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PubMed1989

Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages.

A complement fixing IgM monoclonal antibody (1B10) that reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes was produced. In Western blot analysis of detergent extracts of cultured human foreskin fibroblasts, antibody 1B10 detected protein bands of Mr 43,000 and 72-80,000. We used the 1B10 antibody with complement to eliminate most 1B10 positive nonepithelial cells from thymic epithelial (TE) cell cultures, thereby allowing us to grow highly enriched populations of human TE cells.

Antibodies, Monoclonal↗
PubMed1977

In vitro studies on the protoporphyrin uptake and photosensitivity of normal skin fibroblasts and fibroblasts from patients with erythropoietic protoporphyria.

Fibroblasts derived from patients with erythropoietic protoporphyria (EPP) are not sensitive to violet light and do not contain an excess of protoporphyrin (PP). Fibroblasts from both normal individuals and patients with EPP can take up very low concentrations of PP from culture medium. Cells grown in PP-containing medium showed a gradually increasing but limited uptake of PP and also an increased sensitivity to light. A sensitive scanning microfluorometric method has made it possible to demonstrate that the PP is mainly localized in the perinuclear granules. After exposure to violet light, cells photosensitized by PP in a culture medium showed increased membrane permeability as well as reduced reproductive capacity. Both of these photodamage effects can be repaired during postirradiation incubation in culture medium at 37 degrees C.

Cell Division↗
PubMed1984

Mucopolysaccharidosis III B: hybridization studies on fibroblasts from a mild case and fibroblasts from severe patients.

Cell fusion with polyethylene glycol (PEG) was performed on fibroblasts from a previously studied patient with a mild form of Sanfilippo B disease and altered residual enzyme activity and fibroblasts from several patients with the classical, severe form of the disease. No complementation was found in heterokaryons. This suggests that the mild phenotype in our patient can be ascribed to an allelic mutation.

Acetylglucosaminidase↗
PubMed1995

Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts.

1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that is carried at least partially by Ca2+ ions; this current in turn causes a long-lasting increase in intracellular Ca2+ concentration. The amplitude and time course of these bFGF-activated ionic events are compatible with their involvement in the control of cell proliferation.

3T3 Cells↗
PubMed1981

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.

Animals↗
PubMed1984

In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy. An electron microscopic study.

The effects of laser irradiations have been estimated both from the clinical point of view (rate of reduction of the ulcerated area) and in ultrastructural terms. The modification produced by laser in human fibroblasts in culture have also been investigated. Under our clinical and experimental conditions, laser stimulates the secretion of fibroblasts, both in vivo and in vitro, for reasons that are still the object of research. After irradiation the formation of periodic collagen fibrils is not observed, so that complete ' restitutio ad integrum' of the dermal matrix does not occur.

Collagen↗
PubMed2002

Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis.

Despite their similar spindle-shaped appearance, periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) appear to display distinct functional activities in the maintenance of tissue integrity and during inflammatory/immune responses. We postulated that different characteristics of PDLF and GF are defined by the differential expression of specific genes. To test this, we investigated the possible variance of gene expression profile between cultured PDLF and GF, using DNA microarray technology. One hundred sixty-three genes were found differentially expressed by at least three-fold between PDLF and GF. Genes encoding transmembrane proteins and cytoskeleton-related proteins tended to be up-regulated in PDLF, whereas genes encoding cell-cycle regulation proteins and metabolism-related proteins tended to be up-regulated in GF. We concluded that PDLF and GF appear to display different gene expression patterns that may reflect intrinsic functional differences of the two cell populations and may well coordinate with their tissue-specific activities.

Blotting, Northern↗
PubMed2003

IGF-1 signaling enhances cell survival in periodontal ligament fibroblasts vs. gingival fibroblasts.

The role of insulin-like growth factors (IGFs) in the regulation of apoptosis has been suggested, yet their impact on specific cells such as periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) remains unknown. The purpose of this study was to test the role of IGF-1 signaling in cell survival in PDLF compared with GF. In periodontal tissue sections, a significantly reduced apoptotic rate was first demonstrated in PDLF compared with GF. In vitro, IGF-1 substantially enhanced cell survival in PDLF compared with GF by the up-regulation of anti-apoptotic molecules and the down-regulation of pro-apoptotic molecules. Furthermore, the differential expression of insulin-like growth factor binding protein 5 (IGFBP-5) was observed in vitro, and its differential distribution was confirmed in vivo. Analysis of the present data suggests an enhanced cell survival in PDLF compared with GF by the up-regulation of IGF-1 signaling pathway.

Animals↗
PubMed2000

Fibroblast biology. Effector signals released by the synovial fibroblast in arthritis.

There is mounting evidence indicating that the synovial fibroblast is a direct effector of tissue injury and matrix remodeling in inflammatory synovitis. Through the elaboration of effector signals including cytokines and chemokines, mesenchymal cells stimulate or suppress inflammation via autocrine and paracrine mechanisms. Synovial fibroblasts are the principal cells mediating joint destruction through secretion of metalloproteinases, and recent evidence suggests that they may also promote bone resorption by stimulating osteoclastogenesis. Moreover, they may play an integral role in the initial phases of synovitis by releasing chemokines that recruit leukocytes to the joint, and cytokines that trigger angiogenesis. Studies focusing on synoviocyte-leukocyte interactions mediated via the cytokine network and the role of cell-cell contact in driving synoviocyte activation will help define the complex interplay that leads to the initiation and perpetuation of synovial inflammation.

Animals↗
PubMed1985

Comparative enzymology of eleven acid hydrolases in cultured amniotic fluid cells, skin fibroblasts and embryonic lung fibroblasts, and the respective changes with the increasing age of the cell cultures.

Assay conditions were studied for eleven lysosomal enzymes (beta-D-galactosidase, alpha-D-mannosidase, beta-hexosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, alpha-D-glucosidase, arylsulfatase, beta-D-glucosidase, alpha-L-fucosidase, alpha-D-neuraminidase and alpha-L-iduronidase) in cultured amniotic fluid cells (CAFC), cultured skin fibroblasts (CSF) and cultured embryonic lung fibroblasts (CELF), and the properties of the enzymes were compared among these cultured cells. In addition, changes in these enzymes from the three cell types were investigated between 4-6 earlier passages and 24-26 later passages. With the exception of alpha-D-glucosidase, alpha-D-neuraminidase and alpha-L-fucosidase, all enzymes assayed for the 4-6 earlier passages and the 24-26 later passages had the same Km values and the same pH optima, and were also unchanged with the increasing age of cell cultures, with regard to their points. The specific activities of beta-D-glucuronidase, arylsulfatase, alpha-D-glucosidase and beta-D-glucosidase for the 4-6 earlier passages increased significantly with development, though no change was observed with development in the specific activities of other enzymes. Variations were observed between the levels of these enzymes in the three cell types with the increasing age of cell cultures, such as increases in some, decreases in others and no change in still others.

Amniotic Fluid↗
PubMed2000

Enamel matrix-derived protein stimulates attachment of periodontal ligament fibroblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fibroblasts.

BACKGROUND: Although it is claimed that enamel matrix-derived proteins (EMP) can be used to promote new attachment formation around periodontally involved teeth, the underlying biological mechanism is not understood. It was the aim of the present study to investigate the effects of EMP on the behavior of human periodontal ligament (HPLF) and gingival fibroblasts (HGF) in vitro, with special focus on their attachment properties, the expression of alkaline phosphatase (ALP) activity, the release of transforming growth factor (TGF)beta1, and their proliferative rate. METHODS: Fibroblast populations were obtained from 10 individuals with a healthy periodontium and cultured in chemically defined medium on culture plates coated with EMP, purified collagen type I, or their respective vehicles. Experiments were performed in the absence of serum for periods up to 48 hours. RESULTS: It was shown that HGF barely attached and spread on EMP-coated substrata, whereas HPLF attached and spread within 24 hours. However, when cultured on purified collagen type I, both cell types showed rapid attachment and spreading. Furthermore, the expression of ALP activity was significantly enhanced under the influence of EMP, especially in HPLF. HPLF and HGF both released significantly higher levels of TGFbeta1 in the presence of EMP. EMP did not influence 3H-thymidine incorporation by HPLF and HGF. CONCLUSIONS: Our results indicate that HPLF and HGF respond differently to EMP. A more rapid attachment of HPLF to this substratum might contribute, during the initial stages of periodontal healing, to selective outgrowth and colonization of exposed root surfaces in vivo.

Adolescent↗
PubMed1998

ATP inhibition of proliferation of immortalized human fibroblasts is greater than that of normal human diploid fibroblasts.

It is known that cancers develop by a multi-step process. Normal cells are first immortalized, and then transformed into tumorigenic cells. Normal human cells are very rarely immortalized, but once they are, they are relatively easily transformed into tumorigenic cells. This indicates that the immortalization step plays a critical part in the development of human cancers. Thus, elucidation of the mechanisms of this step would shed light on the process of carcinogenesis in human cells. To understand the causes of immortalization, it is important to determine the differences in cellular phenotype between immortalized and normal human cells. In this study, we found that immortalized human fibroblasts were more sensitive to the growth inhibitory effects of ATP than normal human fibroblasts. ADP was as effective as ATP, but AMP, adenosine, and phosphoric acid were not. These results indicate that a high-energy bound of ATP and ADP may contribute to the growth inhibition of the cells. When the immortalized cells were pulse-labeled with [32P]-ATP, 30-, 31-, 33- and 40-kDa membrane fraction proteins were more prominently labeled in the immortalized cells than in the normal cells. At present, the characteristics of these proteins are being investigated.

Adenosine↗
PubMed2003

[The comparison of biologic character between mouse embryonic fibroblast and human embryonic fibroblast].

To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.

Animals↗
PubMed2004

[The effect of basic fibroblast growth factor on angiogenesis and on the expression of basic fibroblast growth factor and vascular endothelial growth factor in acute infarcted myocardium].

OBJECTIVE: To evaluate the effect of intramyocardial administration of basic fibroblast growth factor (bFGF) on the angiogenesis and on the expression of bFGF and vascular endothelial growth factor (VEGF) in infarcted myocardium. METHODS: Twenty-four mongrel dogs were randomized into control group and therapeutic group. Acute myocardial infarction was made by ligation of the left anterior descending coronary artery distal to its first diagonal branch. After coronary artery was occlused, 50 micrograms of basic fibroblast growth factor in 15 ml of normal saline were injected into the infarcted myocardium and its border zone in therapeutic group, whereas the same volume of normal saline alone was used in control dogs. Every 3 dogs in each group was studied on the 1st day, the 3rd day, the 10th day and the 17th day after surgery respectively. With sensitivity encoded technique, cine MR was performed on each dog before euthanasia to evaluate the cardiac function. Angiogenesis and the expression of VEGF and bFGF were evaluated by immunohistochemical studies with factor VIII and the antibodies of VEGF and bFGF. RESULTS: In therapeutic group, left ventricular ejection fraction, improved markedly since the 10th day after injection of bFGF. Microvessel density was higher in the therapeutic group than in the control group except on the first day. VEGF and bFGF expressed more in the therapeutic group. CONCLUSION: Intramyocardial administration of bFGF is useful in increasing the growth of microvessels and improving the left ventricular function in acute myocardial infarction.

Animals↗
PubMed2004

Fibroblastic reticular cells and fibroblast-like cells determined by monoclonal antibodies B-F45 and B-D46 in humans.

OBJECTIVE: Identification of stromal microenvironmental components of lymphoid organs is relatively harder at light microscopic level as few markers, which are mostly not very specific, are available to be used for such a purpose. We screened a large panel to determine monoclonal antibodies (mAbs) those reactive with fibroblasts/fibroblast-like cells aiming to obtain further evidence for the organization and function of this cell group. METHODS: Tissue samples of forty patients undergoing surgery in Otorhinolaryngology, Obstetrics and Gynecology, Orthopedics and Traumatology, Cardiovascular Surgery and General Surgery Departments, Hacettepe University Medical Faculty Hospital, Ankara, Turkey, due to different pathologies obtained as partial specimens of surgery which were apart from pathological examination were immunostained by indirect immunoperoxidase method in histology and embryology department in 2003. RESULTS: Among the screened monoclonal antibodies, monoclonal antibodies B-F45 and B-D46 reacted with the members of the family, therefore examined in detail in available human organs. Among the unique staining patterns of these mAbs, reactivity on fibroblastic reticular cells, perineural sheet cells pericryptal/perivillous fibroblasts were striking. CONCLUSION: Both mAbs will provide useful tools for further studies on stromal network of peripheral lymphoid organs and peripheral nerves.

Adolescent↗
PubMed1990

[Stimulatory effects of Bacteroides gingivalis fimbriae on production of fibroblasts-derived thymocyte-activating factor (FTAF) by human gingival fibroblasts].

Bacteroides gingivalis (B. gingivalis) is isolated frequently from subgingival plaques of adult periodontal patients. B. gingivalis is a gram-negative anaerobic organism which has fimbriae on its cell surface. In the present study, B. gingivalis fimbriae were examined for their ability to adhere to human gingival fibroblasts (Gin-1), and to stimulate fibroblast-derived thymocyte-activating factor (FTAF) production by Gin-1 cells. The ability of the fimbriae to bind specifically to Gin-1 cells was clearly shown by competition assay between 125I-labeled and unlabeled fimbriae. Significant stimulatory effect of the fimbriae on FTAF production was observed, when the fimbriae were added to Gin-1 cells at a dose of 1 microgram/ml, and this stimulation was observed as early as 24 hr after addition of fimbriae to the cells. It was verified by a spleen cell mitogenic assay for the fimbriae that the stimulatory effect was not due to lipopolysaccharide (LPS) contamination of the fimbriae preparation. The FTAF activity was inhibited about 50% by recombinant human interleukin-1 (IL-1) beta antiserum but not by recombinant human IL-1 alpha antiserum. Therefore, the present study suggests that B. gingivalis fimbriae may play a functional role in the pathogenesis of adult periodontal disease induced by the microorganism.

Bacterial Adhesion↗
PubMed1990

Differences in targeting and secretion of cathepsins B and L by BALB/3T3 fibroblasts and Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts.

BALB/3T3 fibroblasts (3T3) were observed to secrete latent, pepsin-activatable forms of cathepsin B and cathepsin L as well as an active form of beta-glucuronidase when cultured in the absence of serum. The secretion of these proteins was stimulated by the cation ionophore monensin: cathepsin B, 4.3-fold; cathepsin L, 7.2-fold; and beta-glucuronidase, 3.1-fold. These increases were accompanied by a 50% decline in cellular levels of the active forms of these enzymes and by the cellular accumulation of latent forms of cathepsin B and cathepsin L. Latent forms of beta-glucuronidase were not detected. In contrast, Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts (MMSV) secreted greatly increased amounts of latent cathepsin B (17-fold) and latent cathepsin L (27-fold), and moderately increased amounts of active beta-glucuronidase (2-fold) in a manner which was not further increased by monensin. The increased monensin-insensitive secretion of these lysosomal enzymes by MMSV cells may be due to a transformation-induced decrease in mannose 6-phosphate receptors. Thus, 3T3 cells bound the neoglycoconjugate pentamannosyl 6-phosphate-bovine serum albumin at 4 degrees C in a pentamannosyl 6 phosphate and mannose 6-phosphate-inhibitable manner, whereas MMSV cells showed no measurable cell surface mannose 6-phosphate receptor binding activity.

Animals↗
PubMed1986

The mitogenic signaling pathway of fibroblast growth factor is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblasts.

Basic or acidic fibroblast growth factor (FGF), alone, was found to be as potent as alpha-thrombin to reinitiate DNA synthesis in G0-arrested Chinese hamster lung fibroblasts (CCL39). Basic FGF at 50 ng/ml or thrombin at 1 unit/ml rapidly initiated early events such as cytoplasmic alkalinization (0.2-0.3 pH units), rise in cytoplasmic Ca2+, phosphorylation of ribosomal protein S6 and increased c-myc expression, followed by a 30-40-fold increase in labeled nuclei. Whereas thrombin is a potent activator of phospholipase C as judged by the rapid release of inositol trisphosphate, inositol bisphosphate and by the massive accumulation of total inositol phosphate (IP) in the presence of 20 mM Li+, FGF failed to induce the breakdown of polyphosphoinositides in quiescent CCL39 cells. Indeed, no inositol trisphosphate nor inositol bisphosphate could be detected in response to FGF; in presence of Li+ the total IP release never exceeded 8% of the IP released by the action of thrombin. Two additional findings indicated that FGF and thrombin activate different signaling pathways. First, we found that, in contrast to thrombin, the FGF-induced rise in the cytoplasmic free Ca2+ concentration measured by quin-2 fluorescence, is strictly dependent upon the presence of Ca2+ in the external medium. Second, we found that FGF failed to activate protein kinase C as judged by the epidermal growth factor-receptor binding assay. Treatment of the cells with either thrombin or phorbol esters, rapidly inhibited 125I-labeled epidermal growth factor binding (50-60%). Basic or acidic FGF had no effect. We conclude that: the FGF-receptor signaling pathway is not coupled to phospholipase C activation, and early mitogenic events and reinitiation of DNA synthesis can be initiated independently of inositol lipid breakdown and protein kinase C activation.

Animals↗