Biomedical subjects

D Carnes

Publications and source records attributed to D Carnes.

13 recordsLinked to original sources

PubMed2001

Utilization of MO6-G3 immortalized odontoblast cells in studies regarding dentinogenesis.

Tooth formation is the result of reciprocal instructive interactions between oral epithelium and cranial neural-crest-derived ectomesenchymal tissues. These interactions lead to the cytodifferentiation of highly specialized matrix-forming cell types, the ameloblast, odontoblast, and cementoblast, that produce the mineralized tissues enamel, dentin, and cementum, respectively. Our laboratory has been developing immortalized dental cell lines representative of these various cell types to facilitate studies on gene regulation, cell differentiation, matrix formation, and mineralization. Odontoblasts are solely responsible for the synthesis and secretion of the dentin extracellular matrix bilayer that consists of non-mineralized predentin and mineralized dentin. The mouse immortalized MO6-G3 cell line expresses the major matrix proteins associated with the odontoblast phenotype, producing a matrix that is capable of mineralization. This cell line serves as a useful tool in studies designed to explore the various processes of dentinogenesis. In this paper, we present studies using the mouse odontoblast cell line MO6-G3 as examples of the various research applications. Studies highlighted are: in vitro promoter studies investigating the tooth-specific gene regulation of the major non-collagenous dentin matrix protein, dentin sialophosphoprotein; regulation of tertiary dentin formation by cytokines, such as transforming growth factor-Beta 1; and the utilization of dentally relevant cells in dental material biocompatibility testing.

Ameloblasts↗

PubMed2000

Salmonella typhimurium induces epithelial IL-8 expression via Ca(2+)-mediated activation of the NF-kappaB pathway.

Interactions between the enteric pathogen Salmonella typhimurium and the luminal surface of the intestine provoke an acute inflammatory response, mediated in part by epithelial cell secretion of the chemokine IL-8 and other proinflammatory molecules. This study investigated the mechanism by which this pathogen induces IL-8 secretion in physiologically polarized model intestinal epithelia. IL-8 secretion induced by both the prototypical proinflammatory cytokine TNF-alpha and S. typhimurium was NF-kappaB dependent. However, NF-kappaB activation and IL-8 secretion induced by S. typhimurium, but not by TNF-alpha, was preceded by and required an increase in intracellular [Ca(2+)]. Additionally, agonists that increased intracellular [Ca(2+)] by receptor-dependent (carbachol) or independent (thapsigargin, ionomycin) means also induced IL-8 secretion. Furthermore, the ability of S. typhimurium mutants to induce IkappaB-alpha degradation, NF-kappaB translocation, and IL-8 transcription and secretion correlated precisely with their ability to induce an intracellular [Ca(2+)] increase in model intestinal epithelia, but not with their ability to invade these cells. Finally, S. typhimurium, but not TNF-alpha, induced a Ca(2+)-dependent phosphorylation of IkappaB-alpha. These results indicate that S. typhimurium-induced activation of NF-kappaB-dependent epithelial inflammatory responses proceeds by a Ca(2+)-mediated activation of an IkappaB-alpha kinase. These observations raise the possibility that pharmacologic intervention of the acute inflammatory response can be selectively matched to the specific class of initiating event.

Calcium↗

PubMed1997

Prevalence of diabetes and factors associated with diabetic complications in Oneida Indians.

The study objective was to determine the prevalence of diabetes mellitus among Oneida Indians and to identify factors associated with the development of medical complications. After identifying patients with diabetes (N = 373) treated at the Oneida Community Health Center, their medical records were reviewed to determine age, body mass index (BMI), types of medication used, HbAlc, and the presence of diabetic complications. The age and sex-adjusted prevalence of diabetes was 86.5/1000 population. Where type of diabetes could be determined (N = 345) 96% were non-insulin dependent; 67% were afflicted with diabetic complications. 56% had hypertension. Age, BMI, and HbAlc were significant variables with respect to the presence of certain complications. Although lower than the prevalence for some other groups of Native Americans, diabetes and its complications represent a serious problem for the Oneida Indians. Consistent screening, intensive intervention and culture-sensitive prevention efforts focussing on weight control are needed.

Adolescent↗

PubMed1997

Unmasking of intestinal epithelial lateral membrane beta1 integrin consequent to transepithelial neutrophil migration in vitro facilitates inv-mediated invasion by Yersinia pseudotuberculosis.

Idiopathic intestinal disease states characterized by active inflammation associated with transepithelial migration of neutrophils may, paradoxically, be associated with an increased risk of infection by enteric pathogens. Although the specific ligands with which various intestinal pathogens associate remain largely unknown, it is thought that many reside on the basolateral membrane. For example, beta1 integrin, a basolateral membrane protein, mediates the specific interaction between epithelial cells and the inv gene product (invasin) on the surface of Yersinia pseudotuberculosis. Our observations indicate that neutrophil migration across model T84 cell intestinal epithelia produced transient separation of epithelial cells at sites of neutrophil migration, resulting in microdiscontinuities that remained unsealed for several hours. We hypothesized that such sites of microdiscontinuities would yield a potential route for luminal pathogens to gain access to basolateral ligands and, thus, provide a window of risk for enteric infection. The surface biotinylation and fluorescence localization studies reported here revealed that, as in natural intestinal epithelia, beta1 integrin was strictly polarized to the basolateral membrane in confluent T84 monolayers. However, the transient microdiscontinuities resulting from neutrophil migration permitted access to beta1 integrin from the apical reservoir. Coincident with such basolateral exposure of beta1 integrin, monolayers became susceptible to invasion by Y. pseudotuberculosis. Fluorescence localization indicated that Y. pseudotuberculosis selectively associated with monolayers at sites where small discontinuities resulting from neutrophil transmigration were found. An increased risk for Y. pseudotuberculosis infection was specifically related to exposure of beta1 integrin (normally concealed by tight junctions) to the apical compartment, as Y. pseudotuberculosis cells lacking the inv gene were unable to invade following neutrophil transepithelial migration. Following closure of the microdiscontinuities associated with neutrophil migration, a small pool of beta1 integrin remained apically localized, presumably due to incomplete repolarization. However, this small apical pool of beta1 integrin was insufficient to support a detectable increased risk of Yersinia infection. Together, these observations indicate that by transiently perturbing monolayer continuity, neutrophil transepithelial migration is associated with a window of risk in which luminal pathogens can access basolateral ligands such as beta1 integrin.

Adhesins, Bacterial↗

PubMed1997

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 +/- 2% (P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 microM ML-7 or 40 microM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 (P < 0.01), which was inhibited by ML-9 (P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

Animals↗

PubMed1996

Intestinal epithelial cytoskeleton selectively constrains lumen-to-tissue migration of neutrophils.

Migration of neutrophils (polymorphonuclear leukocytes; PMN) across polarized epithelia is asymmetrical: basolateral-to-apical (physiologically directed) migration is far more efficient than migration in the reverse direction, suggesting the presence of luminal retention signal(s). Following pilot observations, we used polarized intestinal epithelial monolayers (T84) to examine whether asymmetrical constraint of migration afforded by the epithelial cytoskeleton might underlie such retention signals. Rearrangement of epithelial cortical F-actin accompanied PMN transepithelial migration (in either direction) and was prevented by preloading monolayers with the F-actin stabilizing agent phallacidin. Although phallacidin preloading did not influence physiologically directed PMN transepithelial migration, such treatment greatly enhanced migration in the reverse direction (i.e., effective loss of luminal retention signal). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) preloading also prevented epithelial cortical actin rearrangements and selectively resulted in loss of luminal retention signal(s). BAPTA preloading did not influence resistance or forskolin-induced Cl- secretion, and phallacidin preloading did not influence resistance or carbachol-induced Cl- secretion, suggesting that barrier function and surface polarity were maintained under these conditions. These and supplementary data suggest that epithelial actin (but not microtubule) cytoskeletal reordering asymmetrically influences PMN migration and underlies, at least in part, the observed signal that biases for retention of PMN in the luminal space.

Actins↗

PubMed1995

Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia.

The rho family of GTP-binding proteins regulates actin filament organization. In unpolarized mammalian cells, rho proteins regulate the assembly of actin-containing stress fibers at the cell-matrix interface. Polarized epithelial cells, in contrast, are tall and cylindrical with well developed intercellular tight junctions that permit them to behave as biologic barriers. We report that rho regulates filamentous actin organization preferentially in the apical pole of polarized intestinal epithelial cells and, in so doing, influences the organization and permeability of the associated apical tight junctions. Thus, barrier function, which is an essential characteristic of columnar epithelia, is regulated by rho.

ADP Ribose Transferases↗

PubMed1995

Transepithelial signaling to neutrophils by salmonellae: a novel virulence mechanism for gastroenteritis.

Salmonella serotypes which elicit human enteritis cannot be distinguished from those that do not on the basis of their in vitro interactions with eukaryotic cells. We have recently reported that an enteritis-producing strain of Salmonella typhimurium signals intact intestinal epithelium to recruit subepithelial neutrophils to migrate across the epithelial (B. A. McCormick, S. P. Colgan, C. D. Archer, S. I. Miller, and J. L. Madara, J. Cell Biol. 123:895-907, 1993). We now utilize a cell culture model of human intestinal epithelium (with T84 cells) to examine whether such transepithelial signaling to neutrophils by salmonellae is predictive of potential to elicit gastroenteritis. Various Salmonella serotypes, including S. typhimurium, S. enteritidis, S. pullorum, S. arizonae, S. typhi, and S. paratyphi, as well as invasion-defective mutants of S. typhimurium, were studied. Strains or serotypes which elicit diffuse enteritis in humans (defined histologically as transepithelial migration of neutrophils) exhibited transepithelial signaling to neutorphils across epithelial cell monolayers, while those which do not elicit diffuse enteritis in humans did not display transepithelial signaling. In contrast, the ability to enter the apical surface of T84 cells did not differentiate strains or serotypes which induce diffuse enteritis from those which do not. These results strongly suggest that the ability of salmonellae to elicit transepithelial signaling to neutrophils is a key virulence mechanism underlying Salmonella-elicited enteritis.

Cells, Cultured↗

PubMed1994

Expression of monocyte chemoattractant protein 1 in human osteoblastic cells stimulated by proinflammatory mediators.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine superfamily of genes that induces chemotaxis of monocytes in inflammatory processes. The effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), platelet-derived growth factor (PDGF-BB), parathyroid hormone (PTH), and 1,25(OH)2D3 on MCP-1 expression in human osteoblastic cells were compared. Inflammatory or proinflammatory cytokines stimulated the production of MCP-1 in normal human osteoblastic cells as determined by RIA. The osteotrophic mediators PTH and 1,25(OH)2D3 and PDGF-BB had no effect on MCP-1 expression. In further studies, the steady-state mRNA and MCP-1 protein levels in two human osteoblastic cell lines, MG-63 and SaOS-2, were examined. MCP-1 expression at both the protein and mRNA levels was greatly increased by IL-1 beta and TNF-alpha. At the mRNA level, IL-1 beta and TNF-alpha strongly induced MCP-1 expression; TGF-beta and IL-6 induced MCP-1 but to a lesser extent. No significant changes in MCP-1 mRNA or MCP-1 protein secretion were observed when cells were treated with PDGF-BB, PTH, and 1,25(OH)2D3. When tested on preosteoclasts, MCP-1 was shown to have no effect on the formation of multinucleated, tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells.

Autoradiography↗

PubMed1988

Computer-assisted densitometric image analysis in periodontal radiography. A methodological study.

A videobased computer assisted densitometric image analysis (CADIA) system to quantify alveolar bone density changes on standardized dental radiographs was tested. An algorithm was used for grey level correction of a subsequent image to the baseline image. Quantitative information regarding positive and/or negative grey level changes were obtained automatically. Comparison of the ability of CADIA to detect surgically induced bone loss with interpretation of digital subtraction images and conventional radiographic interpretation revealed that CADIA was the most sensitive of the 3 methods, followed by interpretation of digital subtraction images which was considerably more sensitive than conventional radiographic interpretation. CADIA was capable of assessing differences in alveolar bone changes due to periodontal surgery between sites exposed to ostectomy/osteoplasty and control sites and sites exposed to periodontal surgery without ostectomy/osteoplasty. Finally, CADIA was capable of assessing differences in remodeling activity over 4-6 weeks after periodontal surgery between 45 surgical sites and 45 control sites. The system offers an objective method to quantitatively follow alveolar bone density changes over time and appears to be the most sensitive of previously described radiographic interpretation techniques.

Absorptiometry, Photon↗

PubMed1987

Bone mineral in the castrated rat model of osteopenia.

The effects of castration on cortical bone structure and chemistry were studied in male Wistar rats of two age groups, 8 and 24 months. Some rats were castrated and some were sham operated (control) in each group. All animals were killed after 4 months. Although no changes in serum chemistry, bone chemistry, or bone histology could be found, the young castrated animals versus controls showed less density of bone mineral as determined by the density fractionation technique and smaller crystallite size of mineral particles as determined by x-ray diffraction line-broadening analysis. These changes, indicative of less mature bone formation, were not observed in mature castrated or control rats despite a decreased amount of bone compared with young rats. In this model, castration appears to affect bone mineral quality in young but not in old animals.

Age Factors↗

PubMed1986

Monocyte bone degradation: in vitro analysis of monocyte activity in patients with juvenile rheumatoid arthritis.

We examined the ability of the mononuclear phagocyte in vitro to degrade 45Ca-labeled bone particles to determine whether this assay allowed us to monitor disease activity in patients with juvenile rheumatoid arthritis. The monocytes from patients with juvenile rheumatoid arthritis receiving no anti-erosive therapy (n = 10) degraded significantly more bone than did cells obtained from normal controls (n = 10, P less than 0.001) or patients with juvenile rheumatoid arthritis receiving either gold thioglucose (n = 4, P less than 0.001) or D-penicillamine (n = 6, P less than 0.005). In two patients monitored for either 8 or 11 months, results of monocyte assays were found to parallel the clinical course. We conclude that in vitro monocyte bone degradation assays may provide a means of assessing joint activity in patients with juvenile rheumatoid arthritis. Further, this study and others indicate that mononuclear phagocytes are capable of causing erosive changes.

Adolescent↗

PubMed1984

Treatment of congenital osteopetrosis with high-dose calcitriol.

We administered high doses of calcitriol (up to 32 micrograms per day) to an infant with malignant osteopetrosis, in an attempt to stimulate bone resorption. The patient was placed on a low-calcium diet to prevent hypercalcemia. Measures of bone turnover increased during calcitriol therapy; hydroxyproline excretion rose from 140 to 1358 micrograms per milligram of creatinine per 24 hours, with parallel increases in the ratio of calcium to creatinine in the urine, urinary gamma-carboxyglutamic acid, serum osteocalcin, and serum alkaline phosphatase. A pretreatment bone-biopsy specimen contained no osteoclasts with ruffled borders, a feature of active osteoclasts. After 11 days of calcitriol, ruffled borders were noted. After three months, numerous osteoclasts with ruffled borders and associated bony disruption were evident. Before therapy, the patient's monocytes were incapable of in vitro bone resorption, but after calcitriol, their resorptive capacity was increased to 3.3 times control levels. These data demonstrate that calcitriol increased bone mineral and matrix turnover in our patient. However, during the three months of calcitriol therapy there was only slight clinical improvement in her severe disease. Early and sustained treatment with calcitriol may be useful in osteopetrosis.

1-Carboxyglutamic Acid↗