Antimetabolite-induced apoptosis in Tenon''s capsule fibroblasts

PURPOSE: To analyze the computed tomographic (CT) features of intestinal Beh?et syndrome and to determine the usefulness of CT in detecting complications. MATERIALS AND METHODS: The CT scans of 28 patients with intestinal Beh?et syndrome were retrospectively reviewed. Five patients had bowel perforation, and two had peritonitis. Patterns of bowel involvement, patterns of contrast enhancement, and ancillary findings were compared in patients with complications (n = 7) and patients without complications (n = 21). RESULTS: Ten patients had polypoid lesions, nine had a thickened bowel wall, and nine had both findings. Lesion enhancement was mild in eight patients (29%) and marked in 20 (71%). Polypoid lesions were more commonly seen in patients without complications (P = .020); a thickened bowel wall was more commonly seen in patients with complications (P = .030). Seventeen of 18 patients (94%) with minimal perienteric infiltration did not have complications (P = .0003), whereas all five patients with severe perienteric infiltration did have complications. CONCLUSION: In patients with known intestinal Beh?et syndrome, CT can be useful in determining the extent of the lesions and in identifying cases in which complications are likely to occur.     

Lack of effect of hematopoietic growth factors on human breast epithelial cell growth in serum-free primary culture

A number of recombinant cytokines believed to regulate normal hematopoiesis are now being used in cancer treatment protocols to reduce the myelosuppressive toxicity of intensive chemoradiotherapy regimens. It is widely assumed that such cytokines are relatively specific for hematopoietic cells, although some cell lines derived from a variety of non-hematopoietic human tumors can respond to some of these factors. However, relatively little is known about their ability to stimulate (or inhibit) the proliferation of freshly isolated normal or malignant non-hematopoietic cells. We have used a serum-free culture medium that selectively supports the growth of human breast epithelial cells (HBEC) obtained directly from normal or malignant tissue samples to evaluate potential stimulatory or inhibitory effects of eight cytokines: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Steel factor, interleukin-2, interleukin-3, interleukin-6, transforming growth factor-beta and macrophage inflammatory protein-1 alpha, on these cells cultured both in the presence of epidermal growth factor, a potent stimulator of HBEC growth, and in its absence. HBEC growth was assessed after 7 and 14 days using the tetrazolium-dye reduction assay. Potential effects on the well studied MCF-7 breast cancer cell line, cultured under the same conditions, were also investigated. None of the cytokines (which were tested over a wide range of concentrations) had any modulating effect on the growth of normal or malignant HBEC under the conditions used with the exception of transforming growth factor-beta, which was consistently and significantly inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long term growth arrest of human Tenon''s fibroblasts following single applications of beta radiation

Aortic stenosis (AS) is a major risk factor for perioperative cardiac events in patients undergoing noncardiac surgery. We previously showed that selected patients with AS who were not candidates for, or refused, aortic valve replacement could undergo noncardiac surgery with acceptable risk. We extended our previous experience over a subsequent 5-year period by retrospectively analyzing the perioperative course of all patients with severe AS (aortic valve area index < 0.5 cm2/m2 or mean gradient > 50 mm Hg), determined with Doppler echocardiography or cardiac catheterization, who underwent noncardiac surgery. Nineteen patients underwent 28 surgical procedures: 22 elective and 6 emergency. The types of these procedures were 12 orthopedic, 6 intraabdominal, 4 vascular, 4 urologic, 1 otolaryngologic, and 1 thoracic. Mean age was 75 +/- 8 years. Of the 19 patients, 16 (84%) had > or = 1 symptom: dyspnea, angina, syncope, or presyncope. Mean left ventricular ejection fraction was 61 +/- 11%. The type of anesthesia was general in 26 procedures and continuous spinal in 2. Intraarterial monitoring of blood pressure was used in 20 of the 28 surgical procedures. Intraoperative hypotensive events were treated promptly, primarily with phenylephrine. In all cases the anesthesia team was aware of the severity of the AS and integrated this into the anesthetic plan. Two patients (elective operation in 1 and emergency in 1) had complicated postoperative courses and died. There were no other intraoperative or postoperative events in any of the other patients. Although aortic valve replacement remains the primary treatment for patients with severe AS, selected patients with severe AS, who are otherwise not candidates for aortic valve replacement, can undergo noncardiac surgery with acceptable risk when appropriate intraoperative and postoperative management is used.     

Effect of growth factors on growth of bovine parathyroid cells in serum-free medium

Published work has suggested a possible role of the epidermal growth factor receptor (EGFr) in parathyroid disease. Bovine parathyroid cells (BPCs) are frequently used as a tissue model for studying parathyroid disorders. We have studied the effect of the EGFr ligands EGF and transforming growth factor alpha (TGFalpha), alone and with insulin-like growth factors IGF-I and IGF-II on BPC growth. Experiments were run in triplicate and repeated three times. Cell numbers were assessed on day 5 by colorimetric MTT assay as well as by tritiated thymidine uptake. Results show that TGFalpha alone (p < 0.05) and IGF-I and IGF-II alone (p < 0.05) significantly stimulated growth over controls (t-test). Furthermore, the combination of TGFalpha with IGF-I and IGF-II exhibited significant enhancement above that seen with IGF-I and IGF-II alone (p < 0.01). EGF did not stimulate growth over controls. EGFr may be expressed in BPCs, but TGFalpha exhibits a more potent growth stimulus than EGF. Addition of IGF-I or IGF-II to the growth medium further enhances this effect.     

Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions

A single Plasmodium falciparum isolate was adapted for growth in serum-free culture medium. The parasitemia increased from 0.5% to 20% on day 7 after thawing. The asexual forms of the parasites appeared morphologically normal and pigment formation was comparable with that seen under standard conditions with serum present. Parasites were coincubated in 96-well plates with serum, peripheral blood mononuclear cells (PBMC), and PBMC in the presence of autologous serum from healthy non-immune individuals (n = 12), healthy semi-immune individuals (n = 12), and malaria patients (n = 7). Growth was monitored for six days. The concentration of interleukin-6 and interferon-gamma (IFN-gamma) in supernatants from the continuous cultures were measured by a bioassay and an enzyme-amplified sensitivity immunoassay. The results of this study showed that parasites cultured in serum-free medium in the presence of PBMC develop more rapidly, particularly with cells from malaria patients, compared with parasites cultured alone. The growth of parasites was different if 10% autologous serum was added to the culture. Parasite growth with sera from acutely infected individuals was similar with that with sera from aparasitemic, nonimmune individuals, and both supported significantly higher parasite growth over the six-day culture period compared with sera from the uninfected semi-immune individuals. Production of IFN-gamma by cells from nonimmune individuals and malaria patients was higher when cultures did not contain autologous serum. Nonimmune donor cells produced high amounts of IFN-gamma, but cells from the semi-immune donors produced little of this cytokine. There was no marked inhibition of parasite growth with any combination of serum and cells over six days of culture. A difference between the groups was observed after two days of culture, when growth with cells and serum from the uninfected, semi-immune group was significantly lower than that from the nonimmune group, but this was not subsequently sustained. The results of the study show that continuous cultivation of P. falciparum in serum-free medium provides a novel in vitro model to study mechanisms of the interplay between components of the human immune system and the malarial parasite, in which any possible influence of human serum is removed.     

The effects of nickel ions on articular chondrocyte growth in monolayer culture

Monolayer cultures of lapine articular chondrocytes were used to study the effects of nickel ions (Ni++) on chondrocyte proliferative capacity, proteoglycan synthesis and cellular morphology. Nickel depressed chondrocyte proliferation at the highest concentration tested (100 mumol/l; P less than 0.01) and also exerted a dose-dependent inhibition of proteoglycan synthesis (50 and 100 mumol/l; P less than 0.01). Despite both these effects, no evidence of chondrocyte damage was detectable at the light-microscopical level. The possible significance of the nickel-induced reduction of articular chondrocyte proteoglycan synthesis for the functional integrity of the residual cartilage following hemiarthroplasty operations is discussed.     

Effects of transforming growth factors on dopaminergic neurons in culture

It was recently reported that TGF-beta 1 either had no significant effect on or increased the survival of dopaminergic neurons in culture. TGF-beta 2 and TGF-beta 3 were reported to cause increased survival or to greatly inhibit survival. The transforming growth factors are a highly pleiotropic group of compounds, and the above results suggest that their actions may be critically dependent on the conditions of the assay. We have therefore tested these compounds under optimal conditions of culture, in a medium containing a low (2.5%) concentration of fetal bovine serum. TGF-beta 2 and 3 inhibited neuronal (MAP2-pos) survival only at the highest concentration (10 ng/ml) tested, while inhibition of survival of dopaminergic neurons was observed at 1.0 and 10 ng/ml. These results therefore suggest that the inhibitory action of TGF-beta 2 and 3 on the survival of dopaminergic neurons in culture, under the experimental conditions outlined, may be relatively specific.     

The effects of fibroblast growth factors in long-term primary culture of dystrophic (mdx) mouse muscle myoblasts

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.     

The effects of growth factors on human normal placental cytotrophoblast cell proliferation

The effects of growth factors were investigated on the proliferation of a normal placental cytotrophoblast cell line (NPC). Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-I (IGF-I) stimulated NPC cell proliferation. In contrast, TGFbeta1 was found to be a negative regulator, inhibiting EGF-induced cell proliferation. When EGF/TGF alpha receptor was analysed by radio-ligand binding, two binding sites of different affinities were revealed in the proliferating NPC cells but only the low affinity binding site was detected in the non-proliferating cytotrophoblast cells in primary cultures. The results suggest that EGF stimulates cytotrophoblast proliferation through high affinity binding sites.     

Transnasal endoscopic approach to expose the medial rectus from the annulus of Zinn to the penetration of Tenon''s capsule

Conventional strabismus surgery employs a conjunctival incision to gain access to Tenon's space where a wide variety of procedures are routinely performed on the tendon and anterior aspect of the extraocular muscles. Recently, transnasal endoscopic surgical techniques have gained acceptance as effective means of decompressing the medial wall and floor of the orbit in patients with thyroid-related orbitopathy. The orbital surface of the medial rectus and inferior rectus are exposed from the annulus of Zinn to a position close to where the muscles penetrate Tenon's capsule. In theory, this technique also provides the exposure necessary to locate and retrieve a "lost" medial rectus when the usual sub-Tenon's approach fails to recover the muscle. Cadaver studies demonstrate the feasibility of exposure and suture placement in the stump of a lost medial rectus with passage of the suture through Tenon's capsule to transmit the force of the muscle to the globe, provided that the lost muscle is retrieved before severe contracture develops.     

Regulatory effects of soluble growth factors on choriocapillaris endothelial growth and survival

The purpose of the following study was a multidimensional assessment of the course of anorexia nervosa. In our follow-up-study we were able to examine 41 out of 51 patients meeting DMS-111 R criteria for anorexia nervosa in our department at a mean follow-up of 5.3 years. At the first evaluation the patients were characterized by an early age of onset (mean 14.2 years), no or very little previous psychiatric treatment, and restrictive eating habits (72.5%); the mean age was 15.2 years. At the time of follow-up, the physical outcome was unfavourable for 50% of the patients; one patient had died. The psychosocial outcome was less favourable for 60%. Against the background of a neurotic structure, social and sexual disturbances were found although the patient's weight was often normal. Depressive symptoms and a "slimness ideal" were found among a large number of patients, just as much as the fear of gaining weight--which prove to be a reliable indicator for the continuation of an eating disorder. A multidimensional evaluation facilitates an assessment of the determinants of prognosis. Previous psychiatric treatment and low body weight were associated with an unfavourable, mention of problems/disturbances in the family environment with a good somatic outcome. Outpatient treatment indicates a favourable psychosocial outcome.     

Effects of serum-free culture media on human liver and fibroblastic cells

The morphological aspects and amino acid variations of human fibroblast and liver cell monolayer cultures were studied in serum free media. Under these conditions, the behaviour of the two cell types differed greatly. The morphological changes for the liver cells, as compared with the fibroblasts, appear more quickly and some of these changes are particular. Furthermore, extracellular amino acid variations are of lesser importance for the liver cells, during the 10 days following the suppression of serum, except for serine.     

Urokinase-type and tissue-type plasminogen activators as growth factors of human fibroblasts

In this study we have verified the mitogenic effect of urokinase-type (u-PA) and tissue-type plasminogen activators (t-PA) on human normal fibroblasts. We report that both PAs can induce DNA replication and cell division in serum-deprived cultured human skin fibroblasts. The activity of u-PA and t-PA is, respectively, three- and twofold more potent than that exerted by epidermal growth factor (EGF) with an activity slightly lower (50-60%) than that induced by basic fibroblast growth factor (bFGF). The u-PA and t-PA, but not plasmin, induced DNA synthesis, which could be neutralized by anti-u-PA and anti-t-PA antibodies, respectively, but was insensitive to aprotinin treatment. The addition of anti-u-PA-receptor (u-PAR) monoclonal antibodies to the assays selectively suppressed the mitogenic effect exerted by u-PA, but not that of t-PA, and the amino-terminal fragment of u-PA, containing the EGF-like domain and the kringle module, did not elicit any mitogenic activity. Anti-bFGF antibodies completely suppressed the mitogenic activity of bFGF, but did not have any effect on that of u-PA and t-PA; the activity of both PAs was inhibited by anti-fibronectin IgG concentrations ineffective on bFGF. These results indicate that PAs may be considered growth factors of human fibroblasts.     

The differential regulation and secretion of proteinases from fetal and neonatal fibroblasts by growth factors

Until recently, research on the pathogenesis of glomerulonephritis has been mainly focused on the characterization of humoral immune responses in the initiation of glomerular injury. However, there is a growing recognition that both cellular and humoral immune responses, in varying proportions, are involved in the pathogenesis of immunologically-mediated glomerulonephritis. T lymphocytes are essential cellular elements of cell-mediated immunity. Both in experimental models of immune-mediated renal disease and in histopathological analyses of human nephropathies, the involvement of T cells has been demonstrated in the immunoregulation of nephritogenic immune responses and in the immune injury in the kidney. T cell activation resulting in either delayed-type hypersensitivity, cytolytic reactions, abnormal expression of major histocompatibility complex (MHC) molecules, or B cell activation can result in glomerulonephritis. These different types of responses are exerted by distinct T cell subsets defined by cell surface markers and cytokine profiles. CD4+ T cells in vivo are functionally heterogeneous with respect to cytokine production and the CD45 isoforms that are found on their surface. Like CD4+ T cells, CD8+ T cells may also be heterogeneous at the level of cytokine production. The roles of CD4+ and CD8+ cells and their cytokine profiles in glomerulonephritis have not been extensively investigated yet, but such studies might improve the understanding of the pathogenesis and possibly lead to new therapeutic approaches of human glomerulonephritis. In this review the role of distinct T lymphocyte subsets in experimental and human glomerulonephritis will be discussed.     

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, G-CSF, and IL-3 were found to be the most important HGF for production of neutrophilic cells. The 4-HGF combination of IL-3, IL-6, G-CSF, and SCF was optimized by performing dose-response experiments and shown to be as potent as 6 HGF for production of nascent CFU-GM and neutrophilic precursors.     

Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts

The effects of acidic and basic fibroblast growth factors (FGFs) on collagen expression by keloid fibroblasts were examined in the absence and presence of heparin. Collagen biosynthesis and gene expression of type I collagen were down-regulated by the FGFs in the presence of heparin. Acidic FGF, in a concentration range of 0.4 to 50 ng/ml, had little or no effect on collagen synthesis after a 4-day incubation. However, in the presence of heparin (100 micrograms/ml) acidic FGF, in concentrations ranging from 2 to 50 ng/ml, decreased [3H]hydroxyproline synthesis by 44 to 68%, compared with untreated control cultures. Total [3H]hydroxyproline synthesis was similar between control and heparin-treated cultures. Basic FGF (2.0 to 50 ng/ml) was effective in suppressing [3H]hydroxyproline synthesis by 50 to 90% after a 4-day incubation without heparin in keloid and normal fibroblast cultures. The steady-state levels of type I collagen messenger RNA were significantly decreased by acidic FGF in the presence of heparin, as well as by basic FGF without heparin. The data suggest that the FGFs are effective in down-regulating excess collagen production by keloid fibroblasts and that this inhibitory effect is apparently associated with pretranslational events. Moreover, acidic FGF is apparently dependent on heparin, whereas basic FGF is not, for potentiation of the down-regulatory effects of the FGFs.     

The growth characteristics of fibroblasts in vitro on lavsan with different contours

Peculiarities of the hen embryo fibroblasts growth on substates and different relief and surface properties (lavsan glass [correction of grass], gauze and pellicle). The most quick and intensive growth with monolayer forming occurs on the glass [correction of grass] surface, although degradation of the culture also starts here earlier. The most slow and less active growth takes place on the gauze but the cells keep growing when fibroblast degenerative changes on the glass [correction of grass] are almost over. Lavsan pellicle is between glass [correction of grass] and gauze as a substrate for fibroblasts growth. Not all but only some cells from the suspension used for the explantation are likely to possess an ability for adhesion and the following growth on lavsan. In other words a natural selection of cells occurs during their adaptation to in vitro conditions.     

Peptide growth factors regulate insulin-like growth factor binding protein production by fetal rat lung fibroblasts

Insulin-like growth factor (IGF) binding proteins (IGFBPs) are expressed in fetal lung and may provide important post-translational regulation of IGF-induced mitogenesis during lung organogenesis. Because of the observation that growth factors can control cell growth through regulation of IGFBPs, we examined IGFBP production by fetal lung fibroblasts following stimulation by peptide growth factors important for fetal lung growth and development. Fetal lung fibroblasts were cultured in serum-free medium supplemented with various growth factors for up to 48 h, and IGFBPs in conditioned medium (CM) were analyzed by ligand blot and immunoblot techniques. Accumulation of CM IGFBP-3 was increased and IGFBP-2 decreased by incubation with either keratinocyte growth factor (KGF) or epidermal growth factor (EGF). The effect of these factors on IGFBP-3 accumulation increased with time but the effects of KGF on CM IGFBP-2 decreased over 48 h of incubation. CM IGFBP-4 was increased by 24 and 48 h incubation with basic fibroblast growth factor (bFGF; 2.1- and 2.7-fold increases at 24 and 48 h, respectively) and platelet-derived growth factor-BB (PDGF-BB; 4.2- and 14.9-fold increases at 24 and 48 h, respectively), and 48 h incubation with EGF (6.3-fold increase). In 48-h coincubation experiments, EGF in combination with PDGF-BB or with bFGF, and bFGF in combination with PDGF-BB, resulted in IGFBP-4 accumulations twice that expected from a summation of the effects of either growth factor alone (IGFBP-4 increased 9.8-, 4.0-, and 1.8-fold by PDGF-BB, EGF, and bFGF, respectively; and 27.1-, 37.3-, and 13.0-fold by PDGF-BB plus EGF, PDGF-BB plus bFGF, and EGF plus bFGF, respectively). These results suggest synergistic effects of these growth factors on IGFBP-4 accumulation in fetal lung fibroblast CM. Because IGFBPs are known to regulate DNA synthesis, we speculate that peptide growth factors may alter cell proliferation in fetal lung, in part through their effect on IGFBPs.     

Regulation of growth of human diploid fibroblasts by factors elaborated by activated lymphoid cells

Fibroblasts growth synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80 degrees C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (alpha-LT). It was not cytotoxic, as shown by the 51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0 or G1 stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.