Cortical fibroblast culture from human biopsies.

BACKGROUND: Tubulointerstitial fibrosis is an integral part of progressive renal disease. Human cortical fibroblasts are believed to be key effector cells in fibrogenesis. Thus, a reliable culture of these cells is necessary for studies of their pathophysiology. METHODS: Cortical fibroblast culture from routine kidney biopsies were analyzed and the cells were characterized. Indirect immunofluorescence staining was done after the first passage for cytokeratin, vimentin, alpha-smooth muscle actin, CD 44, CD 54, CD 68, collagen types I, III, and HLA-DR. We then assessed the utility of the putative fibroblast markers CD 90, prolyl-4-hydroxylase (P4H) and F1b in simultaneous stainings of tubular epithelial cells. RESULTS: During the study period, 49 biopsy cores were cultured and cortical fibroblasts could be successfully established in 21 cases (42.9%). There was no relation between the success rate of culture and the degree of interstitial fibrosis, but an association was seen with the time of completion of the first passage. There was a negative correlation between the extent of scarring and the percentage of cytokeratin positive cells (r = -0.66, p < 0.001). All primary fibroblasts were negative for factor VIII, HLA-DR, CD 68, and cytokeratin. They expressed alpha-smooth muscle actin and collagen types I and III to variable degrees. There was a robust correlation between the percentage of alpha-smooth muscle actin positive cells and interstitial scarring but no such association with collagen type I or type III positive cells. The three putative fibroblast markers did not prove useful in differentiating between tubular epithelial cells and fibroblasts. However, since only fibroblasts stained positive for CD 90 and negative for cytokeratin, these two markers may suffice to distinguish fibroblasts from other renal cellular elements. CONCLUSIONS: Cortical renal fibroblasts can be easily cultured from kidney biopsy cores, though the success rate of pure cultures is below 50%. Staining for CD 90 and cytokeratin may suffice for initial characterization of these cells.

TGF-beta 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2).

BACKGROUND: The prognosis of primary renal disease is often dependent on the degree of tubulointerstitial scarring. Scarring is caused by proliferation and excessive matrix production of renal fibroblasts and possibly other cellular elements. Transforming growth factor-beta (TGF-beta) is the most important cytokine for the induction of matrix synthesis in the kidney. However, its effects on renal fibroblast proliferation have not been determined. We have recently demonstrated that the expression of basic fibroblast growth factor (FGF-2) is robustly up-regulated in human kidneys with tubulointerstitial fibrosis and that FGF-2 is a potent inducer of fibroblast proliferation. The present study examined the interaction between TGF-beta 1 and FGF-2 in human renal fibroblasts. METHODS: Experiments were performed on a transformed medullary fibroblast line and on primary cortical kidney and skin fibroblasts isolated from human biopsies. mRNA levels of FGF-2 and TGF-beta 1 were analyzed by Northern blot analyses. Changes in protein expression were examined by immunoblots and enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine incorporation assays and cell counts were used to analyze cell proliferation. The expression of cell cycle-regulatory proteins cyclin-dependent kinase (cdk) 2 and the cdk inhibitor p27(kip1) were determined by immunoblots. RESULTS: Stimulation of renal fibroblasts with FGF-2 resulted in no change of TGF-beta 1 mRNA expression, whereas incubation of the cells with TGF-beta 1 induced FGF-2 mRNA up to 3.51 +/- 0.21-fold after six hours. This increase could be blocked almost completely by the addition of cyclohexamide, indicating that the process is in large part dependent on protein synthesis. The up-regulation in FGF-2 mRNA expression was paralleled by de novo detection of FGF-2 protein in the supernatant, peaking after 12 to 24 hours, as determined by Western blot and ELISA, whereas cellular protein was only increased up to 2.1-fold. Interestingly, both methods detected release of FGF-2 protein to the supernatant already at three hours, indicating a role for TGF-beta1 in directly releasing preformed FGF-2. Since TGF-beta 1 induced FGF-2, which results in fibroblast proliferation, we hypothesized that TGF-beta1 may cause fibroblast proliferation mediated by FGF-2. This hypothesis was verified by cell proliferation assays demonstrating that stimulation of renal fibroblasts with TGF-beta1 resulted in an up to 3.21 +/- 0.28-fold increase in bromodeoxyuridine incorporation and a 1.95 +/- 0.16-fold increase in cell number after 72 hours. This mitogenic effect of TGF-beta1 could be blocked completely by the addition of a neutralizing antibody to FGF-2 or the tyrosine kinase inhibitor tyrphostin AG1296, which blocks FGF receptor (FGFR) tyrosine kinase activity. Conversely, a neutralizing antibody to epidermal growth factor (EGF) or the tyrphostin B42, which inhibits EGF receptor signal transduction, had no effect. Interestingly, a neutralizing antibody to PDGF had only minor effects in primary kidney fibroblasts but reduced TGF-beta 1-induced proliferation considerably in primary skin fibroblasts. Finally, TGF-beta1-induced proliferation in kidney fibroblasts was paralleled by a robust increase in cdk 2 protein expression up to 72 hours, whereas p27(kip1), whose activity is maintained by TGF-beta in epithelial cells, was down-regulated up to 48 hours. CONCLUSIONS: Our studies demonstrate, to our knowledge for the first time, that TGF-beta1 induces proliferation in human renal fibroblasts and that this process is mediated largely by FGF-2. The induction of proliferation by TGF-beta 1 via induction of FGF-2 may play an important role in the autonomy of renal fibroblast growth and thus in the pathogenesis of human fibrogenesis.

Leukaemia-associated immunophenotypes (LAIP) are observed in 90% of adult and childhood acute lymphoblastic leukaemia: detection in remission marrow predicts outcome.

Analysis of differentiation antigens on leukaemic blasts is routinely done for diagnostic purposes, i.e. determination of stage of differentiation and lineage assignment. Acute lymphoblastic leukaemias are also frequently characterized by a leukaemia-associated immunophenotype (LAIP), either the coexpression of differentiation antigens physiologically restricted to other stages of differentiation (asynchronous LAIP) or cell lineages (aberrant LAIP). We defined LAIP in 241 consecutive unselected B-lineage (n = 193) and T-lineage (n = 48) ALL by three-colour flow cytometry using directly conjugated monoclonal antibodies. The incidence of LAIP was found to be 91.7%. In 63% of patients two to six leukaemia-associated expression patterns were detected. In order to study the specificity of LAIP in a therapy-relevant setting, remission bone marrow samples from patients with B-lineage ALL were analysed for the expression of T-lineage-associated phenotypes on the normal bone marrow cells and vice versa. The frequency of all T-lineage LAIP+ cells and all aberrant B-lineage LAIP+ cells was <1% in regenerating bone marrow samples at different timepoints. The incidence and clinical significance of LAIP+ cells was studied in 196 remission marrows of 70 ALL patients (55 remaining in CCR, 14 with bone marrow relapse, one with isolated CNS relapse). The presence of >1% LAIP+ at two consecutive timepoints predicted 5/8 bone marrow relapses in B-lineage ALL. The occurrence of LAIP+ cells >1% in T-lineage ALL after induction therapy predicted relapse in 7/7 cases. In conclusion, flow cytometric detection of LAIP+ cells appears to be a powerful tool for the prediction of outcome in ALL.

[Comparative follow-up study in vagotomized and conservatively treated duodenal ulcer patients]. / Vergleichende Verlaufsstudie bei vagotomierten und konservativ therapierten Ulcus-duodeni-Patienten.

We observed 87 patients with duodenal ulcer disease about one year. 46 patients (first group) after proximal gastric vagotomy were observed. A second group (41 patients) were treated with antacids only. We compared our patients for their complaints, consumption of antacids, relapse rate and clinical, social and genetic aspects. The clearly better results of the surgically treated patients allow the conclusion for a better quality of life in patients after proximal gastric vagotomy.