Toxicity of cadmium on Sertoli cell functional competence: an in vitro study.

Cadmium (Cd), an ubiquitous environmental metal, mainly used for industrial purposes, may be toxic at level of the reproductive system. Testis tubular-based Sertoli cells (SC), play a major role in constituting the blood-testis barrier and provide a unique microenvironment for the genesis and differentiation of germ cells. Hence SC strictly control sperm qualitative and quantitative parameters. We aimed to assess whether exposure to Cd would adversely affect superior mammal SC viability and function. We isolated and purified SC from pre-pubertal pig testes according to our method and incubated the retrieved cells with three different Cadmium chloride concentrations (5-10-15 microM). Parameters of SC function such as inhibin B and anti-Mullerian hormone (AMH) were depressed by Cd exposure, contrary to what observed in untreated controls. No impairment of the FSH receptor integrity on the SC, as assessed by 17-beta-estradiol production, upon stimulation with FSH, was observed in either 5 microM Cd-treated or untreated controls. Differences, on the contrary, were observed for higher Cd concentrations (10 and 15 mM), in terms of FSH receptor integrity, that was altered, as compared to untreated controls, in terms of lower production of 17-beta-estradiol. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all Cd -treated SC conditions as compared to controls. In conclusion, the Cd -related toxicity on SC, clearly demonstrated by our study, even at low concentrations, is expected to damage spermatogenesis that directly is dependent upon retention of SC viability and function.

Male mating performance and cytoplasmic incompatibility in a wPip Wolbachia trans-infected line of Aedes albopictus (Stegomyia albopicta).

Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae) is a maternally inherited endosymbiont of a large number of insects and other arthropods that induces various effects on host reproductive biology. Among these, cytoplasmic incompatibility (CI) is a form of sterility induced in eggs produced by mating between infected males and females uninfected or infected by an incompatible Wolbachia strain. This phenomenon has been proposed as a potential way to produce functionally sterile males to be used in genetic control programmes. In this paper, we report on experiments carried out to evaluate the mating performances of males of an Aedes albopictus (Stegomyia albopicta) (Diptera: Culicidae) line (ARwP), harbouring a new Wolbachia infection [the wPip strain from Culex pipiens Linnaeus (Diptera: Culicidae)], in comparison with naturally infected males (SR line). ARwP males did not differ from SR males with regard to insemination capacity. Mating competitiveness did not differ significantly between lines in either laboratory or greenhouse conditions. Moreover, crosses with SR females were characterized by a 100% CI regardless of ARwP male age. All of these findings suggest that ARwP males may represent a very efficient tool for control programmes against Ae. albopictus based on the release of functionally sterile males.

Silica particle size and shape: in vitro effects on extracellular matrix metabolism and viability of human bronchial epithelial cells.

Crystal micro-morphology and dimension of silica particles could be responsible for the high prevalence of silicosis as recently found among goldsmiths. In the present study we investigated two samples of silica particles with different surface sizes and shapes for their capacity to induce changes in ECM component production. In addition we investigated if their different effects could be related to cytotoxicity and apoptotic effects. Human bronchial epithelial cells were cultured with or without a sample of Silica used for casting gold jewellery, named in our experiments Silica P or a commercial sample of Silica with different physical and chemical properties, named in our experiments Silica F. After 48 h of exposure PCR analysis determined levels of several matrix components. As induction of the apoptosis cascade, annexin assay, caspase 3 activity and cellular cytoxicity by MTT assay were assayed. Silica F promoted fibronectin, MMP12, tenascin C and Integrins b5 gene expressions more than Silica P. Silica P stimulated more TGFß1 and its TGFßR1 receptor than Silica F. Cytotoxic effects were induced by the two samples of Silica. On the contrary, no alteration in classic apoptotic marker protein expression was observed in presence of either Silica F or Silica P, suggesting silica particles affect ECM production and metalloproteases through a mechanism that does not involve apoptotic activation. Different Silica micromorphology and TGFß signal pathway are linked to lung fibrotic effects but the potential role Silica in apoptotic and toxic reaction remains to be ascertained.

Gamma ray dosimetry and mating capacity studies in the laboratory on Aedes albopictus males.

In Italy, Aedes albopictus Skuse is currently recognized as the most dangerous mosquito, and as currently applied conventional control methods gave unsatisfactory results, we are developing alternative strategies such as the sterile insect technique. To find the optimal sterilizing dose, male pupae were exposed to different doses of gamma rays in the range 20-80 Gy, generated by a Cesium-137 source. The effects of male pupal age at irradiation and gamma ray dose on adult male emergence, sterility level, longevity, and mating capacity were evaluated, and dose-response curves of residual fertility were calculated. Radiation tests were also performed on female pupae to observe their reproductive capacity in case of accidental release. Results confirmed that the age at which the male pupa is irradiated is an important factor that affects the longevity of the adult, whereas the effect of age on the induced sterility level is less pronounced. When male pupae older than 30 h were irradiated, the longevity of the adults was not affected by doses up to 40 Gy. The 40-Gy dose appeared sufficient to induce high level of sterility (>99%) at any male pupal age for all the strains tested. The duration of coupling and the number of mated females per male appeared to be affected by the radiation received by male pupae only at doses higher than 40 Gy. The female pupae were more sensitive to radiation than male pupae, with strong reduction in fecundity and fertility at 20 Gy and complete suppression of oviposition at higher doses.

Development and calibration of a model for the potential establishment and impact of Aedes albopictus in Europe.

The Asian tiger mosquito (Aedes albopictus) is one of the most invasive disease vectors worldwide. The species is a competent vector of dengue, chikungunya, Zika viruses and other severe parasites and pathogens threatening human health. The capacity of this mosquito to colonize and establish in new areas (including temperate regions) is enhanced by its ability of producing diapausing eggs that survive relatively cold winters. The main drivers of population dynamics for this mosquito are water and air temperature and photoperiod. In this paper, we present a mechanistic model that predicts the potential distribution, abundance and activity of Asian tiger mosquito in Europe. The model includes a comprehensive description of: i) the individual life-history strategies, including diapause, ii) the influence of weather-driven individual physiological responses on population dynamics and iii) the density-dependent regulation of larval mortality rate. The model is calibrated using field data from several locations along an altitudinal gradient in the Italian Alps, which enabled accurate prediction of cold temperature effects on population abundance, including identification of conditions that prevent overwintering of the species. Model predictions are consistent with the most updated information on species' presence and absence. Predicted population abundance shows a clear south-north decreasing gradient. A similar yet less evident pattern in the activity of the species is also predicted. The model represents a valuable tool for the development of strategies aimed at the management of Ae. albopictus and for the implementation of effective control measures against vector-borne diseases in Europe.

Effects on male fitness of removing Wolbachia infections from the mosquito Aedes albopictus.

Cytoplasmic incompatibility (CI) induced by maternally inherited Wolbachia bacteria is a potential tool for the suppression of insect pest species with appropriate patterns of infection. The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) is known to be infected by two strains of Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae), wAlb A and wAlb B, throughout its geographical distribution. This infection pattern theoretically restricts the application of CI-based control strategies. However, Wolbachia can be horizontally transferred using embryonic microinjection to generate incompatible transfected lines harbouring a single new strain of Wolbachia. In order to assess the feasibility of this approach, the effects of Wolbachia removal on mosquito fitness need to be clearly evaluated as the removal of natural superinfection is an inescapable step of this approach. Previous research has shown that uninfected females, produced by antibiotic treatment, showed a decrease in fitness compared with those infected with Wolbachia. In this study, the effect of Wolbachia removal on male fitness was investigated. Longevity and reproductive potential (mating competitiveness and sperm capacity) were assessed in both laboratory cages and greenhouses. No differences were observed between uninfected and infected males with respect to longevity, mating rate, sperm capacity and mating competitiveness in either laboratory conditions or greenhouses. The preservation of fitness in males of Ae. albopictus deprived of natural Wolbachia infection is discussed in relation to the development of incompatible insect technique suppression strategies. Finally, the potential application of aposymbiotic males in mark-release-recapture studies is suggested.

Production and characterization of alginate microcapsules produced by a vibrational encapsulation device.

The optimization, through a Design of Experiments (DoE) approach, of a microencapsulation procedure for isolated neonatal porcine islets (NPI) is described. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An alginate/polyornithine encapsulation procedure, developed and validated in our laboratory for almost a decade, was used to embody pancreatic islets. We analyzed different experimental parameters including frequency of vibration, amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. We produced calcium-alginate gel microbeads with excellent morphological characteristics as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology, viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

Comparative in vitro studies on the fibrogenic effects of two samples of silica on epithelial bronchial cells.

The small dimension and particle shape of silica in gypsum used to prepare moulds for lost wax casting might be responsible for the high prevalence of silicosis in gold jewellery. To test this hypothesis, human pulmonary epithelial cell (BEAS-2B) cultures were exposed to two samples of silica with different crystal micro-morphologies: Silica Powder (Silica P) which is used in casting gold jewellery, and no powder Silica (Silica F). Extracellular matrix (ECM) production was evaluated using radio-labelled precursors and quantified by RT-PCR analysis. Expression of basic fibroblast growth factor (FGF2) and its receptor (FGFR2) was also evaluated. The results demonstrated Silica P particles had a very fine lamellar crystalline structure while Silica F was characterized by larger rounded crystals. Silica P stimulated collagen production significantly more than Silica F and downregulated laminin and metalloprotease expression. Both silica samples down-regulated FGF2 but only Silica F enhanced FGF2 receptor expression. In conclusion each Silica sample promoted a profibrotic lung microenvironment in a different manner and also elicited different FGF2 signalling pathways. The data confirm that different micromorphology of Silica particles affects the fibrogenic potential and the molecular mechanisms of dust pathogenicity.

Lung regions differently modulate bronchial branching development and extracellular matrix plays a role in regulating the development of chick embryo whole lung.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.

Thickness of the dental (radicular) cementum: a parameter for estimating age.

The present study used 127 extracted teeth from people aged 16 to 90 years old. The aim of this research was to verify the reliability of the method using a single dental parameter based on the correlation of the radicular cementum thickness and the chronological age of the subject. The thickness was measured both on the lingual side and on the vestibular side of the tooth, at two different levels: apex and one third of the root length from the apex. The data were reported through a Cartesian graph with the X-axis showing the cementum thickness and the Y-axis showing the subject's age. The correlation between age and the increase of the cementum thickness is more statistically evident when the measurement is taken at the apex (R2=0.67), in comparison with the measurement taken at approximately one third of the root length from the apex (R2=0.56).

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

In vitro human osteoblast and extracellular matrix changes after transforming growth factor beta 1 treatment.

AIMS: Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia. METHODS: In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined. RESULTS: Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (p < or = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities. CONCLUSION: TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.

Interleukin-1 and interleukin-6 differentially regulate the accumulation of newly synthesized extracellular matrix components and the cytokine release by developing chick embryo skin fibroblasts.

In the present study, we demonstrate that both interleukin-1 (IL-1) and interleukin-6 (IL-6) induced a significant decrease in glycosaminoglycan (GAG) synthesis and, more strikingly, secretion by 7 and 13 day-old chick embryo skin fibroblasts. We demonstrated that interleukin treatment also inhibited the synthesis of collagenase-digestible proteins (type I collagen). In addition, tissue culture supernatants (conditioned media, CM) were tested for reactivity for IL specific ELISAs and for their ability to stimulate proliferative responses in mouse thymocytes and hybridoma cells. Our findings demonstrate that chick embryo skin fibroblasts spontaneously produce IL-1 and, in even greater amounts, IL-6. Highest levels of interleukin secretion were found in the CM of 13 day-old fibroblasts and the IL-1 beta isoform was predominant over IL-1 alpha. Pretreatment of the fibroblasts with either IL-1 or IL-6 increased the secretion of both cytokines. Increased IL-1 levels were correlated with enhanced IL-1 bioactivity in the CM of IL-6 treated fibroblasts. By contrast, the raised concentrations of IL-1 in the CM of IL-1 treated cells and IL-6 in the CM of IL-1 or IL-6 treated fibroblasts failed to translate into augmented bioactivity. These observations, taken together, indicated that IL-1 and IL-6 are able to regulate the synthesis and secretion of ECM macromolecules of developing connective tissues and the cytokine release by chick embryo skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Acetylcholinesterase at high catalytic efficiency and substrate specificity in the optic lobe of Eledone moschata (Cephalopoda: Octopoda): biochemical characterization and histochemical localization.

In the optic lobe of the cephalopod mollusc Eledone moschata, two acetylcholinesterase forms I and II were detected, both showing a marked active site specificity with differently sized substrates. Catalytic efficiency (kcat/Km) of the prevailing form II is similar to that of acetylcholinesterases from vertebrate nervous system. Enzyme forms I and II were co-purified from a high-salt-Triton X-100 soluble extract of optic lobe by consecutive affinity chromatographies on procainamide- and concanavalin A-Sepharose columns and then separately obtained by preparative density gradient centrifugation. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the major form II is an amphiphilic globular dimer (135-136 kDa, 6.3-7.4 S) of monomers (66 kDa) S-S linked between terminal segments. Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. Form I, characterized only in part owing to its small amount, showed molecular size (129 kDa) and sedimentation coefficient (7.5 S) similar to those of form II; it is likely to be attached to the cell membrane by electrostatic interactions. Both forms behaved similarly with various inhibitors and underwent excess-substrate inhibition. The results obtained suggest a common origin of both form I and II from a single gene. The former could be a degradation product of the prevailing one (II), which is likely to be functional in cholinergic synapses.

Cellular support systems for alginate microcapsules containing islets, as composite bioartificial pancreas.

To improve the functional performance of microencapsulated islets, we examined the effects of putative cellular support systems, consisting of rat purified Sertoli cells (SC) and astrocytes (AA), on coenveloped allogeneic islets. Coincubation of islets with SC but not AA, resulted in significant stimulation of beta cell mitogenesis, coupled with a significant increase in in vitro glucose-stimulated insulin release. Preliminarily, the xenotransplantation of coencapsulated rat islets and homologous SC significantly prolonged remission of hyperglycemia in diabetic mice.

Sertoli cell-induced reversal of adult rat pancreatic islet beta-cells into fetal-like status: potential implications for islet transplantation in type I diabetes mellitus.

BACKGROUND: Clinical success of pancreatic islet allograft (TX) for the therapy of diabetes mellitus is hampered by several pitfalls, primarily including the restricted availability of donor tissue and the immune- and/or non-immune-related TX's early loss, with the latter not necessarily being prevented by the host's general immunosuppression. Finally, adult islet beta-cells normally exhibit minimal proliferation capacity, which would not permit restoration of an eventually declining TX mass. METHODS: To address the limited beta-cell growth capacity, we have examined whether in vitro co-culturing adult rat islets (I) with prepubertal homologous Sertoli cells (SC) would stimulate I beta-cell expansion. SC-derived effects on the islets were studied in vitro, both morphologically (confocal laser microscopy) and functionally (glucose-stimulated insulin release). We have also preliminarily examined the in vivo impact of microencapsulated SC + I co-cultures on TX in diabetic mice. RESULTS: In vitro, we observed that SCs promoted significant beta-cell replication, as I beta-cell mitotic activity increased from 1% to greater than 8%, which coincided with the adult elements reversing into fetal-like status. This finding was coupled with significantly greater insulin release either in basal or in response to glucose, as compared with controls. CONCLUSIONS: Addition of SC to islets promotes reversal of the adult beta-cell elements into fetal-like conditions, thereby providing a new, potentially powerful tool that could significantly enhance the functional performance of islet TX in diabetic recipients.

Silica and its antagonistic effects on transforming growth factor-beta in lung fibroblast extracellular matrix production.

BACKGROUND: Silicosis, a pneumoconiosis marked by interstitial pulmonary fibrosis, is caused by inhalation of free crystalline silica particles. When silica particles are injected into the lower lung, they are translocated across the epithelium into the interstitial space, where macrophage-derived growth factors affect lung fibroblast proliferation and collagen deposition. We hypothesized that silica may act directly on pulmonary fibroblasts modifying extracellular matrix (ECM) synthesis and that the effects of silica may be mediated by transforming growth factor-beta (TGFbeta) overproduction. METHODS: To test this hypothesis, we studied a human lung fibroblast cell line (WI-1003) exposed to silica in vitro. We investigated cell morphology by electron microscopic procedure, cell growth, collagen production, and glycosaminoglycans (GAG) composition by radiolabeled precursors. Cytokine and growth factor synthesis were evaluated by specific enzyme-linked immunoadsorbent assay kits and Northern blotting analysis. RESULTS: Pulmonary fibroblasts internalized silica particles without detectable cell damage. Silica directly stimulated collagen synthesis and decreased the amount of 3H-glucosamine-labeled GAG. Silica-treated fibroblasts secreted less TGFbeta than untreated controls, antagonized the stimulatory effect of TGFbeta on ECM synthesis, and reversed TGFbeta-induced inhibition of cell proliferation. Northern blotting analysis showed increased interleukin-1alpha (IL-1alpha) mRNA after silica treatment. IL-1alpha had no influence on collagen synthesis but increased the number of WI-1003 fibroblasts. CONCLUSIONS: These results support our hypothesis that lung fibroblasts are direct silica targets. However, contradicting our hypothesis, silica antagonized TGFbeta activities through a TGFbeta downregulation and an IL-1alpha upregulation. The complex pattern of TGFbeta and IL-1alpha regulation in pulmonary fibroblasts is imbalanced by silica exposure and might play a key role in silica-mediated pulmonary fibrosis.

Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration.

Human gingival fibroblasts were cultured in vitro using as substrates an extracellular matrix (matrix) and polytetrafluoride (PTFE) membranes, which are used in guided tissue regeneration. To test the degree of biocompatibility of these membranes, the cellular proliferation and the accumulation of extracellular matrix (ECM) macromolecules were considered as parameters. The fibroblasts were cultured in vitro for 24 and 48 hours without serum on plastic, matrix, and PTFE membranes in the presence of 3H-thymidine, 3H-glucosamine, and 3H-proline to study the neo-synthesis of DNA, glycosaminoglycans (GAG), and collagen proteins, respectively. Studies on cell proliferation showed that fibroblasts grown on matrix membrane significantly increased 3H-thymidine incorporation, while fibroblasts grown on PTFE membrane decreased 3H-thymidine incorporation, compared to plastic used as a control. Moreover, the PTFE membrane induced a marked decrease of collagen and GAG accumulation both in the cellular and extracellular pool, while the matrix membrane provoked a decrease of the two macromolecules in the cellular pool and an increase in the extracellular one, compared to the control. The data we obtained demonstrate that matrix membranes are the most suitable to stimulate both cellular proliferation and ECM macromolecule accumulation.