Proteasome inhibition by bortezomib: effect on HLA-antibody levels and specificity in sensitized patients awaiting renal allograft transplantation

BackgroundSensitization to human leukocyte antigen (HLA) prolongs waiting list time and reduces allograft survival in solid organ transplantation. Current strategies for pretransplant desensitization are based on B-cell depletion and extracorporeal treatment. The proteasome inhibitor bortezomib allows direct targeting of the antibody-producing plasma cell and has been used in antibody-mediated rejection (AMR) and recipient desensitization with varying results. Here, we report the effect of bortezomib preconditioning on HLA antibody titers and specificity in highly sensitized patients awaiting renal allograft transplantation.Patients and methodsTwo highly sensitized patients awaiting third kidney transplantation were given one cycle of bortezomib (1.3 mg/m², days 1, 4, 8, 11), as part of recipient desensitization. Time-course and levels of anti-HLA antibodies, as well as specificity to previous transplant antigens were monitored by luminex technology. In addition, measles and tetanus toxoid immunoglobulin G (IgG) was measured.ResultsFollowing bortezomib, overall changes in IgG levels were small and no sustained reduction in anti-HLA class I or II antibody levels was observed over more than 100 days of follow-up to both, donor specific and non-donor specific antigens. Moreover, anti-measles and -tetanus toxoid IgG levels remained unchanged.ConclusionsBortezomib preconditioning alone does not result in sustained reduction of HLA antibody levels or alter protective immunity in sensitized patients. This supports the notion, that bortezomib requires activation of plasma cells, as in AMR, to effectively reduce HLA antibody production. Hence, in a pretransplant setting, combination strategies may be required to derive benefit from proteasome inhibition.     

Modulation of Idiotypic and Antiidiotypic Immunoglobulin G Responses in an Immune Thrombocytopenic Purpura Patient as a Consequence of Extracorporeal Protein A Immunoadsorption

Abstract: Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by the presence of antiplatelet antibody which sensitizes platelets resulting in their clearance by the reticuloendothelial system. Extracorporeal protein A immunoadsorption has been demonstrated to be of benefit in the treatment of this autoimmune disorder. In the present study, a patient with underlying systemic lupus erythematosus (SLE) presented with ITP. The patient received 14 immunoadsorption treatments and responded to therapy. During the course of immunoadsorption treatments, there was a decline in circulating immune complex (CIC) levels, antinu-clear antibody (ANA) levels, and antiplatelet IgG antibody levels. In addition, elevated levels of antiidiotypic IgG antibody detected before initiation of therapy were significantly reduced during the course of immunoadsorption treatments. This study suggests that specific autoimmune idiotypic IgG antibody and corresponding antiidiotypic IgG antibody responses may be modulated in association with extracorporeal immunoadsorption employing protein A/silica columns.     

Studies on the removal of anti-pig xenoantibodies in the human by plasmapheresis/immunoadsorption

Abstract: Removal of human preformed natural anti-pig antibodies from the blood is a prerequisite before xenografting between pig and man can be performed. This work explores the effect of plasmapheresis and immunoadsorption (protein-A sepharose) on the reduction and recurrence of anti-pig antibodies in 14 patients. The anti-pig antibody changes were evaluated by lymphocy to toxic, hemagglutinating, and endothelial cell ELISA techniques. The changes induced showed a similar pattern with all three techniques used. In addition, plasma from plasmapheresis treatments were perfused through pig kidneys and the reduction of anti-pig antibodies was estimated by the mentioned in vitro techniques. The anti-pig antibody titers could be reduced to low levels, but not completely eliminated, by 3–4 plasmapheresis sessions. The titers gradually returned to pretreatment levels or higher in a period of 1–2 weeks. A few patients showed signs of a more rapid resynthesis reaching pretransplant levels in 3–4 days. Protein A immunoadsorption satisfactory removed IgG but not IgM antibodies. In vitro perfusion of pig kidneys at 37°C showed a rapid reduction of anti-pig antibody titers of 3–4 titer steps. The combination of 3–4 plasma exchanges followed by in vitro pig kidney perfusion completely removed all anti-pig antibodies. Reduction of the anti-pig lymphocyte and erythrocyte antibody titers by soluble oligosaccharides carrying terminal Galoc-epitopes was only partly successful. A 40–60% inhibition was achieved by 5–10 mg saccharide/ml serum and no clear inhibition difference between di- and trisaccharides was found. Inhibition of plasma obtained after 3–4 plasmapheresis treatments with soluble Galα1-di- and trisaccharides resulted in very low anti-pig titers. Therefore one feasible pretreatment procedure, before pig to human xenotransplantation could be plasmapheresis for major reduction of anti-pig antibody titer followed by neutralisation of the remaining antibodies by addition of soluble oligosaccharides or immunoadsorption with Galα-1-columns.     

In vitro removal of anti-HLA IgG antibodies from highly sensitized transplant recipients by immunoadsorption with protein A and protein G sepharose columns: a comparison

In the present study we compared the capabilities of sepharose-bound protein A versus protein G columns to remove in vitro lymphocytotoxic anti-HLA antibodies from sera of four highly sensitized renal transplant recipients (PRA70%). In none of the patients were protein A sepharose columns capable of completely removing anti-HLA antibodies, as demonstrated by the presence of residual alloreactive lymphocytotoxic activity in IgG 3 antibodies containing fractions eluted at pH 7. In contrast, no residual anti-HLA lymphocytotoxic antibody activity was found in fractions eluted at pH 7 from protein G columns. These data demonstrate that: (1) IgG 3 antibodies can be partly responsible for lymphocytotoxic anti-HLA reactivity in some sensitized renal transplant recipients and (2) at least in this patient category, in vitro immunoadsorption with protein G is more efficient than protein A sepharose columns in completely removing anti-HLA IgG antibodies from sera.     

Modulation of Idiotypic and Antiidiotypic Immunoglobulin G Responses in an Alloimmune Thrombocytopenic Patient Associated with Extracorporeal Protein A Immunoadsorption

Abstract: In the present case study, a patient with Non-Hodgkins Lymphoma underwent combination chemotherapy resulting in severe pancytopenia requiring transfusion support with blood products. The patient became refractory to random donor platelet transfusions and subsequently received five immunoadsorption treatments. The patient's clinical response to immunoadsorption therapy was assessed by monitoring platelet transfusion recovery and survival. In addition, changes in antibody responses were assessed. Early during the course of immunoadsorption therapy, antiplatelet immunoglobulin G (IgG) alloantibody was detected. There was a decline in antiplatelet IgG alloantibody levels by the last immunoadsorption treatment associated with increases in platelet corrected count increments after completion of immunoadsorption therapy. In addition, elevated levels of antiidiotypic IgG antibody detected early during the course of therapy were significantly reduced by the last immunoadsorption treatment. This case study suggests that specific alloimmune idiotypic IgG antibody and corresponding antiidiotypic IgG antibody responses may be modulated in association with extracorporeal immunoadsorption employing protein A/silica columns.     

Changes of Circulating Antibody Levels Induced by ABO Antibody Adsorption for ABO-Incompatible Kidney Transplantation

ABO-incompatible kidney transplantation using immunoadsorption to remove anti-A/B antibodies has become a successful clinical practice. Since the data on the specificity of the ABO columns are controversial, the present study assessed the efficiency and specificity of the ABO immunoadsorption, the effect on total immunoglobulins and antibodies previously induced by vaccination. Anti-A/B antibodies were measured by agglutination and ABO flow cytometry, total IgG/IgM, carbohydrate- and protein-specific antibodies by nephelometry and ELISA. The first immunoadsorption not only efficiently reduced donor-specific anti-A/B IgM (81%) and IgG (56%) but also reduced compatible anti-A/B IgM (59%) and IgG (34%). The measurements of antidonor A/B antibodies by direct agglutination (IgM) or flow cytometry better represented the effective antibody levels than the indirect agglutination test (IgG). The median reduction of total IgM and total IgG levels after a single immunoadsorption was 34% and 18%, respectively. Antibodies against pneumococcus and haemophilus polysaccharide antigens were significantly reduced, whereas antitetanus and antidiphtheria protein antibodies were not affected. Intravenous immunoglobulin administration restored the protective anticarbohydrate antibody levels. In summary, immunoadsorption efficiently removed antidonor A/B antibodies, but was not specific for A/B antigens. Anti-A/B antibody levels as determined by ABO flow cytometry are useful to establish the minimal number of immunoadsorptions needed for successful ABO-incompatible transplantation.     

Reduction in Platelet-Binding Immunoglobulins and Improvement in Platelet Counts in Patients with HIV-Associated Idiopathic Thrombocytopenia Purpura (ITP) Following Extracorporeal Immunoadsorption of Plasma Over Staphylococcal Protein A-Silica

Platelet-directed antibodies and circulating immune complexes (CIC) were removed from plasma of patients with human immunodeficiency virus (HIV) infection and idiopathic thrombocytopenic purpura (ITP) by extracorporeal immunoadsorption using columns of Staphylococcal protein A-silica (Prosorba columns). In addition, stimulation of a broadly cross-reactive anti-F(ab')2 antibody response was observed. These antibodies also appeared to play a role in the additional removal of platelet-directed immunoglobulins (Igs) and CIC from plasma. Removal of these components from plasma was associated with diminishing levels of antibodies and CIC on patient platelets and significant increases in platelet counts. Extracorporeal immunoadsorption of IgG and CIC from plasma is a beneficial new treatment modality for HIV-associated ITP.     

Differential inhibitory effects of intravenous immunoglobulin preparations on HLA-alloantibodies in vitro

BACKGROUND: Treatment of allosensitized patients with intravenously administered pooled immunoglobulin preparations (IVIG) may lead to a long-lasting reduction of anti-HLA alloantibody titers. An inhibitory response of IVIG preparations on lymphocytotoxicity is suggested to depend on IgG and to predict a successful reduction of anti-HLA alloantibodies upon the administration of high-dose IVIG in vivo. METHODS: In this study, we evaluated different IVIG preparations for their in vitro inhibitory capacity on lymphocytotoxicity and binding of anti-HLA alloantibodies to purified HLA antigens. For that purpose sera from 24 highly sensitized patients awaiting kidney transplantation and serological HLA testing reagents were used. Panel-reactive antibody (PRA) determinations using standard complement-dependent cytotoxicity testing and anti-HLA alloantibody determination by ELISA were carried out in the presence and absence of 50% (v/v) IVIG. RESULTS: The addition of IgG-containing IVIG preparations gave only a moderate inhibitory response judging from the average decrease of PRA levels (absolute DeltaPRA range: -2% to 16%), whereas the largest inhibition of lymphocytotoxicity was seen after the addition of IgM/IgA-containing IVIG preparations (absolute DeltaPRA range: 19% to 44%). For both IgG and IgM/IgA-containing IVIG preparations, the reduction of lymphocytotoxicity occurred in a dose-dependent fashion without a preference for particular anti-HLA class I antibody specificities. Significantly lower inhibitory effects on anti-HLA antibody reactivity were observed when the effects of IVIG preparations were monitored by ELISA (absolute DeltaPRA range: 7% to 22%). CONCLUSIONS: Our data suggest that the immunomodulatory capacity is largely caused by the IgM/IgA fraction of IVIG when analyzed by lymphocytotoxicity. The different effect on ELISA versus complement-dependent cytotoxicity testing suggests that interactions of IVIG with complement rather than anti-idiotypic antibodies may contribute to the inhibitory effects of IVIG in vitro.     

Complement Activation during Tryptophan Immunoadsorption Treatment

Abstract: Antibodies against human lymphocyte antigens (HLA) are frequently seen among patients undergoing repeated renal transplantations. Graft survival can be improved by eliminating these antibodies by plasmapheresis before transplantation. In this study, we have tried a new extracorporeal procedure to remove the anti-HLA antibodies. An immunoadsorption column (IM-TR) with a matrix of polyvinyl alcohol (PVA) gel conjugated with a hydrophobic amino acid tryptophan was utilized. Previous results have shown that repeated IM-TR treatments are at least equally effective as plasmapheresis in reducing levels of specific immunoglobulins in treated patients. In this study, 7 HLA-immunized patients were treated before renal transplantation. Each patient was subjected to a total of 12 treatment sessions divided into 3 sessions per week. After each treatment session, the reduction of the immunoglobulins was less than what has been reported for plasmapheresis. This suggests that mechanisms other than immunoglobulin depletion are involved in the reduction of the total immunoglobulin levels. The IM-TR treatment resulted in a strong complement activation triggered by the alternative pathway. Since the adsorbed plasma was returned to the patient, exceedingly high levels of the activation fragment C3d (C3dg) were found in plasma during and after the treatment. We conclude that the extensive generation of C3dg may be one of the factors that plays a role in the reduction of the antibody levels since the C3dg fragment has been shown to down-regulate the immune response.     

Antibody removal and subsequent transplantation of a highly sensitised paediatric renal patient

We report a successful renal transplant in a highly sensitised paediatric recipient following removal of HLA-specific antibodies by extracorporeal immunoadsorption. The immediate pretransplant cytotoxic titre against the donor was greater than 1:512; this was reduced to negativity by two immunoadsorption sessions prior to transplant surgery. We also describe the presence of unexpected non-HLA-specific antibody activities in this immunoadsorbed patient.     

Panel reactive antibody positivity and associated HLA antibodies in Turkish renal transplant candidates

Pre- and post-renal transplantation panel reactive antibody (PRA) screening is associated with increased incidence of hyperacute or acute graft rejection and graft loss. This study was designed to find any relationship PRA sensitization and associated human leukocyte antigen (HLA)-specific antibodies in Turkish renal transplant candidates. We included 340 patients who were in the renal transplantation waiting list in the study. We determined PRA sensitization ratio and the associated anti-HLA IgG antibody distribution of the patient group. The PRA testing was currently performed and levels above 30% were accepted to be positive. The PRA class I positivity was determined in 24 (7%) and class II in 34 (10%) of the patients. The most frequent HLA antibodies for class I were B56, A2, A34, A1, A23, A24 and B61; and for class II were DR11, DR14, DQ7, DR10, DQ5, DR1 and DR7, respectively. From these, the increase of the numbers of anti-HLA class II antibodies was significantly correlated with the increase of PRA sensitization ratio. In conclusion, the identification of the associated HLA-specific antibodies and correlation with the Turkish population HLA antigen distribution will identify the high-risk patients who are candidates for transplantation.     

BLyS neutralization results in selective anti-HLA alloantibody depletion without successful desensitization

Pre-existing anti-HLA allo-antibodies (allo-Abs) are a major barrier to successful kidney transplantation, resulting in an elevated risk for antibody-mediated rejection (AMR) and eventual graft loss. The cytokine B lymphocyte stimulator (BLyS) promotes B cell maturation and plasma cell survival; consequently, anti-BLyS therapy represents a potential therapeutic opportunity in diminishing pre-existing allo-Abs. Here we report that in our 1-year pilot trial, BLyS neutralization failed to reduce total anti-HLA allo-Ab levels in highly sensitized candidates awaiting kidney transplant in a clinically meaningful way. Additionally, we performed a post hoc analysis using sera from trial candidates which revealed selective depletion of anti-HLA class I and class II Abs in response to belimumab treatment, restricted to certain allele specificities and IgG subclasses. Altogether, we observed that BLyS blockade only results in selective depletion of anti-HLA Abs recognizing a few discrete HLA allele specificities.     

Anti-HLA antibody binding to hla class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome

OBJECTIVE: Development of anti-HLA antibodies is associated with development of bronchiolitis obliterans syndrome after lung transplantation. We sought to determine the mechanism by which anti-HLA antibodies affect the development of bronchiolitis obliterans syndrome. We postulated that anti-HLA antibodies bind to the donor lung epithelium and stimulate phosphorylation and proliferation. METHODS: The A549 lung epithelial carcinoma cell line was cultured in serum-deficient medium to produce static growth. Then the cells were treated with anti-HLA sera from lung transplant recipients, pooled anti-HLA serum from highly sensitized patients, or normal human serum. The cells were also treated with the W6/32 mouse anti-HLA class I monoclonal antibody or control mouse IgG. Tritiated thymidine uptake was determined at 24, 48, and 72 hours. In parallel experiments the cells were treated as described above, and the levels of tyrosine phosphorylation were determined by Western blot analysis. RESULTS: Cells treated with anti-HLA serum or the W6/32 monoclonal antibody exhibited significantly greater proliferation and tyrosine phosphorylation of proteins of approximately 170, 130, 110, and 70 kd compared with cells treated with normal human serum or mouse IgG, respectively. CONCLUSIONS: These data indicate that anti-HLA antibodies have the ability to stimulate airway epithelial cell proliferation and that they may play an important role in the development of bronchiolitis obliterans syndrome. Prevention of HLA sensitization and immunosuppression with agents capable of blocking indirect antigen presentation and the humoral immune response against the allograft may be pivotal in preventing the development of bronchiolitis obliterans syndrome after lung transplantation.     

Immunodepletion in xenotransplantation

Xenograft transplantation is perhaps the most immunologically difficult problem in transplantation today. An overwhelming hyperacute rejection reaction (HAR) occurs within minutes of organ implantation. Preformed antibodies are thought to initiate this process. We used a pig-to-dog renal xenograft transplant model and investigated methods of decreasing the severity of hyperacute rejection. Female pigs weighing 15-20 kg were used as donors. Recipients were mongrel dogs weighing 15-25 kg. Experimental dogs were all given a number of treatments of IgG depletion using an antibody removal system (Dupont-Excorim). This machine immunoadsorbs plasma against a column containing immobilized staphylococcal protein A, which is known to bind the IgG Fc receptor. An 84% reduction in the IgG levels and a 71% reduction in IgM levels was achieved. Postoperative assessment was made of urine output, time to onset of HAR, and histopathological examination of the rejected kidneys. Although cross-matches between donor lymphocytes and recipient sera remained strongly positive in the treated dogs, there was a two- to fourfold reduction in the titers. The time to onset of HAR was prolonged in the experimental group, and the urine output was increased slightly. The histopathologic changes in the experimental group generally showed signs of HAR, but of less intensity than in the nonimmunodepleted control group.     

Different responses of human anti-HLA and anti-αgal antibody to long-term intravenous immunoglobulin therapy

Concentrated human immunoglobulin (IVIG) has been administered intravenously in the treatment of autoimmune disorders and to reduce anti-HLA antibodies in highly sensitized patients awaiting organ transplantation. It has also been shown, in experimental animals, to prevent the hyperacute rejection of discordant xenografts, possibly by anticomplement activity. The aim of the present study was to assess the effect of IVIG therapy on both acquired anti-HLA antibodies and natural antigalactose α1–3 galactose (αGal) antibodies in five patients awaiting heart transplantation. Five patients placed on mechanical circulatory support who had developed high HLA panel-reactive antibodies (PRA) or in whom the percentage of PRA was increasing rapidly were treated weekly with 500 mg/kg IVIG, which contained 1% of anti-αGal IgG. Levels of PRA, anti-αGal IgG and IgM, and serum cytotoxicity to pig cells were measured before, during, and after therapy. PRA percentages in the five patients were initially 85%, 53%, 23%, 19% and 19% (mean 39%). Mean PRA fell by 66% after 3 months of therapy (to a mean PRA of 14%), and by 96% after 6 months therapy (to a mean PRA of 2%). Anti-αGal antibody levels and serum cytotoxicity to pig aortic endothelial cells did not change significantly. These results confirm the effectiveness of IVIG therapy in reducing PRA in HLA highly sensitized patients. It is likely that IVIG does not contain the relevant anti-HLA antibody, resulting in an accelerated catabolism of native alloantibodies. However, as IVIG contains a normal level of anti-αGal IgG, catabolism of anti-αGal IgG is not modified, as it is being continuously replaced. To achieve a decrease in the anti-αGal IgG level it would be necessary to use IVIG depleted of this antibody.     

Rejection of a kidney transplant does not always lead to priming of cytotoxic T cells against mismatched donor HLA class I antigens

BACKGROUND: Previous studies showed that graft rejection is often associated with the presence of primed cytotoxic T cells (CTLs) with a high avidity for donor cells. Similar high avidity CTLs have been found in individuals who have formed IgG anti-HLA antibodies. The presence of such CTLs to a specific HLA mismatch is therefore considered to be a reflection of an activated immune system, and a contraindication for retransplantation with a donor sharing this particular HLA class I mismatch. METHODS: In our study we investigated whether patients have always primed CTLs against all individual HLA class I mismatches present on a rejected graft. Therefore, 14 patients who had undergone transplantectomy after irreversible kidney graft rejection were analyzed with respect to donor-specific CTLp frequencies and the presence or absence of high avidity CTLs directed against HLA class I mismatches present on the rejected graft. RESULTS: Patients, who have not formed anti-HLA antibodies against the donor have mainly naive CTLs. Most of the patients, that have developed IgG anti-HLA antibodies against a donor mismatch, have primed CTLs directed against that particular mismatch. However, patients with IgM anti-HLA antibodies only, and patients with IgG anti-HLA antibodies in historical sera but no IgG anti-HLA antibodies in current sera, have mainly naive CTLs against the donor HLA mismatch. CONCLUSION: Our results suggest that it is not always necessary to exclude repeated HLA class I mismatches for a subsequent transplantation. In addition to good anti-HLA antibody screening, the CTLp-assay may be a useful tool for donor-selection in retransplant candidates.     

Clinical importance of anti-human leukocyte antigen-specific antibody concentration in performing calculated panel reactive antibody and virtual crossmatches

BACKGROUND: Highly sensitized patients develop multi-human leukocyte antigen (HLA) specific antibodies. This study measures concentrations of anti-HLA antibodies in multispecific sera by converting fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units. This was used to determine MESF units required for a positive T and B flow crossmatches (FLXM). METHODS: MESF values of negative controls and sera from patients devoid of HLA antibodies were measured by FLXM and flow panel reactive antibody (PRA) screening beads. Fluorescence intensity values of anti-HLA specific antibodies determined by FlowPRA single antigen beads of highly sensitized patients were converted into MESF units. In addition, endpoint titers, MESF units, and % PRA of 26 sera were established. RESULTS: MESF analysis accurately predicted the outcomes of 100% of T and B FLXM of sera with strong (MESF units>18,000) donor-specific antibody (DSA). The predictive values of T and B FLXM declined to 95% and 88% with weak DSA (6,000 MESF<10,000). Endpoint titers of sera from highly sensitized patients ranged from 1:512 to 1:8 with corresponding MESF values of 452,596 to 20,000 units. However, there was no statistical difference in PRA values among these sera (95%-100%). We successfully transplanted five patients who had weak donor-specific HLA antibodies (MESF units>2,000). The graft survival at 1 year was 100% and there was no evidence of DSA posttransplant. CONCLUSION: MESF analysis is both a time and cost efficient way of measuring antibody strength. The strength of the antibody present in the sera of transplant candidates is critical for crossmatch prediction.     

Immune Modulation Associated with Extracorporeal Immunoadsorption Treatments Utilizing Protein A/Silica Columns

Abstract: The Prosorba column is designed for the removal of IgG and IgG containing immune complexes from plasma. Clinical studies employing patients presenting with idiopathic thrombocytopenic purpura (ITP) indicate that this new form of therapy is effective in approximately 40% of treated patients. Responding patients exhibit a significant increase in platelet numbers associated with decreases in antiplatelet antibody and immune complexes suggesting the induction of immune modulation. Preliminary studies indicate that ITP patients presenting with antiplatelet IgG antibody are those most likely to respond. In addition, this subgroup of ITP patients also exhibit elevated levels of antiidiotypic IgG antibody, which may contribute to an exacerbation of the autoimmune process due to antigen mimicry of the platelet autoantigen. Interestingly, antiidiotypic IgG antibody levels appear to decrease in association with antiplatelet IgG autoantibody levels suggesting that removal of immune complexes composed of IgG autoantibody and platelet autoantigen and/or antiidiotypic IgG antibody may be related to the observed clinical responses. Additional studies with alloimmune patients refractory to platelet transfusion suggest that transfused platelet retention time may be increased as a consequence of immunoadsorption therapy. This clinical response appears to be related to decreases in IgG alloantibody, again suggesting the induction of immune modulation. Alloimmune thrombocytopenic patients also appear to present with elevated levels of antiidiotypic IgG antibody which may contribute to an exacerbation of the alloimmune process due to antigen mimicry of platelet alloantigen(s). Preliminary studies indicate that both IgG alloantibody and corresponding antiidiotypic IgG antibody levels appear to decrease during immunoadsorption therapy, which suggests that removal of these antibodies, possibly in the form of immune complexes, may be related to clinical responses. Finally, studies in rheumatoid arthritis patients suggest that immunoadsorption therapy may be of clinical benefit in this autoimmune disorder. Consistent with the results observed above, preliminary studies in patients responding to immunoadsorption treatments again suggest that there is a concomitant decrease in idiotypic IgG (rheumatoid factor) and antiidiotypic IgG antibodies levels during therapy.     

Angiotensin-converting enzyme genotype is a predictive factor in the peak panel-reactive antibody response

The presence of a high panel-reactive antibody (PRA) level represents an independent risk factor for early graft failure and chronic allograft dysfunction. It has also been reported that patients with the ACE-DD and AGT-AA genotypes display poorer chronic allograft function. We investigated the effects of gene polymorphisms of the renin angiotensin system (RAS) on anti-HLA antibody production among renal transplant candidates. Genotyping was performed on 133 dialysis patients for the ACE (I/D) and AGT (M235T) as well as the type 1 (A1166C) and type 2 (A1223G) angiotensin II receptor genes. Patients with a peak PRA >/= 30% were considered to be positive for anti-HLA antibody (40.6% of 133 patients). Genetic polymorphisms of the RAS were not associated with anti-HLA antibody production at this PRA level. Another analysis comparing the 29 patients with a peak PRA >/=50% with the 104 patients with a peak PRA <50% showed that previous transplants, the presence of ACE-DD genotype, history of blood transfusions, and dialysis duration were all associated with the high levels of antibody production by univariant analysis. A multivariate analysis using a logistic regression model revealed previous transplants, the presence of ACE-DD genotype, and history of blood transfusions to be predictors of anti-HLA antibody production. The ACE-DD genotype is an important risk factor for higher PRA levels. This study suggests that genetic control of RAS activity correlates with production of anti-HLA antibodies, possibly explaining the relationship to chronic allograft outcome.     

Removal of lymphocytotoxic antibodies by pretransplant immunoadsorption therapy in highly sensitized renal transplant recipients

A high level of panel-reactive antibodies (PRA) in potential renal transplant recipients is associated with a long waiting time until transplantation and correlates inversely with graft outcome. We report our experience with the employment of immunoadsorption (IA) using a column composed to sepharose-bound staphylococcal protein A (which has a relatively selective affinity for binding IgG compared with other immunoglobulins) to decrease the PRA levels and expedite transplantation in 6 highly sensitized potential renal transplant recipients (1 primary and 5 awaiting second transplants). All patients had PRA levels of greater than or equal to 70% for a duration of 1 year prior to IA. Only patients with antibody specificity localized to 1 or 2 HLA A or B antigens were accepted for the study. IA procedures were performed on alternate days until a twofold decrease in antibody titer had occurred (maximum: 6 procedures). Repeat procedures were initiated if the HLA antibody titer returned to its baseline value. Intravenous cyclophosphamide (CY) (10 mg/kg/day every 3 weeks) and methylprednisolone (MP) (0.5 mg/kg/day) were provided as adjunctive immunosuppression until transplantation. A total of 44 immunoadsorption procedures were performed (27 primary and 17 repeat) with treatment of 2.49 +/- 0.02 plasma volumes per session. Serum IgG concentration decreased 95 +/- 3% and PRA activity decreased 75 +/- 16% after the primary treatment course. Four patients received cadaveric grafts within 3.7 +/- 1.2 months following the last IA procedure. Three grafts are functioning at 1 year, 8 months, and 8 weeks posttransplant. The remaining graft demonstrated primary nonfunction. All four patients had a past positive crossmatch using pre-IA sera with their respective donors. Patients not transplanted exhibited rapid resynthesis of IgG and a return of the PRA towards baseline levels within a few weeks after IA. We conclude that IA can effectively remove HLA antibodies and expedite graft availability in highly sensitized patients.