Carmen Rodriguez-Sainz

Risk factors for developing de novo autoimmune hepatitis associated with anti-glutathione S-transferase T1 antibodies after liver transplantation.

De novo autoimmune hepatitis (de novo AIH) is a rare form of graft dysfunction that develops after liver transplantation (LT) in patients transplanted for conditions other than autoimmune disorders. Although characterized by biochemical, serological, and histological features of AIH, de novo AIH is sometimes associated with atypical serum autoantibodies, many of which are directed against glutathione S‐transferase T1 (anti‐GSTT1). GSTT1 donor/recipient genotype mismatch has been suggested as a necessary condition for the appearance of autoantibodies and de novo AIH. However, clinically evident disease is not observed in all patients with anti‐GSTT1 antibodies. We examined the incidence of de novo AIH and its conditioning (risk) factors in patients with anti‐GSTT1 antibodies. Anti‐GSTT1 autoantibodies were detected in 29 of 419 [6.9%; 95% confidence interval (CI), 4.9–9.8] consecutive adult LT recipients with donor/recipient GSTT1 mismatch. Twenty of 27 assessable patients (74%) developed de novo AIH after a median follow‐up of 26 months (95% CI, 19.2–32.8). The probability of de novo AIH was 11%, 44%, and 60% 12, 24, and 36 months after LT, respectively. No relationship emerged between de novo AIH and recipient gender, donor and recipient age, rejection episodes, immunosuppressive regime, allelic GSTT1 expression, human leukocyte antigen distribution, or cytomegalovirus infection. Multivariate analysis identified male donor [hazard ratio (HR), 3.3; 95% CI, 1.18–9.26; P = 0.018], nonalcoholic etiology (HR, 4.67; 95% CI, 1.64–13.3; P = 0.002), and high anti‐GSTT1 titer (HR, 2.98; 95% CI, 1.04–8.57; P = 0.035) as independent predictors of de novo AIH. Most patients with anti‐GSTT1 antibodies and donor/recipient GSTT1 mismatch developed clinically evident de novo AIH after LT. The risk of developing the disease was increased by male donor gender, nonalcoholic etiology of original liver disease, and a high anti‐GSTT1 titer. Liver Transpl 15:530–539, 2009.

Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

The potential role of the HIV-1 immunogen (Remune) as a therapeutic vaccine in the treatment of HIV infection.

Immune-based therapies, such as therapeutic vaccination, that might preserve, restore, enhance and induce new HIV-specific immunologic responses are currently being explored. HIV-specific immunotherapy with Remune® (The Immune Response Corp.) offers the opportunity to boost immune responses against HIV-1. The clinical trial program in a wide range of subjects has established the efficacy of Remune in stimulating an appropriate immune response in HIV-positive individuals. Furthermore, a recent unblinded study conducted in Europe has documented a significant effect of Remune on viral load. Evidence regarding clinical end points is more difficult to collect. The same studies have revealed no serious safety issues in a total of more than 2000 Remune-treated patients. It is therefore reasonable to suggest that the risk–benefit ratio of Remune could be positive.

Sublingual therapeutic immunization with a polyvalent bacterial preparation in patients with recurrent respiratory infections: immunomodulatory effect on antigen-specific memory CD4+ T cells and impact on clinical outcome

Recurrent respiratory tract infections (RRTIs) are common clinical conditions in individuals with alterations of the immune function. A prospective open pilot study in a cohort of patients with RRTIs has been performed to assess whether sublingual immunization with a polyvalent bacterial vaccine could exert an immunomodulatory effect on the antigen‐specific immunological responses and have an impact on the clinical outcome. Seventeen patients with RRTIs were recruited. An oral polyvalent bacterial preparation (Bactek®) was administered to all patients daily for 6 months. Immunological assessment was performed at baseline and at the end of immunization. Immunological measurements included: T cell‐specific proliferations of CD3+CD4+ and CD3+CD8+ to Bactek® antigens, total immunoglobulin levels, antibodies to pneumococcal polysaccharide and tetanus toxoid and B, T and natural killer (NK) cell subsets. There was a significant increase in the proliferative capacity of CD3+CD4+ T cells specific to Bactek® antigens at month 6 in comparison to baseline (P < 0·0001). A significant increase in total CD3+ T cells was also observed (P < 0·05). No significant differences were observed between baseline and month 6 in levels of total immunoglobulins, specific antibodies and B, T or NK cell subsets. A significant reduction in the patients rate of RRTIs was observed compared with 1 year prior to initiation of therapy (P < 0·0001). The results demonstrate that long‐term administration of a sublingual polyvalent bacterial preparation in patients with RRTIs exerts an immune stimulating effect on CD4+ T helper cell responses to bacterial antigens which could be associated with clinical benefit.

Prognostic value of peripheral blood mononuclear cell-associated HIV-1 DNA for virological outcome in asymptomatic HIV-1 chronic infection

BACKGROUND Studies in primary HIV-1 infection and advanced HIV-1 disease have demonstrated that HIV-1 DNA associated with peripheral blood mononuclear cells (PBMC HIV-1 DNA) has predictive value for disease progression. OBJECTIVES To analyse in asymptomatic HIV-1 chronic infection the predictive value of PBMC HIV-1 DNA for virological failure. STUDY DESIGN In 115 individuals who had previously participated in study STIR-2102, we retrospectively analysed the PBMC HIV-1 DNA by quantitative real-time PCR. Antiretroviral naïve patients (baseline pre-ART) received 6 weeks of ART prior to randomisation (baseline post-ART). The predictive value of PBMC HIV-1 DNA, HIV-1 RNA in plasma and CD4+ T cells, at baselines pre-ART and post-ART, was determined by Kaplan-Meier and Proportional Hazards Regression analyses. RESULTS At baseline post-ART, 82% of patients showed suppression of HIV-1 RNA, however they maintained significant amounts of HIV-1 DNA (geometric mean: 690 copies/10(6) PBMC). Pre-ART and post-ART levels of HIV-1 DNA and pre-ART levels of HIV-1 RNA showed predictive value (Log-Rank test: p<0.001, p<0.001, p=0.003, respectively). In a multivariate model post-ART PBMC HIV-1 DNA was the stronger predictive variable (adjusted HR, 2.51 [95% CI, 1.33-4.73, p=0.004]) independently of HIV-1 RNA (HR 1.74 [95% CI, 1.16-2.61, p=0.007]). CONCLUSIONS PBMC HIV-1 DNA is an effective prognostic marker for virological outcome in individuals with asymptomatic HIV-1 chronic infection.

Flow Cytometry Analysis with a New FITC-Conjugated Monoclonal Antibody-3E12 for HLA-B*57:01 Rapid Screening in Prevention of Abacavir Hypersensitivity in HIV-1–Infected Patients

Abstract Background: Rapid screening for the detection of HLA-B*57:01 in the prevention of abacavir hypersensitivity in HIV-1–infected patients is a hallmark for clinical services. Objective: The aim of this work was to analyze the utility of flow cytometry with a new FITC-conjugated B-17 monoclonal antibody (mAb3E12) for HLA-B*57:01 screening in a Spanish cohort of 577 HIV-1+ individuals. Methods: Cryopreserved peripheral blood mononuclear cell samples from HIV-1+ individuals were analyzed by flow cytometry with the mAb 3E12 that recognizes both HLA-B*57 and HLA-B*58 alleles (members of the group specificity, HLA-B17). Patients’ DNA samples had been previously typed for HLA-B*57:01 with PCR-SSO or PCR-SSP and additional DNA sequencing (EPI Study). The results obtained by flow cytometry were compared with the results obtained by the DNA-PCR techniques. Results: By flow cytometry, 46 samples (7.97%) were positive for HLA-B17, 530 (91.86%) were negative, and 1 (0.17%) was undetermined. All samples found negative by flow cytometry were negative for HLA-B*57:01 by DNA-PCR. Of the HLA-B17 positive samples, 31 (67.4%) were positive for HLA-B*57:01, 2 (3.25%) were positive for HLA-B*57:03, 11 (26.1%) were positive for HLA-B*58, and 2 (3.25%) were negative for both HLA-B*57 and HLA–B*58 antigens. The undetermined sample was negative for HLA-B*57 and HLA-B*58 alleles by DNA-PCR. Conclusions: This study shows that flow cytometry with mAb3E12 is a highly sensitive method (no false negatives) to implement prior to DNA-PCR analysis for rapid screening of HLA-B*57:01. Additional confirmation by molecular HLA typing method would be required in less than 10% of the cohort of HIV-1–infected individuals.

Prevalencia del polimorfismo C77G en el exón 4 del gen CD45 en la población española

Fundamento y objetivo: El cambio C77G en el exon 4 del gen CD45 produce un splicing anormal frecuente en poblaciones sanas europeas y relacionado con la infeccion por el virus de la inmunodeficiencia humana. El objetivo de este trabajo es analizar la frecuencia de C77G en la poblacion espanola. Pacientes y metodo: Se incluyeron 517 muestras de sangre anticoagulada con acido etilendiaminotetraacetico procedentes de donantes sanos, en las que se determino la expresion de CD45RA y CD45RO sobre linfocitos T circulantes mediante citometria de flujo. Se realizo asimismo la secuenciacion del exon 4 de CD45 en las muestras con coexpresion anormal de ambas isoformas de CD45. Resultados: En 6 de 517 individuos se detecto persistencia de la expresion de CD45RA en los linfocitos T memoria; todos ellos eran heterocigotos para C77G. La frecuencia alelica es del 0,58% (intervalo de confianza del 95%, 0,23-1,32). Conclusiones: La mutacion C77G esta presente en la poblacion sana espanola. Establecer su significado clinico requiere estudios con grupos de pacientes

Mannose Binding Lectin (mbl2) Genotype Frequencies in Solid Organ Transplant Patients

Background Mannose-binding lectin (MBL) is a protein critical in the activation of the lectin complement pathway. Patients with wild-type and variant mbl2 genotypes have high or low concentrations of MBL protein, which have been associated to increase susceptibility to transplant rejection or infection, respectively. Objective Our objective was to determine mbl2 genotype frequencies in a cohort of solid organ transplant recipients and its relationship with clinical outcomes. Materials and Methods A retrospective observational study in a single center. DNA samples were obtained at the time of inclusion in the waiting list for solid organ trasplantation as part of an extended immunological analysis to assess the pre-transplant immunocompetence status of the patients (109 heart transplantation, 3 liver transplantation). DNA was extracted from 1.5-mL ethylene diamine tetraacetic acid–treated whole blood samples. Genotyping of MBL2 was done by a polymerase chain reaction (PCR)/sequence-based typing technique. MBL2 encompassing a region from the promoter to the end of exon 1 was obtained by PCR amplification. Results Frequencies of the MBL genotype in our patients were similar to those of other Spanish populations used as a reference: Low-expressing genotype 16 (14%), intermediate 30 (27%), high 66 (59%). We have confirmed a correlation of genotype and phenotype as patients with the intermediate and deficient mbl2 genotypes disclosed significantly lower concentrations of MBL protein. Patients with low-intermediate expressing genotypes had a higher prevalence of viral infections (p=0.004). Results Conclusion: Low-intermediate expressing genotype is a frequently expressed profile in the population that may predispose solid organ recipients to a greater susceptibility of viral infections. Under immunosuppressive clinical settings these genetic host factors might be associated with distinct clinical outcomes. The potential role of this genetic biomarker warrants further evaluation in prospective multicenter studies. Fondo de Investigación Sanitaria. Project FIS 1501472. With participation of FEDER funds. A way of making Europe.

Impact of the Polymorphism rs9264942 near the HLA-C Gene on HIV-1 DNA Reservoirs in Asymptomatic Chronically Infected Patients Initiating Antiviral Therapy

Several genome-wide association studies have identified a polymorphism located 35 kb upstream of the coding region of HLA-C gene (rs9264942; termed −35 C/T) as a host factor significantly associated with the control of HIV-1 viremia in untreated patients. The potential association of this host genetic polymorphism with the viral reservoirs has never been investigated, nor the association with the viral control in response to the treatment. In this study, we assess the influence of the polymorphism −35 C/T on the outcome of virus burden in 183 antiretroviral-naïve HIV-1-infected individuals who initiated antiviral treatment (study STIR-2102), analyzing HIV-1 RNA viremia and HIV-1 DNA reservoirs. The rs9264942 genotyping was investigated retrospectively, and plasma levels of HIV-1 RNA and peripheral blood mononuclear cell- (PBMC-) associated HIV-1 DNA were compared between carriers and noncarriers of the protective allele −35 C before antiretroviral therapy (ART), one month after ART and at the end of the study (36 months). HIV-1 RNA and HIV-1 DNA levels were both variables significantly different between carriers and noncarriers of the allele −35 C before ART. HIV-1 DNA levels remained also significantly different one month posttherapy. However, this protective effect of the −35 C allele was not maintained after long-term ART.