Carlos Cotorruelo

Comprehensive analysis of RHD alleles in Argentineans with variant D phenotypes.

BACKGROUND: The serologic assignment of the RhD status may be hindered in patients with weak D expression. A comprehensive study of RHD alleles occurring in the mixed population of Argentina is necessary to evaluate the most suitable DNA typing strategy.

Effect of membrane-bound IgG and desialysation in the interaction of monocytes with senescent erythrocytes.

AbstractDetermination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDSPAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked antiimmunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis

Expression of the gene encoding secretor type galactoside 2 α fucosyltransferase (FUT2) and ABH antigens in patients with oral lesions

Objective: The aim of this work was to evaluate the expression of FUT2 gene in saliva and histo ABH antigens of patients with oral lesions. Study Design: In total 178 subjects were examined, half of whom suffered from oral pre-cancerous and cancerous lesions, while the other half were the healthy control group We analyzed the FUT 2 polymorphism by ASO-PCR (allele specific oligonucleotid – polymerase chain reaction) with specific primers for G428 allele and the wild type allele of FUT2 gene. To reveal A, B and H antigens in tissue sections of the patients (n= 89) we used a modified specific red cell adherence technique. Results: We found a high intensity of oral disease in the non-secretor group (OR = 2.43). A total of 58% of the patients with oral pre-cancerous and cancerous lesions was non secretors (se_/_), in contrast with the healthy population (21.5%). A strongly positive reaction was defined as a sheet of indicator erythrocytes adhered to the epithelial cells. In 31 of the 54 samples analyzed the test showed slightly positive results on atypical areas, and there was a complete antigen deletion in areas affected by neoplasia. Nineteen samples showed a total absence of ABH antigens in both histologically normal and pathological areas. Blood group antigens were expressed at a high level in benign and highly differentiated malignant tumors. In poorly differentiated malignant tumors, they were mostly absent. Conclusion: Considering these results we suggest the use of this method to monitor probable preneoplastic lesions in risk population, especially in those with no secretor status (absence of FUT2 gene). Key words: FUT2 gene, ABH antigens, secretor status, oral lesions.

Distribution of the FYBES and RHCE*ce(733C>G) alleles in an Argentinean population: Implications for transfusion medicine

BackgroundThe understanding of the molecular bases of blood groups makes possible the identification of red cell antigens and antibodies using molecular approaches, especially when haemagglutination is of limited value. The practical application of DNA typing requires the analysis of the polymorphism and allele distribution of the blood group genes under study since genetic variability was observed among different ethnic groups. Urban populations of Argentina are assumed to have a white Caucasian European genetic component. However, historical and biological data account for the influence of other ethnic groups. In this work we analyse FY and RH blood group alleles attributed to Africans and that could have clinical implications in the immune destruction of erythrocytes.MethodsWe studied 103 white trios (father, mother and child, 309 samples) from the city of Rosario by allele specific PCRs and serological methods. The data obtained were analysed with the appropriate statistical test considering only fathers and mothers (n = 206).ResultsWe found the presence of the FY*BESand RHCE*ce(733C>G) alleles and an elevated frequency (0.0583) for the Dce haplotype. The number of individuals with a concomitant occurrence of both alleles was significantly higher than that expected by chance. We found that 4.68% of the present gene pool is composed by alleles primarily associated with African ancestry and about 10% of the individuals carried at least one RH or FY allele that is predominantly observed among African populations. Thirteen percent of Fy(b-) subjects were FY*A/FY*BES.ConclusionTaken together, the results suggest that admixture events between African slaves and European immigrants at the beginning of the 20th century made the physical characteristics of black Africans to be invisible nowadays. Considering that it was a recent historical event, the FY*BESand RHCE*ce(733C>G) alleles did not have time to become widespread but remain concentrated within families. These findings have considerable impact for typing and transfusion strategy in our population, increasing the pool of compatible units for Fy(b-) individuals requiring chronic transfusion. Possible difficulties in transfusion therapy and in genotyping could be anticipated and appropriately improved strategies devised, allowing a better management of the alloimmunization in the blood bank.

HLA class II DRB1 polymorphism in Argentinians undergoing chronic Trypanosoma cruzi infection.

DNA typing of human lymphocyte antigen (HLA)-dicloro-1-[beta]-D-ribofuranosyl-benzimidazole 1 (DRB1) alleles in 35 individuals serologically positive for T. cruzi and in 41 healthy controls was performed. DRB1 * 0409 allele was significantly more prevalent in seropositive individuals, with a trend being also observed for the DRB1 * 0701 and DRB1 * 1503 alleles. Although statistically insignificant, the latter was found more frequent in cases with cardiomyopathy.

Modifications of band 3 and oxidation level of membrane proteins in senescent erythrocytes

OBJECTIVES To study membrane proteins modifications in Senescent Red Blood Cells (SeRBC). DESIGN ANDMETHODS: SeRBC were obtained on Percoll gradients. Membrane proteins were analyzed by SDS-PAGE, band 3 by immunoblotting, and protein oxidation by measuring the carbonyl groups. RESULTS Densitometric analysis showed no change in SeRBC while an increase in band 3 and its degradation products was found. An increase of protein oxidation level was found in SeRBC. CONCLUSIONS These findings provide further experimental evidence about protein modifications occurring during the RBC lifespan.

Duffy genotyping facilitates transfusion therapy

The Duffy (FY) blood group system is clinically significant in transfusion medicine because FY antibodies are involved in hemolytic transfusion reactions and hemolytic disease of the newborn. The Fya and Fyb antigens are encoded by the FY*A and FY*B alleles which are responsible for the Fy(a+b+), Fy(a+b−) and Fy(a−b+) phenotypes. The Fy(a−b−) phenotype is found in individuals homozygous for a silent FY*B allele, named FY*BES, which is caused by a mutation in the promoter region of FY*B that result in the loss of FY expression in the erythroid linage. The aim of the present study was to evaluate the role of FY DNA typing as a tool in transfusion compatibility testing. We studied 275 white blood donors from the city of Rosario by serological method and allele specific PCRs. We found that the 106 serologically Fy(a+b+) samples all genotyped as FY*A/FY*B (100%). Among the 94 Fy(a+b−) samples, 81 (86.2%) were FY*A/FY*A and 13 (13.8%) were FY*A/FY*BES. Of the 75 Fy(a−b+) 67 (89.3%) were FY*B/FY*B and 8 (10.7%) were FY*B/FY*BES. No Fy(a−b−) samples were encountered. The frequencies of the FY*A, FY*B and FY*BES alleles clearly revealed that the genetic pool analyzed is comprised of Caucasian and non-Caucasian alleles. These results showed that there is an important proportion of patients phenotyped as Fy(b−) that can be exposed to Fy(b+) blood units with no risk of alloimmunization when they carry the FY*A/FY*BES genotype. Thus, FY genotyping allow increasing the pool of compatible units facilitating transfusion therapy and benefiting patients that require chronic transfusions.

The Dc(G48)es haplotype is frequent among the Dce haplotypes within a white population

BACKGROUND: The absence of hybrid Rhesus boxes denotes an RHD homozygous status and helps to detect the presence of Dce haplotypes instead of dce. RHCE exon 1 C48, characteristic of RHC alleles, and RHCE exon 5 G733, responsible for VS antigenicity, have been noted in many RHce alleles but it was not clearly established whether they occurred in the same allele and/or cosegregate together with RHD.

Molecular structures identified in serologically D– samples of an admixed population

The D– phenotype is mainly caused by the complete deletion of the RHD gene in Caucasians. However, a plethora of allelic variants have been described among D– individuals from different ethnic groups.

Association of leprosy with HLA-DRB1 in an Argentinean population

Abstract Background Previous studies have suggested an influence of HLA molecules on the regulation of the anti Mycobacterium leprae immune response. Methods DNA typing of HLA-DRB1 alleles in 71 leprosy patients and 81 healthy controls was performed. Genomic DNA was extracted from peripheral blood and used as a template to amplify the polymorphic second exon of the HLA-DRBl by the ploymerase chain reaction (PCR). PCR products were hybridized separately with sequence-specific oligonucleotides. Results DRB1*1401 and DRB1*1406 alleles were significantly more prevalent in leprosy patients, whereas a decreased frequency of DRB1*0808 and DRB1*1103 alleles was found, by comparison with the group control. Conclusions The HLA-DRB1 alleles could act alone or in combination with other genes to confer differential susceptibility and also protection to leprosy disease in endemic areas of the American continent.