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Biomedical subjects

G A Müller

Publications and source records attributed to G A Müller.

At least 37 records · Page 2Linked to original sources
PubMed2001

Proteinuria and plasma total homocysteine levels in chronic renal disease patients with a normal range serum creatinine: critical impact of true glomerular filtration rate.

Conflicting data have been reported concerning the independent association between proteinuria and plasma total homocysteine (tHcy) levels, particularly among chronic renal disease (CRD) patients with a normal range serum creatinine. Studies of this potential relationship have been limited by failure to assess true GFR, failure to assess proteinuria in a quantitative manner, or arbitrary restriction of the range of proteinuria examined. We examined the potential independent relationship between plasma tHcy levels and a wide range of quantitatively determined proteinuria (i.e., 0.000-8.340 g/day), among 109 CRD patients with a normal range serum creatinine (range; 0.8-1.5 mg/dl; median=1.2 mg/dl). Glomerular filtration rate (GFR) was directly assessed by iohexol clearance, and plasma status of folate, pyridoxal 5'-phosphate, and B12, along with serum albumin, were also determined. Linear modeling with ANCOVA revealed that proteinuria was not independently associated with tHcy levels (partial R=0.127; P=0.201), after adjustment for potential confounding by GFR (partial R=0.408; P<0.001), age, sex, plasma B-vitamin status, and serum albumin. Moreover, descending across quartiles (Q) [from Q4 to Q1] of GFR, ANCOVA-adjusted (i.e., for age, sex, and folate status) geometric mean tHcy levels (micromol/l) were significantly increased: tHcy Q4 GFR=9.6; tHcy Q3 GFR=10.5; tHcy Q2 GFR=11.9; tHcy Q4 GFR=14.5; P<0.001 for overall Q difference. We conclude that across a broad spectrum of quantitatively determined proteinuria, after adjustment for true GFR, in particular, there is no independent relationship between proteinuria and tHcy levels among CRD patients with a normal range serum creatinine.

Adult↗
PubMed2001

Reduction of hepatitis C virus load by H.E.L.P.-LDL apheresis.

The association of HCV with apolipoprotein B containing lipoproteins has been observed and this led to the assumption that the LDL receptor may also serve as a candidate receptor for HCV. H.E.L.P.-LDL apheresis is suggested to be an effective and rapid tool to safely eliminate apolipoprotein B containing lipoproteins. In this pilot study, we have investigated whether H.E.L.P. treatment would reduce HCV load in five patients, all infected for more than 4 years with HCV and resistant against established anti-HCV therapy (interferon, ribaverin). HCV-RNA was determined by RT-PCR in plasma immediately before the start of apheresis (SA) and after treatment of 2500 mL plasma (AA). H.E.L.P. apheresis led to a mean decrease of 77.3% (16th percentile 36.5%, 84th percentile 89.6%) of HCV-RNA when AA values were compared to SA values. This decline was reproducible during nine treatment procedures, but was not correlated to the decrease in LDL cholesterol. This investigation shows for the first time that HCV load can be reduced by H.E.L.P. apheresis, which is an established and approved therapy for hypercholesterolemia. Even though the efficiency of viral load reduction varied between single procedures and did not correlate to LDL removal, this extracorporeal therapy opens the possibility to treat patients with established immune modulatory and antiviral therapy in the interval between two apheresis procedures.

Blood Component Removal↗
PubMed2001

A novel model to study renal myofibroblast formation in vitro.

BACKGROUND: In chronic renal disease, the decrease of excretory renal function closely correlates with the extent of interstitial fibrosis. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. We studied the transformation of renal fibroblasts into myofibroblasts in vitro as a model to elucidate the mechanisms underlying this process. METHODS: Primary cultures of freshly isolated rat inner medullary fibroblasts were established as reported previously. mRNA expression of myofibroblast markers and interstitial collagens was examined by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Phenotypic expression was investigated by immunolabeling. Endogenous transforming growth factor-beta (TGF-beta) production was inhibited by addition of a neutralizing antibody or by TGF-beta 1 antisense oligodeoxynucleotides (ODNs). RESULTS: Initially and during the first 36 hours, primary culture cells expressed neither alpha-smooth muscle actin nor desmin mRNA. From day 2 of primary culture, we observed a strong increase in these mRNAs, as evaluated by RT-PCR, followed by the phenotypic expression of these myofibroblast markers. Collagen type I mRNA was first detectable from day 4 of primary culture and showed a strong increase in its expression level during the following days, with phenotypic expression predominantly in myofibroblasts. The transformation rate of fibroblasts to myofibroblasts largely decreased in cocultures with collecting duct cells. This effect could be reversed by reducing the seeding density. We examined in this system the effect of TGF-beta 1 to define further its putative fibrogenic activity. However, neither the addition of exogenous TGF-beta 1 nor the inhibition of endogenous TGF-beta production showed any significant effect on fibroblast transformation, suggesting that this cytokine exerts its effects at other levels or requires a cofactor. CONCLUSIONS: We present a novel model to examine the de novo expression of myofibroblast markers and collagen type I in freshly isolated fibroblasts under defined conditions in primary culture. This model could provide a strategy for the molecular characterization of myofibroblast formation and the phenomena associated with this process.

Actins↗
PubMed2001

Effect of heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) apheresis on hepatitis C plasma virus load.

Association of the hepatitis C virus (HCV) with apolipoprotein B containing lipoproteins has been suggested, and this led to the concept that the low-density lipoprotein (LDL) receptor may also serve as a candidate receptor for HCV uptake into the liver. We have investigated whether heparin-induced extracorporeal LDL precipitation (HELP) LDL apheresis treatment reduces HCV plasma load in 6 patients, all infected for more than 4 years with HCV and resistant against established anti-HCV therapy. HELP apheresis treatment caused an HCV-RNA decrease of 77.3% in mean. This decline was not correlated with LDL-cholesterol reduction. HCV-RNA was retained on the HELP filter as shown for 1 patient. The effect of RNA lowering was only transient due to the high turnover of HCV. However, HELP apheresis may open a window of opportunity for an immune-modulating and antiviral therapy in the interval between two apheresis procedures in patients with high virus load.

Anticoagulants↗
PubMed2001

Identification of nucleated cells in urine using lectin staining.

Microscopic examination of urinary sediment is an integral component in the evaluation of nephropathies. However, identification and differentiation of the nucleated nonsquamous cells in urine is often difficult using such conventional techniques as phase contrast or bright field microscopy, even after Papanicolaou staining, and requires a lot of experience. We now report a method to differentiate urinary cell types using lectin staining. Twenty-five lectins were examined with respect to their binding pattern on cryosections of the human kidney and urinary tract, as well as binding to blood cells. The specificity of lectin binding to a cell type both in situ and in urine was confirmed by double labeling with specific antibodies directed against various sections of the nephron or nucleated blood cells. For urine cytologic examinations, acetone-fixed cytopreparations of urinary sediments were incubated with a combination of a fluorescein isothiocyanate (FITC)-coupled and a rhodamine-coupled lectin, followed by staining of the nuclei with 4',6-diamidino-2-phenylindole. Specimens were examined in triple immunofluorescence (FITC/rhodamine/UV). Cell types could be identified by their characteristic lectin-binding pattern. For example, the lectin combination of Sophora japonica agglutinin (aggl; SJA) and Erythrina cristagalli aggl (ECA) permitted a differentiation between cells of the proximal tubules (SJA positive [SJA+], ECA+), distal tubules (SJA negative [SJA-], ECA+), collecting ducts (SJA+, ECA-), and lymphocytes (SJA-, ECA-). In preliminary studies, examination of urinary sediment in various chronic nephropathies by this technique showed differences in their cellular excretion pattern. In summary, staining urinary sediments with combinations of lectins provides a rapid and relatively inexpensive method for a facilitated and reliable differentiation of the various nucleated cell types in urine.

Binding Sites↗
PubMed2001

Renal fibrosis: an update.

Tubulointerstitial fibrosis invariably accompanies the course of chronic renal failure towards end-stage renal disease. Tubular epithelial cells, the predominant cell type in the tubulointerstitium, are increasingly being recognized for playing a dominant role as mediators of renal fibrogenesis. Tubular epithelial cells become activated either by the glomerular ultrafiltrate from their apical side or by mononuclear cells from their basolateral side. They initiate the scarring process by secreting chemokines, which in return attract mononuclear cells as well as growth factors that stimulate interstitial fibroblasts. In later phases of renal fibrogenesis, cellular changes of tubular epithelial cells contribute to the chronic impairment of renal function. Whereas tubular epithelial cells react by proliferation or hypertrophy to initial stimuli, they may undergo apoptosis or transdifferentiate into fibroblasts, and thus contribute to tubular atrophy in later stages of progressive renal disease. Resident interstitial fibroblasts are also important in renal fibrogenesis, and recent research has demonstrated that these cells are much more heterogeneous than expected. Cytokines such as fibroblast growth factor type 2 and epithelial growth factor have been shown to be pro-fibrogenic, whereas hepatocyte growth factor and bone morphogenic protein type 7 may inhibit fibrogenesis. Despite recent progress, further research is mandatory for a better understanding and the development of novel therapeutic approaches.

Fibrosis↗
PubMed2001

Characterization of aldose reductase from the thick ascending limb of Henle's loop of rabbit kidney.

BACKGROUND: The organic osmolyte sorbitol plays an important role in the osmoregulation of immortalized epithelial cells of the thick ascending limb of Henle's loop (TALH) of rabbit. The intracellular sorbitol content seems to depend strongly on the extracellular osmolarity. To investigate the nature of the osmotic regulation we characterized the aldose reductase. METHODS: We determined aldose reductase activity enzymatically and the content of organic osmolytes by HPLC. RESULTS: The aldose reductase activity correlates with the extracellular tonicity. Elevating the osmolarity of the medium from 300 to 600 mosm/l by addition of NaCl or sucrose resulted in a significant increase of maximal velocity (V(max)) of the adapted cells from 8 +/- 1 micromol/g x min (300 mosm/l) to 322 +/- 28 micromol/g x min (600 mosm/l, NaCl) or 54 +/- 9 micromol/g x min (600 mosm/l, sucrose), respectively, while affinity (K(m)) remained unchanged. But we found no rise of aldose reductase activity when extracellular urea concentration was elevated. Similar alterations in V(max) were observed when the activity of the highly enriched enzyme was determined with glucose as substrate. Elevation of the extracellular osmolarity by NaCl and sucrose strongly induced the expression of aldose reductase protein with an apparent molecular weight of 39 kD. The affinity of glucose is characteristically low with a K(m) above 300 mmol/l. Aldose reductase utilizes both NADPH and with lower affinity NADH as coenzymes. In vitro sulfate ions (0.4 mol/l) results in a two-fold activation of the aldose reductase activity whereas sodium (200-400 mmol/l) decreased the activity significantly (22-33%). Potassium and chloride up to 400 mmol/l did not alter the aldose reductase activity in vitro. CONCLUSIONS: These results indicate that the aldose reductase of TALH cells of the outer medulla is osmotically regulated and has many similarities with aldose reductase in renal inner medulla. Therefore, intracellular sorbitol synthesis seems to be of similar importance in the osmoregulation of TALH cells as in the inner medulla.

Adaptation, Physiological↗
PubMed2001

Evidence for a sorbitol transport system in immortalized human renal interstitial cells.

Sorbitol plays a major role in the maintenance of cell volume and functional integrity of several renal cells. Sorbitol synthesis takes place in inner collecting duct cells, whereas sorbitol dehydrogenase activity, which catalyzes the degradation of sorbitol to fructose, could mainly be detected in renal inner medullary interstitial cells. Therefore, we supposed that interstitial cells would require a sorbitol transport into the cells. However, such a transport system has not yet been described. Therefore, we have characterized the uptake of sorbitol in immortalized interstitial TK-173 cells, which were derived from human renal fibroblasts. Comparable to fresh isolated renal fibroblasts of the rat, immortalized TK-173 cells have a high sorbitol dehydrogenase activity. In this report, a temperature-dependent sorbitol uptake with saturation kinetics could be detected in immortalized TK-173 cells. The transport is characterized by a high velocity (Vmax 84 mmol/l x h) and an apparent Km of 10 mmol/l. The sorbitol uptake is independent of membrane potential, sodium, and chloride. Altogether, the physiological characteristics of this sorbitol transport are different from those of the osmotically regulated sorbitol efflux from epithelial cells. These results provide evidence that TK-173 cells derived from renal fibroblasts have a specific sorbitol transport. Furthermore, these data suggest a cooperation between epithelial and interstitial cells concerning osmoregulation.

Biological Transport↗
PubMed2001

Interleukin 16 expression in relation to disease activity in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology characterized by an infiltration of CD4+ T lymphocytes within the rheumatoid synovium. Cytokines have been shown to play a modulatory role in the pathogenesis of RA. We analyzed the expression of a T cell derived cytokine. interleukin 16 (IL-16), in relation to disease activity to characterize its biologic function in RA. METHODS: Secreted IL-16 was measured by enzyme immunoassay in sera and synovial fluids (SF) from 25 patients with RA in comparison to 20 control samples from patients with osteoarthritis (OA). IL-16 expression in peripheral blood mononuclear cells (PBMC) was characterized by flow cytometric analysis after intracellular cytokine staining for IL-16. In synovial tissue specimens, IL-16 mRNA expression was analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In parallel, expression of IL-16 was localized in synovial tissues by in situ hybridization and immunohistochemistry. Results were analyzed in relation to disease activity. RESULTS: IL-16 was detected at significantly higher levels in sera and SF of patients with RA in comparison to OA (p < 0.001). Flow cytometry of PBMC showed that a great proportion of both CD4+ and CD8+ T cells constitutively expressed the IL-16 protein. In synovial tissues, IL-16 mRNA levels were significantly elevated in comparison to OA controls (p < 0.001). In situ hybridization for IL-16 producing cells revealed a predominant accumulation of IL- 16 positive cells within the inflammatory infiltrates. A significant correlation between IL- 16 expression and local inflammatory activity could not be established (r = 0.27, p = 0.19) by microscopic analysis of the synovial cell infiltrate. In addition, no significant association was observed between serum, SF, and synovial tissue expression of IL-16 and clinical disease activity in RA. CONCLUSION: These data suggest IL-16 might play a role in the pathogenesis of chronic inflammation in RA. The lack of significant correlation between IL-16 expression, clinical disease activity, and local inflammatory activity suggests a regulatory rather than a proinflammatory function for IL-16 in the pathogenesis of chronic synovial inflammation in RA.

Adult↗
PubMed2000

Potential-dependent steady-state kinetics of a dicarboxylate transporter cloned from winter flounder kidney.

The two-electrode voltage-clamp technique in combination with tracer uptake experiments was used to investigate the dependence of dicarboxylate transport kinetics on membrane potential in Xenopus laevis oocytes expressing the flounder renal high-affinity-type sodium dicarboxylate cotransporter (fNaDC-3). Steady-state succinate-dependent currents in the presence of Na+ were saturable with an apparent affinity constant for succinate, K0.5,succ, of 60 microM. K0.5,succ was independent of membrane potential, suggesting succinate binding at the surface of the fNaDC-3 protein. The maximal succinate-dependent current, deltaImax, increased with hyperpolarization, suggesting that the empty carrier may translocate net charge. Succinate-induced currents showed sigmoidal dependence on Na+ concentration, and K0.5,Na+ decreased with hyperpolarization, suggesting Na+ binding in an ion well. Lowering the external Na+ concentration to 20 mM increased K0.5,succ approximately threefold. Succinate-induced currents were inhibited by Li+ with an Ki,Li+ of approximately 0.5 mM, and a Hill coefficient of below unity indicating the interaction of one Li+ ion with an inhibitory site at fNaDC-3.

Animals↗
PubMed2000

Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.

Reports of spontaneous regressions of metastases and the demonstration of tumor-reactive cytotoxic T lymphocytes indicate the importance of the host's immune system in controlling the devastating course of metastatic renal cell carcinoma. Recent research indicates that immunization with hybrids of tumor and antigen presenting cells results in protective immunity and rejection of established tumors in various rodent models. Here, we present a hybrid cell vaccination study of 17 patients. Using electrofusion techniques, we generated hybrids of autologous tumor and allogeneic dendritic cells that presented antigens expressed by the tumor in concert with the co-stimulating capabilities of dendritic cells. After vaccination, and with a mean follow-up time of 13 months, four patients completely rejected all metastatic tumor lesions, one presented a 'mixed response', and two had a tumor mass reduction of greater 50%. We also demonstrate induction of HLA-A2-restricted cytotoxic T cells reactive with the Muc1 tumor-associated antigen and recruitment of CD8+ lymphocytes into tumor challenge sites. Our data indicate that hybrid cell vaccination is a safe and effective therapy for renal cell carcinoma and may provide a broadly applicable strategy for other malignancies with unknown antigens.

Aged↗
PubMed2000

Acute effect of H.E.L.P. treatment on radical scavenging enzyme activities, total glutathione concentrations in granulocytes, and selenium in plasma.

BACKGROUND: It has been suggested that granulocytes are activated on artificial surfaces such as dialyzer membranes or by plasma separation procedures resulting in the generation of free radicals. We reported recently that free radical scavenging enzyme (FRSE) activities of red blood cells obtained from patients undergoing hemodialysis and LDL-apheresis (LA) do not reflect an acute oxidative stress. However, because mature red cells are free of DNA and RNA, enzymes cannot be regulated on the gene level. In contrast, granulocytes are nucleated cells in which genes can be regulated, e. g. by redox sensitive transcription factors activated by extracellular oxidative stress. Therefore, granulocyte FRSE may better reflect acute oxidative stress caused by extracorporeal treatment. MATERIALS AND METHODS: Hyperlipidemic patients (n = 18) with coronary heart disease (CHD) were treated with the Heparin-induced-Extracorporeal-LDL-Precipitation (H.E.L.P.) system. Glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), superoxide dismutase (SOD) activities, and total glutathione were determined in granulocytes before and immediately after a single LA treatment. Selenium (Se) concentrations were assessed in plasma. RESULTS: As a result of the H.E.L.P. treatment GSSG-R activity was significantly induced (+ 20%) and the GSH concentration increased (+ 41%) in granulocytes. GSH-Px activity in granulocytes (- 19%) and Se in plasma (- 27%) were significantly reduced whereas SOD activity in granulocytes was not affected by the H.E.L.P. procedure. CONCLUSION: These results show that the defence against oxygen radicals in granulocytes is affected but not severely compromised in patients undergoing regular H.E.L.P-LDL-apheresis treatment, which points to the safety of this system with respect to oxidative stress.

Adult↗
PubMed2000

Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation.

BACKGROUND: Interstitial fibroblasts play a critical role in renal fibrogenesis, and autocrine proliferation of these cells may account for continuous matrix synthesis. Basic fibroblast growth factor (FGF-2) is mitogenic for most cells and exerts intracrine, autocrine, and paracrine effects on epithelial and mesenchymal cells. The aims of the present studies were to localize and quantitate the expression of FGF-2 in normal and pathologic human kidneys and to study the in vitro effects of FGF-2 on proliferation, differentiation, and matrix production of isolated cortical kidney fibroblasts. METHODS: FGF-2 protein expression was localized by immunofluoresence double labelings in normal and fibrotic human kidneys. Subsequently, interstitial FGF-2 labeling was determined semiquantitatively in 8 normal kidneys and 39 kidneys with variable degrees of interstitial fibrosis and was correlated with the morphometrically determined interstitial cortical volume. In addition, FGF-2 expression was quantitated by immunoblot analysis in three normal and six fibrotic kidneys. FGF-2 mRNA was localized by in situ hybridizations. Seven primary cortical fibroblast lines were established, and expression of FGF-2 and FGF receptor-1 (FGFR-1) were examined. The effects of FGF-2 on cell proliferation were determined by bromodeoxyuridine incorporation and cell counts, those on differentiation into myofibroblasts by staining for alpha-smooth muscle actin, and those on matrix synthesis by enzyme-linked immunosorbent assay for collagen type I and fibronectin. Finally, proliferative activity in vivo was evaluated by expression of MIB-1 (Ki-67 antigen). RESULTS: In normal kidneys, FGF-2 expression was confined to glomerular, vascular, and a few tubular as well as interstitial fibroblast-like cells. The expression of FGF-2 protein was increased in human kidneys, with tubulointerstitial scarring correlating with the degree of interstitial fibrosis (r = 0.84, P < 0.01). Immunoblot analyses confirmed a significant increase in FGF-2 protein expression in kidneys with interstitial scarring. In situ hybridization studies demonstrated low-level detection of FGF-2 mRNA in normal kidneys. However, FGF-2 mRNA expression was robustly up-regulated in interstitial and tubular cells in end-stage kidneys, indicating that these cells are the source of excess FGF-2 protein. Primary cortical fibroblasts express FGF-2 and FGFR-1 in vitro. FGF-2 induced a robust growth response in these cells that could be blocked specifically by a neutralizing FGF-2 antibody. Interestingly, the addition of the neutralizing antibody alone did reduce basal proliferation up to 31.5%. In addition, FGF-2 induced expression of alpha-smooth muscle actin up to 1.6-fold, but no significant effect was observed on the synthesis of collagen type I and fibronectin. Finally, staining for MIB-1 revealed a good correlation of interstitial FGF-2 positivity with interstitial and tubular proliferative activity (r = 0.71, P < 0.01 for interstitial proliferation, N = 30). CONCLUSIONS: Interstitial FGF-2 protein and mRNA expression correlate with interstitial scarring. FGF-2 is a strong mitogen for cortical kidney fibroblasts and may promote autocrine fibroblast growth. Expression of FGF-2 correlates with interstitial and tubular proliferation in vivo.

Adult↗
PubMed2000

Soluble CD-4 and CD-8 as markers of immunological activation in renal transplant recipients.

BACKGROUND: T lymphocytes are activated following kidney transplantation in cases of acute graft rejection and viral infections. In plasma, elevated levels of T-cell markers can be measured in soluble form. The reason for this shedding is still not entirely understood. METHODS: Plasma concentrations of soluble CD-4 and CD-8 (sCD-4, sCD-8) were determined in 78 patients following kidney transplantation by commercially available enzyme-linked immunosorbent assay (ELISA) test kits. RESULTS: The concentrations of both soluble T-cell markers increased significantly in the course of acute allograft rejections and cytomegalovirus (CMV) infections. Frequently, the parameters increased shortly before clinical diagnosis and decreased under successful therapy. Additionally, sCD-8 showed significant higher plasma concentrations in cases of CMV infection as compared with acute allograft rejections. Accordingly, the sCD-4/sCD-8 ratio increased in cases of acute allograft rejection and decreased during CMV infections. Cyclosporin A nephrotoxicity caused no significant changes in the sCD-4 and sCD-8 levels in plasma. CONCLUSION: The present study demonstrates that sCD-4 and sCD-8 are markers of immunological activation and may enable a further differentiation of T-cell activation if serial measurements are performed. However, further prospective investigations are necessary to elucidate the diagnostic potential of sCD-4 and sCD-8 for monitoring acute rejection and viral infection in kidney graft recipients.

Adult↗
PubMed2000

Effects of pentoxifylline, pentifylline and gamma-interferon on proliferation, differentiation, and matrix synthesis of human renal fibroblasts.

BACKGROUND: Kidneys that progress to end-stage renal failure are almost invariably characterized by the presence of tubulointerstitial fibrosis. Therapeutic interventions to halt the progressive deterioration of renal function are still limited. Pentoxifylline, pentifylline, and gamma-interferon have shown a potential benefit in the treatment of fibrotic processes in the skin and lung. Thus, the aim of the present study was the analysis of potential anti-fibrotic effects of these substances on human kidney fibroblasts in vitro. METHODS: Primary renal fibroblasts were established from human kidney biopsies and were studied in addition to two renal fibroblast cell lines. Cells were first growth arrested by withdrawal of fetal calf serum (FCS) and subsequently stimulated with 10% FCS in the presence of different concentrations of pentoxifylline (PTX), pentifylline (PTF), or gamma-interferon (IFN-gamma). Fibroblast proliferation was determined by bromodeoxyuridine incorporation and cell counts. Northern and western blot hybridizations for basic fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-beta1 were performed to analyse inhibitory effects. The effects of all three substances on matrix synthesis were evaluated by immunoblot analyses and ELISA for collagen type I and fibronectin after stimulation with TGF-beta1. Finally, differentiation into myofibroblasts was examined by double immunofluorescence staining for alpha-smooth-muscle actin and Hoechst dye H33258. RESULTS: PTX and PTF resulted in a dose- and time-dependent inhibition of proliferation in all fibroblast lines (maximum 78.9+/-6.2% at 500 microg/ml PTX). Conversely, IFN-gamma had only modest effects on fibroblast proliferation, resulting in a maximum of 36.0+/-6.1% inhibition at 500 U/ml. Northern blot hybridizations determined that FGF-2 mRNA levels in fibroblasts were decreased up to 73.7 and 91.5% by PTX (1000 microg/ml) and PTF (100 microg/ml), whereas IFN-gamma led to a reduction of 46.2% at 1000 U/ml, indicating that the inhibitory effects of all three substances may be mediated through inhibition of FGF-2 synthesis. These findings were corroborated by immunoblot analyses where again PTX and PTF had the strongest inhibitory effects. No change in TGF-beta1 mRNA levels was noted. Synthesis of cellular and secreted collagen type I was robustly inhibited by PTX and PTF, whereas IFN-gamma exerted the strongest inhibitory effect on fibronectin synthesis and secretion. In addition, IFN-gamma down-regulated the expression of alpha-smooth-muscle actin up to 73.3% (at 1000 U/ml) whereas PTX and PTF resulted in a down-regulation of up to 49.7+/-1.8 and 80.0+/-4.4% (at 1000 and 100 microg/ml) respectively. PTF was in all experiments about 10 times more potent than equimolar concentrations of PTX. CONCLUSIONS: PTX and PTF exerted robust inhibitory effects on fibroblast proliferation, extracellular matrix synthesis, and myofibroblastic differentiation. Conversely, IFN-gamma caused strong inhibition of fibronectin synthesis and alpha-smooth-muscle cell actin expression but had only weak inhibitory influences on fibroblast proliferation and collagen type I synthesis. Inhibitory effects of all three substances on proliferation may be mediated through inhibition of FGF-2 synthesis.

Cell Differentiation↗