Enhanced cyclooxygenase-2 mRNA expression in human gingival fibroblasts induced by cell contact with human lymphocytes.

Prostaglandins, especially prostaglandin E2 (PGE2), play a crucial role in the pathogenesis of periodontal disease. We have previously reported that inflammatory mediators interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) increase the production of PGE2 in human gingival fibroblasts. In this study, we investigated the effect of cell-to-cell interactions between gingival fibroblasts and lymphocytes on PGE2 production by using co-culture technique. Cell-to-cell contact between gingival fibroblasts and lymphocytes synergistically enhanced the production of PGE2 in co-cultures. In contrast to lymphocytes, the cyclooxygenase-2 (COX-2) mRNA expression in gingival fibroblasts was strongly enhanced following cell contact between gingival fibroblasts and lymphocytes. The level of COX-1 mRNA expression, however, was not affected either in gingival fibroblasts or in lymphocytes by the interactions between fibroblasts and lymphocytes. The study demonstrates that cell contact between gingival fibroblasts and lymphocytes strongly stimulates PGE2 production partly due to enhanced COX-2 mRNA expression in gingival fibroblasts. The cell-to-cell contact between gingival fibroblasts and lymphocytes should be considered as an important regulatory aspect for the enhancement of PGE2 in periodontal disease.

Effect of phenytoin on the production of interleukin-6 and interleukin-8 in human gingival fibroblasts.

The in vitro effect of phenytoin (PHT) on the production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human gingival fibroblasts, challenged with or without interleukin-1beta (IL-1beta), was studied. PHT (20 microg/ml) alone increased the mRNA level for both IL-6 and IL-8, as well as synergistically enhancing the production of IL-6 and IL-8, at both transcriptional and translational level in fibroblasts challenged with IL-1beta (30 pg/ml). The stimulatory effect of PHT on IL-1beta-induced IL-6 production was strongly reduced by the specific cyclooxygenase-2 inhibitor NS-398 (1 microM). The anti-inflammatory drug, dexamethasone (1 microM), abolished the production of both IL-6 and IL-8 in gingival fibroblasts challenged with PHT in the presence or absence of IL-1beta. The ability of PHT, alone as well as in combination with IL-1, to upregulate the production of IL-6 and IL-8 in human gingival fibroblasts may contribute to enhanced recruitment and activation of inflammatory cells. This effect of PHT may thereby give a prerequisite for the establishment of an interaction between cytokines and connective tissue cells in the periodontal tissue, which is suggested to lead to gingival overgrowth.

Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.

Safe biotechnology. 8. Transport of infectious and biological materials. Working Party "Safety in Biotechnology" of the European Federation of Biotechnology.

The transport of infectious and biological material is regulated by a number of international organizations. This mini-review has been compiled to increase awareness within the scientific community of problems caused by differences in terminology (such as infectious materials/substances, biological products, diagnostic specimens, genetically modified microorganisms) and certain technical aspects of the main international guidelines, and to assist policy makers in the creation of harmonized guidelines. A list of relevant Internet resources has been compiled.

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

Effect of phenytoin on interleukin-1 beta production in human gingival fibroblasts challenged to tumor necrosis factor alpha in vitro.

Effects and interaction of tumor necrosis factor alpha (TNF alpha) and the antiepileptic drug phenytoin (PHT) on interleukin-1 beta (IL-1 beta) production as well as on prostaglandin E2 (PGE2) formation were studied in gingival fibroblasts in vitro. TNF alpha, in contrast to PHT, dose-dependently stimulated the production of cell-associated IL-1 beta. The stimulatory effect of TNF alpha on IL-1 beta production was accompanied by enhanced PGE2 formation. When PHT and TNF alpha were added simultaneously, the drug potentiated the stimulatory effect of TNF alpha on both IL-1 beta production and PGE2 formation. The major PHT metabolite, p-HPPH, did not affect IL-1 beta production, either alone or in combination with TNF alpha. The production of IL-1 beta induced by TNF alpha and the combination of TNF alpha and PHT was further enhanced in the presence of the prostaglandin endoperoxide (PGH) synthase inhibitors, indomethacin and flurbiprofen. The PHT-mediated enhancement of TNF alpha-induced IL-1 beta production and PGE2 formation in gingival fibroblasts may be an important link in the pathogenesis of gingival overgrowth induced by PHT.

Safe biotechnology. Part 6. Safety assessment, in respect of human health, of microorganisms used in biotechnology.

The assessment of microorganisms in respect to human health is an important step for the introduction of new natural and genetically modified production strains to biotechnology. This report outlines the potential hazards posed by industrial microorganisms, important considerations related to pathogenicity, such as routes and portals of entry into the human body, mechanisms of spread of biological material and a definition of pathogenicity. Furthermore the most important steps in the assessment of pathogenicity of unknown strains are described. A short overview on characterization and in vitro and in vivo tests is presented. The hazard related to allergens and toxic metabolites is reviewed and the choice of methods and the handling of strains with unknown potential are discussed.

Safe biotechnology (5). Recommendations for safe work with animal and human cell cultures concerning potential human pathogens.

The benefits of using animal or human cell cultures have been clearly demonstrated in diagnostic and therapeutic research and in their application for manufacturing. Cell cultures serve as a tools for the production of vaccines, receptors, enzymes, monoclonal antibodies and recombinant DNA-derived proteins. They represent an integral part of drug development for which corresponding facilities, equipment and manufacturing processes are required. Although the cells themselves offer no particular risk to workers in laboratories and production areas or to the environment, the cell cultures may be contaminated with viruses, mycoplasma, bacteria, yeast and fungi or might contain endogenous viruses. The containment level for animal and human cells is therefore determined by the risk class of these agents. The history of animal and human cell cultures has proved that they can be handled safely. The recommendations in this publication concern the safe handling of cell cultures (tissue explants, primary cell cultures) and permanent cell lines of animal and human origin. A classification system of safety precautions has been elaborated according to the potential for contamination with the pathogenic agents involved.

The phenytoin metabolite p-HPPH upregulates prostaglandin biosynthesis in human gingival fibroblasts challenged to interleukin-1.

The effects of and interactions between the major phenytoin (PHT) metabolite 5-parahydroxyphenyl-5-phenylhydantoin (p-HPPH) and interleukin-1 (IL-1 alpha, IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on prostaglandin biosynthesis in human gingival fibroblasts were studied. IL-1 alpha, IL-1 beta and TNF alpha, dose-dependently, stimulated PGE2 formation in gingival fibroblasts. The metabolite, p-HPPH (1.2-2.4 micrograms/ml), did not induce PGE2 formation itself but potentiated IL-1 alpha and IL1 beta induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentration of both IL-1 and p-HPPH. The metabolite also stimulated IL-1 induced formation of 6-Keto PGF1 alpha, the stable breakdown product of PGI2, in a dose dependent manner. IL-1 beta induces release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts, which was potentiated by p-HPPH (> or = 1.2 micrograms/ml). TNF alpha (> or = 1 ng/ml) significantly stimulated the biosynthesis of PGE2 by a process that was also potentiated by p-HPPH. Addition of exogenous, unlabelled AA (10 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by p-HPPH (1.6 micrograms/ml). The results indicate that treatment with p-HPPH results in upregulation of prostaglandin synthesis in gingival fibroblasts challenged to IL-1 or TNF alpha at the level of phospholipase A2.

Safe biotechnology (4). Recommendations for safety levels for biotechnological operations with microorganisms that cause diseases in plants.

The Working Party on Safety in Biotechnology of the European Federation of Biotechnology has proposed a classification of microorganisms that cause diseases in plants. In this paper appropriate safety levels are proposed for these classes of microorganisms in order to ensure that research, development and industrial fermentation work with plant pathogens will limit the risk of outbreaks of diseases in crops that could result from work with such microorganisms when they are cultivated in laboratories, glasshouses and biotechnology installations.

Bradykinin induces a B2 receptor-mediated calcium signal linked to prostanoid formation in human gingival fibroblasts in vitro.

The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca2+, [Ca2+]i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg9-BK, induced a significant rapid (within seconds) and transient increase in [Ca2+]i, that was not dependent on extracellular Ca2+. The stimulatory effect of BK was seen in concentrations at or above 10(-8) M, with the most pronounced effect at 10(-6) M. D-Arg0-Hyp3-Thi5,8-DPhe7-BK, a BK B2 receptor antagonist, but not des-Arg9-Leu8-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca2+]i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE2 formation. When extracellular Ca2+ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca2+ addition, BK still caused a significant stimulation of PGE2 formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE2 formation. The two phorbol esters phorbol 12,13-dibutyrate and 4-beta-phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE2 formation. The results indicate that BK-induced PGE2 formation in gingival fibroblasts is coupled to an increase in [Ca2+]i mediated by the BK B2 receptor, and which is independent of extracellular Ca2+.

On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts.

Recombinant human interleukin-1 beta (IL-1 beta) and bradykinin (BK) synergistically stimulate prostaglandin E2 (PGE2) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1 beta per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1 beta, caused a rapid, transient rise of intracellular Ca2+ concentration ([Ca2+]i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1 beta did not change the effect of BK on [Ca2+]i. Ionomycin and A23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1 beta on PGE2 formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1 beta on PGE2 formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE2. In IL-1 beta treated cells, the enhancement of PGE2 formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1 beta. These data show that i) the synergistic interaction between IL-1 beta and BK on PGE2 formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca2+]i; ii) the signal transducing mechanism by which BK interacts with IL-1 beta, however, may be linked to a BK induced stimulation of [Ca2+]i and/or protein kinase C; iii) the mechanism involved in the action of IL-1 beta may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.

Phenytoin potentiates interleukin-1-induced prostaglandin biosynthesis in human gingival fibroblasts.

1. The effect of phenytoin (PHT) on prostaglandin E2 (PGE2) biosynthesis in human gingival fibroblasts stimulated by interleukin-1 (IL-1 alpha, IL-1 beta) or by tumour necrosis factor alpha (TNF alpha) was studied. 2. IL-1 alpha (1.5-6.0 ng ml-1) and IL-1 beta (30-300 pg ml-1), dose-dependently, stimulated PGE2 formation, in 24 h cultures, with IL-beta being the most potent agonist. 3. PHT (2.5-20 micrograms ml-1) did not induce PGE2 formation itself but potentiated IL-1 alpha- and IL-1 beta-induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentrations of both IL-1 and PHT. 4. IL-1 beta (0.1-1.0 ng ml-1) induced release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts that was potentiated by PHT (20 micrograms ml-1). 5. TNF-alpha (greater than or equal to 0.01 micrograms ml-1) significantly stimulated the biosynthesis of PGE2 by a process that was potentiated by PHT. 6. Addition of exogenous arachidonic acid (AA) (greater than or equal to 1 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by PHT (20 micrograms ml-1). 7. The results indicate that treatment with PHT results in upregulation of prostaglandin biosynthesis in gingival fibroblasts challenged with IL-1 or TNF alpha, at least partly due to enhanced level of phospholipase A2 activity.

Influence of phenytoin on cytoplasmic free Ca2+ level in human gingival fibroblasts.

Influence of 5,5-diphenylhydantoin (phenytoin; PHT) on the cytoplasmic free Ca2+ concentration, [Ca2+]i, was studied in fura 2 loaded adherent monolayers of human gingival fibroblasts derived from three patients before and after 9 months of PHT therapy. In the patient where gingival overgrowth developed during PHT medication (responder), addition of PHT to gingival fibroblasts derived before PHT medication induced a transient extracellular Ca2+ dependent increase in [Ca2+]i. In a non-responder patient, where gingival overgrowth did not develop during the same period of PHT therapy, addition of PHT to gingival fibroblasts derived before the start of medication did not significantly affect [Ca2+]i. Under extracellular Ca2+ deficient conditions, addition of PHT to serum-starved fibroblasts derived from the two categories of patients before the medication resulted in an increase in [Ca2+]i. In fibroblasts derived from the responder patient during PHT medication, in contrast to those from the non-responders (n = 2), the basal level of [Ca2+]i was significantly decreased. The results indicate that, in the cases studied, there is a relationship between PHT induced alterations in [Ca2+]i in gingival fibroblasts and the clinical development of gingival overgrowth.

Effect of phenytoin on intracellular 45Ca2+ accumulation in gingival fibroblasts in vitro.

Effect of 5,5 diphenylhydantoin (phenytoin; PHT) alone or in combination with epidermal growth factor (EGF) on the intracellular accumulation of the radioisotope 45Ca2+ (4 min labelling period) was determined in gingival fibroblasts. EGF as well as PHT increased the intracellular accumulation of the radioisotope in normal gingival fibroblasts by approximately 2 and 1.6-fold, respectively. In contrast, in fibroblasts derived from the phenytoin-induced gingival overgrowth, neither EGF nor PHT stimulated intracellular accumulation of 45Ca2+. When normal gingival fibroblasts were treated in vitro with EGF in combination with PHT, the EGF-induced increase in intracellular accumulation of the radioisotope 45Ca2+ was abolished. The rate of efflux of the radioisotope 45Ca2+ in prelabelled normal gingival fibroblasts was decreased by PHT treatment in vitro to a level already present in fibroblasts derived from PHT-induced gingival overgrowth. This study indicates that PHT influences the cellular calcium metabolism in fibroblasts which may contribute to the pathogenesis of gingival overgrowth.

An improved standardized procedure for urine mutagenicity testing.

The aim of the present study was to optimize the procedures for urinary mutagenicity testing in order to lower the baseline variation in mutagenic activity found in urine from unexposed subjects and to increase the sensitivity of the method. This was accomplished by using urine from nonsmokers and smokers as well as chemically spiked nonsmokers' urine. Diet was standardized. The number of mutants per ml of urine calculated from the linear portion of the dose-response curve was used as a measure of mutagenicity. The parameters investigated were (i) the total volume of urine per resin volume, (ii) the flow rate, (iii) the pH, (iv) the ionic strength of the urine, and (v) elimination of histidine. XAD-2 and C18 Sep-Pak resins recovered mutagens in smokers' urine and in chemically spiked urine with the same efficiency when an optimized procedure was adopted. The optimized procedure using a maximum volume of 50 ml acidified urine per Sep-Pak cartridge, or equal amount of XAD-2 resin, gave well over ten times greater recovery of mutagens from smokers' urine than in earlier reports. Histidine was effectively eliminated, and the background variation was also lowered.

Mitogenic activity of chloroform and carbon tetrachloride in serum-deficient or calcium-deficient cultures of human embryonic lung fibroblasts.

It was demonstrated that chloroform--studied in the non-toxic concentration interval 0.025-0.1% w/v--as well as carbon tetrachloride at 0.0125-0.05%, can support proliferation of cell cycling human embryonic lung fibroblasts in Dulbecco's modified Eagle's medium (DMEM) complemented with 0.5% foetal calf serum. This serum concentration restricts the growth of the non-solvent treated fibroblast (control) cultures. Even after a pretreatment of the fibroblasts with the low serum-medium for up to 4 days, the cells responded to the mitogenic impulse of the solvents. This indicates that quiescent fibroblasts are also sensitive to the mitosis-stimulating effect of the solvents. The effect of the solvents in serum-free DMEM was followed in a short-term study in order to determine if cofactor(s) from serum are needed for expression of the mitosis-stimulating activity of the solvents. Carbon tetrachloride was shown to be mitogenic for the fibroblasts. At solvent concentrations greater than or equal to 0.00078% w/v the cells were observed to proliferate about as rapidly as control cells incubated in 10% serum-containing DMEM. After change from solvent-containing to solvent-free medium cell proliferation was soon inhibited. Chloroform was not mitogenic in the serum-free medium. The mitogenic activity of CHCl3 or CCl4 in solvent containing 10% serum and reduced content of calcium ions (0.05 mM) was then determined. It was shown that the solvents under the non-physiological conditions support proliferation of the fibroblasts for 2-3 days depending on the solvent concentration.

The effect of Ca2+ on the thermal stability of trypsin in phosphate-buffered saline solution used for harvesting of human embryonic lung fibroblast cultures.

The stabilizing effect of Ca2+ (264.9 mg CaCl2 X 2H2O ml-1) on a 0.5% solution of twice-crystallized bovine trypsin in phosphate-buffered saline (used for harvesting human embryonic lung fibroblasts) was studied at 7-37 degrees C and at -20 and -70 degrees C. It can be concluded that storage of the enzyme in the buffer (with or without Ca2+) is not advisable at temperatures greater than or equal to 20 degrees C. At 7 degrees C, on the other hand, trypsin can be stored for some weeks in the calcium-containing phosphate-buffered saline if a moderate loss of activity is acceptable. At -20 degrees C and -70 degrees C the stability of the enzyme was good. In the presence of Ca2+ about 90% of the activity remained after 18 weeks. Without Ca2+ the activity was approximately 10% lower.

Interaction between Yersinia pseudotuberculosis and the HeLa cell surface.

The adherence of Yersinia pseudotuberculosis to the surface of HeLa cells at 4 degrees C was studied. This temperature allows adhesion of bacteria but prevents engulfment. Adhesion between the bacteria and the cells was not dependent upon the presence of serum, Ca2+ or Mg2+ in the medium. Maximum adhesion was obtained at pH 6.5-7.9 and pretreatment of the cells with formaldehyde or glutaraldehyde inhibited the attachment of the bacteria. The interaction between the bacteria and the cell surface seems to involve cellular processes that are mostly microvilli. An intimate association between the bacteria and the cellular glycocalyx was found. Three virulent bacterial strains adhered more easily to the cell surface than five avirulent strains. Maximum adherence was obtained with bacteria from late logarithmic and early stationary phases of growth. The bacteria gradually lose their adhesive property when cultivated for several generations at 37 degrees C in nutrient broth but not when cultivated at 20 degrees C. Treatment of the bacteria with protease IV from Streptomyces caespitosus markedly reduced the efficiency of attachment.

Variation in cell surface morphology of two 90Sr-induced osteosarcomas serially transplanted in CBA mice.

The induction of skeletal tumours, which can be classified as osteosarcomas of many different types, is considered to be the primary carcinogenic effect of radiostrontium. In the present report the cell surface morphology in vivo of 90Sr-induced osteosarcoma cells was investigated, since a variety of tumour cells--and especially those investigated in vitro--have been shown to possess morphologic changes compared with their normal counterparts. Using scanning electron microscopy, variations in cell surface morphology were observed in 2 tumour series, which were serially transplanted in mice for 45 and 60 transfer generations, respectively. The slow-growing osteosarcoma cells of the early transfer generations, of osteoblastic as well as fibroblastic type, seemed to have more cytopodia than the fast-growing osteosarcoma cells from later generations. This may be due to the fact that slowly growing cell populations have a large proportion of cells in G1, in which stage there is a higher frequency of cellular cytopodia.