[ANCA and anti-MBG double-positive vasculitis: An update on the clinical and therapeutic specificities and comparison with the two eponymous vasculitis]. / Vascularites double-positives ANCA et anti-MBG : mise au point sur les spécificités cliniques et thérapeutiques et comparaison aux deux vascularites éponymes.

Double-positive vasculitis with anti-polynuclear cytoplasm (ANCA) and anti-glomerular basement membrane (GBM) antibodies is a rare entity of systemic vasculitis defined by the presence of ANCA and anti-GBM antibodies. The gradual accumulation of clinical and therapeutic data shows the usefulness of identifying and differentiating this entity from the two vasculitis respectively associated with the isolated presence of each of these two antibodies. Indeed, the double-positive ANCA and anti-GBM vasculitis appears to associate the characteristics of the demography and the extra-renal and pulmonary involvement of the ANCA-associated vasculitis on the one hand, and of the histological type and severe renal prognosis of the anti-MBG vasculitis on the other hand, with the renal involvement which is the only involvement consistently observed in double-positive vasculitis. The aim of this focus is to describe the epidemiological, clinico-biological, histological and prognostic characteristics of this entity, in light of recent literature and ongoing therapeutic changes in the two eponymous vasculitis.

Adenovirus-mediated gene transfer into isolated mouse adult pancreatic islets: normal beta-cell function despite induction of an anti-adenovirus immune response.

The in vitro purification of pancreatic islets offers an opportunity for their modification by ex vivo gene transfer. We investigated the efficiency and functional consequences of adenovirus-mediated gene transfer into adult murine pancreatic islets with a recombinant adenovirus encoding for the beta-galactosidase (beta-Gal) reporter gene. At 10(6) pfu/islet, almost all of the islets were transduced, but maximal transduction was obtained at 10(7) pfu/islet. Histochemical analysis of frozen islet sections showed that transduced cells were only located at the periphery of islets. Transduced islets showed normal insulin secretion in vitro, and were able to normalize in vivo the glycemia of streptozotocin-induced diabetes in syngeneic and allogeneic mice. beta-Gal expression in transduced islets was observed for at least 6 weeks in naive normal recipients and in immunodeficient mice, but was shortened in mice preimmunized to adenovirus. Nevertheless, islets maintained normal control of glycemia in all mice. An early leukocyte infiltrate was observed in syngeneic grafts of transduced islets, but no acceleration in rejection of fully MHC-incompatible islet grafts occurred. In summary, adenovirus-mediated gene transfer in adult mouse islets, although sparing most of the beta-cells, was highly efficient and did not impair insulin secretion by islets. The immune response to the adenovirus and/or to the transgene might be only partially responsible for the decreased expression over time of the transduced gene. Accordingly, adenovirus-mediated gene transfer might allow efficient expression of vectorized sequences with potential immunosuppressive effects in the islet microenvironment.

Effect of cyclosporine on interleukin 2 receptor expression in a human alloreactive T cell clone.

The effect of CsA on antigen-induced IL-2 receptor expression was studied on a human T lymphocyte clone (4AS) obtained from cells infiltrating a rejected human kidney. Stimulation of 4AS clone cells with specific antigen (D.BLCL) was strongly inhibited by CsA (50% inhibition of tritiated thymidine uptake at about 12.5 ng/ml). Addition of recombinant IL-2 only partially restored 4AS growth inhibition, suggesting that another antigen-induced activation signal such as IL-2-receptor expression could be impaired by CsA. Using 125I-labeled human recombinant IL-2 and 125I-labeled 33B3.1 (a MoAb directed against TAC antigen), we found that expression of both high and low affinity sites was decreased when clone cells were stimulated with D.BLCL in the presence of CsA and exogenous IL-2 (about 50% inhibition in the presence of 500 ng/ml of CsA). Northern blot analysis of IL-2-receptor m.RNA (TAC antigen m.RNA) showed that inhibition occurred at least in part at the pretranscriptional level.

Intact pancreatic islet function despite humoral xenorecognition in the pig-to-monkey combination.

BACKGROUND: The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. METHODS: Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. RESULTS: Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. CONCLUSIONS: Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.

Protection of rat endothelial cells from primate complement-mediated lysis by expression of human CD59 and/or decay-accelerating factor.

The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.

Effect of anti-LFA1 (CD11a) monoclonal antibodies in acute rejection in human kidney transplantation.

A murine IgG1 monoclonal antibody, 25-3 (Immunotech, France), directed against the alpha chain (CD11a) of the human LFA1 molecule was used in the treatment of 7 histologically documented first acute rejection in first kidney transplantations under cyclosporine. Four patients (group I) received 20 mg/day for 2 days and 10 mg/day for 8 days of 25-3 MoAb. One developed Quincke's edema after the first injection of 25-3 and was immediately withdrawn from the study. In 2 patients, whose serum creatinine continued to increase, 25-3 MoAb was replaced by steroids, followed by ALG after 3 and 4 days of treatment, respectively. In the last case, rejection was reversed by 25-3 MoAb alone. As the clinical response of rejection to 25-3 was poor, another group of 3 patients (group II) was treated with 25-3 at a dose of 40 mg/day for 2 days, 20 mg/day for 2 days, and 10 mg/day for 6 days, but 25-3 was still unsuccessful in reversing acute rejection, and rescue treatment was initiated between days 5 and 8 in all cases. MoAb tolerance was excellent in 3 patients. With the exception of the one case of Quincke's edema, only minor side effects were noted in the last 3 recipients. 25-3 MoAb serum trough levels peaked between 1.5-3.5 micrograms/ml at day 3 in group I and between 2-9 micrograms/L at day 2 in group II. Surprisingly, only one patient, in group I, exhibited a borderline IgG immune response against 25-3. These findings suggest that the 25-3 anti-CD11a MoAb is ineffective in controlling the course of acute rejection in kidney transplantation. However as already reported for another anti-LFA1 or with an anti-CD4 MoAb in mouse, 25-3 would be the first example in humans of a MoAb that does not elicit a strong immune response against its own determinants. This property might have important applications if 25-3 can prevent rejection in a prophylactic protocol or block the immune response against other MoAbs.

Intact pig pancreatic islet function in the presence of human xenoreactive natural antibody binding and complement activation.

BACKGROUND: The expression of xenogeneic epitopes and the activation of human complement by adult pig islets after prolonged culture have hitherto not been described. MATERIALS AND METHODS: Freshly isolated and cultured islets were analyzed by fluorescence-activated cell sorter analysis, fluorescence microscopy, and immunohistology for expression of Gal(alpha1,3)Gal epitopes, binding of human xenoreactive natural antibodies (XNA), and complement deposition. RESULTS: Freshly isolated and cultured islets showed detectable Gal(alpha1,3)Gal expression and human XNA binding limited to intraislet capillary endothelial cells. No significant modification in Gal(alpha1,3)Gal expression and human XNA binding levels was detected in adult pig islets cultured for up to 4 days compared with freshly isolated islets. Incubation of pig islets with human serum demonstrated the deposition of C3, C4, and membrane attack complex, but not factor B with a similar pattern to XNA. However C3 and C4 showed a more widespread deposition. Despite complement activation, no cytotoxic effect on islets was detected after 4 hr of incubation with human serum capable of killing porcine endothelial cells. Even after 4 days of culture in 50% intact human serum, pig islets retained both their normal morphology and a normal insulin response to glucose stimulation. CONCLUSIONS: Neither islet cell lysis nor, more importantly, any alteration in beta cell function occurred, which suggests that adult pig islets may not be directly damaged by serum after xenotransplantation in humans. Nevertheless, complement activation in vivo could trigger rapid cellular rejection mechanisms through islet cell opsonization and release of bioactive fragments.

Anti-CD4 MoAb therapy in kidney transplantation--a pilot study in early prophylaxis of rejection.

B-F5, a mouse IgG1 anti-CD4 MoAb, was used in recipients of a first cadaveric kidney allograft. Eighteen patients received 30 mg/day MoAb with a quadruple sequential therapy. All but one kidney were functioning at 6 months, with a mean serum creatinine of 153 micromol/L. However, 50% of the patients had an acute rejection episode within the first three months, and most of the early episodes (i.e., < 1 month) occurred in patients with low levels of circulating MoAb. The biological analysis showed a strong depleting effect on the CD4+ cell counts, a saturation by the MoAb of the remaining circulating CD4+ cells, and no detectable immunization against B-F5. Although the biological parameters indicate an action of B-F5 in vivo, the clinical data associated with poor MoAb bioavailability suggest the need for an improved pharmacokinetic behavior of the MoAb to determine its use for prophylaxis of early rejection.

Rat interleukin-2 immunoglobulin M fusion proteins are cytotoxic in vitro for cells expressing the IL-2 receptor and can abolish cell-mediated immunity in vivo.

A hybrid cDNA coding for a fusion protein between rat interleukin 2 (IL-2) and a truncated heavy chain from rat immunoglobulin M (IgM) was constructed. The rat IL-2 and rat IgM CH2-3-4 hybrid gene was subcloned into a vector (PKCR6) for expression of the fusion molecule in Chinese hamster ovary (CHO) cells. Cells transfected with the hybrid cDNA secrete multimeric forms of the fusion protein (IL-2-Mu). Size analysis of the construct revealed that the majority (95%) of the secreted proteins have a high mw (> 500 kDa). The IL-2-Mu construct bind specifically to cells bearing the IL-2 receptors (IL-2R) with a binding affinity around 5 nM. The specific binding to IL-2R leads to T cell proliferation or, if rabbit complement is added, to T cell lysis. Multimeric forms (> 500 kDa) of the fusion protein mediate complement-dependent lysis but trigger only weak proliferation when compared with the low-mw forms (< 500 kDa). In contrast, the latter only efficiently mediate T cell proliferation without inducing complement-dependent lysis. After intravenous administration of CHO supernatant containing IL-2-Mu, or purified IL-2-Mu proteins into rats, the fusion proteins disappeared from the circulation with a t1/2 of 1 hr. The circulating IL-2-Mu constructs in the rat serum retained their capacity to induce complement-dependent lysis of IL-2R-bearing T cells in vitro. Furthermore, the IL-2-Mu construct was able to suppress the delayed-type hypersensitivity (DTH) reaction (an IL-2R, T helper cell-dependent event) in mice. A weak immune response (antirat IL-2-Mu antibodies) was observed when rats received multiple daily injections of the construct.

Anti-interleukin 2 receptor monoclonal antibody in the treatment of ongoing acute rejection episodes of human kidney graft--a pilot study.

Monoclonal antibodies (MoAbs) against human interleukin 2 receptor (IL-2-R) have been shown to prevent early kidney rejection in animals and humans. We report here the effect of an anti-IL-2-R MoAb (33B3.1) inhibiting IL-2 binding high-affinity sites on activated lymphocytes in 10 declared acute rejection episodes of first cadaveric kidney grafts. Six patients were under cyclosporine treatment only at the time of diagnosis of the rejection. All rejection episodes but one were biopsy-proved cellular rejections. Treatment consisted of intravenous infusions of 33B3.1 at 20 mg/day x 2 days, followed by 10 mg/day for 8 additional days. In case of MoAb ineffectiveness at day 5, anti-IL-2-R MoAb was discontinued and a rescue treatment of corticosteroid boluses (CSb) was given. If not, in all cases corticosteroids (CS) were given (1 mg/kg) at the end of MoAb treatment (day 10) and tapered off thereafter. Two rejection episodes immediately responded to 33B3.1 treatment. During 33B3.1 treatment four other patients had only a stabilization of their blood creatinine concentration, which nevertheless returned to prerejection levels after day 10 when anti-IL-2-R was discontinued and CS administered at 1 mg/kg (no rescue treatment). The four remaining patients had an increase of their blood creatinin levels at day 5 despite 33B3.1 treatment, and their renal function only improved with CSb rescue treatment. One of these patients lost the graft despite rescue treatment, as well as a 9-day course of antithymocyte globulin. Trough levels of MoAb reached a plateau as early as day 2 (approximately 6 micrograms/ml). All patients developed antibodies (IgM and IgG) after day 14. In no instance could unresponsiveness be related to low circulating 33B3.1 trough levels or to early host anti-MoAb immune response (IgM or IgG). We conclude that 33B3.1, known to be effective in preventing early rejection, has only inconsistent and/or incomplete effects on the ongoing rejection process. Our data suggest that once IL-2-dependent clones are expanded in the rejected graft, interference with IL-2/IL-2-R signals does not block the effector mechanisms sustaining acute rejection.

Prevention of acute rejection episodes with an anti-interleukin 2 receptor monoclonal antibody. II. Results after a second kidney transplantation.

The focus of progress in transplantation immunosuppression is to achieve more specific immunosuppression with monoclonal antibodies. We have already shown that the efficacy of 33B3.1, a rat monoclonal Ig2A directed against the human IL-2 receptor, was similar to that of rabbit antithymocyte globulin in the prevention of acute rejection in first kidney transplants. A similar comparative analysis has been made in 40-sec renal transplants. ATG (1 mg/kg/day) or 33B3.1 (10 mg/day) was administered during the first 10 days postgrafting in association with corticosteroids and azathioprine. Cyclosporine was introduced on day 9 and azathioprine/CsA constituted the patient's maintenance treatment after day 45. Rejection treatment consisted of equine antilymphocyte globulin in both cases and of steroid boluses when patients were under Cyclosporine. One patient in each group died. Graft survival was 90%, 85%, and 79% in the ATG group (n = 20) and 100%, 89%, and 89% in the 33B3.1 group (n = 20) at 3, 12, and 24 months, respectively. Of the ATG group patients, 45% and 40% in the 33B3.1 group had at least one rejection episode, half the episodes in the MoAb cohort occurring under 33B3.1, vs. none in the ATG group. Transplant function was similar in both groups. Viral infections appeared to be more frequent with ATG (60%) than with 33B3.1 (12%), with CMV accounting for half of these in the ATG group, and none in the MoAb group. Tolerance of both agents was good. Of the 33B3.1 recipients, 70% developed anti-33B3.1 antibodies. From these data, we conclude that this anti-IL-2 receptor MoAb seems less effective than rabbit ATG as induction treatment in second kidney transplant patients.

Prevention of acute rejection episodes with an anti-interleukin 2 receptor monoclonal antibody. I. Results after combined pancreas and kidney transplantation.

A prospective, randomized trial was conducted to evaluate the short-term and long-term effects of induction immunosuppression with the rat IgG 2a monoclonal antibody 33B3.1, directed against the human alpha chain of the interleukin 2-receptor, following primary, cadaveric, combined pancreas and kidney transplantation. Forty patients were randomly assigned to receive 10 mg/day of 33B3.1 (n = 20) or 1.5 mg/kg/day of rabbit antithymocyte globulin (n = 20) for the first 10 postoperative days. Azathioprine, low-dose corticosteroids, and cyclosporine were given in association with either 33B3.1 or ATG. All 40 patients received the entire 10-day bioreagent course and no episode of rejection was observed during this period. Although the incidence of rejection did not significantly differ within the first, second, and third postoperative months (ten 33B3.1 and 6 ATG patients experienced, respectively, 10 and 6 rejection episodes within the first 3 months), the total number of 33B3.1 patients experiencing rejection throughout the follow-up was significantly higher than that of ATG (13 versus 6; P < 0.02). Immunological graft failure accounted for 2 pancreas and 2 kidney losses in the 33B3.1 group versus 1 in the ATG one (P = ns). The total number of infectious episodes was similar in both groups (21 versus 23). Two malignancies were observed in the ATG group (1 responsible for patient's death). One 33B3.1 patient died because of infectious pneumonia and 3 ATG patients died because of 2 cardiovascular diseases and 1 cancer. All patients had functioning grafts at the time of death. The 3-month and 36-month patient, pancreas, and kidney actuarial survival rates were, respectively, 100, 65, and 100%, and 95, 50, and 82% in the 33B3.1 group and 95, 80, and 90%, and 80, 70, and 80% in the ATG one (P = ns). These data suggest that, although a significantly higher rejection episode incidence was observed in patients treated with 33B3.1 monoclonal antibody as compared with ATG, similar long-term results can be obtained following primary cadaveric combined pancreas/kidney transplantation.

Parameters of interaction of a novel monoclonal antibody (33B3.1) with the human IL2-receptors: interrelationship between 33B3.1, anti-Tac, and IL2 binding sites.

This report describes the molecular parameters of interaction of a new antibody (33B3.1) with the human membrane RIL2 expressed by ConA-activated T lymphocytes or allogeneic T-cell clones established from a rejected kidney allograft: the 33B3.1 immunoprecipitates a membrane protein of 55000 MW. It inhibits IL2-driven proliferation of activated T cells. This inhibition occurred in the nanomolar range when low concentrations of recombinant IL2 (rec-IL2) were used. The (125I)-33B3.1 binds in a specific way to a single class of receptor sites on activated T cells. The rate constants of association and dissociation at 37 degrees C of the labeled 33B3.1 were k*1 = 12 X 10(5) M-1 s-1 and k*-1 = 7 X 10(-4) s-1, respectively, and its equilibrium dissociation constant was KD = 0.65 nM. Maximal binding capacities were fairly variable among T-cell clones, as high as 300,000 sites/cell for some of them. Competition experiments demonstrate that the 33B3.1 and anti-Tac interact with the RIL2 in a competitive manner, suggesting that they recognize closely associated epitopes on the RIL2. However, the 33B3.1 inhibits the binding of (35S)-recombinant IL2 to its high affinity RIL2 in a noncompetitive way. The 33B3.1 seems therefore to interact with an epitope close but distinct from the IL2 binding site. Our data could suggest either that the 33B3.1 is able to convert high affinity RIL2 towards low affinity conformations or that there is more than one IL2 binding site per molecule of high affinity RIL2.

A dose-searching trial of an anti-LFA1 monoclonal antibody in first kidney transplant recipients.

25.3, a mouse IgG1 monoclonal antibody (MoAb), directed at the alpha chain of the LFA1 molecule (CD11a) has been used in prophylaxis of rejection in recipients of cadaveric kidney graft. Promising clinical results have been obtained for both tolerance and efficacy [1]. The aim of this trial was to determine the optimal dosage, base on a pharmacokinetic-pharmacodynamic analysis of the data obtained from the 15 patients included in this dose-searching study. Biological parameters, such as circulating levels and functional inhibition (as detected in an adhesion assay of patient lymphocytes), were measured during and after treatment. A Hill relation was calculated between the effect and the concentration measured and led us to select a 15 mg/day dose for further clinical trials, with a loading dose of 30 mg. An additional group receiving this protocol was submitted to the same calculation, and the results from this last group were in agreement with this previous analysis.