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PubMed1996

Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis.

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

Amino Acid Sequence↗
PubMed2005

Coagulation cascade activation causes CC chemokine receptor-2 gene expression and mononuclear cell activation in hemodialysis patients.

Priming of the coagulation cascade during hemodialysis (HD) leads to the release of activated factor X (FXa). The binding of FXa to its specific receptors, effector protease receptor-1 (EPR-1) and protease-activated receptor-2 (PAR-2), may induce the activation of peripheral blood mononuclear cells (PBMC) and promote a chronic inflammatory state that is responsible for several HD-related morbidities. In the attempt to elucidate the mechanisms underlying the coagulation-associated inflammation in HD, 10 HD patients were randomized to be treated subsequently with a cellulose acetate membrane (CA) and Ethylen-vinyl-alcohol (EVAL), a synthetic membrane that has been shown to reduce FXa generation. At the end of each experimental period, surface FXa and thrombin receptors (EPR-1 and PAR-1, -2, and -4) and CCR2 (monocyte chemoattractant protein-1 receptor) gene expression in isolated PBMC were examined. the ability of dialytic membranes to activate protein-tyrosine kinases and the stress-activated kinase JNK and to modulate the generation of terminal complement complex (TCC) was also investigated. EPR-1 and PAR-2 and -4 mRNA expression, barely detectable in normal PBMC, were significantly upregulated in HD patients, particularly in those who were treated with CA. A striking increase of tyrosine-phosphorylated proteins and JNK activation was observed at the end of HD only in CA-treated patients. Simultaneously, an increased gene expression for both splicing isoforms of CCR2, A and B, only in PBMC from CA-treated patients was demonstrated. The increased CCR-2 mRNA abundance was followed by a significant increase in its protein synthesis. The high expression of CCR2 was associated with an increased generation of plasma TCC and a significant drop in leukocyte and monocyte count. By contrast, EVAL treatment slightly lowered TCC generation and normalized leukocyte count. In vitro FXa induced CCR2 A and B expression and JNK activation in freshly isolated PBMC. FXa-induced CCR2 mRNA expression was completely abolished by JNK and tyrosine kinase inhibition. In conclusion, these data suggest that subclinical clotting activation may cause an increased CCR2 gene and protein expression on uremic PBMC, contributing to HD-related chronic microinflammation. The use of the less coagulation-activating membrane, EVAL, may reduce PBMC activation through the modulation of the stress-activated kinase JNK.

Adult↗
PubMed1996

Biological responses differ considerably between endovascular and conventional aortic aneurysm surgery.

OBJECTIVES: To determine the inflammatory responses in endovascular abdominal aortic aneurysm (AAA) repair and their relation to clinical findings. DESIGN: Prospective non-randomised study. SETTING: University Hospital, Department of Surgery. PATIENTS AND METHODS: Seven patients treated with an endoluminal procedure (AAA-E) and seven patients undergoing conventional surgery (AAA-C) were included. Inflammatory parameters were assessed by measurements of the cytokines interleukin (IL)-1 beta, IL-6, IL-8 and Tumour Necrosis Factor-alpha (TNF-alpha); analyses of complement proteins C1q, C4, C3, C5a and Terminal Complement Complexes (TCC); haematologic parameters and determination of C-reactive protein (CRP). RESULTS: In six of seven patients in the AAA-E group blood pressure decreases were recorded during introduction of the device. IL-6 and CRP levels were found to be significantly higher in AAA-C patients compared to the AAA-E group. On the other hand, high TNF-alpha levels were recorded in the AAA-E group. Less consumption of the complement proteins C1q, C4 and C3 was observed in AAA-E compared to AAA-C patients. Increased C5a levels were recorded in the AAA-C group, whereas only slight fluctuations were noticed in the AAA-E group. TCC levels were unchanged in both groups. CONCLUSION: Endovascular aortic aneurysm repair induced a significant inflammatory response, mainly involving TNF-alpha and differing from the findings during open AAA repair. These inflammatory responses were probably related to blood pressure decreases during the procedures. On the other hand, conventional repair induced responses related to the more extensive surgical trauma and reperfusion injury.

Aged↗
PubMed1997

Thrombocytopenia and complement activation under recombinant TNF alpha/IFN gamma therapy in man.

Infusions of recombinant human tumor necrosis-alpha plus recombinant human interferon-gamma (rhTNF alpha/rhIFN gamma) were assessed in two patients with Ewing's sarcoma. We analyzed platelet count, coagulation and the terminal complement complex (TCC). During cycles with continuous rhTNF alpha-infusions we found a rapid, marked decrease of platelet count (minus 90% of initial values) and a simultaneous increase of TCC (plus 84% of initial values) at day 4. At days 5-7 a spontaneous increase of platelet count and decrease of TCC were visible. Short-term infusion led to a milder, continuous decrease of platelet counts and to a moderate, progressive increase of TCC at days 5-7. Bleeding occurred only as petechia and mild hemoglobinuria. There was no effect related to the dosage of rhTNF alpha or rhIFN gamma. Relevant differences were seen only in the variable time courses of rhTNF alpha application. Ex vivo analysis of one patient's platelets showed no cytokine-related effect on induced aggregation according to Born. Additionally, we analyzed in vitro effects of the cytokines on platelet count, platelet aggregation, and the assembly of TCC in platelet membranes. No effects were found after incubation of platelet-rich plasma (PRP) with 1000 pg/ml rhTNF alpha and/or 50 pg/ml rhIFN gamma. Fluid-phase and membrane-bound TCC did not change after incubation of PRP with cytokines. A slightly time-dependent increase of TCC without alteration of platelet count and platelet function did not agree with the assumption of a direct injury to platelets. We assume a cytokine-mediated role of the endothelium in platelet loss.

Adult↗
PubMed1993

Local and systemic activation of the whole complement cascade in human leukocytoclastic cutaneous vasculitis; C3d,g and terminal complement complex as sensitive markers.

We have studied complement activation both in plasma samples and in lesional skin from patients with leukocytoclastic cutaneous vasculitis (LCV). Enzyme immunoassay (EIA) quantification of the complement activation markers, C3d,g and the terminal complement complex (TCC) in plasma, showed that their levels were significantly increased in 66% and 55% of the patients, respectively (n = 29) compared with healthy controls, whereas the standard measurements of C3, factor B, C1q, C4 and C2 were generally within normal range. Elevations of C3d,g and TCC levels in plasma were significantly correlated. Importantly, a significant correlation was found between the severity of the vasculitis and both C3d,g and TCC plasma levels. Immunofluorescence studies of skin biopsy specimens demonstrated simultaneous presence of perivascular dermal deposits of C3d,g and TCC in lesional skin from 96% and 80% respectively of the patients (n = 25). There was a significant correlation between the intensity of the deposits of both markers. Clusterin, a TCC inhibitory protein, was always found at the same sites of perivascular TCC deposits. Immunofluorescence studies at the epidermal basement membrane zone (BMZ) revealed in each case deposits of C3d,g which were accompanied by TCC deposits in 52% of the biopsy specimens. These data demonstrate that there is a local and systemic activation of the whole complement cascade in human LCV. The presence of both C3d,g and clusterin-associated TCC perivascular deposits suggests an intervention of a regulatory mechanism of local complement activation in LCV. Finally, measurement of plasma C3d,g and TCC appears to be a sensitive indicator of systemic complement activation and disease severity in LCV.

Aged↗
PubMed2006

Prognostic role of myocardial tumor necrosis factor-alpha and terminal complement complex expression in patients with dilated cardiomyopathy.

BACKGROUND: In patients with dilated cardiomyopathy (DCM), elevated plasma levels of tumor necrosis factor-alpha (TNF-alpha) are associated with poor prognosis. The terminal complement complex (C5b-9) stimulates myocardial TNF-alpha expression. AIMS: To investigate whether myocardial TNF-alpha and C5b-9 expression correlate with clinical outcome in DCM. METHODS AND RESULTS: 71 patients with DCM underwent myocardial biopsy. Biopsies were analyzed for TNF-alpha, C5b-9, markers of inflammation and for viral genome. Patients were divided into three groups according to biopsy results: group A: no TNF-alpha and no C5b-9; group B: TNF-alpha or C5b-9; and group C: TNF-alpha and C5b-9. NYHA classification, ECG and echocardiography were documented. Patients received conventional treatment of heart failure and, in a few cases, additional treatment with interferon beta(1b) (virus positive) or prednisolone (inflammatory DCM). There were 13 patients (18%) in group A, 19 patients (27%) in group B, and 39 patients (55%) in group C. All groups had a similar and significant improvement in NYHA classification and echocardiographic parameters. TNF-alpha and C5b-9 did not significantly correlate with the presence of viral genome or with markers of inflammation. CONCLUSION: TNF-alpha and C5b-9 are widely distributed in the myocardium of DCM patients. Neither of the antigens correlates with clinical outcome. Myocardial TNF-alpha may not be a useful prognostic marker in DCM.

Adult↗
PubMed1999

The terminal complement complex is generated in chronic leg ulcers in the absence of protectin (CD59).

Loss of membrane complement regulators accompanied by complement activation is suggested to be involved in the pathophysiological processes leading to tissue damage in myocardial ischaemia. In the present study we have investigated whether the same phenomenon may occur in ischaemic and/or venous hypertension leg ulcers. The deposition of complement, plasma complement regulators and expression of membrane regulators were detected by immunohistochemical methods, including immunofluorescence with antibodies against C3d, the terminal complement complex (TCC), vitronectin, clusterin, decay-accelerating factor (CD55) and protectin (CD59). Eleven frozen biopsies from ischaemic leg ulcers, 10 biopsies from venous hypertension leg ulcers, and 10 biopsies from normal skin were studied. In 9 of 11 ischaemic and in 5 of 10 venous hypertension leg ulcers, marked staining for TCC was found around the capillaries, most often at the ulcer margin. No TCC staining was found in normal skin. Staining for TCC was always accompanied by staining for clusterin and vitronectin and C3d. In normal skin, CD59 was found on the elastic fibers in the dermis, on the muscle coat, the Schwann sheath and acinar cells. Semiquantitative measurement of CD59 showed marked increased staining intensity in the endothelium in venous hypertension ulcers and diminished intensity in ischaemic ulcers compared to normal skin. No such difference could be observed for CD55. When TCC was positive in the capillary walls, weak or no staining for CD59 was found. A significantly higher ratio of TCC/CD59 was found in the ischaemic compared to venous ulcers (p = 0.018). This was due to a marked difference between the ulcer margins (p = 0.013). Localized areas in the venous ulcers had the same pattern as that seen in the ischaemic ulcers. Our results suggest that loss of CD59 may enhance deposition of TCC and that complement-dependent inflammation may be an important factor in the tissue-damaging processes seen in chronic leg ulcers.

Adult↗
PubMed2007

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization.

Sublytic C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen-specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b-9 in DC function has not been described. In this report, we use in vitro assembled functional C5b-9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b-9. This was demonstrated by up-regulation of CD83, HLA-antigens and costimulatory molecules, including CD80, D86, B7-H1, B7-H3, B7-H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)-12 and tumor necrosis factor-alpha was increased while the capacity for antigen uptake (FITC-Dextran and Lucifer Yellow) was reduced in C5b-9-treated DC. Mixed lymphocyte reactions indicated that C5b-9-activated DC acted as stimulators that significantly promoted CD4+ T cell activation and elicited production of cytokines, including interferon-gamma and IL-2. Interestingly, C5b-9-treated DC also orient CD4+ CD45RA+ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b-9, suggesting that C5b-9 bridges innate and acquired immunity by inducing DC maturation.

Cell Differentiation↗
PubMed1994

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Amino Acid Sequence↗
PubMed2006

C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1.

Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.

Angioplasty↗
PubMedDate not supplied

Terminal complement complexes in childhood type I membranoproliferative glomerulonephritis.

BACKGROUND: The role of terminal complement complexes (TCCs), which are the final products of complement activation, in the pathogenesis of human glomerulonephritis has not been completely elucidated. To clarify the clinical significance of TCCs in type I membranoproliferative glomerulonephritis (MPGN), we studied TCCs in plasma, renal tissue and urine in pediatric patients with this disease. PATIENTS AND METHODS: We measured the concentrations of TCC in plasma (n=25) and urine (n=13) using enzyme-linked immunosorbent assay. Frozen tissue from 18 renal biopsies were evaluated for the presence of TCC by direct immu-noperoxidase staining. RESULTS: At the early stage of the disease, TCC concentrations in plasma were elevated to above 0.5 arbitrary units (AU)/mL in 14 of 25 patients (high-TCC group), while the remaining 11 patients showed less than 0.5 AU/mL (low-TCC group). In the high-TCC group, TCCs were deposited more diffusely and intensely in the glomerulus, compared with those in the low-TCC group (p=0.034). Furthermore, urinary TCC concentrations in the high-TCC group were higher than those in the low-TCC group (p=0.0001). The high-TCC group showed not only a poorer response to steroid treatment, but also poorer prognosis than the low-TCC group. CONCLUSIONS: These results suggest that, in pediatric patients with type I MPGN, TCCs in circulation may play a particular role in TCC formation in the glomerulus and in urine. The TCC concentration in plasma could be used as a marker of responsiveness to steroid treatment and long-term prognosis.

Adolescent↗
PubMed2003

The heterogeneity of neuropsychiatric systemic lupus erythematosus is reflected in lack of association with cerebrospinal fluid cytokine profiles.

The objective was to study the occurrence of autoantibodies and cytokines in serum and cerebrospinal fluid (CSF) in neuropsychiatric systemic lupus erythematosus (NPSLE). In total, 28 consecutive patients with NPSLE and 16 systemic lupus erythematosus (SLE) patients without neuropsychiatric involvement (non-NPSLE) were studied. IFN-alpha, IL-6, IL-10, soluble terminal complement complex (TCC), anti-ribosomal P protein antibodies (anti-P) and anti-cardiolipin antibodies (aCL) were measured in serum and CSF by immunoassays. Analyses of white blood cell differential count, CSF-albumin/serum-albumin ratio, IgG-index in CSF and isoelectric focusing in serum and CSF were also performed. CSF specimens from 23 healthy individuals were used as controls. IFN-alpha was elevated in the CSF of 5 of 28 NPSLE patients compared to three of 14 among the non-NPSLE patients. IL-6 was elevated in CSF in three of 26 NPSLE patients. Normal concentration of IL-10 was found in CSF in all 27 NPSLE-patients analysed. IFN-alpha in serum was elevated in 18 of 28 NPSLE patients. No distinct clinical phenotype was related to elevated cytokine concentration in serum or CSF. One patient with cerebral involvement complicated by progressive multifocal leukoencephalopathy displayed a very high IFN-alpha concentration in serum. High concentration of TCC was present in CSF from only one patient with systemic vasculitis and focal cerebral symptoms. In conclusion, the results of this study suggest that the diagnostic value of serum and CSF concentrations of IFN-alpha, IL-10, IL-6 and TCC is limited in unselected neuropsychiatric SLE, probably due to the heterogeneity of NPSLE pathogenesis.

Adolescent↗
PubMedDate not supplied

Terminal complement complex in synovial tissue from patients affected by rheumatoid arthritis, osteoarthritis and acute joint trauma.

The C5b-9 complex (Terminal Complement Complex-TCC) is the final product of the terminal complement pathway. In this study, using the monoclonal antibody MCaE11 (specific for a C9 neoantigen) and an immunohistochemical technique, we examined the TCC deposits in synovial tissues from 4 patients affected by rheumatoid arthritis (RA) and 6 patients affected by osteoarthritis (OA). Synovial tissues from 8 patients affected by acute joint trauma were examined as controls. Furthermore, plasma TCC levels were measured in 44 RA patients and 51 controls, using the above mentioned antibody and a sandwich ELISA. Eight synovial fluids were also included in this study. Abundant TCC deposits were detected in the cytoplasm of the synovial lining cells and of large stromal mononuclear cells in all the RA and in 3 out of 6 OA synovial tissues characterized by histological signs of inflammation. No TCC deposits were found in non-inflamed synovial tissues from patients with joint trauma. In agreement with previous observations, the TCC plasma levels found were significantly higher in RA patients than in controls, but no difference was seen between patients with active and non-active disease. The mean TCC level was significantly higher in the synovial fluid than in the plasma, but no correlation emerged between these two series of values. This study shows that: a) the plasma level of TCCs cannot serve as an indicator of disease activity in RA; b) the TCC deposits in synovial tissue correlate well with the extent of inflammatory synovitis, irrespective of whether the synovitis is rheumatoid or osteoarthritic in nature.

Acute Disease↗
PubMed2000

Terminal complement complexes concomitantly stimulate proliferation and rescue of Schwann cells from apoptosis.

The consequences of sublytic terminal complement complex (TCC) assembly on Schwann cell proliferation and apoptosis were examined by using purified complement proteins (C5*-9) or antibody-sensitized Schwann cells in the presence of a serum that was depleted of the seventh component of complement (C7dHS) and reconstituted with purified C7. Stimulation of cultured Schwann cells with antibody plus 10% C7dHS and C7 or C5*-9 induced DNA synthesis over antibody plus 10% C7dHS alone or in Schwann cells in which C5*-9 insertion was inhibited by heat inactivation, respectively. Cell cycle analysis with propidium iodide showed that, at 24 h, viable Schwann cells in defined medium were synchronized in G1/G0 phase. C5*-9 shifted 64% of these cells into S or G2/M phases in a manner similar to beta-neuregulin (beta-NRG), a known Schwann cell mitogen. Furthermore, antibody with 10% C7dHS and C7 or purified C5*-9 induced proliferation of viable Schwann cells. These effects were mediated by signal-transduction pathways involving p44 ERK1 (extracellular-regulated kinase 1), Gi proteins, and protein kinase C. Culturing in defined medium for 24 h resulted in apoptosis of up to 50% of Schwann cells that was prevented by treatment with beta-NRG or TCC. Sublytic C5*-9 significantly inhibited apoptosis 41% by 24 h, as determined by a terminal deoxyuridine triphosphate-biotin nick end labeling assay, and also decreased annexin-V binding at 4 h. Collectively, these data suggest that sublytic TCC, like beta-NRG, is a potent Schwann cell trophic factor that is capable of stimulating mitogenesis and apoptotic rescue. TCC assembly on Schwann cells during inflammatory demyelination of peripheral nerves may promote survival of mature cells to enhance repair and remyelination processes.

Animals↗
PubMed1994

Urinary excretion of cytokines and complement SC5b-9 in idiopathic membranous glomerulonephritis.

Idiopathic membranous glomerulonephritis (iMGN) has previously been shown to be associated with urinary excretion of terminal complement complexes while increased urinary levels of cytokines have been reported in mesangial proliferative glomerulonephritis. In the present cross-sectional study urinary excretion of IL-1 beta, TNF-alpha, IL-6, and soluble C5b-9 (SC5b-9) was examined for 23 patients with iMGN, 16 patients with diabetic nephropathy (DNP), and 17 healthy subjects. IL-1 beta excretion (pg/mg crea) was significantly higher in iMGN patients (375, range 162-11,000) than in DNP patients (39, range 22-59, P < 0.001) or healthy controls (151, range 23-481, P < 0.001). TNF-alpha excretion rate (pg/mg crea) was clearly higher (38, range 21-700) in iMGN patients than in DNP patients (14, range 8-52, P < 0.001) or healthy subjects (11, range 7-26, P < 0.001). Median IL-6 excretion (pg/mg crea) was only marginally higher in iMGN patients (73, range 0-850) than in healthy subjects (64, range 3-158, P = 0.02) but significantly higher than in DNP patients (29, range 17-47, P < 0.001). No significant correlation with corresponding serum values was observed for urinary IL-6 or TNF-alpha excretion. Urinary IL-1 beta and TNF-alpha correlated with decreased renal function. Five of 23 patients showed progression of iMGN over a follow-up of 6 months. The excretion of all cytokines, TNF-alpha in particular, was significantly higher in patients with a progressive disease than in the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

Is platelet-activating factor produced during hemodialysis with AN-69 polyacrylonitrile membrane?

BACKGROUND: Platelet-activating factor (PAF) production during hemodialysis (HD) with cuprophane (CU) membrane has previously been demonstrated, while the results regarding PAF production during HD with AN-69 polyacrylonitrile membrane are dubious. In this study an attempt is made to show that PAF is produced during HD using AN-69 membrane while comparing this production with the corresponding one from HD with CU. Since previous studies have indicated that PAF, like the complement system, could also be implicated in HD-related leukopenia and thrombocytopenia (especially when CU membrane is used), the circulating leukocyte and platelet counts as well as the C3a-desArg and SC5b-9 (soluble, nonlytic form of the terminal complement complex) levels were measured. METHODS: Ten hemodialyzed patients were subjected to HD with CU and AN-69 membranes for 2 consecutive weeks (first week with CU and second with AN-69). During the third HD session of each week and at different times (0, 2, 5, 15, 30, 60, 180 and 240 min), the PAF levels in the blood as well as the leukocyte and platelet counts were measured, while the circulating levels of the C3a-desArg and SC5b-9 were measured at 0, 5, 15, 60 and 240 min. PAF was detected by ethanol extraction, followed by purification by column chromatography and high-pressure liquid chromatography and finally quantified by bioassay. The C3a-desArg and SC5b-9 fractions of the complement were measured by immunoassay while an autoanalyzer gave the leukocyte and platelet counts. RESULTS: Circulating PAF levels were detected at all time intervals during HD with AN-69 (PAF(AN-69)) and CU (PAF(CU)) membranes. At all time intervals PAF(AN-69) < PAF(CU), however, statistically significant differences (s) between the two membranes existed only at 15, 30, 60, 180 and 240 min. The highest PAF(AN-69) and PAF(CU) occurred at 5 and 15 min into dialysis, respectively. The same observations were made for circulating C3a-desArg levels (s existed additionally at 5 min as well). The reduction of the circulating leukocytes had almost a mirror image with the C3a-desArg as well as PAF levels while the maximal reduction of platelets was observed after 2 min into dialysis with both membranes (i.e., simultaneously with the first increase in PAF secretion). CONCLUSIONS: PAF is indeed produced during HD with AN-69 membrane, as it is during HD with CU. At all time intervals during the HD procedure, PAF(AN-69) < PAF(CU). PAF seems to contribute to HD-related leukopenia and thrombocytopenia with both membranes.

Acrylic Resins↗
PubMed1997

Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent.

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.

B-Lymphocytes↗
PubMed1995

Urinary C5b-9 excretion and clinical course in idiopathic human membranous nephropathy.

Recent reports suggested that the presence of terminal complement complex (C5b-9) in urine from patients with idiopathic membranous nephropathy (IMN) may indicate on-going immunological damage. This report documents the relationship between C5b-9 excretion and clinical outcome in 35 adult patients with biopsy-proven IMN and progressively declining renal function. There were two groups of patients. Group I received one of three treatment regimens: prednisolone alone, prednisolone and chlorambucil, or prednisolone and cyclophosphamide (N = 22). Group II received no immunosuppressive therapy (N = 17). Three of the 18 patients receiving immunosuppressive drugs had more than one treatment regimen as they experienced a clinical relapse during the study period; hence 22 treatments were available for analysis. Urine samples were collected regularly and urinary C5b-9 (uC5b-9) was determined by ELISA. Both groups were similar with respect to age, sex distribution, and the duration of follow-up. An improvement in proteinuria and creatinine clearance was noted in the immunosuppressed group. Thirty-five patients were excreting C5b-9 initially (18 from group I and 17 from group II); 17 patients continued to excrete C5b-9 at the end of the observation period. These 17 patients had a significantly worse clinical outcome when compared to the 18 patients whose C5b-9 excretion became negative, either spontaneously or with treatment (P < 0.005). These results indicate that continuing C5b-9 excretion is correlated with a poor clinical outcome. They also suggest that uC5b-9 is a dynamic marker of ongoing immunological injury, and therefore may be useful in the initial assessment and monitoring of patients with IMN and in identifying patients who may derive benefit from immunosuppressive therapy.

Biopsy↗