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Background Bullous pemphigoid (BP) is an autoimmune disease with bullous vesicles and an incidence of 0.2 to 1.4 per 100,000 inhabitants. Many studies have been published demonstrating the association of pemphigoid with HLA class II system alleles in different populations, however there are no data on the BP, one of the most heterogeneous in the world. Objective To typify HLA alleles in Brazilians with Bullous pemphigoid. Methods The study group included 17 Brazilian patients with a confirmed diagnosis of BP from a hospital in Sao Paulo city, southeast Brazil. DNA was extracted from peripheral blood using Qiagen kits and HLA A, B, C, DR and DQ typing was performed using polymerase chain reaction. The control group was composed of a database of 297 deceased donors from the city of Sao Paulo. The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA class I (A, B and C) and class II (DRB1, DQB1 and DQA1). Results Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population, with relative risks of 8.31 (2.46 to 28.16), 3.76 (1.81 to 7.79), 3.57 (1.53 to 8.33), and 4.02 (1.87 to 8.64), respectively (p

It has been related in orthopedic surgeries the HLA sensitization. Thus, we evaluate if the use of fresh-frozen homologous bone (FFHB) for dental implant placement induce anti-HLA sensitization. Six patients were treated with FFHB corticocancellous block grafts. After 6 months, bone biopsies were harvested during implant placement to allow histomorphometric analysis. Vital mineralized tissue (VMT), non-vital mineralized tissue (NVMT) and non-mineralized tissue (NMT) were quantified histomorphometrically. Peripheral blood was collected from the patients before FFHB placement and 6 months after the surgery for anti-HLA analysis. The histomorphometric analysis showed the presence of VMT, NVMT and NMT in 45.56 ± 15.72 %, 14.16 ± 13.39 % and 40.29 ± 12.60 %, respectively. The baseline and 6 months postoperative CTs revealed bone thickness in the order of 5.66 ± 0.67 mm and 8.71 ± 1.52 mm (3.05 ± 1.39 mm). The anti-HLA analysis revealed that two of the six patients (33.3 %) became sensitized, however this was not associated with any FFHB incorporation loss (p > 0.05). A total of 24 implants were placed all of which were osseointegrated after 6 months. Although FFHB-related HLA sensitization does not appear to affect bone incorporation when treating insufficient bone thickness for implant placement, further follow-up is required to determine whether there is an association between HLA sensitization and long-term graft survival.

Melkersson-Rosenthal syndrome (MRS) is a neuromucocutaneous disease that manifests by the triad of recurrent orofacial edema (frequently as cheilitis granulomatosa), relapsing facial paralysis and plicated tongue. The cause of MRS remains unknown, but genetic predisposal and a relationship with inflammatory bowel disease are suspected. The objective of this research was to compare the frequency of class I and II HLA alleles in patients with a confirmed diagnosis of MRS with those of a healthy control group. We conduct a case-control study and typed of HLA A, B, C, DR, and DQ using molecular techniques. The study included 36 patients with MRS and 297 patients in the control group. There was an increase in the expression of HLA A*02 (p = 0.0269; OR: 1,79 [1,045-2,973]), HLA DRB1*11 (p 0,0001; OR: 4,009 [2,214-7,277]), HLA DRB1*13 (not statistically significant) and HLA DQB1*03 (p = 0,0177; OR: 1,829 [1,122-2,978]) and low levels of HLA A*01 (p = 0.0046; OR: 0,097 [0,009-0,538]), HLA DRB1*04 (p = 0.0274; OR: 0,228 [0,053-0,844]), HLA DRB1*07 (p = 0,0091; OR: 0,183 [0,043-0,670]) and HLA DQB1*02 (p = 0.0051; OR: 0,312 [0,143-0,721]) in MRS patients compared with the control group. Crohn disease (CD) patients had disparate genetic profiles versus those with MRS. This single-institution study had a small cohort, because this disease is rare. Conclusions: There is a genetic predisposition toward MRS, involving associated and protective genes.

The impact of the kinetics of the anti-HLA antibodies after KTx on the occurrence of acute rejection as well as the better time-point to monitor anti-HLA Abs after transplantation is not completely defined. This prospective study followed 150 patients over 12 months after transplantation. Serum IgG anti-HLA Abs were detected by single antigen beads after typing donors and recipients for loci A, B, C, DR, and DQ. Before KTx, 89 patients did not present anti-HLA Abs and 2% developed “de novo” Abs during the 1st year, 39 patients were sensitized without DSAs, and 13% developed DSA after surgery; all of them presented ABMR. Sensitized patients presented higher acute rejection rates (36.4% versus 13.5%, p<0.001), although 60% of the patients did not present ABMR. Patients, in whom DSA-MFI decreased during the first two weeks after surgery, did not develop ABMR. Those who sustained their levels presented a rate of 22% of ABMR. 85% of patients developed ABMR when MFIs increased early after transplantation (which occurred in 30% of the DSA positive patients). In the ABMR group, we observed an iDSA-MFI sharp drop on the fourth day and then an increase between the 7th and 14th POD, which suggests DSA should be monitored at this moment in sensitized patients for better ABMR prediction.

To analyse the rate of clinical recurrences in Brazilian patients with Vogt-Koyanagi-Harada (VKH) disease after early high-dose corticosteroid treatment.Retrospective study including patients treated with early high-dose corticosteroids (prednisone, 1-1.5 mg/kg/day, or 3-day 1 g methylprednisolone pulsetherapy) within 1 month from disease onset followed by slow taper (at least 6 months). Patients with a minimum 12-month follow-up were subdivided based on the presence of disease recurrence or persistence after 6 months from initial presentation into: acute-resolved (AR, no recurrences), chronic-recurrent (CR), and chronic-recurrent with subretinal fibrosis (SRF). Recurrences were defined as the presence of clinical and/or fluorescein angiography findings.Twenty-nine patients (58 eyes) with a median follow-up of 65 months were included. Six (21 %), 11 (38 %) and 12 (41 %) patients were allocated to AR, CR, and SRF groups respectively. Though having received treatment within 1 month of onset, median time to initial treatment differed among groups (11, 15, and 25 days, in AR, CR, and SRF groups respectively). Intensity of immunosuppression, cataract development, and longer time to achieve logMAR visual acuity ≤0.8 differed significantly among the groups, being more severe in SRF group. HLA-DRB1*0405 allele followed the same trend, though not reaching significance (0.5 in AR group, 0.6 in CR, and 0.8 in SRF).VKH disease in Brazilian patients evolved to chronic-recurrent disease in 79 % of cases; 38 % developed subretinal fibrosis, in spite of similar initial treatment regimens. Time to initiate treatment influenced outcomes.

The purpose of this study was to sequentially monitor anti-HLA antibodies and correlate the results with antibody-mediated rejection (AMR), graft survival (GS), and graft function (GF). We collected sera from 111 kidney transplant recipients on transplant days 0, 7, 14, 30, 60, 90, 180, and 360 and analyzed PRA levels by ELISA. DSAs were analyzed by single-antigen beads in rejecting kidneys. At pre-transplant, 79.3% of the patients were non-sensitized (PRA = 0%) and 20.7% were sensitized (PRA > 1%). After transplant, patients were grouped by PRA profile: no anti-HLA antibodies pre- or post-transplant (group HLApre-/post-; n = 80); de novo anti-HLA antibodies post-transplant (group HLApre-/post+; n = 8); sensitized pre-transplant/increased PRA post-transplant (group HLApre+/post↑; n = 9); and sensitized pre-transplant/decreased PRA post-transplant (group HLApre+/post↓; n = 14). De novo anti-HLA antibodies were detected at 7-180 d. In sensitized patients, PRA levels changed within the first 30 d post-transplant. Incidence of AMR was higher in HLApre-/post+ and HLApre+/post↑ than in HLApre-/post-, and HLApre+/post↓ (p

Objective: The HLA haplotype has been associated with many autoimmune diseases, but no associations have been described in sepsis. This study aims to investigate the HLA system as a possible marker of genetic sepsis susceptibility. Methods: This is a prospective cohort study including patients admitted to an intensive care unit and healthy controls from a list of renal transplant donors. Patients with less 18 years of age; pregnant or HIV positive patients; those with metastatic malignancies or receiving chemotherapy; or with advanced liver disease; or with end-of-life conditions were excluded. The DNA was extracted from the whole blood and HLA haplotypes determined using MiliPlex® technology. Results: From October 2010 to October 2012, 1,121 patients were included (1,078 kidney donors, 20 patients admitted with severe sepsis and 23 with septic shock). HLA-A*31 positive subjects had increased risk of developing sepsis (OR 2.36, 95%CI 1.26-5.35). Considering a p value

Background Pemphigus vulgaris is a mucocutaneous blistering autoimmune disease that manifests as painful blisters or erosions on the skin and/or mucosal surfaces. IgG autoantibodies target desmoglein, playing a major role in disease pathogenesis. Genetic predisposal to pemphigus vulgaris, especially the HLA DR and DQ alleles, has been known since the 1980s. The unique constitution of the Brazilian population favors exploratory genetic studies. Methods The study group included 51 patients with a confirmed diagnosis of pemphigus vulgaris from a tertiary hospital in Sao Paulo city, Sao Paulo, southeast Brazil. DNA was extracted from peripheral blood, and HLA A, B, C, DR, and DQ typing was performed. The control group was composed of a database of 297 deceased donors from the city of Sao Paulo typed with the same method. The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA A, HLA B, HLA C, HLA DRB1, DQA1, and HLA DQB1. Results The alleles HLA-B*57, HLA-C*15, HLA-DRB1*04:02, HLA-DRB1*08:04, HLA-DRB1*14:01, DQA1*03:01, DQB1*03:02, and DQB1*05:03 were associated with susceptibility. Alleles HLA DRB1*04:02 and HLA-DRB1*14:01 and their respective haplotypes DRB1*04-DQA1*03:01-DQB1*03:02, and DRB1*14-DQA1*01:01-DQB1*05:03 conferred a risk of the disease. Conclusions The DRB1*04:02 and DQB1*05:03 alleles are associated with pemphigus vulgaris in our study as well as in various populations. The association with HLA-DRB1*08:04 in our study was confirmed to be specific to this allele and not to linkage disequilibrium to any adjacent gene. The association between HLA-B*57 and pemphigus vulgaris is reported for the first time in the present study.

Background: Identification of asthma phenotypes enables a better understanding and management of this heterogeneous disease. Studies have reported associations between human leukocyte antigens (HLA) and asthma in different populations, but the results have been inconclusive and they have rarely considered the distinct disease phenotypes. Our objective was to characterize allergic and nonallergic asthma phenotypes and evaluate possible associations with the HLA system. Methods: A total of 109 patients with asthma were prospectively followed during 2 years. They were divided into 2 groups, i.e., allergic and nonallergic asthma, according to their clinical history and skin prick test and serum-specific immunoglobulin E (IgE) results. The control group comprised 297 deceased donors of solid organs. Patients' features and HLA class I and II genotypes were assessed and compared. Results: This study showed different features between asthma phenotypes. Nonallergic patients were older at the onset of asthma symptoms and had a higher rate of intolerance to nonsteroidal anti-inflammatory drugs. Allergic patients had higher total serum IgE levels, reported atopic dermatitis and rhinoconjunctivitis more frequently, and, unexpectedly, had a greater disease severity. New associations between the HLA genotypes and allergic and nonallergic asthma were identified. The HLA-B*42, HLA-C*17, HLA-DPA1*03, and HLA-DPB1*105 genotypes were associated with allergic asthma and the HLA-B*48 genotype with the nonallergic phenotype. The presence of the haplotype HLA-DPA1*03 DQA*05 was associated with allergic asthma, and the presence of HLA-DPA1*03 and the absence of HLA-DQA*05 with nonallergic asthma. Conclusions: Allergic and nonallergic asthma have distinct phenotypic and genotypic features. New associations between asthma phenotypes and HLA class I and II were identified.

Background Sensitized patients (pts) may develop acute antibody-mediated rejection (AMR) due to preformed donor-specific antibodies, undetected by pre-transplant complement-dependent cytotoxicity (CDC) crossmatch (XM). We hypothesized that C4d staining in 1-h post-reperfusion biopsies (1-h Bx) could detect early complement activation in the renal allograft due to preformed donor-specific antibodies. Methods To test this hypothesis, renal transplants (n = 229) performed between June 2005 and December 2007 were entered into a prospective study of 1-h Bx and stained for C4d by immunofluorescence. Transplants were performed against a negative T-cell CDC-XM with the exception of three cases with a positive B-cell XM. Results All 229 1-h Bx stained negative for C4d. Fourteen pts (6%) developed AMR. None of the 14 protocol 1-h Bx stained positive for C4d in peritubular capillaries (PTC). However, all indication biopsies-that diagnosed AMR-performed at a median of 8 days after transplantation stained for C4d in PTC. Conclusions These data show that C4d staining in 1-h Bx is, in general, not useful for the early detection of AMR when CDC-XM is negative.

Há crescente evidência de que os anticorpos direcionados contra antígenos não-HLA presentes em células endoteliais estão associados à rejeição ao enxerto. Objetivos: 1. Verificar a presença de anticorpos anti-célula endotelial (AACE) nos eluatos de rins nefrectomizados após rejeição mediada por anticorpos não anti-HLA; 2. Verificar a possibilidade de detectar AACE em soros coletados no pré-transplante e antes da rejeição; 3. Verificar o potencial efeito inibitório da IVIG in vitro sobre os AACE. Métodos: Soros (absorvidos com pool de plaquetas) e eluatos provenientes de 12 aloenxertos renais foram testados por citometria de fluxo contra a linhagem EAHy.926 (hibridoma de célula epitelial com célula endotelial). Eluatos positivos foram testados com ou sem adição de imunoglobulina polivalente intravenosa (IVIg). Resultados: A ausência de anticorpos anti-HLA contra o doador foi verificada antes do transplante, da rejeição e antes e depois da transplantectomia, através de provas cruzadas utilizando a técnica de citotoxicidade dependente de complemento (CDC) com e sem anti-globulina humana. Em nove eluatos foram detectados anticorpos reativos contra a linhagem. Em 7/9 casos a marcação de C4d foi negativa. Tais anticorpos não foram detectados no soro pré-transplante. Contudo, 2/4 soros pré-rejeição foram positivos para AACE, e um foi considerado positivo fraco. Em 9 casos, a adição de IVIg resultou em forte diminuição na ligação dos AACE. Conclusões: Os AACE parecem estar envolvidos no processo de rejeição humoral. A detecção de tais anticorpos no soro pré-rejeição abre a possibilidade de um monitoramento imune. A utilização de IVIg para bloqueio da ligação desses anticorpos com a célula-alvo pode funcionar como uma possível terapia na rejeição humoral causada por AACE.

Although aromatic anticonvulsants are usually well tolerated, they can cause cutaneous adverse drug reactions in up to 10% of patients. The clinical manifestations of the antiepileptics-induced hypersensitivity reactions (AHR) vary from mild skin rashes to severe cutaneous drug adverse reactions which are related to high mortality and significant morbidity. Genetic polymorphisms in cytochrome P450 genes are associated with altered enzymatic activity and may contribute to the risk of AHR. Here we present a case-control study in which we genotyped SNPs of CYP2C19, 2C9 and 3A5 of 55 individuals with varying severities of AHR, 83 tolerant, and 366 healthy control subjects from Sao Paulo, Brazil. Clinical characterization was based on standardized scoring systems and drug patch test. All in vivo investigation followed the ENDA (European Network of Drug Allergy) recommendations. Genotype was determined by real time PCR using peripheral blood DNA as a template. Of all 504 subjects, 65% were females, 45% self-identified as Afro-American, 38% as Caucasian and 17% as having non-African mixed ascendancy. Amongst 55 subjects with AHR, 44 had severe cutaneous drug adverse reactions. Of the 46 drug patch tests performed, 29 (63%) were positive. We found a strong association between the absence of CYP3A5*3 and tolerant subjects when compared to AHR (p = 0.0002, OR = 5.28 [CI95% 2.09–14.84]). None of our groups presented positive association with CYP2C19 and 2C9 polymorphisms, however, both SNPs contributed to separation of cases and tolerants in a Classification and Regression Tree. Our findings indicate that drug metabolism genes can contribute in the tolerability of antiepileptics. CYP3A5*3 is the most prevalent CYP3A5 allele associated with reduced enzymatic function. The current study provides evidence that normal CYP3A5 activity might be a protective factor to aromatic antiepileptics-induced hypersensitivity reactions in Brazilian subjects.

Objetivo: Haplótipos do HLA têm sido associados a muitas doenças autoimunes, mas não foi descrita qualquer associação na sepse. O objetivo desse estudo é investigar o sistema HLA como um possível marcador de suscetibilidade genética à sepse. Métodos: Estudo prospectivo de coorte, incluindo pacientes admitidos em unidade de terapia intensiva e controles-saudáveis obtidos em lista de doadores de transplante renal. Foram excluídos pacientes abaixo dos 18 anos de idade, gestantes ou HIV positivos, pacientes com doença maligna metastática ou sob quimioterapia, pacientes com hepatopatia avançada, com condições de fim de vida. O DNA foi extraído de sangue total, e a haplotipagem de HLA foi realizada com a tecnologia MiliPlex®. Resultados: Foram incluídos 1.121 pacientes (1.078 doadores de rim, 20 pacientes com sepse grave e 23 pacientes admitidos por choque séptico) entre outubro de 2010 e outubro de 2012. Os participantes positivos para HLA-A*31 tiveram risco aumentado de desenvolver sepse (OR: 2,36 IC95%: 1,26-5,35). Não foi identificada outra associação significativa, quando considerado como nível de significância o valor de p

Preformed donor-specific human leukocyte antigen (HLA) antibodies have been associated with allograft dysfunction and failure. However, recipients of HLA-identical kidneys can develop acute humoral rejection, implicating putative pathogenic antibodies that are directed against non-HLA antigens. We investigated the presence of endothelial cell-reactive antibodies in 11 patients who experienced early loss of their transplanted kidneys owing to humoral rejection and 1 loss from renal venal thrombosis. We examined the potential efficacy of intravenous immunoglobulin to block the binding of these antibodies, as previously suggested for anti-HLA antibodies.

Introduction. We sought to evaluate 2 single-nucleotide polymorphisms (SNPs) in the C-reactive protein (CRP) gene promoter region for their effects on CRP levels in chronic kidney disease (CKD) patients before and after a successful kidney transplantation. Methods. Fifty CKD patients were evaluated before and at the first and second years after the graft. Two SNPs were studied, a bi-allelic (GiA) at the 409 and a tri-allelic (CiTiA) variation at the 390 position in the CRP gene. Results. All patients presented the 409GG genotype. At the 390 position, the “A” allele was not found; there were 15 “CC” patients, 11 “TT” patients, and 24 “CT” patients. CRP levels were different among patients with various genotypes (P .019). Also the presence of the allele “T” was sufficient to determine differences in CRP levels both in pretransplantation (P .045) and at 1 year posttransplantation (P .011), but not at the second year (P .448). Conclusion. SNPs at the 390 position of the CRP gene promoter region influence CRP basal levels in such a way that the “C” allele correlated with the lowest and the “T” with the highest. We did not observe this influence in our patients at the second year posttransplantation.

Objective: To identify promiscuous and potentially protective human CD4 T-cell epitopes in most conserved regions within the protein-coding genome of HIV-1 clade B consensus sequence.Design: We used the TEPITOPE algorithm to screen the most conserved regions of the whole genome of the HIV-1 subtype B consensus sequence to identify promiscuous human CD4 T-cell epitopes in HIV-1. the actual promiscuity of HLA binding of the 18 selected peptides was assessed by binding assays to nine prevalent HLA-DR molecules. Synthetic peptides were tested with interferon-gamma ELISPOT assays on peripheral blood mononuclear cells (PBMC) from 38 HIV-1 infected patients and eight uninfected controls.Results: Most peptides bound to multiple HLA-DR molecules. PBMC from 91% of chronically HIV-1 infected patients recognized at least one of the promiscuous peptides, while none of the healthy controls recognized peptides. All 18 peptides were recognized, and each peptide was recognized by at least 18% of patients; 44% of the patients recognized five or more peptides. This response was not associated to particular HLA-DR alleles. Similar responses were obtained in CD8 T-cell-depleted PBMC.Conclusion: in silico prediction of promiscuous epitopes led to the identification of naturally immunodominant CD4 T-cell epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to HIV-1. This combination of CD4 T-cell epitopes-11 of them not described before-may have the potential for inclusion in a vaccine against HIV-1, allowing the immunization of genetically distinct populations. (c) 2006 Lippincott Williams & Wilkins. Univ São Paulo, Sch Med, Heart Inst Incor, Immunol Lab, BR-05403000 São Paulo, Brazil Univ São Paulo, Sch Med, Div Clin Immunol & Allergy, Dept Med, BR-05403000 São Paulo, Brazil Univ São Paulo, Sch Med, Dept Infect Dis, BR-05403000 São Paulo, Brazil Univ São Paulo, Inst Trop Med, BR-05403000 São Paulo, Brazil Hoffmann La Roche Inc, Dept Genom & Informat Sci, Nutley, NJ 07110 USA Universidade Federal de São Paulo, Div Infect & Parasit Dis, São Paulo, Brazil La Jolla Inst Allergy & Immunol, San Diego, CA USA Universidade Federal de São Paulo, Div Infect & Parasit Dis, São Paulo, Brazil Web of Science

Background This study evaluated the influence of circulating anti-HLA antibodies on outcomes of 97 liver allografts from deceased donors. Methods Human leukocyte antigen (HLA) antibody screening was performed by both complement-dependent cytotoxicity (CDC) and multiparameter Luminex microsphere-based assays (Luminex assay). Results The agreements between T- and B- cell CDC and Luminex assays were 67% and 77% for pre- and posttransplant specimens, respectively. Graft dysfunction was not associated with either positive pretransplant CDC or Luminex panel-reactive antibody (PRA) values. Likewise, positive posttransplant T- or B- cell CDC PRA values were not associated with graft dysfunction. In contrast, posttransplant Luminex PRA values were significantly higher among patients with graft dysfunction compared with subjects with good outcomes ( P = .017). Conclusion Posttransplant monitoring of HLA antibodies with Luminex methodology allowed identification of patients at high-risk for poor graft outcomes.

Background The cytochrome P450 2C9 and 3A5 enzymes are predominantly found in the human liver, and have important functions in the metabolism of antiepileptics. Genetic polymorphisms in these genes are associated with altered enzymatic activities and may give rise to the risk of drug hypersensitivity. Therefore, we analyze the association between CBZ hypersensitivity reactions (CHR) and polymorphisms of CYP2C9 and 3A5 in a population of Sao Paulo, Brazil. Methods Case-control study in which we genotyped the SNP of CYP2C9 (rs1057910, rs1799853) and 3A5 (rs776746) of samples obtained from 52 subjects with varying severities CHR, 82 tolerants, and 366 control subjects, all from Sao Paulo, Brazil. According to the alleles presente, each subject was classified as normal, decrease or increased funcion for the enzyme. The phenotype was evaluated based on standardized scoring systems using an adapted ENDA (European Network of Drug Allergy) questionnaire, medical records and on the clinical follow-up. The patch test with the culprit drug was performed according the ENDA recommendations.

Identification of risk factors influencing graft survival may lead to the development of models to predict graft outcome. Such models may provide guidance for immunosuppressive therapy, measure posttransplantation outcome, and eventually improve graft survival in high-risk patients. A major risk factor influencing graft survival is allosensitization. However, due to the lack of standardization of lymphocytotoxicity assays, the detection of alloantibodies utilizing this current methodology may not correlate with posttransplant events. Recently, a novel standardized enzyme-linked immunosorbent assay (ELISA) for the detection of anti-HLA class I IgG antibodies was developed. To evaluate the predictive value of this diagnostic test, a retrospective analysis of 124 renal allograft recipients with an 18-month follow-up time was performed. A highly significant (P=0.01) correlation between pre-transplant ELISA panel reactive antibody (PRA) results and graft loss was observed. Patients with pre-transplant ELISA PRA of >10% had a three times higher risk of graft loss compared with patients who tested negative. No such correlation was observed with complement-dependent cytotoxicity results independent of the reduction of IgM antibodies with dithiothreitol. Similarly, a highly significant correlation of ELISA results with the occurrence of early graft dysfunction was observed. Almost all patients (88%) with a pretransplant ELISA PRA of >50% required posttransplant dialysis, compared with 45% of patients with a pretransplant ELISA PRA of 10-50% and 27% of patients with a pretransplant ELISA PRA of