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PubMed2003

Hypodermin A, a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection.

BACKGROUND: Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations. METHODS: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA). RESULTS: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 +/- 16 U/ml vs. 700 +/- 63 U/ml, P < 0.05). Sera from rats injected with 3.2 mg/kg of HA were less effective in lysing guinea-pig endothelial cells (12 +/- 7%) than normal rat sera (79 +/- 3%; P < 0.001). Ex vivo, guinea-pig hearts perfused by rat serum supplemented with HA survived longer than those perfused by non-treated serum (210 +/- 34 and 154 +/- 71 min, respectively; P < 0.05). In vivo, guinea-pig hearts transplanted into HA treated rats survived longer than in non-treated rats (27 +/- 5 min vs. 13 +/- 4 min; P < 0.001). In the presence of human serum, smaller amounts of C6 and C5b-9 were deposited onto HA-transfected CHO cells than onto control cells. The mHA-PAEC11 cells were significantly more resistant to lysis by human C than control PAEC11 cells. CONCLUSIONS: These data suggest that transgenic HA could be used to prevent hyperacute xenogeneic rejection.

Acute Disease↗
PubMed1994

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) on human breast cancer cells.

BACKGROUND: Normal human cells resist the lytic activity of homologous complement (C) by expressing inhibitory molecules on their cell membranes. Recently, it has become increasingly evident that information on C inhibitors on malignant tumor cells is crucial before considering any immunotherapeutic attempts with C-activating antibodies. As one of the most potent inhibitors of C lysis is protectin (CD59), we have examined its expression and function on human breast cancer cells. EXPERIMENTAL DESIGN: Immunofluorescence microscopy was used to detect protectin expression on solid breast tumor samples (N = 12). Using immunoaffinity chromatography, protectin was isolated from the membranes of cultured MCF7 and T47D breast cancer cells. The purified proteins were incorporated into heterologous cells to study their C inhibitory activities. The reactivity of tumor cell protectins with terminal C complexes was examined by sucrose density ultracentrifugation analysis. A chromium release assay was used to study the effects of protectin neutralization on the sensitivity of MCF7 and T47D cells to C-mediated cytotoxicity. RESULTS: Protectin was found to be strongly expressed by all human breast cancer tumors examined. The affinity-purified protectins had a glycophosphoinositollipid anchor and migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as glycosylated smears of 19 to 25 kilodaltons. Protectin isolated from T47D cells bound to nascent C5b-9 complexes generated in human sera and inhibited C lysis of guinea pig erythrocytes when incorporated into their cell membranes. C-mediated killing of breast cancer cells could be significantly enhanced after treatment of the cells with F(ab')2 fragments of the anti-protectin monoclonal antibody YTH53.1. CONCLUSIONS: Human breast cancer cells resist C membrane attack by expressing protectin on their cell membranes. Neutralization of protectin on the surface of the tumor cells increases their sensitivity to C lysis.

Adenocarcinoma↗
PubMed1981

Assembly of the functional membrane attack complex of human complement: formation of disulfide-linked C9 dimers.

The 158,000 Mr protein, previously designated C5c, present in fully assembled complement (C) membrane attack complexes (MC5b-9) has been identified as a disulfide-bonded dimer of C9. This conclusion was based on the observations that: (i) a portion of the 125I-radiolabeled precursor C9 incorporated into MC5b-9 complexes comigrated with the 158,000 Mr protein band in NaDodSO4/polyacrylamide slab gels; (ii) monospecific antisera produced against native C9 and the 158,000 Mr protein immunologically crossreacted with monomeric native C9 by double immunodiffusion and with monomeric C9 and the 158,000 Mr protein on immunoreplication procedures; and (iii) two-dimensional NaDodSO4/polyacrylamide slab gel electrophoresis, in which the second dimension was conducted under reducing conditions, revealed that the 158,000 Mr protein contained two identical 71,000 Mr subunits which comigrated with monomeric C9. Molar ratio estimates indicated that 1 mol of C5b, C9 dimer, C6, C7, and C8 and 3-4 mol of C9 monomer were present per MC5b-9 complex. Each fully assembled membrane-bound MC5b-9 complex would therefore have a calculated Mr of 982,000. The presence of C9 dimers in the hemolytically active 29S dimeric form of the MC5b-9 complex and the absence of C9 dimers in the hemolytically inactive 23S monomeric form of the fluid phase SC5b-9 complex strongly suggest an important role for C9 dimer formation in MC5b-9 complex structure and function. The most probable function of C9 dimers would be the formation of intercomplex disulfide crosslinks which would provide a mechanism to stabilize the assembly of MC5b-9 into aggregates of increasing size on the target membrane surface which would thus be responsible for the observed pore size heterogeneity of functional C lesions.

Animals↗
PubMed1999

Cytokine and S100B levels in paediatric patients undergoing corrective cardiac surgery with or without total circulatory arrest.

OBJECTIVES: Neurological damage following cardiopulmonary bypass (CPB) is difficult to objectively evaluate in infants. In adults, serum elevations of astroglial S100B correlate with proven brain injury independent of operative temperature. The deleterious effects of inflammatory cytokines, generated during CPB, on the brain have not been studied in infants using S100B as a marker for cerebral injury. METHODS: Twelve neonates, weighing 3.3 +/- 0.2 kg (total circulatory arrest group (TCA)) and 12 infants weighing 7.0 +/- 1.0 kg (cardiopulmonary bypass group (CPB)) underwent corrective cardiac surgery for various pathologies. Serial blood samples on induction, at the end of CPB, 30 min, 2 h and 24 h after the administration of protamine, were taken. The resultant plasma was frozen to -80 degrees C and stored for batch analysis. Cytokines were measured using ELISAs and S100B using a luminometric assay. RESULTS: The TCA group were younger and experienced a longer perfusion time than the CPB group (137 +/- 8 vs. 113 +/- 7, P = 0.04). The mean TCA time was 23 +/- 4 min. The TCA group had significantly higher levels of IL-6 (P = 0.001), IL-8 (P = 0.005) and S100B (P = 0.002) at 24 h. C5b-9 levels were significantly lower in the TCA group: end of CPB (P = 0.001), 30 min (P < 0.001), 2 h (P = 0.002). There was a weak, but significant correlation between IL-6 levels at the end of CPB and S100B levels 2 h later (r = 0.55, P = 0.03). Long extubation times were associated with high 24-h S100B levels (r = 0.52, P = 0.01). CONCLUSIONS: (1) The TCA group have prolonged rises of IL-6, IL-8 and S100B. (2) The TCA group generates significantly lower complement. (3) Astroglial injury, seen after surgery, may, in part, be cytokine mediated.

Antibodies, Monoclonal↗
PubMed1980

Differences in cell-bound C8 sites on chicken erythrocytes measured by their reactivity with guinea pig and human C9.

Methods for preparing chicken erythrocytes (CE) with C7 bound to their surface were devised by using either the classical pathway (CEA1-7) or an activated 56hu reagent (CE567) derived from inulin-treated human serum. These intermediates were used to study the lysis of CE by functionally purified C8 and C9 isolated from guinea pig and human serum. The results indicated that GPC9 was less efficient in lysing CEA1-8 or CE5678 than HuC9. This finding was observed irrespective of the species of C8 used. Experiments designed to analyze this difference indicated that there were two functionally distinct forms of C5b-8 that were randomly distributed among the cells but differed in their ability to generate a C lesion depending on the species of C9 used to complete the reaction. The implications of these results on the mechanism of generation of C lesions are briefly discussed.

Animals↗
PubMed1997

C5b-7 and C5b-8 precursors of the membrane attack complex (C5b-9) are effective killers of E. coli J5 during serum incubation.

The finding that C9-deficient sera (C9D) can kill serum sensitive strains of Gram-negative bacteria by us and other investigators, questions the role of C9 in the membrane attack complex as necessary for cell death. In these studies we have demonstrated that C5b-8 complexes generated on E. coli J5 during incubation in C9-depleted and C9-neutralized sera are effective in killing Gram-negative bacteria. In the same study, we extended our investigations to show that the deposition of C5b-7 complexes (from C8-deficient [C8D], C8 depleted and C8-neutralized sera) is also effective in killing Gram-negative bacteria. In all cases, these studies demonstrated that when E. coli J5 was incubated with C8D, C9D and pooled normal human serum [PNHS], deposited C5b-9 complexes from PNHS produced more killing than C5b-7 or C5b-8 complexes alone. These experiments clearly demonstrated that C5b-7 and C5b-8 complexes are bactericidal and that multimeric C9 within C5b-9 is not an absolute requirement for inner membrane damage and cell death of Gram-negative bacteria.

Blood Bactericidal Activity↗
PubMed1993

Isolation of erythrocyte membrane inhibitor of reactive lysis type II. Identification as glycophorin A.

When incubated with activated CoF and normal human serum (as the C source), a portion of the E from most patients with PNH are hemolyzed. In contrast, normal E are completely resistant to this form of C-mediated cytolysis called reactive lysis. This observation implies that normal E express membrane proteins that inhibit reactive lysis. In previous studies, we have shown that when E proteins are subjected to anion exchange chromatography, two peaks of inhibitory activity are observed. We isolated the inhibitor from the first peak and identified it as an 18-kDa protein that we call MIRL (CD59). The purpose of the studies presented herein was to isolate the inhibitory factor (MIRL type II) from the second peak. After anion exchange and gel filtration chromatography, aliquots of the fractions containing MIRL II activity were subjected to preparative SDS-PAGE (nonreducing conditions), and protein was eluted from gel slices electrophoretically. Inhibitory activity was found primarily in two adjacent slices, corresponding to an M(r) range of 84-51 kDa. When MIRL II was analyzed by SDS-PAGE and silver staining, two prominent bands representing proteins with M(r) of 77 and 39 kDa were observed under both reducing and nonreducing conditions. This behavior in SDS-PAGE is characteristic of GP-A, which migrates primarily as a nondisulfide-linked homodimer in equilibrium with a monomeric form. Immunoblotting studies confirmed that MIRL II is GP-A. The protein caused a concentration-dependent inhibition of reactive lysis, with approximately 100 ng producing 50% inhibition. GP-A inhibited reactive lysis by blocking the formation or binding of C5b-7, and this inhibitory activity was immunoprecipitated by monoclonal anti-GP-A. Furthermore, GP-A that was isolated by affinity chromatography also inhibited reactive lysis. These studies demonstrate that the major E sialoglycoprotein functions as an inhibitor of the membrane attack complex of C.

Animals↗
PubMed2007

Myopathy associated with anti-signal recognition peptide antibodies: clinical heterogeneity contrasts with stereotyped histopathology.

We report three patients with anti-signal recognition particle antibodies who had different presenting clinical pictures, mimicking acute polymyositis, limb-girdle muscular dystrophy, and acute rhabdomyolysis. Muscle biopsies typically showed necrotizing myopathy with little or no inflammation and deposits of membrane attack complex (C5b-9) in endomysial capillaries. The clinical course was severe in two patients and mild in one. The combination of corticosteroid with either an immunosuppressive agent or intravenous immunoglobulins was required to improve the condition of these patients.

Adrenal Cortex Hormones↗
PubMed2004

Parvoviral infection of endothelial cells and stromal fibroblasts: a possible pathogenetic role in scleroderma.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disease (CTD) which differs from other CTDs by progressive irreversible fibrosis in lung, kidney, skin, and heart. It has a worse prognosis compared to several other CTDs. The pathogenesis may reflect a humorally mediated microangiopathy in concert with the overproduction of collagen triggered by immune-mediated cytokine production. Having previously demonstrated parvovirus B19 (B19) DNA in bone marrow and skin biopsies of SSc patients in the absence of B19 viremia, we sought to further elucidate a role for B19 in the pathogenesis of SSc. DESIGN: Twelve patients who fulfilled American College of Rheumatology criteria for a diagnosis of SSc were encountered. Ten were serologically screened for B19 infection. Solution phase polymerase chain reaction (PCR) for B19 DNA was performed on skin tissue from six patients, and in all biopsies, reverse transcriptase in situ PCR (RT in situ PCR) for B19 and tumor necrosis factor (TNF)-alpha mRNA was performed. B19 viral protein (VP2) expression was sought by immunohistochemistry and correlated to PCR findings and to light microscopy of hematoxylin and eosin-stained sections. Frozen tissue was also available on five of the patients. Two control groups were assessed for B19 and TNF expression comprising one with irrelevant primers and the other representing 18 cases of inflammatory skin lesions where the etiology was known and unrelated to B19 infection. In addition, frozen and paraffin-embedded tissues procured from skin lesions unrelated to B19 infection were assessed for B19 genome. In all cases, pretreatment with RNase was also performed to verify that any positive signal was indeed RNA based. RESULTS: Diffuse SSc was seen in seven patients, and limited disease in five. All patients had an antinuclear antibody--specifically, an antinucleolar, anticentromere, and/or anti-Scl 70 antibody. Eleven of the 12 had lung involvement, whereas eight patients had myocardial disease. Of 12 patients tested serologically, nine had B19-specific antibodies, which included immunoglobulin M (IgM)-specific antibodies in two cases. Solution phase PCR showed B19 DNA in the skin in three cases and in the bone marrow in three cases, including two in whom skin-based B19 DNA was observed. In all cases, RT in situ PCR demonstrated B19 and TNF-alpha mRNA in endothelia, fibroblasts, mast cells, and perivascular inflammatory cells. Immunohistochemistry to assess VP2 was either negative or equivocal. Immunofluorescent studies revealed prominent deposition of C5b-9 within the cutaneous vasculature from biopsies of all patients tested. The control samples were negative for B19 and TNF RNA and DNA. CONCLUSIONS: Parasitism of endothelia and fibroblasts by B19 with resultant enhanced TNF-alpha expression may be of pathogenetic importance in SSc even in the absence of demonstrable viremia. The vascular deposition of C5b-9 suggests a role for humoral immunity possibly induced by a state of endothelial neoantigenicity evoked by virally mediated cell injury. Treatment strategies include anti-viral therapy, including in the context of intravenous gamma-globulin and anti-TNF therapy.

Adult↗
PubMed2006

Deficiency of functional mannose-binding lectin is not associated with infections in patients with systemic lupus erythematosus.

Infection imposes a serious burden on patients with systemic lupus erythematosus (SLE). The increased infection rate in SLE patients has been attributed in part to defects of immune defence. Recently, the lectin pathway of complement activation has also been suggested to play a role in the occurrence of infections in SLE. In previous studies, SLE patients homozygous for mannose-binding lectin (MBL) variant alleles were at an increased risk of acquiring serious infections in comparison with patients who were heterozygous or homozygous for the normal allele. This association suggests a correlation between functional MBL level and occurrence of infections in SLE patients. We therefore investigated the biological activity of MBL and its relationship with the occurrence of infections in patients with SLE. Demographic and clinical data were collected in 103 patients with SLE. Functional MBL serum levels and MBL-induced C4 deposition were measured by enzyme-linked immunosorbent assay using mannan as coat and an MBL- or C4b-specific monoclonal antibody. The complete MBL-dependent pathway activity was determined by using an assay that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected with a specific monoclonal antibody against C5b-9. Charts were systematically reviewed to obtain information on documented infections since diagnosis of SLE. Major infections were defined as infections requiring hospital admission and intravenous administration of antibiotics. In total, 115 infections since diagnosis of lupus, including 42 major infections, were documented in the 103 SLE patients (mean age 41 +/- 13 years, mean disease duration 7 +/- 4 years). The percentage of SLE patients with severe MBL deficiency was similar to that in 100 healthy controls: 13% versus 14%, respectively. Although deposition of C4 to mannan and MBL pathway activity were reduced in 21% and 43% of 103 SLE patients, respectively, neither functional MBL serum levels nor MBL pathway activity was associated with infections or major infections in regression analyses. In conclusion, SLE patients frequently suffer from infections, but deficiency of functional MBL does not confer additional risk.

Adult↗
PubMed1993

Visceral glomerular epithelial cells can proliferate in vivo and synthesize platelet-derived growth factor B-chain.

In glomerular diseases associated with antibody- and complement-mediated injury to endothelial and mesangial cells, cell proliferation is an important early response that precedes matrix accumulation and glomerulosclerosis. In contrast, in diseases in which the visceral glomerular epithelial cell (vGEC) is the principal target of injury, cell proliferation is not a recognized consequence, although vGECs respond in vitro to a variety of growth factors and inflammatory mediators. To explore the possibility that low levels of vGEC proliferation may occur and participate in such diseases, serial studies were done in the passive Heymann nephritis model of membranous nephropathy, in which the vGEC is the primary target of antibody- and C5b-9-mediated injury. The results showed mitotic figures and positive staining for the proliferating cell nuclear antigen in cells whose location defined them as vGECs. The proliferating cell nuclear antigen-positive cells at the edge of the capillary wall were confirmed to be vGECs by double-immunostaining with antibodies to SPARC/osteonectin, which preferentially label vGECs in passive Heymann nephritis. Proliferation of vGECs in vivo was preceded by increased glomerular expression of platelet-derived growth factor (PDGF) B-chain protein and messenger RNA, both of which localized to vGECs. PDGF B-chain protein and messenger RNA were also detected in cultured vGECs. PDGF receptor beta-subunit protein or messenger RNA could not be demonstrated in vGECs in vivo or in vitro, and no growth response of cultured vGECs to PDGF was noted. These results suggest that proliferation of vGECs does occur in a model of glomerular injury induced by antibody and C5b-9 on vGECs. VGEC proliferation and production of PDGF may be involved in the restoration of the capillary wall but could also contribute to local capillary wall injury and proliferation of other cells in Bowman's capsule, interstitium, and tubules.

Animals↗
PubMed1997

"Low-dose" aprotinin modifies hemostasis but not proinflammatory cytokine release.

BACKGROUND: Cytokines are implicated in the pathogenesis of the "whole-body inflammatory response" that may complicate the period after cardiopulmonary bypass (CPB). Low-Dose aprotinin in the pump during CPB has been shown to improve postoperative hemostasis and platelet preservation. We tested the hypothesis that low-dose aprotinin influences the inflammatory reaction (in terms of cytokine release) after CPB. METHODS: In a prospective, randomized study, 36 patients undergoing elective coronary artery bypass grafting were investigated. Nineteen patients received low-dose aprotinin (2 x 10(6) KIU (280 mg] in the pump), and a control group of 19 did not. Complement activation, cytokine production, leukocyte elastase release. D-dimer level, full blood count, postoperative blood loss, and transfusion requirements were analyzed before, during, and after after CPB. RESULTS: Interleukin-1 beta was not detected in either group, whereas traces of tumor necrosis factor-alpha were infrequently observed. Plasma elastase, interleukin-6, interleukin-8, and neutrophil count increased (p < 0.001) during and after CPB compared with the baseline levels, reaching a peak at 2 hours after protamine administration in both groups before returning toward baseline at 24 hours. Proinflammatory cytokine markers did not differ significantly (p > 0.1) between the groups throughout the study period. The C5b-9 level increased (p < 0.001) in both groups perioperatively, reaching its peak 15 minutes after protamine. Twenty-four-hour postoperative blood loss was significantly (p < 0.001) reduced in the aprotinin group in association with markedly reduced D-dimer levels (p < 0.001). Patients in the aprotinin group also received significantly less banked blood postoperatively than the control group (p < 0.01). CONCLUSIONS: Low-dose aprotinin fails to modify proinflammatory cytokine release, yet confers hemostatic improvement through reduced fibrinolysis in patients undergoing routine coronary artery bypass grafting.

Aged↗
PubMed2005

Phosphodiesterase type 4 inhibitor rolipram inhibits activation of monocytes during extracorporeal circulation.

OBJECTIVE: Cardiopulmonary bypass is associated with systemic inflammatory response syndrome and risk of multiorgan injury mediated by activated leukocytes. Phosphodiesterase type 4 is the predominant phosphodiesterase isozyme in leukocytes and plays a key role in the regulation of leukocyte activation. The aim of this study was to examine the effect of rolipram, a selective phosphodiesterase type 4 inhibitor, on functional changes of monocytes during simulated extracorporeal circulation. METHODS AND RESULTS: Simulated extracorporeal circulation was established by recirculating heparinized human blood for 120 minutes on a membrane oxygenator with or without 10 micromol/L of rolipram. L-selectin and CD11b expression of monocytes were measured with flow cytometry. C4d fragment, Bb fragment, C5b-9, and interleukin-6 were measured with enzyme immunoassay. Rolipram reduced the increase in CD11b expression and the decrease in L-selectin expression of monocytes in response to simulated extracorporeal circulation. Rolipram inhibited the increase in C4d fragment and interleukin-6, but it did not affect the increase in Bb fragment or C5b-9. CONCLUSION: Rolipram inhibited changes in adhesion molecule expression and interleukin-6 release by activated monocytes in simulated extracorporeal circulation. This study suggests that phosphodiesterase type 4 inhibition could be feasible therapeutic strategy to prevent exaggerated inflammatory response and organ injury in patients undergoing cardiopulmonary bypass.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Immunohistochemical localization of cytokines, C5b-9 and ICAM-1 in peripheral nerve of Guillain-Barré syndrome.

The spectrum of the Guillain-Barré Syndrome (GBS) has recently been widened by the newly identified forms of acute motor axonal neuropathy (AMAN) and acute motor sensory axonal neuropathy (AMSAN). An important question has been raised regarding the possibility for the axon to be a target in immune-mediated damage. Although myelin breakdown is the characteristic feature of classic acute inflammatory demyelinating polyradiculoneuropathy (AIDP), axonal degeneration may occasionally be observed in this form, especially in cases with explosive onset and severe clinical course. Immunohistochemical findings of five frozen sural nerves biopsies of patients with GBS (AIDP variant) tested with a panel of monoclonal antibodies raised against different molecules implicated in immune-mediated processes have principally disclosed an immunoreactivity of tumor necrosis factor-alpha (TNF-alpha) on both Schwann cell membranes and in myelinated and unmyelinated axons. On the other hand, interleukin 1-beta (IL1-beta) labeled Schwann cells, endothelial cells and macrophages; interferon-gamma (IFN-gamma) was observed both in endothelial cells and lymphocytes. Membrane attack complex (C5b-9) deposits were observed on Schwann cell membranes and finally intercellular adhesion molecule-1 (ICAM-1) was localized both on endothelial cells and macrophages. Our findings strongly suggest that TNF-alpha is an important factor in the cascade of events leading to immune-mediated demyelination and axonal damage in GBS.

Adult↗
PubMed1982

Cutaneous localization of the membrane attack complex in discoid and systemic lupus erythematosus.

Biopsy specimens of skin lesions from three patients with discoid lupus erythematosus and six patients with systemic lupus erythematosus contained the membrane attack complex, which comprises C5b through C9, as well as immune complexes at the dermal-epidermal junction. The basilar epithelium in these areas was vacuolated and edematous, and the dermis contained an inflammatory infiltrate. In contrast, 19 of 29 specimens of normal-appearing skin from patients with discoid or systemic lupus erythematosus showed only immune complexes at the dermal-epidermal junction, without the membrane attack complex. The other 10 specimens, all from patients without cutaneous involvement, showed neither immune complexes nor membrane attack complexes. These data suggest that immune complexes within skin lesions selectively generate the assembly of the membrane attack complex, which mediates membrane injury. A synergistic interaction of immune complexes and cofactors may be required to activate complement in areas of skin that are predisposed to tissue injury.

Adult↗
PubMed1991

Quantitative analysis of adenine nucleotides during the prelytic phase of cell death mediated by C5b-9.

The nucleated cell death mediated by C5b-9 depends on the extent of C fixation and parameters that affect the ability of the cell to eliminate C5b-9. When C5b-9 formation exceeds elimination, cell death can be initiated. High Ca2+ in the medium accelerates Ehrlich ascites cell death induced by a large number of C5b-9, whereas osmotic prevention of cell swelling has little effect in protecting Ehrlich cells from killing by C5b-9. In the present study, we investigated the interrelationship between intracellular Ca2+, intra- and extracellular adenine nucleotides, and mitochondrial membrane potential, to understand the mechanism of acute cell death induced by C5b-9. When Ehrlich cells carrying C5b-8 were exposed to C9, rapid and profound ATP depletion in the cell was observed before cell death. Leakage of the adenine nucleotides ATP, ADP, and AMP also began during the prelytic phase. Studies using digital imaging fluorescence microscopy showed that loss of mitochondrial membrane potential was noted immediately after C9 addition but before nuclear staining with propidium iodide. These findings suggest that an increase in intracellular Ca2+ through C5b-9 channels and loss of mitochondrial membrane potential may initiate rapid cell death. The prelytic leakage of ATP precursors may also contribute to cell death by decreasing nucleotide pools, because recovery of ATP production was observed after a similar degree of ATP loss in cells exposed to sublethal doses of KCN, in which ADP and AMP leakage was not present.

Adenine Nucleotides↗
PubMed1989

Isolation of homologous restriction factor from human urine. Immunochemical properties and biologic activity.

A soluble form of homologous restriction factor (HRF-U) was isolated from normal human urine. With respect to m.w. (65,000) and immunoblotting characteristics, it resembled membrane HRF (HRF-M) that had been isolated from human E membranes. The protein exhibited limited cross-reactivity with the channel-forming proteins of C and cytotoxic lymphocytes. It inhibited reactive lysis of E by human C5b-9. Inhibition occurred at the attachment stage of C5b-7 to target cells, rather than at the C8 or C9 stage of membrane attack complex assembly which is inhibited by HRF-M. In this respect, HRF-U acts analogously to S protein of serum, but no immunochemical relationship between these two proteins was detected. HRF-U might be derived from the soluble HRF detected in cytoplasmic granules of killer lymphocytes.

Animals↗
PubMed2003

Use of C4d as a diagnostic adjunct in lung allograft biopsies.

PURPOSE: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4d's role as a prognostic adjunct in lung allografts. MATERIAL AND METHODS: Twenty-three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. RESULTS: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p <.0001) compared to other the immunoreactants C1q, C5b-9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon-BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. CONCLUSIONS: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.

Adult↗