Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease

Activated components of the complement system are potent mediators of inflammation that may play an important role in numerous disease states. For example, they have been implicated in the pathogenesis of inflammatory joint diseases including rheumatoid arthritis (RA). To target complement activation in immune-mediated joint inflammation, we have utilized monoclonal antibodies (mAbs) that inhibit the complement cascade at C5, blocking the generation of the major chemotactic and proinflammatory factors C5a and C5b-9. In this study, we demonstrate the efficacy of a mAb specific for murine C5 in the treatment of collagen-induced arthritis, an animal model for RA. We show that systemic administration of the anti-C5 mAb effectively inhibits terminal complement activation in vivo and prevents the onset of arthritis in immunized animals. Most important, anti-C5 mAb treatment is also highly effective in ameliorating established disease. These results demonstrate a critical role for activated terminal complement components not only in the induction but also in the progression of collagen-induced arthritis and suggest that C5 may be an attractive therapeutic target in RA.     

Surveillance study for clinical stage I testicular seminomas and nonseminomatous germ cell tumors

Intravenous immunoglobulin (IVIG) (Octagam), was used to determine the effect on hyperacute rejection in an ex vivo xenograft model. Six pig kidneys were perfused with IVIG and fresh human AB blood, and six control pig kidneys were simultaneously perfused with albumin and blood from the same donation. The survival of the IVIG-perfused xenografts (median, 6.5 h) was significantly (P = 0.03) longer than the albumin-perfused xenografts (median, 3.5 h). Complement was activated in both groups. The administration of IVIG to the perfused blood resulted in immediate and significantly higher complement activation in the fluid phase as compared with the albumin group. At rejection the fluid phase complement activation was higher in the IVIG group than in the albumin group for C1rs/C1inh complexes, C4bc, Bb and TCC. At the time of rejection both the albumin and the IVIG group demonstrated interstitial tubular haemorrhage, vasculitis or necrosis of glomerular capillaries and glomerular microthrombi. IgM, C1q, C3c, C4 and fibrin were located in arteries and glomeruli and IgG in the interstitium in both groups at rejection. The fluid phase findings are consistent with a modulatory effect of IVIG on complement activation by deviating the classical pathway activation towards the fluid phase. The prolonged survival of the IVIG-perfused kidneys suggests that IVIG may be useful to dampen hyperacute rejection.     

Reduced complement and granulocyte activation with heparin-coated cardiopulmonary bypass

Plasma concentrations of the complement activation products C3b, iC3b, and C3c; the terminal C5b-9 complement complex; and the granulocyte proteins calprotectin, myeloperoxidase, and lactoferrin were assessed in two groups of patients undergoing aortocoronary bypass procedures. In 10 patients operated on, the bypass circuits were coated by the Carmeda Bio-Active Surface and systemic heparinization was reduced to 1.5 mg/kg; in another 10, the systems were uncoated and the dosage of systemic heparinization was 4 mg/kg. In both groups, significant complement activation was observed after the onset of cardiopulmonary bypass, but the maximum levels of C3b, iC3b, and C3c and the terminal C5b-9 complement complex were significantly lower in the heparin-coated group. In both groups, a significant increase in calprotectin, myeloperoxidase, and lactoferrin release was observed by the end of operation. The maximum myeloperoxidase levels were significantly lower in the heparin-coated group than those in the uncoated group (p = 0.03). There was a correlation of borderline significance between the formation of terminal C5b-9 complement complex and lactoferrin release, as well as between the formation of terminal C5b-9 complement complex and myeloperoxidase release (p = 0.05). The postoperative blood loss did not differ significantly between the two groups. We conclude that coating by end point-attached and functionally active heparin allows a significant reduction in the amount of systemic heparinization, and significantly reduces complement and granulocyte activation.     

The use of a stereological method to estimate the volume of Schistosoma mansoni granulomas: the effect of zinc deficiency

Kidney xenotransplantation is not yet a realistic clinical treatment modality. However, during the last decades more than 30 kidneys from other species have been transplanted into humans; some of the kidneys sustained some function up to 60 days. Recent progress in genetic engineering has raised the possibility to create large transgenic animals which express human complement regulatory proteins (CRP). Since early complement activation is believed to be the main triggering event for xenograft destruction, complement regulation by species-specific CRP should avoid hyperacute rejection in transspecies transplantation. The perfusion of hDAF-transgenic pig kidneys with human blood was not associated with the morphological signs of hyperacute rejection when compared to non-transgenic control organs. Specific immunohistology could demonstrate that the transgene was sufficient to regulate complement activation beyond C3 despite the endothelial deposition of xenoantibodies. In the future, these organs could be further optimized and ultimately tested in a clinical pilot protocol under appropriate immunosuppression.     

Genetic engineering as an approach to xenotransplantation

A discordant organ xenograft, such as a heart or kidney transplanted from pig to human, is rejected within 1 to 2 hours by a process called hyperacute rejection (HAR) caused in large or full measure by preexisting host xenoreactive natural antibodies (XNAs) and complement. HAR can be averted by blocking either complement or XNAs; but the xenograft is then rejected at 3 to 4 days by a process called delayed xenograft rejection (DXR), which can occur without the presence of T cells. DXR involves type II endothelial cell (EC) activation (the phase of activation that includes up-regulation of proinflammatory genes in the EC), infiltration into the graft of host monocytes and natural killer cells (both of which appear to be activated), and a complex cytokine picture. We discuss in this paper various approaches that might help prevent DXR, after which we assume that one would still have to deal with a xenograft counterpart of the T cell-mediated allograft rejection response. The approach to avoiding DXR focuses largely on genetic engineering of the ECs in the graft.     

A novel 22q11.2 microdeletion in DiGeorge syndrome

BACKGROUND: Decreased red blood cell survival contributes to the anemia of chronic renal failure patients. Because patients on chronic dialysis therapy are frequently exposed to excessive complement activation, we investigated the susceptibility of this patient population to erythrocyte C5b-9 deposition, complement-mediated lysis, and ghost formation. METHODS: We developed a flow cytometric assay using antibodies to both glycophorin and the C5b-9 complex to detect C5b-9 deposition on intact erythrocytes and erythrocyte ghosts. Serum C5b-9 levels and C5b-9 deposition on erythrocyte ghosts were measured by enzyme-linked immunosorbent assay. RESULTS: A significant increase in C5b-9 deposition on intact erythrocytes was demonstrated in patients with advanced chronic renal failure (2.2 +/- 0.5%) and in patients on chronic maintenance hemodialysis (2.3 +/- 0.4%) compared with normal volunteers (0.9 +/- 0.1%, P = 0.005 vs. chronic renal failure, P < 0.001 vs. chronic hemodialysis patients). There was also a significantly higher percentage of C5b-9-positive erythrocyte ghosts in patients with advanced chronic renal failure (20.6 +/- 5%) and in chronic hemodialysis patients (15.5 +/- 3.1%) compared with normal controls (2.6 +/- 0.9%, P < or = 0.001 vs. advanced chronic renal failure and chronic hemodialysis patients). Treatment of erythrocyte preparations with cobra venom factor, which activates the complement cascade, resulted in dramatic increases in the percentages of C5b-9-positive erythrocyte ghosts in patients with chronic renal failure (49.9 +/- 6.9%) and in chronic hemodialysis patients (45.0 +/- 4.2%) compared with normal volunteers (22.3 +/- 2.7%, P < 0.001 vs. chronic renal failure and chronic hemodialysis patients). Erythrocyte membrane expression of the complement regulatory proteins CD59 and CD55 did not significantly differ between normal controls and hemodialysis patients. Plasma C5b-9 levels after cobra venom factor stimulation were higher in chronic renal failure patients (538 micrograms/ml) compared with normal controls (345 micrograms/ml, P < 0.001). CONCLUSIONS: Patients with chronic renal failure and on hemodialysis therapy are susceptible to erythrocyte C5b-9 deposition with subsequent lysis and ghost formation. Susceptibility to complement-mediated erythrocyte injury may contribute to the anemia of chronic renal disease.     

Complement-fixing elicited antibodies are a major component in the pathogenesis of xenograft rejection

Hamster to rat cardiac xenografts undergo delayed rejection as compared with the hyperacute rejection of discordant xenografts. Elicited xenoreactive Abs (EXA) are thought to initiate hamster to rat cardiac xenograft rejection. In this study, we demonstrate that following transplantation of a hamster heart, rats generated high levels of EXA. Adoptive transfer into naive recipients of purified IgM, IgG2b, or IgG2c, but not IgG1 or IgG2a EXA, induced xenograft rejection in a complement-dependent manner. Ability of EXA to cause rejection correlated with complement activation, platelet aggregation, and P-selectin expression in the xenograft endothelium. Cyclosporin A (CyA) administration, after transplantation, totally suppressed IgG1, IgG2a, IgG2b, and IgG2c EXA, and inhibited IgM EXA production, but failed to overcome rejection. Administration of cobra venom factor (CVF), 1 day before and at the time of transplantation, resulted in complement inhibition during 3 days after transplantation, which failed to overcome rejection. Combination of CyA and CVF, which we have previously shown to overcome rejection, resulted in suppression of IgG EXA production and in the return of IgM XNA to preimmunization serum levels, 3 to 7 days after xenotransplantation, while complement remained inhibited. Thus, under CyA/CVF treatment, complement activation by hamster cells was suppressed following xenotransplantation, and presumably for this reason xenograft rejection did not occur. In conclusion, our data demonstrate that EXA play a pivotal role in the pathogenesis of xenograft rejection and that CyA and CVF suppress xenograft rejection by preventing exposure of xenograft endothelial cells to complement activation by EXA.     

The natural history of cigarette smoking from adolescence to adulthood: demographic predictors of continuity and change

In this study a modified experimental kidney xenograft model was developed, which reproduced, in a reliable way, the course of hyperacute rejection. In this model guinea-pig kidneys were transplanted to rats using end-to-side anastomoses with recipient aorta and vena cava, respectively, and ureter drainage for diuresis monitoring. The aim of this study was to investigate the protective effects of complement modulation by soluble complement receptor 1 (sCR1) on the xenografts. Twenty-four xenotransplantations were performed and recipients randomized for treatment either by 3 ml saline or 50 mg/kg sCR1 as a 3-ml bolus. It was found that sCR1 was highly efficient in delaying hyperacute rejection from 10.5 +/- 2.1 min in the control group to at least 2 h in the therapy group and in prolongation of graft function. The complement activity was significantly reduced in the sCR1-treated rats, even at the time of rejection, as a result of complement modulation in this group of xenograft recipients. Xenografts from saline-treated animals showed necroses, interstitial haemorrhages and platelet aggregates occluding the vessels as soon as 10 min after the reperfusion started. No such changes could be seen even after 120 min in the xenografted kidneys of sCR1-treated rats. Also C3 deposits in the glomeruli and interstitium were markedly reduced.     

Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion

BACKGROUND: Human natural xenoantibodies represent a major hurdle to the clinical application of pig lungs in transplantation by initiating hyperacute rejection within minutes to hours. OBJECTIVE: The object was to compare pig organ perfusion and specific depletion of anti-alpha-galactosyl xenoantibodies for prevention of hyperacute rejection in the pig to human lung combination. METHODS: Large White pig (20-25 kg) left lungs were removed and continuously ventilated and reperfused ex vivo either with (1) whole human blood previously perfused in situ through pig right lung (group I), liver (group II), or spleen (group III) or with (2) human plasma in vitro immunoabsorbed on columns containing alpha-galactosyl disaccharide (Gal-alpha-(1-3)Gal-beta-(CH2)3NH2; B disaccharide) (group IV). Each study group included 6 animals. RESULTS: The in situ and in vitro preperfusions depleted anti-alpha-galactosyl xenoantibodies and all in situ perfused pig organs showed histologic signs of hyperacute rejection. After the ex vivo reperfusion, group I xenografts had a significantly (P < .001) longer functional and histologic survival than did xenografts in groups II, III, and IV. Human blood reperfusing group I xenografts had a significantly (P < 0.05) lower (1) decline of clotting factors and total circulating immunoglobulins, (2) total and membrane attack complex (C5b,6,7,8,9) complement activation, and (3) hemolysis. By Western blot analysis, the in situ lung preperfusion removed antibodies against non-alpha-galactosyl proteins of low molecular weight that were not eliminated by the alpha-galactosyl column. CONCLUSIONS: Results demonstrate that specific depletion of anti-alpha-galactosyl antibodies alone incompletely protects pig lungs from hyperacute rejection. It is speculated that the more complete prevention of this rejection afforded by pig lung preperfusion relates to the removal of other, non-alpha-galactosyl antibodies.     

Postoperative inflammatory response after autologous and allogeneic blood transfusion

BACKGROUND: Allogeneic blood transfusions cause immunosuppression. The aim of this study was to determine whether complement anaphylatoxins, cytokines, or both are released in the recipient, after blood transfusions in general, and after autologous blood transfusions in particular. METHODS: Thirty-one patients having total hip joint replacement surgery were randomized to receive either allogeneic red blood cells (n = 15) or predeposited autologous whole blood transfusion (n = 16). Plasma concentrations of the anaphylatoxins C3a and C5a, the terminal C5b-9 complement complex, and cytokines IL-6 and IL-8 in the recipients were repeatedly analyzed before, during, and after surgery. RESULTS: Significantly increased concentrations of IL-6 and IL-8 appeared in both groups, with a significantly greater increase in the autologous blood group. Patients in both groups developed a moderate but significant increase of C3a without a significant difference between them. C5a and terminal C5b-9 complement complex were not greatly changed. CONCLUSIONS: The study showed a greater increase in cytokine concentration after autologous blood transfusion than after allogeneic blood transfusion. The lower response in the latter may result from transfusion-induced suppression of cellular immunity.     

Ragweed allergy: correlation between skin reactivity and in vitro complement activation

Recently we have reported on several observations which indicate that allergen-induced complement activation contributes to the development of the symptoms of ragweed allergy. In the present paper a new finding that supports this assumption is summarized. In 48 ragweed-allergic patients individual skin reactivity to ragweed allergen extract (RWA) was assessed using dilution skin prick testing. Sera of these patients were incubated with 20, 100, and 400 U/ml RWA and generation of two complement activation products, alternative pathway C3-convertase (C3bBbP) and terminal pathway activation complex (C5b-9) was measured by ELISA methods. A strong positive correlation (Spearman correlation coefficient r = 0.495, P = 0.0004, and r = 0.454, P = 0.0012, respectively) was found between individual skin reactivity to RWA and C3bBbP generation induced by 20 and 100 A allergological units/ml (U/ml) RWA. This finding further supports the role of complement activation products in the aggravation of the basic IgE-mediated immunopathology of ragweed allergy.     

Life-supporting pig-to-baboon heart xenotransplantation

BACKGROUND: The aim of this study was to investigate the effect of the expression of human decay-accelerating factor in transgenic pigs on hyperacute rejection in a pig-to-baboon heterotopic heart transplantation model and to assess the ability of such transgenic pig hearts in supporting the life of a primate when transplanted orthotopically. METHODS: Hearts from pigs transgenic for human decay-accelerating factor were transplanted heterotopically (n = 3) and orthotopically (n = 5) into the baboon. All animals received cyclosporine, steroids, and cyclophosphamide. Blood was sampled regularly for total antipig antibody titers, trough cyclosporine levels, full blood count, electrolytes, and creatinine. Rejection of the heterotopic hearts was defined as the absence of palpable cardiac pulsation. Explanted hearts were examined histologically with hematoxylin and eosin and with immunochemistry for complement components C3, C4, C9, and immunoglobulin M. RESULTS: None of the hearts were hyperacutely rejected. In the heterotopic group one heart underwent acute vascular rejection on day 13, and the remaining two recipients with beating xenografts were killed on days 2 and 21. In the orthotopic group, one recipient with a life-supporting xenograft was killed on day 9 because of poor general condition. Histologic examination demonstrated no evidence of rejection. Two xenografts stopped beating on day 5, and histologic study showed acute vascular rejection in both. There were also two graft failures for technical reasons in this group. CONCLUSIONS: Hyperacute rejection is abrogated in pig-to-baboon heart xenotransplantation with the expression of the human decay-accelerating factor transgene. The human decay-accelerating factor transgenic pig heart is able to support primate life for a prolonged period.     

Inhibition of complement activation by soluble recombinant CR1 under conditions resembling those in a cardiopulmonary circuit: reduced up-regulation of CD11b and complete abrogation of binding of PMNs to the biomaterial surface

The influence of soluble recombinant CR1 (sCR1) on complement activation, and its indirect effects on the coagulation system and cellular responses were assessed in two models for the study of blood/surface and blood/air interactions, as are encountered in e.g. cardiopulmonary bypass circuits. The concentrations of C3a and sC5b-9 and the amount of bound C3/C3 fragments were analyzed as indicators of complement activation. Thrombin-antithrombin complexes, the platelet count, surface-ATP, beta-thromboglobulin, and the expression of CD11b on leukocytes were the parameters analyzed to reflect coagulation and cellular responses. In addition, immunochemical analyses of the phenotypes of surface-bound leukocytes and platelets were performed. Recombinant sCR1, at doses ranging between 0.1-0.25 mg/ml, was found to completely inhibit the generation of sC5b-9, and of C3a by two thirds; the binding of C3 and/or C3 fragments to the surface was almost entirely abolished. As a result of the inhibition of complement activation, the expression of CD11b on PMNs, and the binding of these cells to the biomaterial surface was almost completely lost. In contrast, the thrombin-antithrombin complexes, the platelet count, and the adherence of platelets to the surface, as reflected by the ATP binding and the release of beta-thromboglobulin, were not affected. These data show that complement activation, in association with extra-corporeal treatment, causes activation and binding of PMNs to the biomaterial and that these effects can be completely abolished by the addition of soluble recombinant sCR1.     

The contribution of terminal complement components to acute and hyperacute allograft rejection in the rat

Acute rejection and antibody-mediated hyperacute allograft rejection are affected by activation of the complement cascade. Split products of early complement components influence the localization, activation, and effector functions of platelets, granulocytes, monocytes, and lymphocytes, while the formation of membrane attack complex (C5b-C9) can lead to rapid cell destruction. Therefore, we compared acute and Ab-mediated hyperacute allograft rejection in a recently described model of C6 deficient PVG (C-) (RT1c) rats and their normal counterpart PVG (C+) (RT1c) rats. Cardiac allografts from fully MHC disparate ACI donors were heterotopically grafted into naive and skin graft sensitized PVG (C-) and PVG (C+) rats. ACI cardiac allografts were rejected acutely (8.3 +/- 2 days; n = 7) by naive PVG (C+) recipients, but survived significantly longer in PVG (C-) recipients (22 +/- 10 days; n = 10). Presensitized PVG (C+) rats rejected ACI cardiac allografts hyperacutely in 6.1 +/- 2.4 hr (n = 5). In contrast, ACI cardiac allografts transplanted into presensitized (PVG (C-) rats had markedly longer survival of 91 +/- 14 hr (n = 5). The alloantibody responses of naive PVG (C+) and PVG (C-) recipients 7 days after cardiac allografting, and of presensitized PVG (C+) and PVG (C-) recipients at time of cardiac allografting were not significantly different as measured by flow cytometry against ACI lymphocytes. Immunofluorescence demonstrated deposition of IgM, IgG and C3 in ACI allografts in PVG (C-) as well as in PVG (C+) recipients. Deposition of C6 was only found in grafts rejected by PVG (C+). The significantly longer survival of ACI cardiac allografts in C6-deficient PVG (C-) rats indicates that the membrane attack complex contributes to acute as well as antibody-mediated hyperacute allograft rejection.     

The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis

Persistent proteinuria and tubulointerstitial lesions are important signs of progressive renal disease. The purpose of this study was to assess the role of complement in the development of tubulointerstitial lesions in rats with proteinuria due to primary glomerulonephritis. Mesangial proliferative glomerulonephritis was induced in mononephrectomized rats by intravenous injection of monoclonal antibody (mAb) 1-22-3 (Clin Exp Immunol 102: 181-185, 1995). As early as 24 h after the injection, proteinuria became evident, persisted throughout the observation period, and was associated with mesangial cell proliferation and tubulointerstitial lesions when examined at 7 and 14 d after mAb administration. Deposition of rat C3 and C5b-9 was observed at the luminal surface of proximal tubules and in cellular debris present in the tubular lumen (group I). Rats injected with mAb 1-22-3 and depleted of complement by injections of cobra venom factor starting at day 3 developed glomerulonephritis and proteinuria comparable to rats of group I, but complement deposition in the tubules and the tubulointerstitial lesions were markedly reduced (group II). Rats in group III were injected with mAb and, from day 3, with soluble complement receptor type 1, which became detectable at the luminal surface of proximal tubules and in the urine. Deposition of C5b-9 in tubular cells was not detectable, and the severity of tubulointerstitial lesions was reduced compared with rats in group I. These results indicate that, in this model of primary mesangial proliferative glomerulonephritis with proteinuria, the development of tubulointerstitial lesions is associated with activation of serum complement at the level of tubular brush border, and tubulointerstitial lesions can be reduced by inhibition of complement activity.     

The immunological barriers to xenotransplantation

There is an increasing interest in the potential clinical application of xenotransplantation. This interest derives in part from the need to identify a more abundant source of organs for transplantation and in part from rapid progress in understanding the cellular and molecular changes that contribute to hyperacute and acute vascular xenograft rejection. Recent areas of progress in understanding the immunological hurdles to xenotransplantation include the characterization of xenoreactive antibodies and the antigens they recognize, the role of complement regulatory proteins in immune recognition, the mechanism of complement activation in a xenograft, and the pathophysiologic changes in endothelial cells caused by the activation of complement.     

Complement activation during CAPD

Complement activation was monitored in 20 CAPD patients and 20 normal individuals using markers of the alternative (Bb fragment), classical (C4d fragment), common (iC3b) and terminal pathways (SC5b-9, the soluble form of the membrane attack complex, MAC), together with C3, C4 and factor B. CAPD plasma SC5b-9 was higher than normal although this was not due to increased complement activation in the plasma. The calculated cleavage for C3, C4 and factor B to iC3b, C4d and Bb respectively, due to spontaneous activation, was similar in both groups. C3, C4 and factor B in dialysate were less than 1% of plasma concentration, consistent with vascular leakage, whereas iC3b, Bb and SC5b-9 were at higher concentrations, suggesting generation in the peritoneum by the alternative pathway. 2.4% C4d is consistent with leakage of this small molecule but may indicate slight classical activation. It is concluded that complement activation occurs in the peritoneum during CAPD. MAC and the anaphylatoxins which are also generated may contribute to an increased risk of infection and other inflammatory complications.     

Removal of activated complement from shed blood: comparison of high- and low-dilutional haemofiltration

BACKGROUND: Perioperative blood salvage is associated with release of inflammatory mediators. Depending on type of processing, the complement system is activated to some extent in the final blood product. The aim of the present study was to evaluate a haemofiltration technique concerning complement system activation and whether the volume of added saline will have an influence on the elimination of activated complement during processing. METHODS: Sixteen patients undergoing total hip arthroplasty received wound blood salvaged intraoperatively with a haemofiltration technique. Saline was added to the reservoir for washing in a ratio of 1:1 or 5:1 of estimated blood volume. Samples for determination of the anaphylatoxins C3a and C5a, and the terminal SC5b-9 complement complex (TCC) were drawn from the patients, the collected blood, the ultrafiltrate and the processed blood. RESULTS: Increased concentrations of C3a, C5a and TCC were found in aspirated and processed blood. Haemofiltration did not reduce the concentrations of these factors, except that of C3a in the group where saline was added in a ratio of 5:1. There were no increased concentrations of C3a, C5a or TCC in the patient plasma after reinfusion. No differences in blood pressure, heart rate, pH, arterial oxygen tension, arterial carbon dioxide tension, or base excess were found in association with reinfusion of the blood. CONCLUSION: Collected shed blood washed through haemofiltration contained moderately elevated concentrations of C3a, C5a and TCC. Reinfusion of the blood neither led to increased systemic concentrations of complement activation products, nor to disturbances in haemodynamic or biochemical parameters.     

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi

Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 +/- 3.8% and 21.2 +/- 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 +/- 5.9% and 2.6 +/- 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 +/- 3.7 and 21.6 +/- 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 +/- 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 +/- 0.5%). Preincubation of SchC/6d with an F(ab)2 fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 +/- 3.0% but only 1.7-fold (maximum cytolysis 37.4 +/- 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.     

Crossed quadrant homonymous hemianopsia in a case of multiple sclerosis

Prevention of hyperacute rejection is a difficult and unsolved problem in xenotransplantation. Natural antibodies and complement activation have been known to play an important role in the xenotransplantation between discordant species pairs. In the present study, total blood exchange (TBE) was performed with pyridoxalated-hemoglobin-polyoxyethylene conjugate (PHP) solution (Ajinomoto Co., Inc., Kawasaki, Japan) before cardiac xenotransplantation in order to remove the immunoglobulins and prolong xenograft survival time. Guinea pigs and rats were used as the discordant species combination for donor and recipient. Two groups were established: Group 1, untreated control (n = 8) and Group 2, TBT with PHP solution (n = 8). The exchange blood transfusion was carried out at the rate of 15-20 ml/h utilizing PHP solution using a blood pump. After the blood exchange was processed, hematocrit (Ht) levels dropped to 4 or 5%, and a cardiac xenotransplantation was performed within 24 h. The levels of serum IgA, IgM, and IgG were decreased to less than 25, 25, and 10% of the base line, respectively, after blood exchange. A mean xenograft survival time in Group 2 was prolonged to 472 +/- 74 min and to 10.4 +/- 1.8 min in Group 1 (p < 0.01). A titer of the anti-guinea pig lymphocytotoxic antibody in rat serum was decreased to almost nil. The data from this study suggest that total blood exchange with PHP solution may be useful in preoperative removal of xenograft antibodies in xenotransplantation.