Manuel Muro

HLA polymorphism in the Murcia population (Spain): in the cradle of the archaeologic Iberians.

Human leukocyte antigen (HLA) study in Murcian individuals was performed in order to provide information of their historical origins and relationships with other Iberian and Mediterranean populations. HLA class I and class II alleles were determined in 173 unrelated Caucasoid donors from Murcia Region in the Southeast of Spain by serologic and DNA based polymerase chain reaction (PCR) typing. Class I antigen and class II allele frequencies of our series were not very different to those found in Spaniards. The analysis of extended haplotypes showed that the three haplotypes most frequent in our population were respectively, A29-B44-Cwb-DRB1*0701-DRB4*0101-DQA1*0201-DQB1*0202, A1-B8-Cw7-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201 and A30-B18-Cw5-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201. They were followed by A26-B38-Cwb-DRB1*1301-DRB3*0202-DQA1*0103-DQB1*0603, which could point to an ancestral relationship between Murcian and Portuguese Iberian populations, and by A2-B7-Cw7-DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 also present in all Iberian Peninsula populations. Allelic frequencies, populations distance dendrogram and correspondence analysis were used to study the relationships between Murcian and other populations. The closest relation was observed with Spaniards and Portuguese, followed in decreasing order by French, Italians, Algerians, Germans, Catalans, Basques, Cretans, Sardinians, and Greeks. Thus, Murcian population seems to belong to the European genetic pool, revealing a lesser genetic distance with the North Africans and the rest of populations from the Iberian Peninsula.

Association analysis of MICA gene polymorphism and MICA-129 dimorphism with inflammatory bowel disease susceptibility in a Spanish population

MICA is located at 46 kb centromeric of HLA-B, is highly polymorphic and interactions with NKG2D, its receptor on the surface of NK, Tgammadelta, and T CD8 lymphocytes. A variation at amino acid position 129 of the alpha2-heavy chain domain seems to categorize MICA alleles into strong and weak binder of NKG2D receptor, and thereby to influence effector cell function. Our aim was to study allele polymorphism of MICA and the functionally relevant dimorphism (129val/met) of MICA gene in inflammatory bowel disease (IBD) patients in our population. DNA was obtained from IBD patients (n = 88) and unrelated healthy Murcians (n = 154) and used to MICA genotyping using polymerase chain reaction-sequence-specific oligonucleotides. We did not find statistical differences in the distribution of MICA alleles between the IBD and control groups. However, we found a higher frequency of MICA-129met/met and a lower frequency of MICA-129val/met genotypes in IBD patients (mainly in ulcerative colitis) than in controls (pc = 0.02). These preliminary data could suggest a relevant role of MICA-129-val/met SNP (weak/strong binders of NKG2D receptor) in the pathogenesis of IBD.

Effect of partial HLA class I match on acute rejection in viral pre-infected human liver allograft recipients

BACKGROUND Acute rejection in liver transplants is one of the commonest causes of liver dysfunction in the early postoperative period. However, the factors involved in liver graft rejection are still unknown. Our study was aimed at ascertaining whether the degree of HLA class I and class II compatibility or pretransplant viral infection have any influence on early acute liver graft rejection. METHODS We reviewed clinical and laboratory data in 190 consecutive patients who underwent a liver transplant. HLA-A, HLA-B, and HLA-DR typing for the establishment of an HLA match score was performed by a standard microcytotoxicity method. The existence of pretransplant viral infection was investigated in sera and biopsy tissue by serologic (hepatitis B virus, hepatitis C virus) and polymerase chain reaction (cytomegalovirus) techniques, respectively. The influence of these two factors in acute rejection and the interaction between them was also analyzed. RESULTS A strong association between viral infection and acute rejection in the group with partial class I matching was found (odds ratio=7.75; P<0.0009), whereas no correlation was observed in the group with zero class I matching (odds ratio=0.98; P=0.81). The rejection percentage in the group in which partial class I match and viral infections coexisted was 60%, whereas in the partially class I-matched group without pretransplant viral presence it was 16%. CONCLUSIONS These findings suggest a participation of partial HLA class I compatibility in triggering acute rejection in recipients suffering preoperative viral infections and support the idea that HLA class I antigen matching could play a role as a linking element between the MHC-restricted T cell-mediated response to viral infection and the allogenic response in liver transplantation.

CD28 EXPRESSION ON PERIPHERAL BLOOD T LYMPHOCYTES AFTER ORTHOTOPIC LIVER TRANSPLANT : UPREGULATION IN ACUTE REJECTION

Despite immunosuppressive treatments, acute rejection remains a significant cause of graft loss. Efficient allorecognition implicates cognate T-cell interactions and requires costimulatory signals such as those delivered via CD28. Therefore, we have studied CD28 peripheral blood T-cell expression, analyzing its possible implications in liver allograft acute rejection. Fifty-five CsA-immunosuppressed orthotopic liver recipients, with or without acute rejection (AR and NAR) were immunocytometrically monitored after transplant and thirty healthy volunteers were studied as controls. In liver recipients the absolute number of CD28+ cells fell sharply immediately after transplant, but no significant differences were detected between the AR and NAR groups either in the absolute number or in the percentage of CD28+ lymphocytes. By contrast, both CD4+CD28+ and CD8+CD28+ T-cell subsets displayed a significant increase in CD28 intensity expression in AR recipients, whereas CD28 expression was significantly downregulated in the NAR recipients. This data suggests that CD28 molecule can be important in the immunologic events preceding acute rejection and that CD28 up- or downregulation could become a useful predictive marker for acute rejection or tolerance development in liver recipients.

CD28/CTLA-4 and CD80/CD86 costimulatory molecules are mainly involved in acceptance or rejection of human liver transplant

CD28/CTLA-4 interactions with their specific B7-ligands (CD80 and CD86) have decisive roles in antigenic and allogenic responses. Recently, experimental transplant studies demonstrated that donor-specific tolerance is achieved by blocking these interactions. The present study analyzes the expression of these co-stimulatory molecules in peripheral blood cells from 74 liver recipients and in 16 liver biopsies, which were classified into acute-rejection (AR, n = 27) and nonacute-rejection (NAR, n = 47) groups, as well as their influence on the in vitro response of in vivo allosensitized cells. The results clearly indicate that in human liver transplant too, B7 and CD28/CTLA-4 expression on B and CD4(+) peripheral lymphocytes respectively, contributes to graft acceptance or rejection, and appears to be of crucial importance in modulating the host alloresponse and specific-CTL generation. In the NAR-group, costimulatory molecule expression remained at basal levels after transplant, whereas in the AR-group these molecules were significantly upregulated on days of AR. CTLA-4 was observed in the infiltrating lymphocytes in most of the biopsies, but CD80 or CD86 were not. Moreover, specific cytotoxicity from the in vivo primed cells was clearly suppressed in the NAR-patients with low co-stimulatory molecule expression, whereas this activity was not modified but rather stimulated in the AR-group. Together, these findings indicate that intervention of CD28/CTLA-4/B7 signaling could be therapeutically useful in clinical transplantation.

Allelic diversity of MICA gene and MICA/HLA-B haplotypic variation in a population of the Murcia region in southeastern Spain

Major histocompatibility complex class I-related chain A (MICA) is located at 46 kb centromeric of HLA-B. It is highly polymorphic and interacts with NKG2D, its receptor on the surface of NK, Tgammadelta and T CD8 lymphocytes. Data on MICA polymorphism in different populations are still limited. Our aim was to establish allelic diversity of MICA gene and linkage disequilibrium with HLA-B in our population. DNA was obtained from 154 unrelated healthy individuals from the Murcia region in southeastern Spain. HLA-B genotyping was performed using polymerase chain reaction (PCR)-sequence-specific oligonucleotide probes and allele-specific PCR-sequence-specific primers, and MICA genotyping by using PCR-sequence-specific oligonucleotide probes. A total of 19 MICA alleles were detected on this study. MICA*008 was the most frequent allele (25.3%), followed by MICA*002 (16.1%), MICA*004 (14.9%), MICA*001 (7.8%), MICA*009 and MICA*016 (7.1%), and MICA*010 (4.6%). Eleven alleles had frequencies of <1%. In the haplotype analysis, MICA*008-B*0702 was found to be the most common, followed by MICA*004-B*4403 and MICA*001-B*1801, MICA*002-B*3501, MICA*008-B*4402, MICA*004-B*4901, MICA*008-B*0801, and MICA*002-B*3801. The frequency of MICA*010-B*1501, MICA*008-B*1302, MICA*015-B*4501, and MICA*008-B*4001 was remarkable inasmuch as these two last haplotypes have not been reported in Spanish population. Indeed, MICA*016 linked to B*1402 has also not been reported in the literature. In conclusion, the allelic diversity in our population is similar to other Caucasian populations; however we found a series of less frequent alleles, in addition to as-yet-undescribed haplotypic associations in other populations of Caucasian origin.

Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture

The one‐way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable.

Should IFN-γ, IL-17 and IL-2 be considered predictive biomarkers of acute rejection in liver and kidney transplant? Results of a multicentric study.

Acute rejection (AR) remains a major challenge in organ transplantation, and there is a need for predictive biomarkers. In the present multicenter study, we prospectively examined a series of biomarkers in liver and kidney recipients. Intracellular expression of IFN-γ, IL-17 and IL-2 and IL-17 soluble production were evaluated both pre-transplantation and post-transplantation (1st and 2nd week, 1st, 2nd and 3rd month). 142 transplant patients (63 liver/79 kidney) were included in the study. Twenty-eight recipients (14 liver/14 kidney) developed AR. Pre- and post-transplantation intracellular expression of %IFN-γ(+) in CD4(+)CD69(+) and in CD8(+)CD69(+) and soluble IL17 identified liver and kidney transplant patients at high risk of AR. Pre-transplantation, %IL-2(+) in CD8(+)CD69(+) also identified kidney patients at high risk. We constructed pre- and post-transplantation risk prediction models, based on a composite panel of biomarkers, which could provide the basis for future studies and will be a useful tool for the selection and adjustment of immunosuppressive treatments.

HLA-DRB1 and -DQB1 Polymorphism in Liver Recipients: Relationship Between HLA-DQB10302 Allele Frequency and Acute Rejection

Polymorphism of HLA-DRB1 and HLA-DQB1 loci was performed in fifty-three orthotopic liver graft recipients as well as in 108 unrelated healthy controls. Nonradioactive SSOPs were used to study PCR-amplified DNA from peripheral blood lymphocytes and biopsied material. The comparison frequency for DQB1 alleles did not reveal any significant differences between the total group of liver recipients and controls. However, when the liver recipients were subgrouped according to their rejection episode manifestations, increased and significant frequencies were observed for HLA-DQB1*0302 allele in patients showing acute rejection episodes compared to healthy controls or patients without acute rejection. This relationship did not appear influenced by the amino acid beta alanine residue in the 57th position. On the other hand, the study of the DRB1 allele frequencies did not show significant differences in any study. These results suggest that HLA-DQB1 genes could be important in the liver graft alloresponses, opening a way to a better understanding of the special tolerance state, normally observed in this type of transplant, leading us to consider the possible HLA-DQB1*0302 allele effect on tolerance rupture.

HLA-DRB1 and HLA–DQB1 genes on susceptibility to and protection from allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease that affects both patients with cystic fibrosis (CF) and those with asthma. HLA‐DRB1 alleles have previously been associated with ABPA–CF susceptibility; however, HLA‐DQB1 allele associations have not been clearly established. The aim of the present study was to investigate HLA class II associations in patients with ABPA–CF and determine their roles in susceptibility or protection. Patients with ABPA–CF, patients with CF without ABPA, patients with asthma without ABPA (AST), and healthy controls were included in this study. DNA was extracted by automatic extractor. HLA‐DRB1 and ‐DQB1 genotyping was performed by the Luminex PCR‐SSOP method (One Lambda, Canoga Park, CA, USA). Allele specific PCR‐SSP was also performed by high‐resolution analysis (One Lambda). Statistical analysis was performed with SSPS and Arlequin software. Both HLA‐DRB1*5:01 and ‐DRB1*11:04 alleles occurred with greater frequency in patients with ABPA–CF than in those with AST and CF and control subjects, corroborating previously published data. On the other hand, analysis of haplotypes revealed that almost all patients with ABPA–CF lacking DRB1*15:01 or DRB1*11:04 carry either DRB1*04, DRB1*11:01, or DRB1*07:01 alleles. In the HLA‐DQB1 region, the HLA‐DQB1*06:02 allele occurred more frequently in patients with ABPA–CF than in those with AST and CF and healthy controls, whereas HLA‐DQB1*02:01 occurred less frequently in patients with ABPA–CF. These data confirm that there is a correlation between HLA‐DRB1*15:01, –DRB1*11:04, DRB1*11:01, –DRB1*04 and –DRB1*07:01 alleles and ABPA–CF susceptibility and suggest that HLA‐DQB1*02:01 is an ABPA–CF resistance allele.