Anti-glomerular basement membrane (anti-GBM) disease often results in end-stage renal failure des... more Anti-glomerular basement membrane (anti-GBM) disease often results in end-stage renal failure despite therapy with plasma exchange and immunosuppressive drugs. The newly discovered streptococcal enzymes IgG-degrading enzyme of S.pyogenes (IdeS) and endoglycosidase S (EndoS) act with remarkable specificity on circulating IgG. In this study, we investigate their ability in vivo to prevent damage mediated by kidney-bound antibodies in a mouse model of anti-GBM disease. Anti-GBM disease was induced in mice by injection of subnephritogenic doses of rabbit anti-mouse GBM, followed a week later by injection of monoclonal mouse anti-rabbit IgG antibodies. By administrating IdeS or EndoS as fusion partners with GST between these antibody injections, we tested their ability to prevent damage by acting on kidney-bound rabbit anti-GBM. Control animals received placebo injections. All animals in the positive control groups developed severe albuminuria immediately after the second antibody injection (mean, 2.51 mg/24 h; range, 0.13-8.20). This was significantly diminished by EndoS (1.3 +/- 1.3 mg/24 h) and completely prevented by IdeS (0.017 +/- 0.014 mg/24 h). Immunofluorescence studies showed that IdeS treatment effectively removed the Fc fragments of the rabbit IgG. This was accompanied by a significant reduction of the deposition of the complement components C3 and C1q, and this diminished the recruitment of leukocytes to the glomeruli. IdeS degrades IgG bound to the GBM in vivo, thereby preventing renal damage in this animal model. Most likely, IdeS would degrade both circulating and kidney-bound anti-GBM in patients with Goodpasture's disease. Whether this would lead to a halt in disease progression and a better prognosis remains to be determined.
Proximal tubular epithelial cells (PTEC) from human renal tissue obtained from biopsy or nephrect... more Proximal tubular epithelial cells (PTEC) from human renal tissue obtained from biopsy or nephrectomy were grown in monoculture and evaluated in vitro at passage 2-4 for interleukin 6 (IL-6) production in response to medium alone or to interleukin 1 alpha (IL-1 alpha), tumour necrosis factor alpha (TNF alpha), interleukin 2 (IL-2), interferon gamma (INF gamma) or lipopolysaccharide (LPS). IL-6 bioactivity was quantitated using the IL-6-dependent murine hybridoma cell line (B9) and expressed as IL-6 units/ml/10(5) PTEC. PTEC cell lines exposed to medium alone produced intermediate amounts of IL-6 with substantial variability between cell lines. Introduction of IL-1 alpha resulted in a dose- and time-dependent increase in IL-6 production by PTEC that was maximal at 1 ng/ml IL-1 alpha at 24 h. All PTEC cell lines showed an increased IL-6 production on exposure to IL-1 alpha varying from 1.3- to 24-fold increase over baseline production. This response was completely blocked by anti-rIL-1 alpha. No significant IL-6 production by PTEC could be induced by TNF alpha, IL-2, IFN gamma, or LPS over a broad dosage range. Cycloheximide inhibited IL-6 production without irreversible cell toxicity, indicating de-novo synthesis. IL-6 produced by PTEC had a molecular weight of 26-29 kDa as demonstrated by Western blot analysis. Using PCR analysis we could demonstrate upregulation by IL-1 alpha of IL-6 mRNA in a dose-response fashion, indicating that IL-1 alpha regulates IL-6 production at a pretranslational value of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity fo... more The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity for C3bBb formation, which is present in human umbilical vein endothelial cells (HUVEC). Therefore, human endothelial culture supernatants (HECS) were analyzed for the presence of human complement factor H by ELISA. It was found that H was present in HECS. An immunoblot analysis of affinity purified H from HECS showed that the size of HUVEC H was identical to that of plasma H. The mean production of H by HUVEC of first passage cultures was 40 ng/10(6) cells/day. The synthesis of HUVEC H was fully inhibitable by the addition of cycloheximide to the cultures, suggesting that H is de novo synthesized. Additional evidence for de novo synthesis was obtained by using biosynthetic labeling with [35S] methionine, immunoprecipitation, and SDS-PAGE. It was demonstrated that, indeed, HUVEC produce and secrete factor H. Two forms of the protein were identified, the 150-kDa form and also a 45-kDa form, both forms have been identified in plasma. The functional activity of HUVEC H is identical to that of plasma H. IFN-gamma induced enhanced synthesis of H by HUVEC, whereas it had no effect on C3 synthesis. Supernatant from stimulated PBMC, T cell growth factor, enhanced synthesis of both H and C3. The present studies indicate that H is produced by HU-VEC and that H may function as an inhibitor of complement activation at the endothelial cell level and, thereby, together with molecules like decay accelerating factor and membrane cofactor protein, may influence resistance of endothelial cells to complement mediated damage.
Human umbilical vein endothelial cells (HUVEC) synthesize and secrete C component factors H and C... more Human umbilical vein endothelial cells (HUVEC) synthesize and secrete C component factors H and C3. Addition of T cell growth factor to HUVEC enhanced factor H production, and caused a profound increase in C3 production. In the present study, investigations were initiated to characterize the effects of purified rIL-1 and rIFN-gamma on the production of factor H and C3 at the protein and mRNA level. IFN-gamma enhanced factor H production in a dose-dependent fashion and a two-fold increase was observed with an optimal dose of 200 U/ml, whereas IFN-gamma had no effect on C3 production. IL-1 inhibited factor H secretion, but the production of C3 was increased 10-fold at an optimal dose of 500 pg/ml of IL-1. Kinetic experiments demonstrated that addition of IL-1 to HUVEC resulted in an induction of C3 production after more than 24 h, whereas IFN-gamma already had a significant effect on factor H production after 8 h of culture. IL-1 in combination with IFN-gamma had a synergistic effect on C3 production. The effects of IL-1 and IFN-gamma on factor H and C3 production by HUVEC could be blocked by using neutralizing amounts of antibodies specific for these cytokines. Northern blot analysis showed that factor H mRNA expression was enhanced in IFN-gamma-treated HUVEC and C3 mRNA was induced in IL-1-treated HUVEC, indicating that the observed increase of factor H and C3 probably is controlled by enhancement of transcription or stability of the transcript.
A family is described in which 3/11 children showed a homozygous deficiency of C3, and both paren... more A family is described in which 3/11 children showed a homozygous deficiency of C3, and both parents and six other children had subnormal levels of C3. The three children with selective C3 deficiency suffered repeatedly from bacterial infections, whereas the parents and the other siblings were clinically healthy. During infectious episodes the patients showed a maculopapular skin rash, and at such times immune complexes were present in the serum. Biopsy specimens of the skin lesions showed the picture of leukocytoclastic vasculitis.
Peritoneal dialysis international: journal of the International Society for Peritoneal Dialysis
During continuous ambulatory peritoneal dialysis (CAPD), activation of complement in the peritone... more During continuous ambulatory peritoneal dialysis (CAPD), activation of complement in the peritoneal cavity may theoretically occur, with inappropriately high or low levels of certain complement factors in dialysate as a consequence. In a group of children on CAPD, it was tested whether levels of a number of complement factors in dialysate were in the range that was predicted on the basis of their molecular weight. Serum and dialysate levels of C1q, C3, C4, C3d, B, D, and P were measured after a night dwell in children on CAPD. Simultaneously, four non-complement proteins (beta 2-microglobulin, albumin, IgG, and alpha 2-macroglobulin) were also measured in dialysate and serum. Assuming a linear relationship between the log base 10 of the dialysate/serum ratio of these non-complement proteins and the log base 10 of their molecular weight, the expected ratios of all complement factors were determined. The differences between actual and predicted ratios were tested using a modified t-test, taking into account the inaccuracy of the estimate. University hospital. A group of 14 children on CAPD, with a median age of 7.8 years (range 2.1 - 13.2). These children had been on CAPD for a median period of 42.4 months (range 0.4 - 89.1). The ratios of factor D (p < 0.001) and C3d (p = 0.035) were elevated, whereas those of C3 (p < 0.001), C4 (p < 0.001), and factor P (p = 0.012) were decreased. Relatively low dialysate/serum ratios of C4, C3, and factor P could be caused by intraperitoneal consumption of complement. High levels of C3d are compatible with this. High dialysate/serum ratios of factor D indicate intraperitoneal production of factor D. These results provide evidence for activation of complement in the peritoneal cavity in children on CAPD. A further reduction of already low levels of complement factors in dialysate as a result of this may impair host defense.
The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the ... more The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the alpha, beta, and gamma chains. Previous studies have shown that human proximal tubular epithelial cells (PTEC) express IL-2R alpha during inflammation, such as in the kidneys after renal transplantation or in crescentic glomerulonephritis. Furthermore, in this study it is shown that PTEC in culture produce more complement C3 after stimulation with IL-2, suggesting the presence of a functional IL-2R on PTEC. In these experiments, the binding of IL-2 to PTEC was analyzed. PTEC stimulated with IL-2 exhibited very low binding of digoxigenin-labeled IL-2; however, stimulation of PTEC with IL-2, in combination with interferon (IFN)-gamma, resulted in increased binding of the digoxigenin-labeled IL-2. In addition, it was found that IL-2, together with IFN-gamma, enhanced the production of C3 by PTEC from baseline levels of 69.6 +/- 3.4 to 198.6 +/- 3.3 ng of C3 per 10(6) cells. The surface expr...
An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is thei... more An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is their ability to block complement activation. The purpose of this study was to compare the complement-inhibitory activity of four IVIG preparations differing in isotype composition. The preparations were: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2) Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L IgA); (3) IVIgM (35 g/L IgM, 12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG), all from Biotest Pharma GmbH, Dreieich, Germany. Their complement inhibitory activity was assessed in vitro by measurement of the blocking of C1q-, C4-, and C3 deposition on solid-phase aggregated rabbit IgG by enzyme-linked immunosorbent assay (ELISA). Complement inhibition in this ELISA was best for IVIgM, followed by Pentaglobin and IVIgG; IVIgA did not exhibit an inhibitory effect. Control experiments with excess concentrations of C1q as well as with C1q-depleted serum showed that the inhibitory effect...
Mononuclear phagocyte system (MPS) Fc-receptor function in 20 patients with seropositive rheumato... more Mononuclear phagocyte system (MPS) Fc-receptor function in 20 patients with seropositive rheumatoid arthritis (RA) was investigated using radiolabelled autologous erythrocytes coated with an average of 5,800 molecules of anti-rhesus IgG (E. IgG). Although clearance times (T1/2) of E. IgG tended to be longer in RA patients than those in healthy controls (46 +/- 6 min vs 38 +/- 5 min, mean +/- s.e.m., P = 0.38), this did not reach statistical significance. Liver spleen uptake ratios (LS ratios) were increased in patients with RA (13/100 +/- 1/100 vs 7/100 +/- 1/100, P less than 0.05). There was no correlation of T1/2 or LS ratios with articular disease activity, vasculitis, ESR, IgM containing immune complex levels or Clq-binding immune complex levels. Although Clq-binding immune complex levels were significantly higher in patients with vasculitis than in those without (P less than 0.01), T1/2 and LS ratios did not differ in these two groups of patients. The T1/2 and LS ratios of E.Ig...
Anti-glomerular basement membrane (anti-GBM) disease often results in end-stage renal failure des... more Anti-glomerular basement membrane (anti-GBM) disease often results in end-stage renal failure despite therapy with plasma exchange and immunosuppressive drugs. The newly discovered streptococcal enzymes IgG-degrading enzyme of S.pyogenes (IdeS) and endoglycosidase S (EndoS) act with remarkable specificity on circulating IgG. In this study, we investigate their ability in vivo to prevent damage mediated by kidney-bound antibodies in a mouse model of anti-GBM disease. Anti-GBM disease was induced in mice by injection of subnephritogenic doses of rabbit anti-mouse GBM, followed a week later by injection of monoclonal mouse anti-rabbit IgG antibodies. By administrating IdeS or EndoS as fusion partners with GST between these antibody injections, we tested their ability to prevent damage by acting on kidney-bound rabbit anti-GBM. Control animals received placebo injections. All animals in the positive control groups developed severe albuminuria immediately after the second antibody injection (mean, 2.51 mg/24 h; range, 0.13-8.20). This was significantly diminished by EndoS (1.3 +/- 1.3 mg/24 h) and completely prevented by IdeS (0.017 +/- 0.014 mg/24 h). Immunofluorescence studies showed that IdeS treatment effectively removed the Fc fragments of the rabbit IgG. This was accompanied by a significant reduction of the deposition of the complement components C3 and C1q, and this diminished the recruitment of leukocytes to the glomeruli. IdeS degrades IgG bound to the GBM in vivo, thereby preventing renal damage in this animal model. Most likely, IdeS would degrade both circulating and kidney-bound anti-GBM in patients with Goodpasture's disease. Whether this would lead to a halt in disease progression and a better prognosis remains to be determined.
Proximal tubular epithelial cells (PTEC) from human renal tissue obtained from biopsy or nephrect... more Proximal tubular epithelial cells (PTEC) from human renal tissue obtained from biopsy or nephrectomy were grown in monoculture and evaluated in vitro at passage 2-4 for interleukin 6 (IL-6) production in response to medium alone or to interleukin 1 alpha (IL-1 alpha), tumour necrosis factor alpha (TNF alpha), interleukin 2 (IL-2), interferon gamma (INF gamma) or lipopolysaccharide (LPS). IL-6 bioactivity was quantitated using the IL-6-dependent murine hybridoma cell line (B9) and expressed as IL-6 units/ml/10(5) PTEC. PTEC cell lines exposed to medium alone produced intermediate amounts of IL-6 with substantial variability between cell lines. Introduction of IL-1 alpha resulted in a dose- and time-dependent increase in IL-6 production by PTEC that was maximal at 1 ng/ml IL-1 alpha at 24 h. All PTEC cell lines showed an increased IL-6 production on exposure to IL-1 alpha varying from 1.3- to 24-fold increase over baseline production. This response was completely blocked by anti-rIL-1 alpha. No significant IL-6 production by PTEC could be induced by TNF alpha, IL-2, IFN gamma, or LPS over a broad dosage range. Cycloheximide inhibited IL-6 production without irreversible cell toxicity, indicating de-novo synthesis. IL-6 produced by PTEC had a molecular weight of 26-29 kDa as demonstrated by Western blot analysis. Using PCR analysis we could demonstrate upregulation by IL-1 alpha of IL-6 mRNA in a dose-response fashion, indicating that IL-1 alpha regulates IL-6 production at a pretranslational value of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity fo... more The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity for C3bBb formation, which is present in human umbilical vein endothelial cells (HUVEC). Therefore, human endothelial culture supernatants (HECS) were analyzed for the presence of human complement factor H by ELISA. It was found that H was present in HECS. An immunoblot analysis of affinity purified H from HECS showed that the size of HUVEC H was identical to that of plasma H. The mean production of H by HUVEC of first passage cultures was 40 ng/10(6) cells/day. The synthesis of HUVEC H was fully inhibitable by the addition of cycloheximide to the cultures, suggesting that H is de novo synthesized. Additional evidence for de novo synthesis was obtained by using biosynthetic labeling with [35S] methionine, immunoprecipitation, and SDS-PAGE. It was demonstrated that, indeed, HUVEC produce and secrete factor H. Two forms of the protein were identified, the 150-kDa form and also a 45-kDa form, both forms have been identified in plasma. The functional activity of HUVEC H is identical to that of plasma H. IFN-gamma induced enhanced synthesis of H by HUVEC, whereas it had no effect on C3 synthesis. Supernatant from stimulated PBMC, T cell growth factor, enhanced synthesis of both H and C3. The present studies indicate that H is produced by HU-VEC and that H may function as an inhibitor of complement activation at the endothelial cell level and, thereby, together with molecules like decay accelerating factor and membrane cofactor protein, may influence resistance of endothelial cells to complement mediated damage.
Human umbilical vein endothelial cells (HUVEC) synthesize and secrete C component factors H and C... more Human umbilical vein endothelial cells (HUVEC) synthesize and secrete C component factors H and C3. Addition of T cell growth factor to HUVEC enhanced factor H production, and caused a profound increase in C3 production. In the present study, investigations were initiated to characterize the effects of purified rIL-1 and rIFN-gamma on the production of factor H and C3 at the protein and mRNA level. IFN-gamma enhanced factor H production in a dose-dependent fashion and a two-fold increase was observed with an optimal dose of 200 U/ml, whereas IFN-gamma had no effect on C3 production. IL-1 inhibited factor H secretion, but the production of C3 was increased 10-fold at an optimal dose of 500 pg/ml of IL-1. Kinetic experiments demonstrated that addition of IL-1 to HUVEC resulted in an induction of C3 production after more than 24 h, whereas IFN-gamma already had a significant effect on factor H production after 8 h of culture. IL-1 in combination with IFN-gamma had a synergistic effect on C3 production. The effects of IL-1 and IFN-gamma on factor H and C3 production by HUVEC could be blocked by using neutralizing amounts of antibodies specific for these cytokines. Northern blot analysis showed that factor H mRNA expression was enhanced in IFN-gamma-treated HUVEC and C3 mRNA was induced in IL-1-treated HUVEC, indicating that the observed increase of factor H and C3 probably is controlled by enhancement of transcription or stability of the transcript.
A family is described in which 3/11 children showed a homozygous deficiency of C3, and both paren... more A family is described in which 3/11 children showed a homozygous deficiency of C3, and both parents and six other children had subnormal levels of C3. The three children with selective C3 deficiency suffered repeatedly from bacterial infections, whereas the parents and the other siblings were clinically healthy. During infectious episodes the patients showed a maculopapular skin rash, and at such times immune complexes were present in the serum. Biopsy specimens of the skin lesions showed the picture of leukocytoclastic vasculitis.
Peritoneal dialysis international: journal of the International Society for Peritoneal Dialysis
During continuous ambulatory peritoneal dialysis (CAPD), activation of complement in the peritone... more During continuous ambulatory peritoneal dialysis (CAPD), activation of complement in the peritoneal cavity may theoretically occur, with inappropriately high or low levels of certain complement factors in dialysate as a consequence. In a group of children on CAPD, it was tested whether levels of a number of complement factors in dialysate were in the range that was predicted on the basis of their molecular weight. Serum and dialysate levels of C1q, C3, C4, C3d, B, D, and P were measured after a night dwell in children on CAPD. Simultaneously, four non-complement proteins (beta 2-microglobulin, albumin, IgG, and alpha 2-macroglobulin) were also measured in dialysate and serum. Assuming a linear relationship between the log base 10 of the dialysate/serum ratio of these non-complement proteins and the log base 10 of their molecular weight, the expected ratios of all complement factors were determined. The differences between actual and predicted ratios were tested using a modified t-test, taking into account the inaccuracy of the estimate. University hospital. A group of 14 children on CAPD, with a median age of 7.8 years (range 2.1 - 13.2). These children had been on CAPD for a median period of 42.4 months (range 0.4 - 89.1). The ratios of factor D (p < 0.001) and C3d (p = 0.035) were elevated, whereas those of C3 (p < 0.001), C4 (p < 0.001), and factor P (p = 0.012) were decreased. Relatively low dialysate/serum ratios of C4, C3, and factor P could be caused by intraperitoneal consumption of complement. High levels of C3d are compatible with this. High dialysate/serum ratios of factor D indicate intraperitoneal production of factor D. These results provide evidence for activation of complement in the peritoneal cavity in children on CAPD. A further reduction of already low levels of complement factors in dialysate as a result of this may impair host defense.
The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the ... more The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the alpha, beta, and gamma chains. Previous studies have shown that human proximal tubular epithelial cells (PTEC) express IL-2R alpha during inflammation, such as in the kidneys after renal transplantation or in crescentic glomerulonephritis. Furthermore, in this study it is shown that PTEC in culture produce more complement C3 after stimulation with IL-2, suggesting the presence of a functional IL-2R on PTEC. In these experiments, the binding of IL-2 to PTEC was analyzed. PTEC stimulated with IL-2 exhibited very low binding of digoxigenin-labeled IL-2; however, stimulation of PTEC with IL-2, in combination with interferon (IFN)-gamma, resulted in increased binding of the digoxigenin-labeled IL-2. In addition, it was found that IL-2, together with IFN-gamma, enhanced the production of C3 by PTEC from baseline levels of 69.6 +/- 3.4 to 198.6 +/- 3.3 ng of C3 per 10(6) cells. The surface expr...
An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is thei... more An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is their ability to block complement activation. The purpose of this study was to compare the complement-inhibitory activity of four IVIG preparations differing in isotype composition. The preparations were: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2) Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L IgA); (3) IVIgM (35 g/L IgM, 12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG), all from Biotest Pharma GmbH, Dreieich, Germany. Their complement inhibitory activity was assessed in vitro by measurement of the blocking of C1q-, C4-, and C3 deposition on solid-phase aggregated rabbit IgG by enzyme-linked immunosorbent assay (ELISA). Complement inhibition in this ELISA was best for IVIgM, followed by Pentaglobin and IVIgG; IVIgA did not exhibit an inhibitory effect. Control experiments with excess concentrations of C1q as well as with C1q-depleted serum showed that the inhibitory effect...
Mononuclear phagocyte system (MPS) Fc-receptor function in 20 patients with seropositive rheumato... more Mononuclear phagocyte system (MPS) Fc-receptor function in 20 patients with seropositive rheumatoid arthritis (RA) was investigated using radiolabelled autologous erythrocytes coated with an average of 5,800 molecules of anti-rhesus IgG (E. IgG). Although clearance times (T1/2) of E. IgG tended to be longer in RA patients than those in healthy controls (46 +/- 6 min vs 38 +/- 5 min, mean +/- s.e.m., P = 0.38), this did not reach statistical significance. Liver spleen uptake ratios (LS ratios) were increased in patients with RA (13/100 +/- 1/100 vs 7/100 +/- 1/100, P less than 0.05). There was no correlation of T1/2 or LS ratios with articular disease activity, vasculitis, ESR, IgM containing immune complex levels or Clq-binding immune complex levels. Although Clq-binding immune complex levels were significantly higher in patients with vasculitis than in those without (P less than 0.01), T1/2 and LS ratios did not differ in these two groups of patients. The T1/2 and LS ratios of E.Ig...
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Papers by Mohamed Daha