Effect of neutralizing antibodies to IL-10 and C5 on the renal damage caused by a pathogenic human anti-dsDNA antibody.

OBJECTIVES: There is considerable evidence suggesting that anti-double-stranded deoxyribonucleic acid (anti-dsDNA) antibodies are involved in the pathogenesis of lupus nephritis. It was shown previously using severe combined immune deficient (SCID) mice that when the hybridomas secreting human immunoglobulin G (IgG) anti-dsDNA antibodies, RH14 and DIL-6, were implanted intraperitoneally the antibodies produced by RH14, but not DIL-6, deposited in the kidneys, caused pathological changes in the renal tissues and induced proteinuria. In this study we have further analysed the effect of activated terminal complement proteins and interleukin-10 (IL-10) in the pathogenesis of glomerulonephritis caused by the RH-14. METHODS: SCID mice implanted with RH-14 or DIL-6 cell lines were treated with neutralizing antibodies to IL-10 (mAb B-S10) or anti-complement factor 5 (anti-C5) (mAb BB5.1) intraperitoneally. Control groups received either an isotype control antibody (135.8) or phosphate-buffered saline (PBS). Serum human IgG levels and proteinuria were estimated and the extent of renal involvement was examined by histopathological and electron microscopic techniques. RESULTS: While there was a tendency to reduce proteinuria in the anti-IL-10 injected group the anti-C5 injected group showed a significant reduction in proteinuria (P<0.01) compared with the groups injected with either the control mAb or PBS. There was a considerable reduction in the serum human IgG levels in the anti-IL-10 but not in the anti-C5 treated animals. Both anti-IL-10 and anti-C5 treated groups showed significantly reduced renal impairment as revealed by histopathological examination and proteinuria assessment. CONCLUSION: The findings, while confirming the role of IL-10 and activated terminal complement component in the production of antibody at the cellular level and at the site of glomerular immune deposition in this model, respectively, also suggest the beneficial effect of a combined therapy using both anti-IL-10 and anti-C5 mAb to prevent or reduce the effect of the humoral immune response in lupus disease.

Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease.

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.

Effective antigen-specific immunotherapy in the marmoset model of multiple sclerosis.

Mature T cells initially respond to Ag by activation and expansion, but high and repeated doses of Ag cause programmed cell death and can suppress T cell-mediated diseases in rodents. We evaluated repeated systemic Ag administration in a marmoset model of experimental allergic encephalomyelitis that closely resembles the human disease multiple sclerosis. We found that treatment with MP4, a chimeric, recombinant polypeptide containing human myelin basic protein and human proteolipid protein epitopes, prevented clinical symptoms and did not exacerbate disease. CNS lesions were also reduced as assessed in vivo by magnetic resonance imaging. Thus, specific Ag-directed therapy can be effective and nontoxic in primates.

Role of porcine P-selectin in complement-dependent adhesion of human leukocytes to porcine endothelial cells.

BACKGROUND: Rapid leukocyte adherence to donor organ vasculature is a hallmark of hyperacute xenograft rejection. However, the molecular interactions required for leukocyte binding to vascular endothelium have not been characterized. METHODS AND RESULTS: Binding assays performed between human neutrophils and porcine aortic endothelial cells (PAEC) after exposure to human complement demonstrated that adhesion was mediated by both surface-bound C3b and C5b-9 activity. C5b-9-dependent adhesion was blocked by neuraminidase treatment of the neutrophils, suggesting that this binding was mediated by porcine P-selectin. Porcine P-selectin was isolated from a PAEC cDNA library. The porcine P-selectin primary sequence contained an open reading frame encoding 646 amino acids with 82% identity to human P-selectin. Recombinant soluble porcine P-selectin specifically bound to human neutrophils and HL-60 cells. Transfection of COS cells with the full-length porcine P-selectin cDNA resulted in surface expression of the protein and markedly increased the binding of human neutrophils to these cells. The binding of both soluble and COS-expressed porcine P-selectin to human neutrophils was blocked by pretreatment of the neutrophils with neuraminidase or the addition of EDTA. Finally, treatment of PAEC with human thrombin or normal human serum but not purified human C5a- or C8-deficient human serum resulted in the rapid expression of porcine P-selectin on the cell surface. CONCLUSIONS: This report establishes that porcine P-selectin supports the binding of human neutrophils to PAEC in vitro. Further, these data suggest that sublytic deposition of C5b-9 during hyperacute rejection results in the expression of porcine P-selectin, which may contribute to the rapid adhesion of neutrophils to porcine xenografts.

Primary structure and functional characterization of a soluble, alternatively spliced form of B7-1.

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7-1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

Significant role for Fas in the pathogenesis of autoimmune diabetes.

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.

Fas-mediated apoptosis in clinical remissions of relapsing experimental autoimmune encephalomyelitis.

PLP139-51-induced experimental autoimmune encephalomyelitis (R-EAE) displays a relapsing-remitting paralytic course in female SJL mice. We investigated the role of apoptosis/activation-induced cell death (AICD) in the spontaneous recovery from acute disease. Clinical EAE was significantly enhanced in Fas (CD95/APO-1)-deficient SJL lpr/lpr mice, which displayed significantly increased mean peak clinical scores, reduced remission rates, and increased mortality when compared with their SJL +/lpr littermates. PLP139-151-specific proliferative responses were fairly equivalent in the 2 groups, but draining lymph node T cells from SJL lpr/lpr mice produced dramatically increased levels of IFN-gamma. Central nervous system (CNS) Fas and FasL mRNA levels in wild-type SJL (H-2(s)) mice peaked just before spontaneous disease remission and gradually declined as disease remitted. We applied the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay to detect apoptosis in situ in spinal cords of mice at various clinical stages of EAE. Most TUNEL(+) cells were found during active periods of inflammation: the acute, peak, and relapse time points. Significantly fewer apoptotic cells were observed at preclinical and remission time points. Collectively, these findings indicate that Fas-mediated apoptosis/AICD plays a major role in the spontaneous remission after the initial acute inflammatory episode and represents an important intrinsic mechanism in regulation of autoimmune responses.

STESYS2: extended STESYS software for MS Windows.

The STESYS2 software is a new version of the IBM PC software supporting interactive stereological measurements. In comparison with the previous STESYS, it is enhanced by a number of useful options, e.g. on-line image input via a TV camera coupled with a microscope operating under MS Windows OS. The main advantage, when compared with other such software packages, is the design of the STESYS2 as a module of the freeware image processing system Image Tool which provides a user-friendly environment including a number of image processing and preprocessing routines. Capabilities of the STESYS2 are illustrated by a practical example: estimation of the surface area of capillaries in the terminal villi of human placenta by the Sandau spatial grid method.

A role for complement in antibody-mediated inflammation: C5-deficient DBA/1 mice are resistant to collagen-induced arthritis.

Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with significant similarities to human rheumatoid arthritis that can be induced upon immunization with native type II collagen. As in rheumatoid arthritis, both cellular and humoral immune mechanisms contribute to disease pathogenesis. Genotypic studies have identified at least six genetic loci contributing to arthritis susceptibility, including the class II MHC. We have examined the mechanism of Ab-mediated inflammation in CIA joints, specifically the role of complement activation, by deriving a line of mice from the highly CIA-susceptible DBA/1LacJ strain that are congenic for deficiency of the C5 complement component. We show that such C5-deficient DBA/1LacJ animals mount normal cellular and humoral immune responses to native type II collagen, with the activation of collagen-specific TNF-alpha-producing T cells in the periphery and substantial intra-articular deposition of complement-fixing IgG Abs. Nevertheless, these C5-deficient mice are highly resistant to the induction of CIA. These data provide evidence for an important role of complement in Ab-triggered inflammation and in the pathogenesis of autoimmune arthritis.

Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response that causes substantial clinical morbidity. Activation of complement during CPB contributes significantly to this inflammatory process. We examined the capability of a novel therapeutic complement inhibitor to prevent pathological complement activation and tissue injury in patients undergoing CPB. METHODS AND RESULTS: A humanized, recombinant, single-chain antibody specific for human C5, h5G1.1-scFv, was intravenously administered in 1 of 4 doses ranging from 0.2 to 2.0 mg/kg before CPB. h5G1.1-scFv was found to be safe and well tolerated. Pharmacokinetic analysis revealed a sustained half-life from 7.0 to 14.5 hours. Pharmacodynamic analysis demonstrated significant dose-dependent inhibition of complement hemolytic activity for up to 14 hours at 2 mg/kg. The generation of proinflammatory complement byproducts (sC5b-9) was effectively inhibited in a dose-dependent fashion. Leukocyte activation, as measured by surface expression of CD11b, was reduced (P<0.05) in patients who received 1 and 2 mg/kg. There was a 40% reduction in myocardial injury (creatine kinase-MB release, P=0.05) in patients who received 2 mg/kg. Sequential Mini-Mental State Examinations (MMSE) demonstrated an 80% reduction in new cognitive deficits (P<0.05) in patients treated with 2 mg/kg. Finally, there was a 1-U reduction in postoperative blood loss (P<0. 05) in patients who received 1 or 2 mg/kg. CONCLUSIONS: A single-chain antibody specific for human C5 is a safe and effective inhibitor of pathological complement activation in patients undergoing CPB. In addition to significantly reducing sC5b-9 formation and leukocyte CD11b expression, C5 inhibition significantly attenuates postoperative myocardial injury, cognitive deficits, and blood loss. These data suggest that C5 inhibition may represent a novel therapeutic strategy for preventing complement-mediated inflammation and tissue injury.

Complement inhibition with an anti-C5 monoclonal antibody prevents hyperacute rejection in a xenograft heart transplantation model.

BACKGROUND: The present study was undertaken to determine whether anti-complement 5 (C5) monoclonal antibodies (mAb) prevent hyperacute rejection (HAR) in a rat-to-presensitized mouse heart transplantation model and whether these mAb, combined with cyclosporine (CsA) and cyclophosphamide (CyP), can achieve long-term graft survival. METHODS: BALB/c mice were presensitized with 2x10(7) splenocytes from Lewis rats 14 days before grafting. Heart grafts from Lewis rats were heterotopically transplanted into BALB/c mice. Presensitized mice were treated with either anti-C5 mAb or a combination of anti-C5 mAb, CsA, and CyP. Controls included: presensitized mice with no treatment, presensitized mice treated with either CsA + CyP or IgG, and nonpresensitized mice with either no treatment or with CsA + CyP treatment. RESULTS: Although typical features of HAR were evident in the presensitized grafts, the mAb completely inhibited complement activation and successfully prevented HAR. Despite complement inactivation, the graft was rejected on postoperative day 6 with acute vascular rejection (AVR) also known as delayed xenograft rejection (DXR). Notably, this type of rejection cannot be effectively overcome by CsA and CyP. CONCLUSIONS: We conclude that (1) anti-C5 mAb prevents HAR, (2) AVR/DXR still occurs when HAR is prevented by complement inactivation, and (3) AVR/DXR cannot be overcome by conventional immunosuppression. These data suggest that anti-C5 mAb may be valuable for preventing HAR in future clinical xenotransplantation and that additional interventions may be required to address AVR/DXR.

Serial MR imaging of experimental autoimmune encephalomyelitis induced by human white matter or by chimeric myelin-basic and proteolipid protein in the common marmoset.

BACKGROUND AND PURPOSE: Experimental autoimmune encephalomyelitis (EAE) in the marmoset was monitored by serial MR imaging to determine correlates to the natural-history MR studies in multiple sclerosis (MS). The relationships of MR-revealed lesions to clinical status and histopathologic findings were also explored. METHODS: We induced EAE by subcutaneous inoculation in two marmosets by human white matter (HWM) and in seven marmosets by MP4 (a chimeric recombinant fusion protein of myelin-basic and proteolipid protein) in adjuvant along with intravenous inactivated pertussis vaccine to facilitate the disease process. The HWM-inoculated animals were induced with Freund's adjuvant as the established model of marmoset EAE. The MP4-inoculated animals were induced with either Freund's incomplete adjuvant or TiterMax as part of a preclinical treatment trial. MR imaging was performed at 1.5 T at baseline, and repeated at 1- to 2-week intervals for a period of up to 16 weeks in six EAE-induced marmosets, and intermittently for up to 70 weeks in three EAE-induced and two control marmosets. Proton density- (PD-) and T2-weighted, pre- and postgadopentetate dimeglumine enhancement, T1-weighted, and magnetization transfer (MT) images were obtained. The brains were prepared for histologic evaluation of lesion distribution and counts, characterization of lesions as demyelinating or inflammatory, and histopathologic scoring. The clinical, MR, and pathologic scoring were done on grading systems, and correlated for evaluation. RESULTS: White matter (WM) changes after EAE induction were observed first at 9 days in the HWM-induced animals and at 2.5 weeks in the MP4-induced animals, with subsequent week-to-week fluctuations on PD- and T2-weighted images. Contrast-enhancing lesions were not observed in all animals. MR-revealed WM lesions correlated to histopathologic analysis of EAE lesions, measuring from 0.5 mm to 1.5 mm. The lesion count and extent of demyelination was greater in the HWM-induced animals than in the MP4-induced animals. Some MR-revealed lesions correlated directly to clinical symptoms, but the majority of lesions were clinically silent. CONCLUSION: On MR images, lesions in the EAE marmoset model were confined to the WM, and their development, resolution, distribution, and enhancing characteristics fluctuated over the duration of the study. The dynamic presentation of MR-revealed lesions confirms the parallels between EAE in the marmoset and relapsing-remitting MS. Clinical symptoms alone were not representative of ongoing pathologic brain lesions. Therefore, serial MR imaging serves as a very important adjunct to clinical and histologic surveillance of the development of new and the persistence of existing brain lesions in this animal model of MS.

Determinant spreading associated with demyelination in a nonhuman primate model of multiple sclerosis.

Definition of the immune process that causes demyelination in multiple sclerosis is essential to determine the feasibility of Ag-directed immunotherapy. Using the nonhuman primate, Callithrix jacchus jacchus (common marmoset), we show that immunization with myelin basic protein and proteolipid protein determinants results in clinical disease with significant demyelination. Demyelination was associated with spreading to myelin oligodendrocyte glycoprotein (MOG) determinants that generated anti-MOG serum Abs and Ig deposition in central nervous system white matter lesions. These data associate intermolecular "determinant spreading" with clinical autoimmune disease in primates and raise important issues for the pathogenesis and treatment of multiple sclerosis.

Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-C5 therapy.

BACKGROUND: Myocardial ischemia and reperfusion (MI/R)-induced tissue injury involves necrosis and apoptosis. However, the precise contribution of apoptosis to cell death, as well as the mechanism of apoptosis induction, has not been delineated. In this study, we sought to define the contribution of the activated terminal complement components to apoptosis and necrosis in a rat model of MI/R injury. METHODS AND RESULTS: Monoclonal antibodies (mAbs; 18A and 16C) raised against the rat C5 complement component bound to purified rat C5 (ELISA). 18A effectively blocked C5b-9-mediated cell lysis and C5a-induced chemotaxis of rat polymorphonuclear leukocytes (PMNs), whereas 16C had no complement inhibitor activity. A single dose (20 mg/kg i.v.) of 18A blocked >80% of serum hemolytic activity for >4 hours. Administration of 18A before myocardial ischemia (30 minutes) and reperfusion (4 hours) significantly reduced (91%) left ventricular free wall PMN infiltration compared with 16C treatment. Treatment with 18A 1 hour before ischemia or 5 minutes before reperfusion significantly reduced infarct size compared with 16C treatment. A significant reduction in infarct size (42%) was also observed in 18A-treated rats after 30 minutes of ischemia and 7 days of reperfusion. DNA ladders and DNA labeling (eg, TUNEL assay) demonstrated a dramatic reduction in MI/R-induced apoptosis in 18A-treated compared with 16C-treated rats. CONCLUSIONS: Anti-C5 therapy in the setting of MI/R significantly inhibits cell apoptosis, necrosis, and PMN infiltration in the rat despite C3 deposition. We conclude that the terminal complement components C5a and C5b-9 are key mediators of tissue injury in MI/R.

Presentation of proteolipid protein epitopes and B7-1-dependent activation of encephalitogenic T cells by IFN-gamma-activated SJL/J astrocytes.

There is controversy regarding the possible role of glial cells as APCs in the pathogenesis of central nervous system (CNS) demyelinating diseases such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Microglia have been clearly shown to present Ag in the CNS, and due to the proximity of activated astroglial cells to infiltrating T cells and macrophages in demyelinating lesions, it is also possible that astrocytes positively or negatively regulate disease initiation and/or progression. We examined the capacity of IFN-gamma-treated astrocytes from EAE-susceptible SJL/J mice to process and present myelin epitopes. IFN-gamma activation up-regulated ICAM-1, VCAM-1, MHC class II, invariant chain, H2-M, CD40, and B7-1 as determined by FACS and/or RT-PCR analyses. B7-2 expression was only marginally enhanced on SJL/J astrocytes. Consistent with the expression of these accessory molecules, IFN-gamma-treated SJL/J astrocytes induced the B7-1-dependent activation of Th1 lines and lymph node T cells specific for the immunodominant encephalitogenic proteolipid protein (PLP) epitope (PLP139-151) as assessed by proliferation and activation for the adoptive transfer of EAE. Interestingly, IFN-gamma-activated astrocytes efficiently processed and presented PLP139-151, but not the subdominant PLP178-191, PLP56-70, or PLP104-117 epitopes, from intact PLP and a recombinant variant fusion protein of PLP (MP4). The data are consistent with the hypothesis that astrocytes in the proinflammatory CNS environment have the capability of activating CNS-infiltrating encephalitogenic T cells specific for immunodominant epitopes on various myelin proteins that may be involved in either the initial or the relapsing stages of EAE.

Immune tolerance mediated by recombinant proteolipid protein prevents experimental autoimmune encephalomyelitis.

Proteolipid protein (PLP), a transmembrane protein expressed only in the central nervous system (CNS), is a candidate target autoantigen for autoimmune-mediated demyelination. We have evaluated the effect of a recombinant form of the PLP protein, delta PLP4, in a murine model of experimental autoimmune encephalomyelitis (EAE). PLP-specific T-cell responses were observed following immunization of SJL/J, PL/J and SWR mice with delta PLP4, demonstrating processing of the protein to several distinct antigenic epitopes. Clinical EAE associated with inflammation and demyelination in the CNS also developed after sensitization of mice with delta PLP4 in adjuvant. Conversely, tolerance to delta PLP4 in adult mice and prevention of PLP peptide 139-151-induced EAE was induced by intravenous injection of soluble delta PLP4. The prevention of disease onset was paralleled by a significant reduction in demyelination and CNS inflammatory cell infiltration and diminished PLP139-151-specific T-cell proliferative responses. These results are consistent with the establishment of peripheral T-cell tolerance and reinforce the notion that recombinant myelin antigens and intravenous tolerance induction may prove useful in the modulation of the human demyelinating disease, multiple sclerosis (MS).

Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells.

Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity. We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min before, but not 3 h after local antigen challenge. Among C components, local C5 were thought crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings were consistent with our previous finding of the importance of C5 for CS elicitation, using congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving CS skin extracts, as well as late elaboration of IFN-gamma, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin was suggested by demonstration of impaired CS responses in B cell-deficient mice. In summary, these results suggest that C was activated locally, perhaps via a B cell product, in an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-gamma, and subsequently leading to cell infiltration, for development of T cell-dependent CS.

Humanized porcine VCAM-specific monoclonal antibodies with chimeric IgG2/G4 constant regions block human leukocyte binding to porcine endothelial cells.

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.

The role of Fas in autoimmune diabetes.

Immunologically privileged sites express Fas ligand (FasL), which protects them from attack by activated T cells that express Fas and die upon contact with FasL. In an attempt to protect nonobese diabetic mice (NOD) from autoimmune diabetes, we made FasL transgenic NOD mice using the beta cell-specific rat insulin-1 promoter. Surprisingly, these transgenic mice showed heightened sensitivity to diabetogenic T cells, which was due to self-destruction of beta cells upon T cell-mediated induction of Fas. Fas-negative NOD(lpr/lpr) animals were resistant to diabetogenic T cells and to spontaneous diabetes. Thus, induction of Fas expression on beta cells and their subsequent destruction constitutes the main pathogenic mechanism in autoimmune diabetes.

Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.