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Biomedical subjects

M F Graham

Publications and source records attributed to M F Graham.

At least 19 recordsLinked to original sources
PubMed1999

Palmitoyl ascorbate: selective augmentation of procollagen mRNA expression compared with L-ascorbate in human intestinal smooth muscle cells.

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.

Ascorbic Acid↗
PubMed1999

Intestinal macrophages display reduced permissiveness to human immunodeficiency virus 1 and decreased surface CCR5.

BACKGROUND & AIMS: Because the role of intestinal mononuclear cells in the pathogenesis of human immunodeficiency virus 1 (HIV-1) disease has not been elucidated, we determined the biological properties of HIV-1 infection in primary intestinal macrophages. METHODS: Mucosal macrophages purified from normal human jejunum were infected with well-characterized macrophage-tropic isolates of HIV-1 (ADA, DJV, and Ba-L). RESULTS: Productive HIV-1 infection of intestinal macrophages was demonstrated by the release of p24 antigen, the presence of proviral DNA, and zidovudine inhibition of infection. Surprisingly, the titer of virus needed to establish infection of intestinal macrophages was 100-1000-fold higher than that required to infect peripheral blood derived macrophages. This marked reduction in the permissiveness of intestinal macrophages to HIV-1 was not caused by the isolation procedure or differences in CD4 expression. Instead, intestinal macrophages expressed almost no CCR5, the principal coreceptor for macrophage-tropic HIV-1, compared with blood-derived macrophages, although both cell types contained comparable levels of CCR5 messenger RNA. Exposure of blood-derived but not intestinal macrophages to HIV-1 or gp120 led to increased surface expression of CCR5. CONCLUSIONS: Intestinal macrophages express reduced levels of HIV-1, probably because of impaired permissiveness to HIV-1 entry associated with the near absence of cell surface CCR5.

CD4 Antigens↗
PubMed1998

Development of pseudointima and stenosis after transjugular intrahepatic portasystemic shunts: characterization of cell phenotype and function.

The clinical utility of transjugular intrahepatic portasystemic shunts (TIPS) is frequently complicated by the ingrowth of tissue into the stent lumen, causing stent stenosis. These studies were undertaken to define the cellular and matrix components of the pseudointima, define the phenotype and function of the mesenchymal cells in the pseudointima and maintain them in culture, and to study the differences between stenotic and nonstenosed stents. A total of 35 stents were evaluated. TIPS pseudointima were examined histologically, by immunohistochemistry and in situ hybridization to determine the cellular and connective tissue constituents. Mesenchymal cells were grown from tissue within the TIPS and around it, and their phenotype was studied and compared with control smooth muscle cells and fibroblasts. Masson's trichrome staining of histological sections demonstrated that TIPS tissue was composed of collagen and palisades of mesenchymal cells and was lined by an endothelium. Immunostaining demonstrated strong and uniform alpha-smooth muscle staining in TIPS mesenchymal cells and peri-TIPS cells. Type I procollagen mRNA expression was demonstrated in mesenchymal cells in and around the stent by in situ hybridization. TIPS mesenchymal cells secreted less radiolabeled fibronectin, and far more type III, relative to type I, collagen compared with peri-TIPS cells. TIPS cells also expressed high levels of type III procollagen mRNA compared with peri-TIPS cells. There was no difference between stenotic stents and nonstenosed stents with respect to clinical features, time from stenting, gross morphology, histology, presence of bile fistulae, and cell phenotype. However, smooth muscle cells (SMC) from stenotic stents demonstrated both greater cell proliferation and collagen I and III secretion compared with those from nonstenosed stents. These data demonstrate that TIPS stenosis results from an accumulation of collagen and proliferation of SMC within the stent lumen.

Cells, Cultured↗
PubMed1998

Recombinant Helicobacter pylori urease activates primary mucosal macrophages.

Helicobacter pylori urease is absorbed into the gastric mucosa at sites of inflammation, but whether the enzyme activates mucosal macrophages is not known. Because mucosal macrophages differ phenotypically and functionally from blood monocytes, whether recombinant H. pylori urease (rUrease) activated purified lamina propria macrophages in vitro was investigated. rUrease (1-10 microgram/mL) induced primary mucosal macrophages to produce interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha but not IL-8 proteins in a dose-dependent manner (P<.05 to P<.001). Quantitative reverse transcriptase-polymerase chain reaction using capillary electrophoresis laser-induced fluorescence showed that rUrease (0.1-10 microgram/mL) also induced dose-dependent expression of IL-1beta, IL-6, and TNF-alpha but not IL-8 mRNA (P<.05), suggesting that rUrease-induced production of certain cytokines is regulated at the level of gene transcription. These findings indicate that the ability of H. pylori urease to activate mucosal macrophages, resulting in production of proinflammatory cytokines, may be involved in the pathogenesis of H. pylori-associated mucosal inflammation.

Helicobacter pylori↗
PubMed1997

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide.

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.

Amino Acid Sequence↗
PubMed1997

Isolation and purification of CD14-negative mucosal macrophages from normal human small intestine.

Mucosal macrophages play a fundamental role in the regulation of immunological events and inflammation in the small intestine. Because no information is available on normal small intestinal macrophages, we developed a technique for the isolation and purification of jejunal lamina propria macrophages in order to study their phenotype and activity. From sections of normal human jejunum, lamina propria mononuclear cells were isolated by neutral protease digestion and then subjected to counterflow centrifugal elutriation to purify the macrophages. The cells isolated by this procedure contained < 1% CD3+ lymphocytes and displayed the size distribution, morphological features, ultrastructure and phagocytic activity of mononuclear phagocytes. In contrast to blood monocytes, however, mucosal macrophages from the jejunum did not exhibit adherence properties or express CD14, a receptor for the lipopolysaccharide-binding protein. The purification of large numbers of lamina propria macrophages by this procedure offers the opportunity to define the role of this cell in the physiological inflammation characteristic of normal intestinal mucosa and the pathological inflammation associated with small intestinal diseases.

Cell Separation↗
PubMed1997

Corticosteroids repress the interleukin 1 beta-induced secretion of collagenase in human intestinal smooth muscle cells.

BACKGROUND & AIMS: The cytokine interleukin (IL)-1 beta induces collagenase expression and inhibits collagen expression in human intestinal smooth muscle (HISM) cells. Corticosteroids cause transrepression of certain genes, including the collagenase gene. The aim of this study was to determine if corticosteroids repress the induction of collagenase expression and the inhibition of collagen secretion by IL-1 beta in HISM cells. METHODS: HISM cells were exposed to IL-1 beta (1-100 pmol/L) for 24 hours in the presence or absence of dexamethasone (10(-6) mol/L). Collagenase messenger RNA (mRNA) levels were determined by ribonuclease protection assay. Collagenase and tissue inhibitor of metalloproteinase protein secretion were determined by enzyme-linked immunosorbent assay of culture medium. Procollagen and collagen secretion were determined by polyacrylamide slab gel analysis of radiolabeled proteins in culture medium. RESULTS: A 30-fold induction of collagenase mRNA and collagenase protein secretion by IL-1 beta was completely abrogated by dexamethasone. Dexamethasone at 10(-6) mol/L also reduced basal levels of collagenase mRNA by 50% and blocked the IL-1 beta-induced inhibition of collagen secretion. CONCLUSIONS: Corticosteroids repress the collagenolytic action of IL-1 beta on HISM cells in vitro and therefore should promote healing in the inflamed intestine.

Collagen↗
PubMed1997

Sutureless technique: second version.

Mesh repairs have revolutionized hernia surgery. When used to patch or plug a musculoaponeurotic abdominal wall defect, the results have been much better than traditional pure tissue repairs. The difference is simple: patch and plug techniques avoid tension on tissues. The improved sutureless repair not only avoids tissue tension, it obviates the need to suture the mesh. Fixation is achieved by intra-abdominal pressure, the same force that caused the hernia. Thorough dissection of the inguinal canal and the indirect sac is essential to avoid early failure. Whereas various repairs can be used with excellent results, there is no substitute for a complete dissection of the peritoneal sac well into the iliac fossa. The improved sutureless repair offers 2 advantages over the original version: (a) type III hernias can now be repaired without opening the canal's posterior wall, and (b) the incidence of clinically evident seroma has been reduced by 90%. Most primary and recurrent groin hernias can be repaired under local or regional anesthesia on an outpatient basis. Immediate ambulation and prompt recovery accompany this technique. Most patients resume full activity and employment by the end of the first week. The procedure is simple to learn, easy to perform and less costly than other techniques.

Anesthesia, Conduction↗
PubMed1996

Interleukin 1 beta down-regulates collagen and augments collagenase expression in human intestinal smooth muscle cells.

BACKGROUND & AIMS: Smooth muscle cells resident in the intestinal wall play a significant role in the healing of the injured intestine and in the fibrosis that complicates Crohn's disease. The cytokine interleukin 1 beta (IL-1 beta) is involved in inflammatory bowel disease. The aim of this study was to determine the action of IL-1 beta on proliferation and collagen metabolism in human intestinal smooth muscle cells. RESULTS: IL-beta caused a three-fold increase in [3H]thymidine uptake at 100 pmol/L. This mitogenic effect was equipotent with that of platelet-derived growth factor when cells were exposed to IL-beta for 48 vs. 24 hours. IL-beta inhibited the secretion of procollagen into culture medium by 70% and the accumulation of newly synthesized procollagen in cells by 55%. In addition, IL-beta caused a concentration-dependent inhibition of steady-state levels of procollagen I and III messenger RNA (85% inhibition at 100 pmol/L) and a 3-5-fold augmentation of collagenase messenger RNA levels. CONCLUSIONS: IL-beta is mitogenic for human intestinal smooth muscle cells, but this action is associated with a concomitant down-regulation of collagen synthesis and secretion and an augmention of collagenase expression.

Blotting, Northern↗
PubMed1996

Helicobacter pylori cytotoxin induces vacuolation of primary human mucosal epithelial cells.

We investigated whether Helicobacter pylori cytotoxin induces vacuolation in primary epithelial cells from normal human mucosa. Epithelial cells purified by enzyme digestion and elutriation were evaluated for vacuolation in a blinded protocol by light and electron microscopy before and after incubation with culture supernatant (CS) from H. pylori 60190, which has vacuolating activity for HeLa cells (Tox+), and isogenic H. pylori mutant 60190-v1, which lacks this activity (Tox-). Primary epithelial cells (>98% pure) exposed to CS from Tox+ H. pylori exhibited marked vacuolation (52% +/- 5% of cells) compared with epithelial cells exposed to either CS from Tox- H. pylori (23% +/- 3.2%) or uninoculated control broth (23% +/- 3.7%) (P < 0.05) by light microscopy, which was confirmed by electron microscopy and antibody inhibition studies. These are the first data to show that H. pylori cytotoxin causes vacuolation of primary human mucosal epithelial cells.

Bacterial Proteins↗
PubMed1995

Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells.

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L-[5-3H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polyacrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate-replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady-state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two- to fourfold. L-ascorbate phosphate did not increase the activity of L-ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady-state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L-ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L-ascorbate-phosphate is not an adequate substitute for L-ascorbate in this cell line.

Ascorbic Acid↗
PubMed1995

Corticosteroids increase procollagen gene expression, synthesis, and secretion by human intestinal smooth muscle cells.

BACKGROUND & AIMS: Collagen synthesis by smooth muscle cells plays an important role in intestinal fibrosis. Corticosteroids inhibit collagen synthesis in fibroblasts. The aim of this study was to examine the effect of corticosteroids on the expression of collagen by human intestinal smooth muscle (HISM) cells in vitro. METHODS: Collagen synthesis was determined by the sensitivity of radiolabeled protein to collagenase. Secretion was determined by polyacrylamide gel electrophoresis of radiolabeled procollagen in the medium. Procollagen messenger RNA was determined by Northern blot analysis. RESULTS: Collagen synthesis by confluent HISM cells was not affected by corticosteroids at 10(-10) to 10(-5) mol/L but, in subconfluent cultures, was nonspecifically increased 50% at 10(-5) mol/L. Procollagen secretion was nonspecifically increased 60% at 10(-6) mol/L dexamethasone without any effect on the type III/I ratio. Procollagen I and III messenger RNA levels responded in a biphasic manner: a 45%-65% increase at 10(-10) mol/L and a 15% and 30% decrease at 10(-8) and 10(-6) mol/L. In fibroblasts, collagen synthesis was inhibited 85% by dexamethasone, procollagen secretion was decreased 70%, the type III/I ratio decreased from 70:1 to 18:1, and procollagen messenger RNA was inhibited 25% and 60% (types I and III). CONCLUSIONS: Collagen expression by HISM cells is refractory to corticosteroids and, at certain concentrations, is augmented.

Blotting, Northern↗
PubMed1992

Collagen production by human smooth muscle cells isolated during intestinal organogenesis.

The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [3H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen alpha chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.

Collagen↗
PubMed1990

A microassay to quantitate collagen synthesis by cells in culture.

A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [3H]proline. After incubation, the plates were heated to 90 degrees C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble 3H-labeled collagen-derived peptides and the remaining insoluble 3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and non-collagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.

Cells, Cultured↗
PubMed1990

Transforming growth factor beta 1 selectively augments collagen synthesis by human intestinal smooth muscle cells.

Intestinal smooth muscle cells play a major role in the stricture formation that complicates chronic intestinal inflammation, by proliferating and producing collagen. Transforming growth factor beta 1 has been identified as an important inflammatory mediator in the fibrotic response of human tissue to inflammation. To determine whether this mediator might be involved in intestinal fibrosis, the effect of transforming growth factor beta 1 on collagen production and proliferation by human intestinal smooth muscle cells was studied in vitro. Cells in the second passage were grown to subconfluence in medium containing 10% Nu-Serum (Collaborative Research Inc., Bedford, MA), after which the concentration of Nu-Serum was decreased. Forty-eight hours later, transforming growth factor beta 1 was added to the culture medium to achieve concentrations of 1-500 pmol/L. After 24 hours exposure to the transforming growth factor beta 1, cellular collagen synthesis was determined by the uptake of [3H]proline into collagenase-sensitive protein. Transforming growth factor beta 1 caused a 100% increase in collagen production and a 40% increase in noncollagen protein production per cell, reflecting an increase in relative collagen synthesis of 58%. This effect was maximal at a concentration of 10 pmol/L. Epidermal growth factor, by comparison, had no significant effect on relative collagen synthesis. Transforming growth factor beta 1 caused a significant increase in the uptake of methylaminoisobutyric acid, a nonmetabolized amino acid analog, into the cells at 10 pmol/L. However, this effect was small (20% increase) compared with the effect on the uptake of proline into collagen (100% increase) at this concentration. When cell proliferation was examined by the uptake of [3H]thymidine, transforming growth factor beta 1 had no effect, whereas epidermal growth factor (1000 pmol/L) caused a 94% increase. Transforming growth factor beta 1 selectively augments collagen production by human intestinal smooth muscle cells in vitro. This effect is potent and is not related to effects on either cell proliferation or amino acid uptake. These data suggest that transforming growth factor beta 1 has an important role as an inflammatory mediator in the pathogenesis of intestinal fibrosis.

Cells, Cultured↗