Helcio Rodrigues

The impact of pretransplant donor-specific antibodies on graft outcome in renal transplantation: a six-year follow-up study

OBJECTIVE: The significance of pretransplant, donor-specific antibodies on long-term patient outcomes is a subject of debate. This study evaluated the impact and the presence or absence of donor-specific antibodies after kidney transplantation on short- and long-term graft outcomes. METHODS: We analyzed the frequency and dynamics of pretransplant donor-specific antibodies following renal transplantation from a randomized trial that was conducted from 2002 to 2004 and correlated these findings with patient outcomes through 2009. Transplants were performed against a complement-dependent T- and B-negative crossmatch. Pre- and posttransplant sera were available from 94 of the 118 patients (80%). Antibodies were detected using a solid-phase (Luminex®), single-bead assay, and all tests were performed simultaneously. RESULTS: Sixteen patients exhibited pretransplant donor-specific antibodies, but only 3 of these patients (19%) developed antibody-mediated rejection and 2 of them experienced early graft losses. Excluding these 2 losses, 6 of 14 patients exhibited donor-specific antibodies at the final follow-up exam, whereas 8 of these patients (57%) exhibited complete clearance of the donor-specific antibodies. Five other patients developed “de novo” posttransplant donor-specific antibodies. Death-censored graft survival was similar in patients with pretransplant donor-specific and non-donor-specific antibodies after a mean follow-up period of 70 months. CONCLUSION: Pretransplant donor-specific antibodies with a negative complement-dependent cytotoxicity crossmatch are associated with a risk for the development of antibody-mediated rejection, although survival rates are similar when patients transpose the first months after receiving the graft. Our data also suggest that early posttransplant donor-specific antibody monitoring should increase knowledge of antibody dynamics and their impact on long-term graft outcome.

Non–Human Leukocyte Antigen Antibodies Reactive with Endothelial Cells Could Be Involved in Early Loss of Renal Allografts

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.

Dynamics of anti-human leukocyte antigen antibodies after renal transplantation and their impact on graft outcome.

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 < 0.001) groups. One‐yr death‐censored GS was 36% in group HLApre+/post↑, compared with 98%, 88% and 100% in groups HLApre−/post−, HLApre−/post+, and HLApre+/post↓, respectively (p < 0.001). Excluding first‐year graft losses, GF and GS were similar among the groups. In conclusion, post‐transplant antibody monitoring can identify recipients at higher risk of AMR.

HLA-DRB1*04:02, DRB1*08:04 and DRB1*14 alleles associated to pemphigus vulgaris in southeastern Brazilian population

We report here for the first time a strong association of the DRB1*08:04 allele and pemphigus vulgaris (PV) in a Brazilian highly admixed cohort. The authors recently performed a prospective case–control study, including 36 unrelated patients with PV from São Paulo, Brazil. All patients were diagnosed according to the standard criteria: (i) clinical features: a mucocutaneous blistering disorder; (ii) histopathology: a skin or a mucosal biopsy showing suprabasal acantholysis and clefting; and (iii) immunopathology: direct or indirect immunofluorescence to show IgG antibodies that bind to the intercellular spaces of the epidermis (1). Patients with neoplasia or paraneoplastic pemphigus (2) were not included. All patients were non-Jewish, and all except one patient had both parents and grandparents born in Brazil. DNA extraction was performed using the DTAB/CTAB method (3). Human leukocyte antigen (HLA) typing at the DRB1 locus was performed after DNA extraction using polymerase chain reaction (PCR) amplification with sequence-specific oligonucleotides (PCR-SSO) using LABType kits (One Lambda, Inc., Canoga Park, CA) (4, 5). HLA phenotypes were assigned to each individual based on the oligotyping results. Phenotypic and allelic frequencies were compared with those from a collected DNA bank of healthy volunteers donors registered in the national registry of bone marrow donors (REDOME) (6) from Sao Paulo, typed in the same laboratory between January 2008 and April 2009 using the same methods. One hundred sixty-two unrelated individuals were randomly chosen from the data bank (preserving the same ethnicity proportions of study group) and formed the control group. The allele frequencies found in the control group were similar to those reported by previous studies in samples of population from southeastern Brazil (7–9). Unlike other researchers, the authors of this study decided to not select individuals – cases or controls – from a homogeneous ethnic group, based on the premise that the Brazilian population is one of the most heterogeneous populations in the world as a result of five centuries of admixture between Europeans, Africans, and Amerindians, which produces, at the individual level, a significant dissociation of color and genomic ancestry (10). Moreover, evidence shows that the possible effect of population stratification is likely to be small in most situations and decreases as the number of admixed ethnicities increases (11, 12). Two-tailed Fisher’s exact test was used to detect significant deviations in allele frequency from controls; a P value ≤0.05 was considered a significant result. Odds ratios (OR) were calculated by the Woolf method (13) (or by the modified method described by Haldane (14)). Statistical analysis was performed using spss for Mac version 16.0 (SPSS Inc, Chicago, IL). A correction for multiple testing with the false discovery rate method (15, 16) was applied after the identification of alleles in PV patients displaying significant deviation in frequency from controls. The distribution of DRB1 genotypes in the patients and the controls was in Hardy–Weinberg equilibrium for patients (P = 0.82876) and for the controls (P = 0.83123). After correction for multiple comparisons, the null hypothesis was rejected for the association of PV with the alleles DRB1*04:02, DRB1*08:04, and DRB1*14, with OR (confidence interval 95%) 44.6 (19.2–103), 18.6 (8.3–41.7), and 4.8 (2.2–10.4), respectively. The authors report here for the first time a strong association of the DRB1*08:04 allele and PV in a Brazilian highly admixed cohort. Previous studies may have failed to identify this association because they did not include populations that have individuals of African origins. High-resolution typing of nearby loci DQA1 and DQB1 is necessary to differentiate if the association is inherent to the HLA-DRB1*08:04 allele or to a specific haplotype.

C4d staining in post-reperfusion renal biopsy is not useful for the early detection of antibody-mediated rejection when CDC crossmatching is negative

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.

The Absence of CYP3A5*3 Is a Protective Factor to Anticonvulsants Hypersensitivity Reactions: A Case-Control Study in Brazilian Subjects

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 São 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.

Study of the association between human leukocyte antigens (HLA) and pemphigus vulgaris in Brazilian patients

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.

HLA-A*31 como marcador de suscetibilidade genetica em sepse

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 <0.01, no other significant association was identified. Conclusion: HLA-A*31 expression is associated to risk of developing sepsis.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 <0.01, no other significant association was identified. Conclusion HLA-A*31 expression is associated to risk of developing sepsis.

Pre- and Posttransplant Monitoring of Alloantibodies by Complement-Dependent Cytotoxicity and Luminex Methodologies in Liver Transplantation

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.

The Kinetics of Anti-HLA Antibodies in the First Year after Kidney Transplantation: In Whom and When Should They Be Monitored?

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.