3+ (in the form of BSS) on [Ca2+]i, MAP-kinase activity, and proliferation of gastric cells. We found that BSS dose dependently increased [Ca2+]i, p44/p42 and p38 MAP-kinase activites, and gastric mucous epithelial cell growth. The addition of BAPTA to chelate intracellular Ca2+ blocked BSS-induced p44/p42 MAP-kinase activities but not p38 MAP-kinase activity. The p44/p42 MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580 dose dependently decreased gastric mucous cell growth over a 24 hr. All of the BSS-induced changes in [Ca2+]i, MAP-kinase activity, and gastric cell proliferation could be reproduced with the CaSR-agonist gadolinium (Gd3+). Our data suggest that BSS may possess additional novel effects by increasing gastric mucous epithelial cell growth through a Ca2+/MAP-kinase-dependent pathway.

A method for the isolation of human gastric mucous epithelial cells for primary cell culture: A comparison of biopsy vs surgical tissue

We have developed a method for the isolation and growth of normal human gastric mucous epithelial cells using biopsies or surgically resected tissues as the source of the cells. The attachment and growth of cells were dependent upon: (1) cell planting density, ∼50,000 cells/cm2; (2) extracellular matrix (fibronectin); and (3) and the use of a porous filter. In all experiments we found better cells attachment and growth of human gastric mucous cells isolated from surgical specimens compared with those gastric mucous cells isolated from gastric biopsies. The initial cell viability (as measured by Trypan-blue) was the same in both populations of gastric mucous epithelial cells isolated from either gastric biopsies or surgical specimens. After 4–5 days in culture one could detect various amounts of mucin in all the cells using either periodic acid Schiff (PAS) staining or a specific anti-mucin antibody. A similar pattern of much straining was also found in primary cultures of guinea pig gastric mucous epithelial cells. Immunohistochemical staining for chief cells (anti-pepsinogen) or parietal cells (anti-H+/K+ ATPasc) in the gastric mucous cuboidal-like epithelial cells with tight junctions, desmosomes,short microvilli, a filamentous terminal web, mucous granules, and basal lamina-like structure. We could not detect the presence of fibroblasts during the 7–9 days that the primary cells were in culture. This cell culture method will prove useful in the isolation of normal human gastric mucous epithelial cells for in vitro studies of gastric mucosal injury and repair.

Treatment of Burn and Surgical Wounds With Recombinant Human Tropoelastin Produces New Elastin Fibers in Scars.

Tropoelastin (TE), the soluble precursor of insoluble elastin fibers, is produced in minimal amounts in adults. Burn injuries result in inflexible collagen-rich scars because of lack of elastin fiber formation. We studied the feasibility of using recombinant human tropoelastin to enable elastin fiber production in burn and surgical scars to improve skin flexibility. In a swine hypertrophic burn scar model, normal skin and 3 × 3-cm2 partial thickness thermal burns underwent dermatome resection at 1 week post burn and randomized to four subcutaneous injections of saline or TE (either 0.5, 5, or 10 mg/ml) spaced 3 days apart. Two burn sites received TE injections after wound closure (0.5 or 10 mg/ml). At 90 days, skin hardness, flexibility, and histology were evaluated. All injury sites developed hypertrophic scars. New elastin fibers were found in burn scars in all injuries injected after skin closure with low (5/5) and high (6/6) TE doses (P < .05). No elastin fibers were observed without TE treatment. No significant differences in skin hardness, flexibility, or inflammation were observed. This is the first report demonstrating that subcutaneous injections of TE into surgical and burn injuries can safely produce new elastin fibers in scars. Despite the development of new elastin fibers, skin flexibility was not improved, possibly because of insufficient elastin fiber maturation or the hypertrophic model used. The ability to restore elastin fiber formation in adult skin after burns, trauma, and surgery may improve skin regeneration and reduce disabling complications of scar formation.

Prolonged survival of adult rat pancreatic islets transfected with E1A-12S adenovirus

It has been reported that various mutants of the E1A-adenovirus can activate quiescent differentiated cells to start proliferating. The aim of this study was to determine whether transfection with E1A-12S could extend the life span and functionality of pancreatic islets in culture. Rat pancreatic islets were isolated and transfected with retrovirus containing the adenovirus E1A-12S, E1A-13S, or control vectors. Transfection with the retroviral E1A-13S mutant produced extensive islet necrosis compared with nontransfected islets. Islets transfected with the control E1A mutant Ad5-dl312 vector (containing no E1A-12S or E1A-13S segments) were similar to nontransfected islets in their characteristics. We found that the E1A-12S transfected islets maintained greater viability, insulin granule structure, and glucose-induced insulin responsiveness over a 6-week period compared with mock or control islets. At 6 weeks of culture, the E1A-12S transfected islets also had fewer apoptotic cells compared with nontransfected islets. These data suggest that adenovirus E1A-12S can extend the functional life span of cultured rat pancreatic islets.

Development of a functional schwann cell phenotype from autologous porcine bone marrow mononuclear cells for nerve repair.

Adult bone marrow mononuclear cells (BM-MNCs) are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6–8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF) expression. Addition of neuregulin (1–25 nM) increased p75(NGF) levels at 24–48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca2+]i, with nucleotide potency being UTP = ATP > ADP > AMP > adenosine. Suramin blocked the ATP-induced [Ca2+]i but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca2+]i sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

Multipotent adult progenitor cells, but not TIMP3, increase tissue sparing and reduce urological complications following spinal cord injury

Following spinal cord injury (SCI), inflammation amplifies damage beyond the initial insult, providing an opportunity for targeted treatments. An ideal protective therapy would reduce both edema within the lesion area and the activation/infiltration of detrimental immune cells. Previous investigations demonstrated the efficacy of intravenous injection of multipotent adult progenitor cells (MAPC®) to modulate immune response following SCI, leading to significant improvements in tissue sparing, locomotor and urological functions. Separate studies have demonstrated that tissue inhibitor of matrix metalloproteinase-3 (TIMP3) reduces blood-brain barrier permeability following traumatic brain injury in a mouse model, leading to improved functional recovery. This study examined whether TIMP3, delivered alone or in concert with MAPC cells, improves functional recovery from a contusion SCI in a rat model. The results suggest that intravenous delivery of MAPC cell therapy 1 day following acute SCI significantly improves tissue sparing and impacts functional recovery. TIMP3 treatment provided no significant benefit, and further, when co-administered with MAPC cells, it abrogated the therapeutic effects of MAPC cell therapy. Importantly, this study demonstrated for the first time that acute treatment of SCI with MAPC cells can significantly reduce the incidence of urinary tract infection (UTI) and the use of antibiotics for UTI treatment.

MP41-06 MULTIPOTENT ADULT PROGENITOR CELL TREATMENT PROMOTES BLADDER FUNCTION RECOVERY AND DECREASES MORBIDITY AND DURATION OF URINARY TRACT INFECTION IN SPINAL CORD INJURED RATS

groups compared with PBS group (Figure 1). Hematoxylin/eosin staining demonstrated that loss of urothelial integrity in PBS group was restored in IV and BL group. Neutrophil infiltration was also decreased in IV and BL group compared with PBS group. Histological examination revealed a significant decrease in the total number of infiltrated mast cells in IV and BL rats compared with the PBS rats (p<0.05), (Figure 2). CONCLUSIONS: This is the first study to investigate the efficacy of CHA1 for chronic IC model. Submucosal injection of CHA1 in chronic IC showed improvement of voiding pattern and histological restoration and this suggest that CHA1 might have a therapeutic potential for IC. Further studies are required to evaluate the mechanism of CHA1 treatment.