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R Sgonc

Publications and source records attributed to R Sgonc.

At least 19 recordsLinked to original sources
PubMed2001

Heat shock protein expression in the transplanted human kidney.

Heat shock proteins (HSPs) have been shown to represent potential target molecules for T-cell-mediated allograft rejection in heart and kidney transplants. In the present study, we therefore investigated the expression of HSP subtypes 60, 72, and 73 in normal kidneys and qualitative and/or quantitative changes in rejected renal allografts. Six normal kidney tissue specimens, three biopsies from patients with minimal change nephritis, as well as 37 biopsies and eight transplant nephrectomy specimens of patients with renal allograft rejection were studied. Type and severity of rejection were assessed according to the Banff classification. Immunohistochemical demonstration of HSP expression was performed using specific monoclonal antibodies after wet autoclave antigen retrieval on sections from either Carnoy-fixed (biopsies) or formalin-fixed (transplant nephrectomies) and paraffin-embedded tissue. The expression was scored in a semiquantitative manner. All three subtypes were found to be constitutively expressed in normal kidney tissue and in noninflammatory minimal change nephritis, albeit with a characteristic compartmental and cellular distribution. Rejection resulted in a higher immunohistochemical scoring for all three HSP subtypes in compartments in which they were normally present; in addition, a de novo expression of HSP60 was found in the vascular compartment and, moreover, infiltrating mononuclear cells were strongly immunoreactive for HSP60 and HSP73. Only quantitative differences were observed for HSP72 immunoreactivity. These results indicate that rejection episodes are paralleled by an increased but differential expression of HSPs in the glomerular, tubular, and vascular compartments of the kidney. This enhancement as well as the de novo appearance of HSP60 on vascular endothelial cells might explain the presence of HSP-reactive T lymphocytes in rejected allografts.

Carrier Proteins↗
PubMed2000

Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95.

OBJECTIVE: Apoptosis of endothelial cells is a key event in the pathogenesis of systemic sclerosis (SSc). The aim of the present study was to analyze in vitro the mechanism causing endothelial cell apoptosis in SSc. METHODS: Human dermal microvascular endothelial cells (HDMEC) or human umbilical vein endothelial cells (HUVEC) were cultured with native or heat-inactivated serum from SSc patients or controls with or without interleukin-2-activated natural killer (NK) cells or peripheral blood mononuclear cells. SSc and control sera were tested for the presence or absence, respectively, of anti-endothelial cell antibodies (AECA) by indirect immunofluorescence. Apoptosis was detected by the TUNEL technique. RESULTS: Native sera alone had no effect. Apoptosis induction was observed on HDMEC, but not on HUVEC, in the presence of AECA-positive SSc sera and activated NK cells, and could be inhibited by an anti-Fas ligand antibody. Inhibition of the perforin/granzyme pathway with concanamycin A had no effect on apoptosis induction in this in vitro model. Immunofluorescence analysis of cryosections from SSc skin showed Fas (CD95) expression by endothelial cells, supporting the in vitro findings. CONCLUSION: The results suggest that endothelial cell apoptosis in SSc is induced by antibody-dependent cell-mediated cytotoxicity via the Fas pathway. These data not only provide insight into the pathogenesis of SSc, but also may open new ways to rational therapy for this disease.

Adult↗
PubMed2000

Endothelial injury in internal organs of University of California at Davis line 200 (UCD 200) chickens, an animal model for systemic sclerosis (Scleroderma).

Systemic sclerosis (SSc) is a multisystem disorder characterized by mononuclear cell infiltration and fibrosis. Using skin samples from human SSc and UCD 200 chickens, which spontaneously develop a hereditary disease closely resembling human SSc, we have shown previously that endothelial cell apoptosis is a primary event in the pathogenesis of SSc. The aim of the present study was to investigate the initial disease stage in visceral organs of UCD 200 chickens with special emphasis on endothelial apoptosis, mononuclear cell infiltration and collagen deposition using tissue samples from oesophagus, lung, heart, kidney and liver. Apoptotic endothelial cells were detected by terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling (TUNEL), mononuclear cell infiltrates were stained with hematoxylin and eosin, and increased collagen deposition was demonstrated by Goldner staining. Apoptotic endothelial cells were detected in oesophagus, lung and kidney of UCD 200 chickens at the initial stage of the disease. No apoptotic endothelial cells were found in heart or liver of UCD 200 or in visceral organs of healthy normal UCD 058 control chickens. Oesophagus of UCD 200 chickens, which was the most affected internal organ, showed mononuclear cell infiltrations and increased deposition of collagen. Perivascular inflammatory infiltrates and collagen deposition appeared later than endothelial cell apoptosis. These data support the hypothesis that endothelial cell apoptosis initiates the disease process, followed by mononuclear cell infiltration and fibrosis.

Animals↗
PubMed1999

The vascular perspective of systemic sclerosis: of chickens, mice and men.

Systemic sclerosis (SSc), or scleroderma, is an autoimmune connective tissue disease characterized by structural and functional vascular abnormalities, perivascular mononuclear cell infiltration, and increased deposition of extracellular matrix in skin and internal organs. The initial stages of SSc are generally not accessible for analysis in man, therefore, the availability of appropriate animal models is of great importance for the elucidation of the pathogenesis of this disease. UCD-200 chickens show the entire clinical, histopathological and serological spectrum of SSc, whereas tight skin (Tsk)1/+ and Tsk2/+ mice, other animal models of scleroderma, lack the vascular injury. A parallel comparative study of skin biopsies of UCD-200 chickens and human SSc patients revealed that endothelial cell apoptosis, induced by anti-endothelial cell antibody (AECA)- dependent cellular cytotoxicity, is a primary event in the pathogenesis of SSc. This review focuses on recently established data on endothelial cell injury in animals with spontaneous disease and humans, AECA, adhesion molecules and cytokine profiles that support a vascular pathogenesis in scleroderma.

Animals↗
PubMed1998

Neuroendocrine-immune disturbances in animal models with spontaneous autoimmune diseases.

According to our concept, the development of autoimmune disease depends on the presence of two sets of essential genes, one coding for an abnormal autoreactivity of the immune system, the other for a primary susceptibility of the target organ/structure for the immune attack. The final outcome of the disease in a given individual is then fine tuned by modulatory factors, such as diet or hormones. With regard to the latter, the immuno-endocrine interaction via the hypothalamo-pituitary-adrenal (HPA) axis has proven to be of special importance. Investigating the so-called Obese strain (OS) of chickens, an animal model with a spontaneously occurring Hashimoto-like autoimmune thyroiditis, we have first shown an impaired surge of glucocorticoid hormones after stimulation of the HPA axis by antigens or certain cytokines (glucocorticoid-increasing factors--GIFs). More recently, we have found a similar behavior in models with systemic autoimmune diseases, that is, murine lupus erythematosus and avian scleroderma. More detailed studies have, however, proven that the mechanisms underlying this altered immuno-endocrine communication via the HPA axis differs in different models. Finally, recent data point to the possibility that the classical pathways of glucocorticoid-T-cell interactions also take place in the thymus itself, which has been shown to be a site of steroid hormone production.

Animals↗
PubMed1998

Apoptosis detection: an overview.

As the analysis of apoptosis is of interest in many basic and clinically oriented investigations, we intend to give a brief overview on the recently most-used methods for detection of apoptotic cells, including the study of morphology, analysis of DNA degradation, DNA end-labeling techniques, flow cytometric analysis, and nuclease assays. Features and advantages of the different methods are discussed.

Apoptosis↗
PubMed1998

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression.

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.

Apoptosis↗
PubMed1998

Apoptosis of tubular epithelial cells in familial juvenile gouty nephropathy.

Two patients, a 47-year-old woman suffering from chronic renal failure, hyperuricemia and gout, and her 26-year-old son with hyperuricemia and chronic renal failure, are described. The father and two siblings of the woman had died of chronic renal failure. Both patients had a markedly reduced fractional excretion of urate, which was significantly increased by both benzbromarone and probenecid. A renal biopsy of the son revealed an unspecific chronic tubulointerstitial nephropathy. By light microscopy, many proximal tubular epithelial cells showed signs of apoptosis, which was confirmed with the specific TUNEL assay. We propose a hypothesis based on a gain-of-function mutation of the luminal anion exchanger of the proximal tubulus to explain reduced uric acid excretion, dominant inheritance and apoptosis of tubular epithelial cells in this rare disease. Treatment with a combination of allopurinol to reduce the renal urate load and benzbromarone to block the tubular anion exchanger and normalize fractional uric acid excretion is suggested.

Adult↗
PubMed1998

Induction of apoptosis in human dermal microvascular endothelial cells and infantile hemangiomas by interferon-alpha.

BACKGROUND: Hemangioma of infancy is an angiomatous disorder characterized by the proliferation of capillary endothelium. It has been shown that interferon-alpha (IFN-alpha) may induce involution of proliferating, life-threatening hemangiomas in children. This IFN-alpha-induced regression of hemangiomas is not accompanied by any T cell response nor by the occurrence of necrosis. METHODS: To determine whether IFN-alpha may induce apoptosis, we cultured human dermal microvascular endothelial cells (HDMEC) with IFN-alpha at concentrations of 100, 500, 1,000 and 2,000 U/ml for 24-72 h and detected apoptosis by terminal deoxynucleotidyl transferase mediated FITC-dUTP nick end labeling (TUNEL). Quantitative analysis was performed using the FACScan and morphological alterations were studied by confocal laser scanning microscopy. In addition to the in vitro study we also analyzed frozen skin sections from proliferating, spontaneously regressing, and IFN-alpha-treated hemangiomas by simultaneous determination of endothelial cells and apoptotic nuclei using an indirect immunofluorescence test in combination with TUNEL. RESULTS: Apoptosis was detected in up to 20% of the IFN-alpha-treated endothelial cells compared to the untreated controls. A maximum of apoptosis was observed after 48 h of stimulation with IFN-alpha in a dose-dependent manner. The analysis of hemangioma biopsies revealed apoptotic endothelial cells in IFN-alpha-treated as well as in spontaneously regressing hemangiomas, but not in proliferating ones. CONCLUSION: These data indicate that the therapeutic effect of IFN-alpha on hemangiomas is based on apoptosis induction of endothelial cells, which might also explain the clinically and histologically observed involution without any sign of inflammation or necrosis.

Antibodies↗
PubMed1997

p53-induced apoptosis in the human T-ALL cell line CCRF-CEM.

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.

Alleles↗
PubMed1997

Influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells.

BACKGROUND: Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. The aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (RPEC) following viral infection, with special emphasis on those having immune-regulatory functions. METHODS: Cultured RPEC were infected with cytomegalovirus (CMV), coxsackie-virus B3 (CVB) or herpes simplex virus type I (HSV). Double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. RESULTS: CMV downregulated MHC class I antigens on RPEC, whereas CVB and HSV did not alter MHC class I antigen expression. No induction of class II antigens was observed in RPEC infected with CVB, HSV or CMV. The intercellular adhesion molecule ICAM-1 (CD54) was strongly expressed in uninfected RPEC, and a slight increase was observed after virus infection. Vascular cell adhesion molecule 1 (VCAM-1) was expressed in low amounts in both uninfected and infected RPEC. No expression of intercellular adhesion molecule 2 (ICAM-2), E-selectin ELAM-1 or lymphocyte-function-associated antigen 1 (LFA-1) was observed on RPEC before or after virus infection. CONCLUSION: Downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. Our observation in cultured human RPEC indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue.

Animals↗
PubMed1997

Induction of apoptosis in Kaposi's sarcoma spindle cell cultures by the subunits of human chorionic gonadotropin.

OBJECTIVES: Elucidation of the mechanisms of the previously shown growth-inhibitory action of human chorionic gonadotropin (hCG) on Kaposi's sarcoma (KS) cells and the role of the luteinizing hormone/hCG receptor (hCGR). DESIGN AND METHODS: Analysis of KS tissues and cultured spindle-type KS cells for the presence of the hCGR using 125I-hCG binding and reverse transcriptase-polymerase chain reaction; analysis of several hCG preparations (urinary, recombinant, isolated alpha and beta subunits); analysis of apoptosis mechanisms by several assays including using z-Val-Ala-Asp-fluoromethylketone (zVAD-FMK), a known apoptosis-inhibitory drug. RESULTS: First, we found that some urinary preparations of hCG (e.g., CG-10, Steris Profasi) were indeed KS-killing but others (such as Pregnyl, Choragon, Serono Profasi) were not. Secondly, recombinant subunits (alpha as well as beta) of hCG were KS cell-killing but recombinant intact hCG was not. Thirdly, the hCGR message and protein were undetectable in KS. Fourthly, CG10-induced cell death occurred by apoptosis and KS cells could be rescued by preincubation with zVAD-FMK. Finally, we also found that normal peripheral blood lymphocytes (PBL) were killed by CG-10. CONCLUSION: It is proposed that the action of subunits or subunit fragments of hCG, mediated by a putative orphan receptor (as opposed to the hCGR) and executed by interleukin-1-converting enzyme (ICE)-like protease(s), constitutes a novel apoptosis mechanism effective towards KS cells, but PBLs and possibly other cells as well. These results provide a basis for testing in vitro the therapeutic efficacy of hCG preparations which, in turn, should improve current clinical trials with 'hCG' in patients who have KS.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Altered procollagen mRNA expression during the progression of avian scleroderma.

BACKGROUND: Spontaneous animal models of human autoimmune diseases provide the means to study the very first pathogenetic events, which is not possible in their human counterparts. This is particularly true for connective tissue diseases in which clinical symptoms become manifest only after a long and still obscure course of immunologic, inflammatory, and fibrotic processes. University of California at Davis line 200 chickens (UCD-200) develop a hereditary scleroderma-like disease resembling the entire spectrum of human systemic sclerosis, such as early endothelial cell damage, severe lymphocytic infiltration, and accumulation of collagen in skin and internal organs. MATERIALS AND METHODS: In the present study, we investigated mRNA levels of alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III), alpha 1(VI), alpha 2(VI), and alpha 3(VI) procollagen and GAPDH using digoxigenin-labeled antisense probes in a nonradioactive ribonuclease protection assay (RPA). We analyzed tissue samples from comb, esophagus, heart, lung, and liver of UCD-200 chickens at different stages of the disease, and healthy UCD-058 chickens. RESULTS: During the early inflammatory stage of the disease, the ratios of procollagen types VI/I and types VI/III increased 7-fold in comb tissue, followed by a 3-fold elevation in type I procollagen transcripts in the late acute stage. In the chronic stage, alpha 1(III) procollagen message was increased 2-fold. Additionally, hybridization with the 180 bp alpha 2(I) antisense probe resulted in two bands of 180 bp and 115 bp, respectively, in the RPA. The ratio of these two previously undescribed bands changes in the early stage of the disease both in comb and esophagus. CONCLUSIONS: In an animal model with a spontaneous scleroderma-like disease we found a characteristic, sequential increase in type VI, type I, and type III procollagen transcripts, and we found evidence for the presence and altered ratio of two mRNA variants of alpha 2(I) procollagen, possibly caused by alternative splicing. Comparative analysis of alpha 2(I) procollagen variants in early stages of avian scleroderma and human SSc might provide answers to unresolved questions concerning the molecular basis for generalized fibrosis in scleroderma.

Acute Disease↗
PubMed1996

Endothelial cell apoptosis is a primary pathogenetic event underlying skin lesions in avian and human scleroderma.

The mechanism that may cause degenerative fibrotic skin lesions was studied in situ using skin biopsies from patients with systemic sclerosis (SSc), localized scleroderma, or keloids, and at the initial disease stage in the University of California at Davis (UCD) lines 200/206 chickens, which develop a hereditary systemic connective tissue disease resembling human SSc and permit study of disease stages not accessible in humans. Frozen skin sections were analyzed simultaneously for apoptosis by terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling and indirect immunofluorescence staining of cell markers with tetramethylrhodamine isothiocyanate conjugates. The results showed that endothelial cells are clearly the first cells to undergo apoptosis in the skin of UCD-200/206 chickens, a process that seems to be induced by anti-endothelial cell antibodies. In human fibrotic skin diseases, apoptotic endothelial cells could only be detected in early inflammatory disease stages of SSc and localized scleroderma.

Adolescent↗
PubMed1996

2',2'-Difluorodeoxycytidine (gemcitabine) induces apoptosis in myeloma cell lines resistant to steroids and 2-chlorodeoxyadenosine (2-CdA).

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10-6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 microM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 microM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells. The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.

2-Chloroadenosine↗
PubMed1995

Effect of neopterin and 7,8-dihydroneopterin on tumor necrosis factor-alpha induced programmed cell death.

Tumor necrosis factor-alpha and the formation of reactive oxygen intermediates are central mediators of apoptosis. Recent data indicated a role of neopterin and 7,8-dihydroneopterin in oxygen radical mediated processes. We have therefore investigated the effect of neopterin-derivatives on TNF alpha induced apoptosis of the monocyte-like cell line U937. At an elevated concentration 7,8-dihydroneopterin was found to superinduce TNF alpha mediated programmed cell death due to the formation of reactive oxygen intermediates. Our results imply that in combination with TNF alpha high concentrations of 7,8-dihydroneopterin enhances apoptosis due to oxidative stress on cells.

Acetylcysteine↗