Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

Pulmonary macrophages alter the collagen phenotype of lung fibroblasts

Cultured lung fibroblasts produced and secreted interstitial collagen types I and III. The relative proportion of type III collagen increased as a linear function of cell density, with confluent cultures producing 8.6% type III collagen. When human lung fibroblasts were cultured in the presence of newly harvested lung macrophages, the proportion of type III collagen secreted rose to 15.5%. This high level of type III collagen synthesis was greater than could be induced by withdrawal of serum, a perturbation known to alter the proportion of types I and III collagen synthesized by fibroblasts. This effect on fibroblast phenotype was independent of cell density, as both low and high density cultures of fibroblasts responded similarly when cultured with macrophages. There was no evidence that fibroblasts synthesize new or different collagen types (such as type I trimer) in response to macrophages. Optimal conditions for eliciting an effect on fibroblast connective tissue metabolism required interaction of the two cell types for 5–8 days. These in vitro changes are analogous to the sequence of interactions and changes in connective tissue metabolism seen during recovery from tissue injury.     

Proliferation of fibroblasts and endothelial cells is enhanced by treatment with Phyllocaulis boraceiensis mucus

Previously, mucus of some molluscs has been studied as a potential source of new natural compounds capable of inducing cell proliferation and of remodelling tissue. Here, the focus of the study is possible use of mucus released by Phyllocaulis boraceiensis – a compound inducing cell proliferation and enhancing collagen synthesis in dermal fibroblasts and inducing proliferation human endothelial cell cultures. Fibroblasts treated with P. boraceiensis mucus at concentrations below 0.012 μg/μl developed high rates of proliferation, as evaluated using MTT assay; the proliferative effect was dose‐dependent. Production and secretion of extracellular matrix components and collagen type I fibres were enhanced after 24 h of treatment, revealing a hormesis effect, biphasic dose response – low dose for proliferation yet toxic at high dose. No significant change in proliferation was observed in treated endothelial cells and production of lipid polyunsaturated free radicals was low in both cell types. Treatment with P. boraceiensis mucus produced pronounced changes in fibroblast cell number and morphology, and in quantities of well‐ordered collagen deposition. These results support the premise that Phyllocaulis boraceiensis mucus demonstrates proliferative properties in cells involved in the healing process.     

Histochemical demonstration of collagen fibers in ascorbic-acid-fed cell cultures

Summary Nine cultures of fibroblast cell types and 13 epithelial-like cell types were maintained for 1 week in media supplemented with L-asborbic acid (50 μg per ml). All fibroblast-like cultures produced extracellular fibers that stained positively by a silver-impregnation reticulin stain. Nine of the 13 epithelial-like cultures produced fibers that stained positively for reticulin. Nearly all cultures not supplemented with ascorbic acid showed no fiber staining. Those few lines that stained positively for reticulin in the absence of ascorbic-acid supplementation demonstrated only slight reticulin formation. Reticulin from one fibroblast culture and one epithelial culture was examined by electron microscopy, and the silver-impregnated fibrils were morphologically identical to collagen. The reticulin was digestible with collagenase, providing further evidence that the silver-impregnation reticulin stain identifies collagen in culture. The demonstration of collagen can be performed easily in histology laboratories using Formalin-fixed cells, and provides a means of assaying a functional property of cells in culture which is characteristic of connective tissue fibroblasts in general as well as certain specialized epithelia.     

Direct and tumor microenvironment mediated influences of 5′‐deoxy‐5′‐(methylthio)adenosine on tumor progression of malignant melanoma

Recent studies have shown that a loss of methylthioadenosine phosphorylase (MTAP) gene expression exerts a tumor‐promoting effect, including induction of invasiveness, enhanced cell proliferation, and resistance against cytokines. To date, the molecular mechanisms underlying these effects remain unknown. Since the loss of MTAP expression resulted in induced secretion of 5′‐deoxy‐5′‐(methylthio)adenosine (MTA), we hypothesized that MTA might modulate the observed effects. We first determined MTA levels produced by tumor cells in vitro and in situ by means of stable isotope dilution liquid chromatography tandem mass spectrometry. Subsequently, we revealed induction of matrix metalloproteinase (MMP) and growth factor gene expression in melanoma cells accompanied by enhanced invasion and vasculogenic mimicry. In addition, MTA induced the secretion of basis fibroblast growth factor (bFGF) and MMP3 from fibroblasts and the upregulation of activator protein‐1 (AP‐1) activity in melanoma cells and fibroblasts. In summary, we demonstrated a tumor‐supporting role of MTA. J. Cell. Biochem. 106: 210–219, 2009. © 2008 Wiley‐Liss, Inc.     

Histochemical demonstration of collagen fibers in ascorbic-acid-fed cell cultures.

Nine cultures of fibroblast cell types and 13 epithelial-like cell types were maintained for 1 week in media supplemented with L-asborbic acid (50 microgram per ml). All fibroblast-like cultures produced extracellular fibers that stained positively by a silver-impregnation reticulin stain. Nine of the 13 epithelial-like cultures produced fibers that stained positively for reticulin. Nearly all cultures not supplemented with ascorbic acid showed no fiber staining. Those few lines that stained positively for reticulin in the absence of ascorbic-acid supplementation demonstrated only slight reticulin formation. Reticulin from one fibroblast culture and one epithelial culture was examined by electron microscopy, and the silver-impregnated fibrils were morphologically identical to collagen. The reticulin was digestible with collagenase, providing further evidence that the silver-impregnation reticulin stain identifies collagen in culture. The demonstartion of collagen can be performed easily in histology laboratories using Formalin-fixed cells, and provides a means of assaying a functional property of cells in culture which is characteristic of connective tissue fibroblasts in general as well as certain specialized epithelia.     

Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes'' syndrome.

Lysyl oxidase activity against both collagen and elastin substrates has been examined in the culture medium of skin fibroblasts derived from unrelated patients with Menkes' syndrome and from control subjects. The medium of three Menkes' fibroblast lines showed 3--30% of the activity present in the medium of control fibroblasts, against a purified collagen substrate. Lysyl oxidase activity in the culture medium of two of the Menkes' fibroblast lines was also examined by using a crude aortic-elastin substrate and was similarly decreased in comparison with that in the medium of control fibroblasts. Lysyl oxidase activity in the medium of a fourth fibroblast line, derived from a foetus with Menkes' syndrome, was 42% of that in the medium of control fibroblasts derived from a 1-day-old baby against a collagen substrate, and 26% of that in control fibroblast medium against an elastin substrate. The copper content of the cell layers of the Menkes' fibroblast cultures was elevated in comparison with normal fibroblast cultures, as has previously been reported to be characteristic of such cells. It is suggested that the decrease in lysyl oxidase activity would help to explain the connective tissue defects observed in Menkes' syndrome, and that this reduction, in conjunction with the elevated concentrations of cellular copper, would support the hypothesis that a functional intracellular copper deficiency exists in Menkes' syndrome.     

Surfactant components modulate fibroblast apoptosis and type I collagen and collagenase-1 expression

During lung injury, fibroblasts migrate into the alveolar spaces where they can be exposed to pulmonary surfactant. We examined the effects of Survanta and surfactant protein A (SP-A) on fibroblast growth and apoptosis and on type I collagen, collagenase-1, and tissue inhibitor of metalloproteinase (TIMP)-1 expression. Lung fibroblasts were treated with 100, 500, and 1,000 microg/ml of Survanta; 10, 50, and 100 microg/ml of SP-A; and 500 microg/ml of Survanta plus 50 microg/ml of SP-A. Growth rate was evaluated by a formazan-based chromogenic assay, apoptosis was evaluated by DNA end labeling and ELISA, and collagen, collagenase-1, and TIMP-1 were evaluated by Northern blotting. Survanta provoked fibroblast apoptosis, induced collagenase-1 expression, and decreased type I collagen affecting mRNA stability approximately 10-fold as assessed with the use of actinomycin D. Collagen synthesis and collagenase activity paralleled the gene expression results. SP-A increased collagen expression approximately 2-fold and had no effect on collagenase-1, TIMP-1, or growth rate. When fibroblasts were exposed to a combination of Survanta plus SP-A, the effects of Survanta were partially reversed. These findings suggest that surfactant lipids may protect against intraluminal fibrogenesis by inducing fibroblast apoptosis and decreasing collagen accumulation.     

Establishment and comparison of fibroblast cell lines from the medial collateral and anterior cruciate ligaments of the rabbit

Summary We have developed a procedure to explant fibroblasts from the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) of the rabbit knee, and have optimized conditions for maintaining them in culture. Maximal growth for both ACL and MCL cells was obtained with Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 250 μM ascorbate. ACL and MCL fibroblasts displayed intrinsic differences in their responses to changes in culture parameters. Specifically, they displayed different growth responses when plated at different densities and responded to RPMI 1640 medium in very different ways. There were also biochemical differences between the cell types. Both cell types produced similar amounts of collagen in culture, but the ratio of type I to type III, the major collagen subtypes produced by these cells, were different. ACL fibroblasts produced 86.7% type I and 13.3% type III, and MCL fibroblasts produced 71.1% type I and 28.9% type III. In addition, total protein produced by ACL fibroblasts was higher than that produced by MCL cells. This confirms the suggestions of previous researchers that such differences might exist. This work was funded by a grant-in-aid from Medtronic of Canada, by an R&D Grant from the Alberta Ministry of Technology, Research and Telecommunications, and by the Alberta Heritage Foundation for Medical Research.     

Naftopidil reduced the proliferation of lung fibroblasts and bleomycin‐induced lung fibrosis in mice

Naftopidil, an α‐1 adrenoceptor antagonist with few adverse effects, is prescribed for prostate hyperplasia. Naftopidil inhibits prostate fibroblast proliferation; however, its effects on lung fibroblasts and fibrosis remain largely unknown. Two normal and one idiopathic pulmonary fibrosis human lung fibroblast lines were cultured with various naftopidil concentrations with or without phenoxybenzamine, an irreversible α‐1 adrenoceptor inhibitor. We examined the incorporation of 5‐bromo‐2?‐deoxyuridine into DNA and lactic acid dehydrogenase release by enzyme‐linked immunosorbent assay, cell cycle analysis by flow cytometry, scratch wound‐healing assay, and mRNA expressions of type IV collagen and α‐smooth muscle actin by polymerase chain reaction. Effects of naftopidil on bleomycin‐induced lung fibrosis in mice were evaluated using histology, micro‐computed tomography, and surfactant protein‐D levels in serum. Naftopidil, dose‐dependently but independently of phenoxybenzamine, inhibited 5‐bromo‐2?‐deoxyuridine incorporation in lung fibroblasts. Naftopidil induced G1 cell cycle arrest, but lactic acid dehydrogenase release and migration ability of lung fibroblasts were unaffected. Naftopidil decreased mRNA expressions of type IV collagen and α‐smooth muscle actin in one normal lung fibroblast line. Histological and micro‐computed tomography examination revealed that naftopidil attenuated lung fibrosis and decreased serum surfactant protein‐D levels in bleomycin‐induced lung fibrosis in mice. In conclusion, naftopidil may have therapeutic effects on lung fibrosis.     

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun‐protected and sun‐exposed body sites

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun‐protected) and photoaged (sun‐exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun‐protected and sun‐exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun‐exposed‐derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.     

Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-I collagen levels in human lung carcinoma fibroblasts

Matrix metalloproteinases (MMP's) and tissue inhibitors of metalloproteinases (TIMP's) possess a preponderant role in the metabolism of the major extracellular matrix protein, collagen, and are thought to be important in the mechanism of tumor invasion. Lung cancer occupies the first position in mortality and the second position in incidence, among all cancers. In the present investigation, we studied the effect of basic fibroblast growth factor (bFGF) on collagen, matrix metalloproteinase-2 (MMP-2), and tissue metalloproteinase inhibitor-2 (TIMP-2) levels in normal and carcinoma lung tissue fibroblast cultures. MMP-2 was selected because of its high specificity in the degradation of type IV collagen, major component of the basal membrane. The effect of bFGF on MMP-2, TIMP-2, total collagen, and type I collagen levels of normal and carcinoma lung fibroblast cultures was investigated at 0, 10, and 100 ng/ml. Statistical analysis was carried out using the Mann-Whitney-U test and possible correlations were searched using the Spearman correlation analysis method. MMP-2, TIMP-2, total collagen, and type-1 collagen levels based on cell counts (10(3) cells) showed no statistically significant differences between the carcinoma and normal fibroblast cultures. However, positive correlations were found between MMP-2 and TIMP-2 in normal (P = 0.016) and carcinoma (P = 0.001) tissue fibroblast cultures. Positive correlations were also found between total collagen and TIMP-2 levels in normal and carcinoma tissue fibroblast cultures (P = 0.002 and P = 0.032). Total collagen and TIMP-2 levels also showed positive and strong correlations in all cultures except in 100 ng/ml bFGF concentrations. In addition, type I collagen and MMP-2 levels showed positive significant correlations only in normal and carcinoma control cultures, while type I collagen and TIMP-2 levels showed positive correlations in all cultures except carcinoma fibroblasts at 100 ng/ml bFGF. It may be concluded that bFGF does not affect MMP-2, TIMP-2, total collagen, and type-1 collagen levels in fibroblast cultures grown from human carcinoma and normal lung tissues. However, bFGF was noted, in vitro, to disturb the equilibrium which normally exists between the four parameters, both in normal and carcinoma tissue fibroblasts.     

Sera of patients with spontaneous tumour regression and elevated anti‐CA I autoantibodies change the gene expression of ECM proteins

Spontaneous tumour regression after high‐dose therapy and autologous stem cell transplantation is associated with the aplastic anaemia‐like syndrome and the presence of polyclonal autoantibodies against carbonic anhydrase I (CA I). When tumour cells were grown in vitro in the presence of patients’ sera positive for anti‐CA I autoantibodies, their morphological pattern was altered. These changes were accompanied by modifications in the gene expression profile. We observed downregulation of genes of the basal lamina assembly (collagen type IV alpha 4, the laminin subunit gamma 2), the extracellular matrix (collagen type I alpha 1), the cytoskeleton (keratin 14 type I), the collagen triple helix repeat containing 1 and the proto‐oncogene WNT7B. On the other hand, the expression of the CA 1 gene was increased in the tumour cells. It was also noticed that the presence of anti‐CA I autoantibodies did not impair tumour cell proliferation and cell viability in vitro. These findings were observed only in the presence of patients’ sera positive for anti‐CA I autoantibodies.     

Cell-type-specific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3

Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.     

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.     

Antibacterial activity of hinokitiol against both antibiotic‐resistant and ‐susceptible pathogenic bacteria that predominate in the oral cavity and upper airways

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin‐resistant and ‐susceptible Staphylococcus aureus, antibiotic‐resistant and ‐susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin‐resistant S. aureus, methicillin‐susceptible S. aureus, antibiotic‐resistant S. pneumoniae isolates, antibiotic‐susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3–1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9‐22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9‐22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.     

Human recombinant gamma-interferon stimulates proliferation and inhibits collagen and fibronectin production by human dental pulp fibroblasts

Human recombinant-gamma-interferon was tested on human dental pulp fibroblast activity in vitro. Fibroblast proliferation was estimated by a colorimetric test. Type I and type III collagens and fibronectin were quantified by radioimmunoassay in culture supernatant from confluent fibroblasts. A dose dependent stimulation of the proliferation was observed when fibroblasts were treated with recombinant-gamma-interferon. In contrast, an inhibition of the synthesis of soluble types I and III collagen and fibronectin by confluent cell cultures treated with recombinant-gamma-interferon occurred without apparent modification of the insoluble collagen level in the cell layer. Quantimetric analysis of type I collagen immunoperoxidase labelling have demonstrated that there was no intracellular storage of type I collagen in these cultured fibroblasts. These data support the view that human recombinant-gamma-interferon can affect human dental pulp fibroblast functions and thus may play an important part in the regulation of fibrosis.