Fibroblasts In Tissue Culture Research Articles

Nucleotide ectoenzyme activities of human and Chinese hamster fibroblasts in tissue culture

We have previously assigned human ecto-5'-nucleotidase (NT) to chromosome 6 on the basis of conversion of exogenously supplied [14C]AMP to adenosine by whole cells of human and Chinese hamster hybrids carrying chromosome 6. In this paper we demonstrate that the activity on human MRC-5 fibroblasts is typical of previously described and purified ecto-5'-nucleotidases. In contrast to MRC-5 cells, Chinese hamster V79A2 cells weakly express an AMPase activity that is not NT. The cytosolic form of NT in human and hybrid fibroblasts is similar to the ectoenzyme in substrate specificity. Hybrids that lack chromosome 6 express neither the ecto- nor the cytosolic enzyme, suggesting that both forms may be coded by the same gene on chromosome 6. Ecto-ATPase, ecto-ADPase, and ecto-ADP kinase activities are each expressed at similar levels in MRC-5 and V79A2. The ATPase, ADPase and NT activities of MRC-5 cells act sequentially to generate adenosine. A similar cascade acts on V79A2 cells but the lack of NT causes the accumulation of AMP.

In vitro effects of folate inhibitors on Toxoplasma gondii

Three sulfonamides and four dihydrofolate reductase inhibitors were tested alone and in combination to determine their in vitro effects on two strains of Toxoplasma gondii grown in MRC5 fibroblast tissue culture. Toxoplasma growth was quantitated by an enzyme immunoassay performed directly on the fixed cultures, and linear regression models were used to quantify the relationship between the optical density values generated by the enzyme-linked immunosorbent assay and the concentrations of the antimicrobial agents in the culture medium. The cytopathic effects of antimicrobial agents on T. gondii were examined in Giemsa-stained cultures. Sulfonamides and dihydrofolate reductase inhibitors exhibited similar patterns of inhibition, consisting of an important increase of the inhibitory effect within a narrow range of concentrations. Sulfadiazine, sulfamethoxazole, and sulfisoxazole were all found to have important inhibitory effects on T. gondii; the 50% inhibitory concentrations estimated from the regression models were 2.5 micrograms/ml for sulfadiazine, 1.1 micrograms/ml for sulfamethoxazole, and 6.4 micrograms/ml for sulfisoxazole. This inhibition of growth was associated with a reduction of the number of parasitized cells and intracellular parasites that were morphologically normal. With dihydrofolate reductase inhibitors, including pyrimethamine, trimethoprim, trimetrexate-glycuronate, and piritrexim, a strong inhibition of Toxoplasma growth was observed, which was associated with striking morphological changes of the parasites. The 50% inhibitory concentrations were 0.04 microgram/ml for pyrimethamine, 2.3 micrograms/ml for trimethoprim, 0.16 ng/ml for trimetrexate-glycuronate, and 6.9 ng/ml for piritrexim. When sulfonamides and dihydrofolate reductase inhibitors were used in combination, a synergistic effect was observed with sulfadiazine combined with pyrimethamine, trimetrexate-glycuronate, and piritrexim; sulfisoxazole combined with pyrimethamine; and trimethoprim combined with sulfamethoxazole. These results were analyzed in comparison with human pharmacokinetics data.

Adhesion of lymphoid cells to fibroblasts in tissue culture

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.

On the use of fibroblasts in tissue culture to study the neurochemistry of brain mechanisms controlling blood pressure.

Fibroblasts grown in tissue culture from skin biopsies taken from the genetic model of spontaneously hypertensive rats (SHR), show a reduction in the accumulation of choline when compared to fibroblasts grown from skin biopsies taken from normotensive Wistar Kyoto rats. Choline uptake by fibroblasts from both strains of rats is sodium dependent but there is no difference between the strains in the effects of sodium depletion. These results suggest that fibroblasts in tissue culture may be a useful source of tissue for analyzing differences between hypertensive and normotensive individuals that are genetically determined rather than due to prolonged exposure to elevated blood pressure. In addition, the fibroblast shares several biochemical characteristics with the neuron and, at least as far as these common characteristics are concerned, appears to be a good model for neurochemical study.

Cell-surface binding sites for high density lipoproteins do not mediate efflux of cholesterol from human fibroblasts in tissue culture.

The present investigation was designed to test the hypothesis that binding sites for high density lipoproteins (HDL3) on cell surfaces of peripheral tissues mediate cholesterol efflux from these cells. This hypothesis had been formulated to explain two observations: 1) HDL3 binding to peripheral cells and HDL3-mediated cholesterol efflux from these cells had both been found to saturate at similar unbound (free) HDL3 concentrations; and 2) both of these processes had been found to be similarly "up-regulated" by loading the cells with cholesterol. In the present study, however, we found that the "specific" binding of HDL3 to cholesterol-loaded human fibroblasts was saturated at a free HDL3 concentration of approximately 20 micrograms protein/ml, whereas efflux of cholesterol from these cells to HDL3 did not "saturate" even at a free HDL3 concentration of 2000 micrograms protein/ml. In addition, we found that the increase in cholesterol efflux caused by loading the fibroblasts with cholesterol was no greater when the acceptor particles were HDL3 than when albumin or phospholipid vesicles served as acceptors, despite a marked increase in HDL3 binding to these cells. Because HDL3 binding to these cells and HDL3-mediated cholesterol efflux from these cells do not saturate at similar free HDL3 concentrations, and because the cholesterol-induced increase in HDL3 binding is not accompanied by a similar increase in cholesterol efflux that is specific for HDL3, we conclude that the described HDL3 binding sites on human fibroblasts do not mediate cholesterol efflux.

Steady-state and nuclear run-on analyses of transcription in a temperature-sensitive Chinese hamster cell mutant with a defect in RNA metabolism

We have further characterized a temperature-sensitive mutant of Chinese hamster lung fibroblasts in tissue culture with a defect in RNA metabolism. The mutant phenotype is reflected in transcription in crude extracts or in isolated nuclei, when these are made from cells shifted to the nonpermissive temperature; however, differential heat inactivation between mutant and wild-type extracts cannot be demonstrated with cell-free systems. We tentatively conclude that the mutation may affect initiation of transcription which cannot be observed in our in vitro systems. Partially purified RNA polymerase I, II, and III fractions are indistinguishable from wild type. A temperature shift does not affect transcription by RNA polymerase III measured with intact cells or by nuclear run-on experiments. The nuclear run-on and other experiments suggest that RNA polymerase II-dependent transcription is inhibited before RNA polymerase I-dependent transcription. This conclusion is also supported by Northern analyses of selected mRNAs in nonsynchronized and synchronized cells after a shift to the nonpermissive temperature.

Detection of prion protein mRNA in normal and scrapie-infected tissues and cell lines.

Prion protein (PrP) forms the fibrils or prion rods isolated from scrapie-infected brain and has been proposed as the major component of the infectious agent of this slowly progressive spongiform encephalopathy. In previous Northern blot analyses PrP-specific mRNAs have been found in both normal and scrapie-infected brains but not in spleen, an organ which harbours large titres of infectivity. In the present study, mouse PrP DNA was used to probe for PrP mRNA in assorted tissues and cells. A reexamination of mouse and hamster spleens revealed that they contained low levels of PrP mRNA (approx. 0.8% of that in brain mRNA). No consistent differences were observed between normal and scrapie-infected tissues. Also positive for PrP mRNA under stringent hybridization conditions were mouse epithelial, neuroblastoma, erythroid, B-lymphocytic and embryo fibroblast tissue culture cell lines, a hamster ovary cell line, a rat glioma cell line, and human T lymphocytic and neuroblastoma cell lines. In contrast, no PrP mRNA was detected in two mouse myeloid cell lines and one T cell lymphoma. These results provide evidence that PrP is a protein common to numerous, but not all, cell types besides those of the brain.

Radiation inactivation of binding sites for high density lipoproteins in human fibroblast membranes.

Radiation inactivation and target analysis were used to determine the molecular mass of the binding sites for high density lipoproteins (HDL) on membranes prepared from human fibroblasts. These membrane binding sites shared characteristics with the previously described HDL binding sites on whole fibroblasts in tissue culture. They exhibited the same affinity for HDL, the same ligand specificity, and the same sensitivity to proteolytic agents. They were also up-regulated by cholesterol loading of the cells. Kinetics of HDL dissociation from membrane binding sites could not be described by a single exponential function, indicating that HDL probably bind to multiple classes of sites on fibroblast membranes. After exposure to ionizing radiation, these sites decreased in number as an apparent single exponential function of radiation dose, corresponding to an average molecular mass of 16,000 +/- 1,000 Da, which is smaller than any known cell-surface receptor protein. These data indicate that HDL binding sites on fibroblast membranes are not "classical" receptors in that they are kinetically heterogeneous and small in molecular mass.

Toxicity of bilirubin and bilirubin diglucuronide to rat tissue culture fibroblasts.

The effects of obstructive and non-obstructive jaundice on rat tissue culture fibroblasts have been compared. Tissue culture fibroblasts were grown in medium containing unconjugated bilirubin and medium containing conjugated bilirubin (bilirubin diglucuronide). The addition of bilirubin to the culture medium caused morphological changes in the fibroblasts and significantly decreased their rate of multiplication. The addition of bilirubin diglucuronide had no effect. As the fibroblast plays an important role in wound healing, patients with obstructive jaundice (where surgery is most frequently required) may be less likely to undergo wound failure than patients with non-obstructive jaundice.

Purification of two smooth muscle glycoproteins related to integrin. Distribution in cultured chicken embryo fibroblasts.

We have purified two membrane glycoproteins from chicken gizzard smooth muscle. In the presence of reducing agents, these proteins have molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 165,000 and 130,000, but they migrate at 165,000 and 110,000 without reduction. The two proteins can also be isolated as a complex in buffers containing physiologic salt concentrations. This complex has physical properties similar to two proteins of the integrin family of receptors for extracellular matrix proteins, the cell substratum attachment antigen from chicken embryos, and the glycoprotein IIb IIIa complex from mammalian platelets. When the smooth muscle complex is visualized by electron microscopy, it has a striking resemblance to both avian integrin and the glycoprotein IIb IIIa complex. Smooth muscle is a good source of the 165,000 and 130,000 proteins, and purification of both the individual subunits and the complex is achieved using conventional biochemical techniques. Antibodies directed against the 130,000 protein cross-react with integrin but do not cross-react with the 165,000 protein. Immunofluorescence microscopy using these antibodies reveals staining of fibroblast focal contacts and fibrillar streaks which coalign with fibronectin. Whereas monoclonal antibodies against integrin label the periphery of the focal contact more intensely than the center, the anti-130,000-protein serum stains the entire focal contact. Antibodies directed against the 165,000 protein also stain focal contacts and fibrillar streaks of fibroblasts in tissue culture. On the basis of similar physical properties, biochemical characteristics, and immunological cross-reactivity we conclude that the 165,000/130,000 complex is a smooth muscle integrin.

Perigraft seroma: Clinical, histologic, and serologic correlates

A 14 year retrospective study of perigraft seroma, defined as an enlarging sterile fluid collection at the site of a prosthetic graft, revealed well-documented cases in 5 of 118 extraanatomical bypasses (4.2 percent), 3 of 248 aortic reconstructive procedures (1.2 percent), and 1 of 395 femoropopliteal bypasses (0.3 percent). These nine cases involved four polytetrafluoroethylene and five Dacron grafts. There were five graft thromboses, one instance of limb loss, two graft infections, two deaths, and 13 separate surgical procedures related to the perigraft seroma. Histologic studies revealed a fibrous pseudomembrane lining the perigraft seroma wall and immature fibroblasts lining the graft. Sera from three patients with perigraft seroma, five patients with well-incorporated prosthetic grafts, and three healthy volunteer subjects were tested for in vitro evidence of fibroblast inhibition against fibroblast tissue cultures derived from the pseudomembrane of a perigraft seroma. Control fetal calf serum, sera from all three healthy subjects, and sera from all five patients with well-incorporated grafts allowed fibroblast proliferation. In contrast, sera from all three patients with perigraft seroma inhibited fibroblast growth. Furthermore, sera collected 1, 2, and 3 months after graft removal from one patient and serum collected 3 months after spontaneous resolution of a perigraft seroma from another patient failed to inhibit fibroblasts. We have concluded that patients with perigraft seroma have a high rate of graft and limb loss and require multiple reoperations. The pathogenesis of perigraft seroma appears to involve a humoral fibroblast inhibitor which prevents maturation and proliferation of perigraft fibroblasts, leading to poor graft incorporation. The decrease of inhibition below detectable levels after graft removal or spontaneous resolution of the perigraft seroma suggests that the graft may induce host production of the inhibitor. Effective therapy of perigraft seroma may include fibroblast modulation, removal of the inciting graft, or both.

Improvement in efficacy of corticosteroid therapy in an animal model of proliferative vitreoretinopathy by pretreatment.

Intraocular injection of the corticosteroid triamcinolone acetonide reduces the incidence of retinal detachment in rabbit eyes injected with tissue-cultured fibroblasts. When the steroid was injected simultaneously with the cells, a reduction of retinal detachment from 93% (control) to 75% (treated) was achieved on day 28. When the steroid was injected 24 h preceding cell injection, the reduction of retinal detachment was from 85% (control) to 43% (treated). The development of retinal detachment is caused by proliferation of injected fibroblasts. Reduction of this proliferation is probably achieved partially through direct inhibition of mitosis, but more important may be the reduction of the reactive inflammatory process.

Classification of the stages of proliferative vitreoretinopathy in a refined experimental model in the rabbit eye.

Eighty-four rabbit eyes injected intravitreously with tissue-cultured fibroblasts following gas compression of the vitreous were examined clinically over a period of 1 month. Detailed clinical descriptions of the extent of retinal changes were recorded. Clinical landmarks were determined and arranged into a new classification of this model of proliferative vitreoretinopathy (PVR). The proposed classification will allow easier communication between research groups using this model to study the treatment and prevention of PVR.

Mutagenic Potential of a 193-nm Excimer Laser on Fibroblasts in Tissue Culture

In response to concerns regarding possible DNA damage by far ultraviolet radiation during excimer laser corneal surgery, the mutagenic potential of an argon fluoride excimer laser (193 nm) on BALB/3T3 mouse fibroblasts grown in tissue culture was investigated. The cumulative incidence of anaplastic transformation after subablative radiant exposures from 3.5 mJ/cm2/pulse to 13.4 mJ/cm2/pulse was 3.6% of all cell cultures. The incidence of anaplastic transformation in nonirradiated controls was 4.2%. Transformation after exposure to x-ray radiation (60.9 rad) was 98.8%. The difference between the incidence of transformation of nonirradiated controls or excimer-treated cultures compared with x-ray radiation-treated cells was significant P < 0.0001 (chi square test). In this standard cell line, 193-nm laser energy does not appear to have substantial mutagenic potential.

The effect of beta irradiation on monkey Tenon's capsule fibroblasts in tissue culture.

Fibroblast proliferation with subsequent bleb scarring is a major cause of filtering surgery failure. To investigate possible antiproliferative effects of beta radiation, owl monkey Tenon's capsule fibroblasts in tissue culture were irradiated to doses of 30, 100, 300, 1,000, and 3,000 rads with a linear accelerator and to doses of 95,285, and 950 rads with a Strontium-90 applicator. The irradiated cell proliferation expressed as the percentage of the non-irradiated control growth on the third and seventh days, respectively, after beta irradiation was: 97% and 96% after 30 rads; 72% and 90% after 100 rads; 48% and 44% after 300 rads; 39% and 14% after 1,000 rads; and 39% and 14% after 3,000 rads. Similar effects on cell proliferation were observed with the Strontium-90 applicator. The inhibitory effect of beta irradiation on fibroblast proliferation in tissue culture suggests that beta irradiation after filtering surgery may reduce postoperative bleb scarring.

The Toxicity of Commonly Used Antiseptics on Fibroblasts in Tissue Culture

We have allowed the liberal use of antiseptics for wound care to become an established practice with little consideration of any toxicity on exposed healing tissues. The care of open wounds and chronic ulcers is largely delegated to nursing staff with little help or advice from their medical colleagues. Alexander Fleming felt that antiseptics would only be of benefit in septic wounds if they stimulated or conserved natural defence mechanisms! He also regarded the assessment of tissue toxicity as being more important than any antimicrobial effect but there has been little research into this field.

86] Measurement of antagonistic effects of growth factors and interferons

This chapter describes techniques for the measurement of interactions between interferons (IFNs) and growth factors (GFs) in vitro, in tissue culture of either human diploid fibroblasts or blasts of the cortisone resistant mouse thymocytes, and also in vivo, in mice infected with Moloney sarcoma virus. The proliferation of cells and tissues probably cannot occur without the participation of GFs or substances that either mimic or substitute for GFs. GFs have been defined as endogenous substances produced by various tissues that have multiplication stimulating activity and/or other trophic effects but are not nutrients, antibodies, enzymes, or attachment factors. The majority of GFs are polypeptides of various molecular weight and structure that have a hormone-like mode of action. These include the interleukins that stimulate the proliferation of cells of the immune system. IFNs, as well as GFs, fulfill the main biological and biochemical criteria of hormones. However, the response of various cells and tissues to IFNs is inhibition of the proliferation and other complex reactions not necessarily connected with the cell cycle but clearly antagonistic to GFs. Thus, it has been suggested that IFNs and GFs can be grouped together as nonclassical hormones with opposite actions. Human embryonic diploid fibroblasts (HEF) strains are used between fifth and twentieth passage levels in culture. The cells are found to be sensitive to both human IFN and several GFs.

Radiosensitization of Human Fibroblasts by 3-Aminobenzamide: An Inhibitor of Poly(ADP-Ribosylation)

The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide (3AB) increases the radiosensitivity of normal human fibroblasts in tissue culture in a dose-dependent manner at noncytotoxic concentrations. 3AB was also found to inhibit the repair of potentially lethal damage as well as the recovery occurring when radiation was delivered as fractionated vs single exposures. However, repair as measured by an increased survival when radiation is delivered at low dose rate compared to high dose rate was not effected by 3AB administration.

Toxoplasma gondii reactivation identified by detection of parasitemia in tissue culture.

Three patients developed acute toxoplasmosis after allogeneic marrow transplantation for treatment of leukemia. In each case, Toxoplasma gondii was isolated from peripheral blood buffy-coat cells inoculated into fibroblast tissue culture after 10 to 39 days of incubation. Serologic studies did not suggest acute toxoplasmosis, but complete autopsies done in two patients showed active invasive disease. Serologic tests done before the transplants showed that these cases resulted from reactivation of latent infection rather than primary disease. Parasitemia is a feature of reactivation toxoplasmosis in immunosuppressed patients that may be identified by recovery of T. gondii from peripheral blood buffy-coat cells in tissue culture.

Comparison of the transcriptional properties of the friend and moloney retrovirus long terminal repeats: Importance of tandem duplications and of the core enhancer sequence

The effects of sequence differences within the long terminal repeats (LTRs) of various murine retroviruses on transcription are examined by linking genetically engineered recombinant LTRs to the protein coding region of the herpes simplex virus thymidine kinase (TK) gene and assaying TK expression in tissue culture fibroblasts. The Goldberg/Hogness box region and the enhancer region are examined independently. We find that the Friend and Moloney Goldberg/Hogness boxes (TATAAAA and AATAAAA, respectively) give similar results in this assay, whereas differences between the sequences in the enhancer region have a marked effect. Tandem duplications increase the transcription level of the LTR. A single nucleotide transition in the core enhancer sequence has as large an effect on transcription as the presence of a tandem duplication: thymidine in the fifth position of the core enhancer (TCTGTGGTAAG) leads to a much higher transcriptional activity than cytidine. The LTRs have been implicated by others in the oncogenic potential of murine retroviruses, which is perhaps dependent on the transcriptional properties endowed in part by the core enhancer and tandem duplications.