Antonio Núñez-Roldán

Interleukin-28B genetic variants and hepatitis virus infection by different viral genotypes.

Genetic host factors may modify the course of the hepatitis C virus (HCV) infection. Very recently, a genome‐wide scan that reported association of the IL28B locus with response to treatment in HCV infection was published. The aim of the current study was to investigate the relationship of this locus with outcome of HCV infection in a cohort constituted by a total of 731 Spanish individuals. From these, 284 were subjects with persistent infection, 69 were individuals who naturally cleared the virus, and 378 were noninfected subjects. Genotyping of the rs12979860 (C>T) in the IL28B locus was performed using a TaqMan 5′ allelic discrimination assay. The CC genotype was overrepresented among patients infected with viral genotypes non‐1 (66.7% versus 39.1% in patients infected with viral genotype‐1, P = 8.5 × 10−5, odds ratio [OR] = 0.32, 95% confidence interval [CI] 0.17‐0.60); patients with spontaneous resolution of infection (72.5% versus 45.6% of the individuals with persistent infection, P = 6.2 × 10−5, OR = 0.32; 95%CI, 0.18‐0.57); and lastly, patients with sustained response (60.2% versus 32.1% found in patients with nonsustained response, P = 3.1 × 10−5, OR = 0.31; 95%CI, 0.17‐0.56). Conclusion: We have found different rates of viral genotype infection depending on the IL28B variant as well as an association of this locus with natural and treatment‐mediated response. HEPATOLOGY 2010

Antibodies against glutathione S-transferase T1 (GSTT1) in patients with de novo immune hepatitis following liver transplantation

Four patients of 283 liver‐transplant recipients (1·4%) developed de novo immune‐mediated hepatitis approximately 2 years after transplantation. Antibodies showing an unusual liver/kidney cytoplasmic staining pattern were detected in the sera of all four patients and one of them was used to screen a human liver cDNA expression library with the aim of identifying the antigenic target of these newly developed antibodies. After cloning and sequencing the gene, it was identified as the gene encoding the glutathion‐S‐transferase T1 (GSTT1), a 29‐kD molecular weight protein, expressed abundantly in liver and kidney. Sera from the other three patients also contained anti‐GSTT1 antibodies, two of them demonstrated by immunoblot analysis against the recombinant antigen and the other, which was negative by immunoblot, gave a positive reaction when used directly to screen the same library, suggesting it to be directed to a conformational epitope. The GSTT1 enzyme is the product of a single polymorphic gene that is absent from 20% of the Caucasian population. When we analysed the GSTT1 genotype of the four patients described above, we found that this gene is absent from all of them. Three donor paraffin embedded DNA samples were available and were shown to be positive for GSSTT1 genotype. In accordance with these results, we suggest that this form of post‐transplant de novo immune hepatitis, that has been reported as autoimmune hepatitis by others, could be the result of an antigraft reaction in individuals lacking the GSTT1 phenotype, in which the immune system recognizes the GSTT1 protein as a non‐self antigen, being the graft dysfunction not the result of an autoimmune reaction, but the consequence of an alo‐reactive immune response.

Glutathione S‐transferase T1 mismatch constitutes a risk factor for de novo immune hepatitis after liver transplantation

A new form of autoimmune hepatitis referred to as de novo, has been reported after liver transplantation during the past 5 years. The features are identical to those of classical autoimmune hepatitis (AIH), but the facts involved in the onset and outcome of this type of graft dysfunction are still unclear. The identification of antibodies directed to glutathione S‐transferase T1 (GSTT1) in the sera of patients with de novo immune hepatitis led us to the description of an alloimmune reaction due to a GSTT1 genetic incompatibility between donor and recipient. We analyzed a cohort of 110 liver transplant patients treated in the liver transplant unit of our hospital during a period of 1 year, from September 2002 to October 2003. We found the following distribution of the GSTT1 genotypes (recipient/donor): +/+ = 66, +/− = 23, −/+ = 15, −/− = 6. Six of these patients were diagnosed with de novo immune hepatitis; all of them belong to the group of negative recipients with positive donors, and all produced anti‐GSTT1 antibodies. This genetic combination is associated with a statistically significant increased risk of de novo immune hepatitis (IH) in liver transplant patients (P < .0001 by the Fisher exact test). In conclusion, our results clearly establish the importance of the GSTT1 genotype from donor and recipient of a liver transplant as a predictive marker for de novo IH. At the same time, we confirmed our initial results that only this particular donor/recipient combination triggers the anti‐GSTT1 antibody production. (Liver Transpl 2004;10:1166–1172.)

Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation

BACKGROUND Clinical relevance of donor-specific antibodies (DSAs) detected by a single antigen Luminex virtual crossmatch in pre-transplant serum samples from patients with a negative cytotoxicity-dependent complement crossmatch is controversial. The aim of this study was to analyse the influence of a pre-transplant positive virtual crossmatch in the outcome of kidney transplantation. METHODS A total of 892 patients who received a graft from deceased donors after a negative cytotoxicity crossmatch were included. Presence of anti-human leucocyte antigen (HLA) antibodies was investigated using a Luminex screening assay and anti-HLA specificities were assigned performing a Luminex single antigen assay. RESULTS Graft survival was significantly worse among patients with anti-HLA DSA compared to both patients with anti-HLA with no DSA (P = 0.001) and patients without HLA antibodies (P < 0.001) using a log-rank test. No graft survival differences between anti-HLA with no DSA and no HLA antibodies patient groups were observed (P = 0.595). Influence of both anti-Class I and anti-Class II DSA was detected (P < 0.0001 in both cases). When the fluorescence values were stratified in four groups, no significant differences in graft survival were observed among the groups with fluorescence levels >1500 (global P > 0.05). CONCLUSIONS The presence of preformed HLA DSA in transplanted patients with a negative cytotoxicity crossmatch is associated with a lower allograft survival. The detection of anti-HLA with no DSA has no influence in the graft outcome. Finally, there were no demonstrable effects of mean fluorescence intensity (MFI) values >1500 on graft survival.

Association of vitamin D receptor genotypes with early onset rheumatoid arthritis.

The presence of certain vitamin D receptor (VDR) genotypes has been associated with low bone mineral density (BMD) in elderly populations as well as with accelerated bone loss in patients with rheumatoid arthritis (RA). In the present study, VDR genotypes from 120 Spanish patients with RA were investigated. Three VDR gene polymorphisms (BsmI, ApaI and TaqI) were investigated using polymerase chain reaction followed by enzymatic digestion. The distributions of VDR allelic frequencies were similar in patients and controls and therefore no influence of VDR polymorphisms on rheumatoid arthritis susceptibility could be demonstrated. However, in an analysis of the clinical features of the different VDR-related genetic subgroups, the BB/tt genotype, defined by the BsmI and TaqI restriction site polymorphisms, was identified to be weakly associated with an early onset RA in female patients. This VDR genotype has been associated with a low BMD level in various studies. When patients were stratified according to the presence of the shared HLA epitope SE, it was found that SE + female patients bearing the BB/tt genotype showed the earliest disease onset. The mechanisms by which the VDR polymorphism is associated with RA is unknown, but they could be related to the immunoregulatory properties of vitamin D.

Molecular structure of eight human autoreactive monoclonal antibodies

The heavy (H) and light (L) chain V‐region sequences of eight human autoreactive immunoglobulin M (IgM) monoclonal antibodies (mAbs: BY‐4, BY‐7, BY‐12, IRM‐3, IRM‐7, IRM‐8, IRM‐10 and CDC‐1) were determined at the cDNA level. All VH and VL families were identified. Four different VH families were represented, VH3 being the most common as five of the mAbs (BY‐7, BY‐12, IRM‐3, IRM‐8 and CDC‐1) used genetic elements of this family, whereas VH1, VH2 and VH4 were only present in IRM‐7, BY‐4 and IRM‐10, respectively. BY‐4, BY‐7, BY‐12, IRM‐7 and IRM‐10 reacted with a variety of self as well as non‐self antigens, thus exhibiting polyreactive behaviour. Comparison of the gene segments utilized by these mAbs with their germline counterparts revealed that the gene segments were close to germline configuration. The length of H‐CDR3 was found to be relatively long (27–60 nucleotides) among the polyreactive mAbs and the presence of Tyr and Trp residues in this region seems to be of vital importance for polyreactivity. We have analysed the utilization of gene elements and the presence of amino acid residues in regions particularly important for antigen binding, such as CDR. Common molecular features relating to the function of the mAbs are discussed.

CTLA4 polymorphism in Spanish patients with systemic lupus erythematosus.

The cytotoxic T-lymphocyte antigen 4 (CTLA4, CD152) gene is a positional and functional candidate gene to susceptibility to systemic lupus erythematosus (SLE) because CTLA4 gene maps in the described SLE risk region 2q33 and CTLA4 molecule has an inhibitory effect on T-cell activation. Several polymorphisms have been described in CTLA4 gene, among them, a T/C change at position -1722, a C/T transition at position -319, and another A/G transition at position +49. The aim of this study was to investigate the possible association of these polymorphisms with the susceptibility to SLE in 276 Spanish autochthonous patients using a healthy control group composed of 194 ethnically matched volunteer bone marrow donors. Genotyping of these CTLA4 positions was performed in SLE patients and controls using a polymerase chain reaction amplification refractory mutation system. The genotypic frequencies were in Hardy-Weinberg equilibrium in all patients. No differences in the distribution of the genotype frequencies between patients and controls were found in any case. Our results from the Spanish autochthonous population differ from those found in the Korean population regarding the involvement of the polymorphism located at -1722 in the susceptibility to SLE.

Donor-specific antibodies against HLA, MICA, and GSTT1 in patients with allograft rejection and C4d deposition in renal biopsies.

Background. Production of antibodies against donor-specific antigens is one of the central mechanisms of allograft rejection. This antibody-mediated rejection (AMR) is evidenced by the presence of circulating donor-specific antibodies and deposition of complement component C4d on renal endothelium. Although anti-human leukocyte antigen (HLA) antibodies account for a high proportion of AMR, in many cases anti-HLA antibodies cannot be demonstrated. In liver transplant, antibodies against glutathione-S-transferase T1 (GSTT1) expressed on the graft may induce an antibody response leading to a severe graft dysfunction. In addition, presence of antibodies against major-histocompatibility-complex class I chain-related gene A (MICA) has been associated with a poor graft survival in kidney transplantation. Methods. Pre- and posttransplantation sera from 19 patients fulfilling the criteria for AMR including C4d deposition in renal biopsies were included. Donor-specific antibodies against HLA-I and -II and MICA were studied using Luminex. Anti-GSTT1 antibodies were analyzed by indirect immunofluorescence and by an ELISA method. A control group of 39 patients with graft dysfunction negative for C4d was also included. Results. At the time of the biopsy, 4 (21%) patients had only anti-HLA class I antibodies; 3 (15.8%) had anti-GSTT1, 2 (10.5%) had anti-HLA-class II, and 2 (10.5%) had anti-MICA; four patients had combination of antibodies: HLA-I+MICA (n=1), HLA-I+GSTT1 (n=2), and GSTT1+MICA (n=1). No antibodies were found in 4 (21%) patients. In total, 6 (31.6%) C4d+ patients had anti-GSTT1 antibodies, whereas, among the 39 C4d-negative patients, only 3 (7.7%) had anti-GSTT1 antibodies (P=0.027). Conclusion. Besides anti-HLA antibodies, donor-specific antibodies against MICA and GSTT1 antigens could be responsible for the occurrence of antibody-mediated kidney graft rejection.

Specific changes in peripheral blood lymphocyte phenotype from burn patients. Probable origin of the thermal injury-related lymphocytopenia.

T-lymphocyte subsets from 27 severely burned patients and 32 controls were analysed using monoclonal antibody immunofluorescent staining and flow cytometry. Compared to normal controls, burn patients showed a remarkable reduction in absolute number of CD3-lymphocytes in the 48 h following injury, which was accounted for by a decrease in both CD4 and CD8 subsets. Activated lymphocytes, as defined by expression of CD25, CD69 and CD71, were significantly increased in burned patients. Additionally, a moderate increase in lymphocytes bearing simultaneously CD4 and CD8 was observed in some burned patients. The expression of CD11c, CD49a and CD54, members of the integrin family of cell surface molecules, was shown to be increased on lymphocytes from thermally injured patients. We conclude that thermal injury produces a profound T-cell lymphopenia with features of extensive T-cell activation, and postulate that depletion of circulating T-cells could be related with the expression of surface adhesion molecules and cell redistribution from blood to the tissues.

MCP-1 promoter polymorphism in spanish patients with rheumatoid arthritis

To investigate the possible role of the polymorphism located in the regulatory region of monocyte chemoattractant protein-1 (MCP-1) gene in the susceptibility to rheumatoid arthritis (RA), a total of 141 Spanish RA patients and 194 controls, previously typed for human leukocyte antigen DRB1* (HLA-DRB1*), were genotyped for -2518 (A/G) MCP-1 gene polymorphism using polymerase chain reaction-restriction fragment length polymorphism. No association between -2518 (A/G) MCP-1 polymorphism and susceptibility to RA was found. Nevertheless, when patients and controls were stratified according to their HLA shared epitope (SE) status, a significant increase in the frequency of genotype GG was found among SE negative (SE-) patients with respect to both SE positive (SE+) patients and SE- controls (16% versus 4% in SE+ patients, pFisher=0.04, odds ratio [OR]=4.4, 95% confidence interval [95%CI]=1.03-21.48; and 4% in SE- controls, pFisher=0.02, OR=4.13, 95%CI=1.10-15.72). In conclusion, MCP-1 polymorphism is slightly associated with the susceptibility to RA in patients lacking the HLA SE.