Extensive clinical, serologic and molecular studies lead to the first reported Rhmod phenotype in Argentina.

BACKGROUND: A highly reduced expression of Rh antigens in the erythrocyte membrane is the main feature of Rhmod , an extremely rare phenotype. Mutations within RHAG gene, which encodes RhAG glycoprotein and modulates Rh antigen expression and Rh complex formation, are the molecular events responsible for the Rhmod phenotype. Here we report a clinical, serologic, and molecular study of an Argentinean proband with Rh-deficiency syndrome. MATERIALS AND METHODS: Rh antigens, RhAG and CD47 glycoproteins were studied by serologic methods in the proband, her parents and sister. Osmotic fragility and viscoelastic parameters were also examined. RHD zygosity was analyzed by RFLP-PCR. RHD, RHCE, and RHAG genes were studied by Sanger sequencing. RESULTS: No Rh antigens were detected in the proband by standard techniques. However, adsorption-elution and anti-RhAG tests showed that the proposita was Rhmod . Reduced expression of CD47, enhanced osmotic fragility, and surface viscosity alterations giving rise to spherocytes were observed in the patient. Sequencing analysis showed that a c.920C>T mutation in RHAG Exon 6 was present in a homozygous state in the proband and in a heterozygous state in the rest of the family. This novel missense mutation caused the p.Ser307Phe amino acid substitution in Transmembrane Segment 10 of the RhAG glycoprotein. CONCLUSION: This comprehensive study determined the causes of the proband's anemia allowing the diagnosis of Rh-deficiency syndrome.

Characterization of RHD locus polymorphism in D negative and D variant donors from Northwestern Argentina.

BACKGROUND: A notable RHD variability has been observed in Central Argentina's current population attributed to the intermixing of different ethnic groups. The Northwestern region of the country is characterized by a markedly Amerindian genetic contribution. In this sense, the definition of the RHD polymorphism in individuals from this area was lacking. STUDY DESIGN AND METHODS: A total of 757 donors from Northwestern Argentina, with D negative C and/or E positive (n = 526), and D variant (n = 231) phenotype defined by standard hemmaglutination tube techniques were genotyped using in-house PCR strategies, commercial SNP arrays and Sanger sequencing. RESULTS: Among D negative C and/or E positive samples, RHD null (15.40%) and DEL alleles (3.23%) were identified. One unreported SNP c.1001T>A responsible for a null allele was found. RHD*01N.75 (4.18%) and RHD*DEL43 (2.66%) were the most prevalent variants following RHD*03N.01 (8.75%). The characterization of serologic weak D phenotypes showed that RHD*weak D type 1, 2, and 3 variants were found only in 37.24% of the samples, whereas RHD*weak D type 93 was the most prevalent allele (25.11%). Also, a previously unreported missense variation c.764G>A was identified. CONCLUSIONS: A RHD genotyping strategy for patients and donors from Northwestern Argentina must consider the detection of the frequently found RHD*01N.75, RHD*DEL43, and RHD*weak D type 93 variants. Taking into account that RHD*DEL43 has scarcely been found in North Americans and Europeans whereas RHD*01N.75 and RHD*weak D type 93 have never been described in populations other than Argentineans, these RHD variants could be attributed to Native Amerindian genetic influence.

Clinical Significance of an Alloantibody against the Kell Blood Group Glycoprotein.

BACKGROUND: Kell null (K0) individuals can produce anti-Ku, an antibody against many epitopes in the Kell glycoprotein, after transfusion and/or pregnancy. Since sensitized K0 patients are rare, little is known about anti-Ku clinical relevance and in particular about its association to hemolytic disease of the fetus and newborn. CASE REPORT: This work describes a case of neonatal hyperbilirubinemia due to immune-mediated erythrocyte destruction by an alloantibody directed against the Kell glycoprotein. Serologic and molecular approaches identified an anti-Ku alloantibody in maternal serum. A homozygous IVS3 + 1g>a point mutation (KEL*02N.06 allele) was found to be responsible for the lack of Kell antigen expression in the mother's red blood cell and subsequent alloimmunization after a previous pregnancy. Even though in most cases Kell antibodies are clinically severe and may cause suppression of erythropoiesis, in our case the newborn had a moderate anemia and hyperbilirubinemia that was successfully treated with phototherapy without requiring exchange transfusion. Serological and molecular studies performed in the proband's family members allowed us to provide them with proper counseling regarding alloimmunization after transfusion and/or pregnancy. CONCLUSIONS: This case enlarges the understanding of the clinical significance of alloantibodies against Kell blood group antigens.

Genotyping approach for non-invasive foetal RHD detection in an admixed population.

BACKGROUND: Non-invasive foetal RHD genotyping can predict haemolytic disease of the foetus and the newborn in pregnancies with anti-D alloantibodies and also avoid antenatal anti-D prophylaxis in pregnant women carrying an RHD negative foetus. Considering that the Argentine genetic background is the result of generations of intermixing between several ethnic groups, we evaluated the diagnostic performance of a non-invasive foetal RHD determination strategy to guide targeted antenatal RhD immunoprophylaxis. This algorithm is based on the analysis of four regions of the RHD gene in cell-free foetal DNA in maternal plasma and maternal and paternal RHD genotyping. MATERIALS AND METHODS: DNA from 298 serologically D negative pregnant women between 19-28 weeks gestation were RHD genotyped. Foetal RHD status was determined by real-time PCR in 296 maternal plasma samples. In particular cases, RHDΨ and RHD-CE-Ds alleles were investigated in paternal DNA. Umbilical cord blood was collected at birth, and serological and molecular studies were performed. RESULTS: Of the 298 maternal samples, 288 were D-/RHD- and 10 D-/RHD+ (2 RHD*DAR; 5 RHD-CE-Ds; 3 RHDΨ). Plasma from RHD*DAR carriers was not analysed. Real-time PCR showed 210 RHD+ and 78 RHD- foetuses and 8 inconclusive results. In this latter group, paternal molecular studies were useful to report a RHD negative status in 5 foetuses while only 3 remained inconclusive. All the results, except one false positive due to a silent allele (RHD[581insG]), agreed with the neonatal typing performed in cord blood. DISCUSSION: The protocol used for non-invasive prenatal RHD genotyping proved to be suitable to determine foetal RHD status in our admixed population. The knowledge of the genetic background of the population under study and maternal and paternal molecular analysis can reduce the number of inconclusive results when investigating foetal RHD status.

Molecular structures identified in serologically D- samples of an admixed population.

BACKGROUND: 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. STUDY DESIGN AND METHODS: A cohort of 1314 routine serologically D- samples from white Argentineans was studied by molecular methods. RESULTS: Among the 1314 D- samples, 2.1% showed RHD-specific amplifications. One hybrid Rhesus box was detected in all D-/RHD+ samples, suggesting a hemizygous status. The RHDΨ was found in 0.7% of rr samples while DEL and null variants were detected in 16.7% of the D- samples expressing C and/or E antigens. The variants associated with the C antigen were seven RHD-CE-D(s) , two RHD(1-2)-CE(2-9)-D(10), two previously unreported RHD(329T>C)-CE(3-9)-D null alleles, one RHD(M295I), and one new RHCE(1-2)-RHD(3361del11 -10) null allele whereas those associated with the E antigen were five RHD(46T>C) and one novel RHD(581insG) null allele responsible for a premature stop codon. CONCLUSIONS: The prevalence of D-/RHD+ samples is higher than that observed in Europeans. More than 50% of the RHD alleles found were represented by RHDψ and RHD-CE-D(s) showing the African contribution to the genetic pool of the admixed population analyzed. Interestingly, three new alleles were found, two of them being hybrid structures between previously described RHD variants recombined with RHCE sequences. The knowledge of the RHD allele repertoire in our population allowed the implementation of reliable typing and transfusion strategies for a better management of patients and pregnant women.

Expression of the FUT2 gene and CD44 marker in patients with oral lesions

The aim of this work was to investigate the FUT 2 gene, the secretor status and the expression of CD44 protein in epithelial cells obtain from saliva samples from patients with oral lesions (benign, pre-cancerous and cancerous lesions, n = 94). We analyzed polymorphisms of the FUT2 gene by allele specific oligonucleotide¨Cpolymerase chain reaction. The FUT2 gene encodes the ¦Á(1,2) fucosyltransferase (Se enzyme) that regulates the expression of ABH antigens in secretions. Generally speaking, being a non-secretor has several disadvantages with regard to metabolism and immune function. In this study, we found that oral pre-cancerous and cancerous lesions were increased among individuals with non-secretor status and nonsense mutation 428G¡úA. Fifty-one percent of the patients with oral pre-malignant and malignant lesions were non-secretors, in contrast with the healthy population (OR = 3.44). We observed a marginal association between secretor status and these lesions. Our study suggests that the lack of wild type FUT2 gene and a non-secretor status appear to be an associated risk marker for the development of oral cancer in patients with oral lesions (AU)

El objetivo del presente trabajo fue investigar la expresión del gen FUT 2 y del marcador

Desenmascaramiento del antígeno T por larvas de Trichinella spiralis y Ascaris lumbricoides / T antigen unmasking by Trichinella spiralis and Ascaris lumbricoides larvae / Desmascaramento do antigeno T por larvas de Trichinella spiralis e Ascaris lumbricoid

La activación T es causada por cambios en la estructura de la membrana de los glóbulos rojos (GR), que producen la aglutinación de esas células transformadas con la mayoría de los sueros de adultos ABO compatible. La unión del antígeno T con su anticuerpo específico desencadena poliaglutinación, hemólisis, trombocitopenia y trombosis. El objetivo del trabajo fue estudiar el efecto de larvas de A. lumbricoides y T. spiralis sobre el desenmascaramiento del antígeno T eritrocitario. Se trabajó con 3 concentrados de larvas L1/ L2 de A. lumbricoides (CLAL), y 6 de Larvas Musculares de T. spiralis (LM): CLAL1 y LM1: 450-500 larvas/mL; CLAL2 y LM2: 900-1000 larvas/mL; CLAL3 y LM3: 1800-2000 larvas/mL; LM4: 3000-3500 larvas/mL; LM5: 7500-8000 larvas/mL; LM6: 20.000 larvas/mL. Se utilizaron GR Grupo O en medio enzimático. Los GR se incubaron con igual volumen de CLAL/ LM y los GR Controles con solución fisiológica durante 120 minutos a 37 ºC. Se realizaron Pruebas de Aglutinación en Placa y en Tubo, enfrentando GR Tratados y Controles a sueros de adulto y de cordón. Los resultados mostraron que 2 de las 5 suspensiones de GR Tratados con CLAL3 y 1 de las 5 Tratadas con LM6, aglutinaron con suero de adulto, pero no con suero de cordón. Los GR incubados con los concentrados restantes y los Controles no aglutinaron con ninguno de los sueros. Se concluye que es importante continuar estos estudios para relacionar la activación T con las dosis infectantes en ascariosis y triquinosis, particularmente en patologías que cursen con déficit de ácido siálico.

T activation is caused by changes in the structure of red blood cells (RBC) membrane producing the agglutination of these transformed cells with the majority of adult ABO compatible sera. The union of T antigen with its specific antibody unleashes polyagglutination, haemolysis, thrombocytopenia, and thrombosis. The aim of the present work was to study the effect of A. lumbricoides and T. spiralis larvae on erythrocyte T antigen unmasking. Work was performed on 3 L1/ L2 larvae concentrates of A. lumbricoides (ALLC) and 6 muscle larvae concentrates of T. spiralis (ML): ALLC1 and ML1: 450-500 larvae/ mL; ALLC2 and ML2: 900-1,000 larvae/ mL; ALLC3 and ML3: 1,800-2,000 larvae/ mL; ML4: 3,000-3,500 larvae/ mL; ML5: 7,500-8,000 larvae/ mL; ML6: 20,000 larvae/ mL. Group O RBC in enzymatic medium were used. RBC were incubated with an equal volume of ALLC/ ML and the Controls with physiological solution for 120 minutes at 37 ºC. Plate and Tube Agglutination Tests were made, facing Treated RBC and Controls against adult and cord human sera. The results showed that 2 of the 5 RBC suspensions treated with ALLC3 and 1 of the 5 RBC suspensions treated with LM6 agglutinated with serum from adult, but not cord serum. RBC incubated with the remaining concentrates and Control suspensions were not agglutinated with any of the sera. It can be concluded that it is important to continue these studies to correlate T activation with infective dose in ascariosis and trichinosis, particularly in pathologies that course with sialic acid deficiency.

Ativação T é causada por alterações da estrutura do membrana dos glóbulos vermelhos (GV), que produz a agregação dessas células transformadas com a maioria dos soros de adultos ABO compatível. A União de antígeno T com o anticorpo específico desencadeia poliaglutinação, hemólise, trombocitopenia e trombose. O objetivo do trabalho foi estudar o efeito de larvas da A. lumbricoides e T. spiralis sobre o desmascaramento do antígeno T eritrocitário. Trabalhou-se com 3 concentrados de larvas L1 / L2 da A. lumbricoides (CLAL) e 6 de larvas musculares da T. spiralis (LM): CLAL1 y LM1: 450-500 larvas/mL; CLAL2 LM2: 900-1000 larvas/mL; CLAL3 y LM3: 1800-2000 larvas/mL; LM4: 3000-3500 larvas/mL; LM5: 7500-8000 larvas/mL; LM6: 20.000 larvas/mL. Foram utilizados eritrócitos Grupo O em meio enzimático de bromelina O sedimento globular foi incubado com igual volume de CLAL / LM e o de GR Controles com solução fisiológica durante 120 minutos a 37 ºC. Foram realizados Testes de Aglutinação em Placa e em Tubo, enfrentando os GV Tratados e Controles a soros humanos de adulto e de cordão. Se utilizaron GV Grupo O en medio enzimático. Os GV foram incubados com igual volume de CLAL/ LM e os GV Controles com solução fisiológica durante 120 minutos a 37 ºC. Realizaram-se Provas de Aglutinação em Placa e em Tubo, enfrentando GV Tratados e Controles a soros de adulto e de cordão. Os resultados mostraram que 2 das 5 suspensões de GV Tratadas com CLAL3 e 1 das 5 Tratadas com LM6, aglutinaram com soro de adulto, mas não com soro de cordão. Os GV incubados com as concentrações restantes e os Controles não aglutinaram com nenhum dos soros. Conclui-se que é importante continuar estes estudos para relacionar a ativação T com as doses infectantes em ascaríase e triquinose, particularmente em patologias que cursem com déficit de ácido siálico. controles, não aglutinaram com nenhum dos soros.

Desenmascaramiento del antígeno T por larvas de Trichinella spiralis y Ascaris lumbricoides / T antigen unmasking by Trichinella spiralis and Ascaris lumbricoides larvae / Desmascaramento do antigeno T por larvas de Trichinella spiralis e Ascaris lumbricoid

La activación T es causada por cambios en la estructura de la membrana de los glóbulos rojos (GR), que producen la aglutinación de esas células transformadas con la mayoría de los sueros de adultos ABO compatible. La unión del antígeno T con su anticuerpo específico desencadena poliaglutinación, hemólisis, trombocitopenia y trombosis. El objetivo del trabajo fue estudiar el efecto de larvas de A. lumbricoides y T. spiralis sobre el desenmascaramiento del antígeno T eritrocitario. Se trabajó con 3 concentrados de larvas L1/ L2 de A. lumbricoides (CLAL), y 6 de Larvas Musculares de T. spiralis (LM): CLAL1 y LM1: 450-500 larvas/mL; CLAL2 y LM2: 900-1000 larvas/mL; CLAL3 y LM3: 1800-2000 larvas/mL; LM4: 3000-3500 larvas/mL; LM5: 7500-8000 larvas/mL; LM6: 20.000 larvas/mL. Se utilizaron GR Grupo O en medio enzimático. Los GR se incubaron con igual volumen de CLAL/ LM y los GR Controles con solución fisiológica durante 120 minutos a 37 ºC. Se realizaron Pruebas de Aglutinación en Placa y en Tubo, enfrentando GR Tratados y Controles a sueros de adulto y de cordón. Los resultados mostraron que 2 de las 5 suspensiones de GR Tratados con CLAL3 y 1 de las 5 Tratadas con LM6, aglutinaron con suero de adulto, pero no con suero de cordón. Los GR incubados con los concentrados restantes y los Controles no aglutinaron con ninguno de los sueros. Se concluye que es importante continuar estos estudios para relacionar la activación T con las dosis infectantes en ascariosis y triquinosis, particularmente en patologías que cursen con déficit de ácido siálico.(AU)

T activation is caused by changes in the structure of red blood cells (RBC) membrane producing the agglutination of these transformed cells with the majority of adult ABO compatible sera. The union of T antigen with its specific antibody unleashes polyagglutination, haemolysis, thrombocytopenia, and thrombosis. The aim of the present work was to study the effect of A. lumbricoides and T. spiralis larvae on erythrocyte T antigen unmasking. Work was performed on 3 L1/ L2 larvae concentrates of A. lumbricoides (ALLC) and 6 muscle larvae concentrates of T. spiralis (ML): ALLC1 and ML1: 450-500 larvae/ mL; ALLC2 and ML2: 900-1,000 larvae/ mL; ALLC3 and ML3: 1,800-2,000 larvae/ mL; ML4: 3,000-3,500 larvae/ mL; ML5: 7,500-8,000 larvae/ mL; ML6: 20,000 larvae/ mL. Group O RBC in enzymatic medium were used. RBC were incubated with an equal volume of ALLC/ ML and the Controls with physiological solution for 120 minutes at 37 ºC. Plate and Tube Agglutination Tests were made, facing Treated RBC and Controls against adult and cord human sera. The results showed that 2 of the 5 RBC suspensions treated with ALLC3 and 1 of the 5 RBC suspensions treated with LM6 agglutinated with serum from adult, but not cord serum. RBC incubated with the remaining concentrates and Control suspensions were not agglutinated with any of the sera. It can be concluded that it is important to continue these studies to correlate T activation with infective dose in ascariosis and trichinosis, particularly in pathologies that course with sialic acid deficiency.(AU)

AtivaþÒo T é causada por alteraþ§es da estrutura do membrana dos glóbulos vermelhos (GV), que produz a agregaþÒo dessas células transformadas com a maioria dos soros de adultos ABO compatível. A UniÒo de antígeno T com o anticorpo específico desencadeia poliaglutinaþÒo, hemólise, trombocitopenia e trombose. O objetivo do trabalho foi estudar o efeito de larvas da A. lumbricoides e T. spiralis sobre o desmascaramento do antígeno T eritrocitário. Trabalhou-se com 3 concentrados de larvas L1 / L2 da A. lumbricoides (CLAL) e 6 de larvas musculares da T. spiralis (LM): CLAL1 y LM1: 450-500 larvas/mL; CLAL2 LM2: 900-1000 larvas/mL; CLAL3 y LM3: 1800-2000 larvas/mL; LM4: 3000-3500 larvas/mL; LM5: 7500-8000 larvas/mL; LM6: 20.000 larvas/mL. Foram utilizados eritrócitos Grupo O em meio enzimático de bromelina O sedimento globular foi incubado com igual volume de CLAL / LM e o de GR Controles com soluþÒo fisiológica durante 120 minutos a 37 ºC. Foram realizados Testes de AglutinaþÒo em Placa e em Tubo, enfrentando os GV Tratados e Controles a soros humanos de adulto e de cordÒo. Se utilizaron GV Grupo O en medio enzimático. Os GV foram incubados com igual volume de CLAL/ LM e os GV Controles com soluþÒo fisiológica durante 120 minutos a 37 ºC. Realizaram-se Provas de AglutinaþÒo em Placa e em Tubo, enfrentando GV Tratados e Controles a soros de adulto e de cordÒo. Os resultados mostraram que 2 das 5 suspens§es de GV Tratadas com CLAL3 e 1 das 5 Tratadas com LM6, aglutinaram com soro de adulto, mas nÒo com soro de cordÒo. Os GV incubados com as concentraþ§es restantes e os Controles nÒo aglutinaram com nenhum dos soros. Conclui-se que é importante continuar estes estudos para relacionar a ativaþÒo T com as doses infectantes em ascaríase e triquinose, particularmente em patologias que cursem com déficit de ácido siálico. controles, nÒo aglutinaram com nenhum dos soros.(AU)

Exposición del antígeno T eritrocitario por acción de Ascaris lumbricoides / Exposure of erythrocyte T antigen by Ascaris lumbricoides action / Exposição do antígeno T eritrocitário por ação de Ascaris lumbricoides

La poliaglutinabilidad de los glóbulos rojos puede deberse al desenmascaramiento del antígeno T críptico debido a la acción de neuraminidasas microbianas que eliminan residuos terminales de ácido siálico en la membrana del hematíe. En experiencias preliminares se demostró que Ascaris lumbricoides capta ácido siálico del eritrocito y que las suspensiones globulares en medio enzimático, incubadas con este parásito, pierden totalmente la capacidad de agregación. El objetivo fue estudiar la exposición del antígeno T eritrocitario, por acción de A. lumbricoides sobre la carga aniónica de glóbulos rojos deficientes en ácido siálico. Se trabajó con 48 extractos de ejemplares adultos del parásito ([EA]) y un concentrado de larvas L1/ L2 ([CLAL]):1300-1500 larvas/mL). Se utilizaron eritrocitos Grupo O en medio enzimático de bromelina (GRB) y eritrocitos Control en medio salino (GRC). El sedimento globular de GRB se incubó con igual volumen de [EA]/ [CLAL] y el de GRC con solución fisiológica durante 120 minutos a 37 ºC. Se realizaron Pruebas de Aglutinación en Placa y en Tubo, enfrentando los GRB y GRC a sueros humanos de adulto y de cordón. Los resultados mostraron que el 33,33% de los GRB incubados con [EA] y el 66,67% de los GRB incubados con [CLAL] aglutinaron con suero de adulto, pero no con suero de cordón. Los GRC no aglutinaron con ninguno de los sueros. Es la primera vez que se comunica la exposición del antígeno T eritrocitario por acción de un parásito. La activación T podría producir autoaglutinación y hemólisis en el hombre adulto y representaría un factor de riesgo transfusional en la población infantil.

Red cell polyagglutination may be due to the unmasking of the cryptic T antigen by the action of microbial neuraminidases, which remove terminal sialic acid residues in the erythrocyte membrane. Previous experiences showed that Ascaris lumbricoides capture erythrocyte sialic acid and thatglobular suspensions in enzymatic medium, incubated with this parasite, completely lost the ability to aggregate. The aim of this work was to study the erythrocyte T antigen exposure by A. lumbricoides action on the anionic charge of sialic acid deficient red cells. Studies were done on 48 adult specimen parasite extracts ([AE]) and an L1/ L2 larvae concentrate ([ALLC]: 1300-1500 larvae/mL). Group O red cells in bromelain enzymatic medium (RCB) and Control erythrocytes in saline medium (RCC) were used. The RCB sediment was incubated with an equal volume of [AE]/ [ALLC] and the RCC sediment with physiological solution during 120 minutes at 37 ºC. Plate and Tube Agglutination Tests were performed, contrasting RCB and RCC with adult and cord human sera. The results showed that 33.33% of the RCB incubated with [AE] and 66.67% of the RCB incubated with [LCAL] agglutinated with serum from adult, but not cord serum. RCB were not agglutinated with any of the sera. It is the first time that erythrocyte T antigen exposure by a parasite action is communicated. T activation could produce autoagglutination and haemolysis in the adult and would represent a transfusion risk factor in the child population.

A poliaglutinabilidade dos glóbulos vermelhos pode ser resultado do desmascaramento do antígeno críptico T devido à ação de neuraminidases microbianas que eliminam resíduos terminais de ácido siálico na membrana da hemácia. Em experiências preliminares foi demonstrado que Ascaris lumbricoides capta ácido siálico do eritrócito e que as suspensões globulares em meio enzimático, incubadas com esta parasita, perdem totalmente a capacidade de agregação. O objetivo foi estudar a exposição do antígeno T eritrocitário, por ação de A. lumbricoides sobre a carga aniônica de glóbulos vermelhos deficientes em ácido siálico. Trabalhou-se com 48 extratos de exemplares adultos da parasita ([EA]) e um concentrado de larvas L1/ L2 ([CLAL]):1300-1500 larvas/mL). Foram utilizados eritrócitos Grupo O em meio enzimático de bromelina (GVB) e eritrócitos Controle em meio salino (GVC). O sedimento globular de GVB foi incubado com igual volume de [EA]/ [CLAL] e o de GVC com solução fisiológica durante 120 minutos a 37 ºC. Foram realizados Testes de Aglutinação em Placa e em Tubo, enfrentando os GVB e GVC a soros humanos de adulto e de cordão. Os resultados mostraram que 33,33% dos GVB incubados com [EA] e 66,67% dos GVB incubados com [CLAL] aglutinaram com soro de adulto, mas não com soro de cordão. Os GVC não aglutinaram com nenhum dos soros. É a primeira vez que se comunica a exposição do antígeno T eritrocitário por ação de uma parasita. A ativação T poderia produzir autoaglutinação e hemólise no homem adulto e representaria um fator de risco transfusional na população infantil.

Exposición del antígeno T eritrocitario por acción de Ascaris lumbricoides / Exposure of erythrocyte T antigen by Ascaris lumbricoides action / ExposiþÒo do antígeno T eritrocitário por aþÒo de Ascaris lumbricoides

La poliaglutinabilidad de los glóbulos rojos puede deberse al desenmascaramiento del antígeno T críptico debido a la acción de neuraminidasas microbianas que eliminan residuos terminales de ácido siálico en la membrana del hematíe. En experiencias preliminares se demostró que Ascaris lumbricoides capta ácido siálico del eritrocito y que las suspensiones globulares en medio enzimático, incubadas con este parásito, pierden totalmente la capacidad de agregación. El objetivo fue estudiar la exposición del antígeno T eritrocitario, por acción de A. lumbricoides sobre la carga aniónica de glóbulos rojos deficientes en ácido siálico. Se trabajó con 48 extractos de ejemplares adultos del parásito ([EA]) y un concentrado de larvas L1/ L2 ([CLAL]):1300-1500 larvas/mL). Se utilizaron eritrocitos Grupo O en medio enzimático de bromelina (GRB) y eritrocitos Control en medio salino (GRC). El sedimento globular de GRB se incubó con igual volumen de [EA]/ [CLAL] y el de GRC con solución fisiológica durante 120 minutos a 37 ºC. Se realizaron Pruebas de Aglutinación en Placa y en Tubo, enfrentando los GRB y GRC a sueros humanos de adulto y de cordón. Los resultados mostraron que el 33,33% de los GRB incubados con [EA] y el 66,67% de los GRB incubados con [CLAL] aglutinaron con suero de adulto, pero no con suero de cordón. Los GRC no aglutinaron con ninguno de los sueros. Es la primera vez que se comunica la exposición del antígeno T eritrocitario por acción de un parásito. La activación T podría producir autoaglutinación y hemólisis en el hombre adulto y representaría un factor de riesgo transfusional en la población infantil.(AU)

Red cell polyagglutination may be due to the unmasking of the cryptic T antigen by the action of microbial neuraminidases, which remove terminal sialic acid residues in the erythrocyte membrane. Previous experiences showed that Ascaris lumbricoides capture erythrocyte sialic acid and thatglobular suspensions in enzymatic medium, incubated with this parasite, completely lost the ability to aggregate. The aim of this work was to study the erythrocyte T antigen exposure by A. lumbricoides action on the anionic charge of sialic acid deficient red cells. Studies were done on 48 adult specimen parasite extracts ([AE]) and an L1/ L2 larvae concentrate ([ALLC]: 1300-1500 larvae/mL). Group O red cells in bromelain enzymatic medium (RCB) and Control erythrocytes in saline medium (RCC) were used. The RCB sediment was incubated with an equal volume of [AE]/ [ALLC] and the RCC sediment with physiological solution during 120 minutes at 37 ºC. Plate and Tube Agglutination Tests were performed, contrasting RCB and RCC with adult and cord human sera. The results showed that 33.33% of the RCB incubated with [AE] and 66.67% of the RCB incubated with [LCAL] agglutinated with serum from adult, but not cord serum. RCB were not agglutinated with any of the sera. It is the first time that erythrocyte T antigen exposure by a parasite action is communicated. T activation could produce autoagglutination and haemolysis in the adult and would represent a transfusion risk factor in the child population.(AU)

A poliaglutinabilidade dos glóbulos vermelhos pode ser resultado do desmascaramento do antígeno críptico T devido O aþÒo de neuraminidases microbianas que eliminam resíduos terminais de ácido siálico na membrana da hemácia. Em experiÛncias preliminares foi demonstrado que Ascaris lumbricoides capta ácido siálico do eritrócito e que as suspens§es globulares em meio enzimático, incubadas com esta parasita, perdem totalmente a capacidade de agregaþÒo. O objetivo foi estudar a exposiþÒo do antígeno T eritrocitário, por aþÒo de A. lumbricoides sobre a carga ani¶nica de glóbulos vermelhos deficientes em ácido siálico. Trabalhou-se com 48 extratos de exemplares adultos da parasita ([EA]) e um concentrado de larvas L1/ L2 ([CLAL]):1300-1500 larvas/mL). Foram utilizados eritrócitos Grupo O em meio enzimático de bromelina (GVB) e eritrócitos Controle em meio salino (GVC). O sedimento globular de GVB foi incubado com igual volume de [EA]/ [CLAL] e o de GVC com soluþÒo fisiológica durante 120 minutos a 37 ºC. Foram realizados Testes de AglutinaþÒo em Placa e em Tubo, enfrentando os GVB e GVC a soros humanos de adulto e de cordÒo. Os resultados mostraram que 33,33% dos GVB incubados com [EA] e 66,67% dos GVB incubados com [CLAL] aglutinaram com soro de adulto, mas nÒo com soro de cordÒo. Os GVC nÒo aglutinaram com nenhum dos soros. E a primeira vez que se comunica a exposiþÒo do antígeno T eritrocitário por aþÒo de uma parasita. A ativaþÒo T poderia produzir autoaglutinaþÒo e hemólise no homem adulto e representaria um fator de risco transfusional na populaþÒo infantil.(AU)

Acción de Ascaris lumbricoides sobre la carga aniónica de eritrocitos y eritrocitos desializados / Ascaris lumbricoides action on the erythrocyte and desyalated erythrocyte anionic charge / Ação de Ascaris lumbricoides sobre a carga aniônica de eritrócitos e eritrócitos desializados

El objetivo del presente trabajo fue estudiar el efecto de A. lumbricoides sobre la carga aniónica de eritrocitos y eritrocitos desializados. Se trabajó con 30 extractos ([EA]) y 2 concentrados larvales parasitarios ([CLAL] 1: 500- 600/ [CLAL]2: 200- 300 larvas/ mL) y eritrocitos Grupo "O" en medio salino (GR) y enzimático (GRb). Se incubó el sedimento globular con igual volumen de parásito y el Control (GRc) con solución fisiológica, a 37 ºC. Se aplicaron los Métodos de Polibrene y Azul Alcian y se implementó una Titulación de la Agregación a partir del Polibrene. Se definieron coeficientes para cada método que fueron analizados estadísticamente. Los resultados mostraron que a mayor tiempo de tratamiento y a mayor concentración larvaria, aumentó la captación de ácido siálico (AS) disminuyendo la agregación de GR y GRb. El efecto fue mayor en la incubación del parásito con GRb en relación de GR. El tratamiento enzimático redujo la carga porcentual en los glóbulos incubados con ambos [CLAL] de forma similar (16%-17% de la inicial) y la Diferencia de Carga de GR y GRb debida a ambos fue aproximadamente del 10% indicando que la pérdida de carga producida por [CLAL]1 fue significativa con respecto a [CLAL]2. Se concluye que A. lumbricoides puede captar AS eritrocitario. Este fenómeno contribuiría a explicar la anemia y trombos producidos en ascariosis, destacando que en pacientes con patologías que cursen con déficit de AS estas complicaciones serían más relevantes.

The aim of this work was to study the A. lumbricoides' effect on the anionic charge of erythrocytes and desyalated erythrocytes. Work was carried out on 30 extracts ([AE]) and 2 larval concentrates ([ALLC] 1: 500-600; ([ALLC] 2: 200-300 larvae/ mL) of the parasite and Group O erythrocytes in saline (RC) and enzymatic medium (RCb). The red cell sediment was incubated with an equal volume of parasite and the Control (RCc) with saline solution, at 37C. Polybrene and Alcian Blue Methods were applied and Aggregation Titulation from Polybrene was implemented. The coefficients for each method were defined,which were analyzed statistically. The results showed that a longer treatment and also at higher larval concentration,increased the capture of sialic acid (AS),while decreasing the RC and RCb aggregation. The effect was greater on the incubation of the parasite with RCb in relation to RC. The enzymatic treatment reduced similarly the percentage charge in the globules incubated with both [ALLC] (16% -17% of initial) and the variation of RC and RCb charge due to both was about 10%,indicating that the charge loss caused by [ALLC]1 was significant with regard to [ALLC]2. We concluded that A. lumbricoides can capture erythroctyte SA. This phenomenon would contribute to explain the anaemia and thrombi produced in ascariosis,noting that in patients with AS deficit pathologies,these complications would be more relevant.

O objetivo foi estudar o efeito de A. lumbricoides sobre a carga aniônica de eritrócitos e eritrócitos desializados. Trabalhou-se com 30 extratos ([EA]) e 2 concentrados larvais parasitários ([CLAL]1: 500- 600/ [CLAL]2: 200- 300 larvas/ mL) e eritrócitos Grupo "O" em meio salino (GV) e enzimático (GVb). Foi incubado o sedimento globular com igual volume de parasita e o Testemunha (GVc) com solução fisiológica,a 37ºC. Foram aplicados os Métodos de Polybrene e Azul Alciano e se implementou uma Titulação da Agregação a partir do Polybrene. Definiram-se coeficientes para cada método que foram analisados estatisticamente. Os resultados mostraram que a maior tempo de tratamento e a maior concentração larvária,aumentou a captação de ácido siálico (AS) diminuindo a agregação de GV e GVb. O efeito foi maior na incubação da parasita com GVb em relação de GV. O tratamento enzimático reduziu a carga percentual nos glóbulos incubados com ambos [CLAL] de forma similar (16%-17% da inicial) e a Diferença de Carga de GV e GVb devida a ambos foi aproximadamente de 10% indicando que a perda de carga produzida por [CLAL]1 foi significativa com relação a [CLAL]2. A conclusão é que A. lumbricoides pode captar AS eritrocitário. Este fenômeno contribuiria a explicar a anemia e trombos produzidos em ascaridíase,destacando que em pacientes com patologias que cursem com déficit de AS estas complicações seriam mais relevantes.

Acción de Ascaris lumbricoides sobre la carga aniónica de eritrocitos y eritrocitos desializados / Ascaris lumbricoides action on the erythrocyte and desyalated erythrocyte anionic charge / AþÒo de Ascaris lumbricoides sobre a carga ani¶nica de eritrócitos e eritrócitos desializados

El objetivo del presente trabajo fue estudiar el efecto de A. lumbricoides sobre la carga aniónica de eritrocitos y eritrocitos desializados. Se trabajó con 30 extractos ([EA]) y 2 concentrados larvales parasitarios ([CLAL] 1: 500- 600/ [CLAL]2: 200- 300 larvas/ mL) y eritrocitos Grupo "O" en medio salino (GR) y enzimático (GRb). Se incubó el sedimento globular con igual volumen de parásito y el Control (GRc) con solución fisiológica, a 37 ºC. Se aplicaron los Métodos de Polibrene y Azul Alcian y se implementó una Titulación de la Agregación a partir del Polibrene. Se definieron coeficientes para cada método que fueron analizados estadísticamente. Los resultados mostraron que a mayor tiempo de tratamiento y a mayor concentración larvaria, aumentó la captación de ácido siálico (AS) disminuyendo la agregación de GR y GRb. El efecto fue mayor en la incubación del parásito con GRb en relación de GR. El tratamiento enzimático redujo la carga porcentual en los glóbulos incubados con ambos [CLAL] de forma similar (16%-17% de la inicial) y la Diferencia de Carga de GR y GRb debida a ambos fue aproximadamente del 10% indicando que la pérdida de carga producida por [CLAL]1 fue significativa con respecto a [CLAL]2. Se concluye que A. lumbricoides puede captar AS eritrocitario. Este fenómeno contribuiría a explicar la anemia y trombos producidos en ascariosis, destacando que en pacientes con patologías que cursen con déficit de AS estas complicaciones serían más relevantes.(AU)

The aim of this work was to study the A. lumbricoides effect on the anionic charge of erythrocytes and desyalated erythrocytes. Work was carried out on 30 extracts ([AE]) and 2 larval concentrates ([ALLC] 1: 500-600; ([ALLC] 2: 200-300 larvae/ mL) of the parasite and Group O erythrocytes in saline (RC) and enzymatic medium (RCb). The red cell sediment was incubated with an equal volume of parasite and the Control (RCc) with saline solution, at 37C. Polybrene and Alcian Blue Methods were applied and Aggregation Titulation from Polybrene was implemented. The coefficients for each method were defined,which were analyzed statistically. The results showed that a longer treatment and also at higher larval concentration,increased the capture of sialic acid (AS),while decreasing the RC and RCb aggregation. The effect was greater on the incubation of the parasite with RCb in relation to RC. The enzymatic treatment reduced similarly the percentage charge in the globules incubated with both [ALLC] (16% -17% of initial) and the variation of RC and RCb charge due to both was about 10%,indicating that the charge loss caused by [ALLC]1 was significant with regard to [ALLC]2. We concluded that A. lumbricoides can capture erythroctyte SA. This phenomenon would contribute to explain the anaemia and thrombi produced in ascariosis,noting that in patients with AS deficit pathologies,these complications would be more relevant.(AU)

O objetivo foi estudar o efeito de A. lumbricoides sobre a carga ani¶nica de eritrócitos e eritrócitos desializados. Trabalhou-se com 30 extratos ([EA]) e 2 concentrados larvais parasitários ([CLAL]1: 500- 600/ [CLAL]2: 200- 300 larvas/ mL) e eritrócitos Grupo "O" em meio salino (GV) e enzimático (GVb). Foi incubado o sedimento globular com igual volume de parasita e o Testemunha (GVc) com soluþÒo fisiológica,a 37ºC. Foram aplicados os Métodos de Polybrene e Azul Alciano e se implementou uma TitulaþÒo da AgregaþÒo a partir do Polybrene. Definiram-se coeficientes para cada método que foram analisados estatisticamente. Os resultados mostraram que a maior tempo de tratamento e a maior concentraþÒo larvária,aumentou a captaþÒo de ácido siálico (AS) diminuindo a agregaþÒo de GV e GVb. O efeito foi maior na incubaþÒo da parasita com GVb em relaþÒo de GV. O tratamento enzimático reduziu a carga percentual nos glóbulos incubados com ambos [CLAL] de forma similar (16%-17% da inicial) e a Diferenþa de Carga de GV e GVb devida a ambos foi aproximadamente de 10% indicando que a perda de carga produzida por [CLAL]1 foi significativa com relaþÒo a [CLAL]2. A conclusÒo é que A. lumbricoides pode captar AS eritrocitário. Este fen¶meno contribuiria a explicar a anemia e trombos produzidos em ascaridíase,destacando que em pacientes com patologias que cursem com déficit de AS estas complicaþ§es seriam mais relevantes.(AU)

Expression of the gene encoding secretor type galactoside 2 á 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 his to 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) (AU)

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. STUDY DESIGN AND METHODS: A total of 18,379 patients from two stratified groups, Group 1 (G1; public hospital) and Group 2 (G2; private laboratory), were RhD phenotyped, and 88 samples with reduced D expression underwent molecular characterization. RESULTS: The frequencies of D+, D-, and variant D phenotypes differed significantly (p < 0.001) between G1 and G2 (94.49% vs. 87.66%, 5.15% vs. 11.58%, and 0.36% vs. 0.75%, respectively). Eleven alleles were responsible for the weak D expression. Approximately 60% of the variant D phenotypes from G1 and G2 were weak D Types 1 through 4.0/4.2 and 25% were DVII. RHD alleles associated with African ancestry were encountered in G1. A new -282G>A mutation within the promoter region of DAU-4 and DOL alleles was identified. Three weak D Type 1 samples on R(0) haplotypes were found in G1. CONCLUSIONS: The D phenotype distribution in G2 resembles that in Europeans while the frequencies in G1 account for the Amerindian and African genetic contribution. The genotyping strategy described here is suitable to study D variants in the overall population and could allow a better use of the few available D- units and a rational administration of anti-D immunoprophylaxis. The results also show that weak D Type 1 alleles do not exclusively segregate with a Ce allele, as assumed until present.

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).

Alteración de la carga eritrocitaria utilizando el método de azul Alcian en extractos de Ascaris lumbricoides / Change of erythrocyte charge with the use of Alcian blue method in Ascaris lumbricoides extracts

Introducción: el estudio de las interacciones hospedador-parásito es un nuevo desafío para comprender aspectos del metabolismo parasitario, los mecanismos de invasión, escape inmunológico y daño. Ascaris lumbricoides puede provocar anemia y trombosis. Previamente se demostró que este parásito altera la carga superficial eritrocitaria, lo cual indica que puede captar ácido siálico del glóbulo rojo. Objetivo: estudiar el efecto producido por extractos del parásito adulto sobre la carga eritrocitaria, utilizando el método de azul Alcian y comparar su sensibilidad con el método de Polibrene. Métodos: se trabajó con 55 extractos parasitarios [EA] y con suspensiones de eritrocitos grupo O. Se realizó el tratamiento de los glóbulos incubando el sedimento con igual volumen de [EA] a 37 ºC durante 1 h. El Control (eritrocitos sin contacto con [EA]) fue incubado con tampón fosfato pH 7,4. Se aplicó el método de azul Alcian y se determinó la carga aniónica eritrocitaria porcentual (CAE por ciento) en el Control y en los glóbulos tratados. Se definió el coeficiente experimental de carga aniónica eritrocitaria (Cexp CAE), como el cociente entre CAE por ciento final e inicial. Resultados: se observó que 27 de los 55 [EA] (49,1 por ciento) modificaron la carga de los glóbulos rojos, el Cexp CAE para estos eritrocitos resultó 0,75 ± 0,1144 y para los que no mostraron variación de carga de 0,94 ± 0,04450. El análisis estadístico concluyó que los métodos de Polibrene y de azul Alcian tienen sensibilidades comparables (p> 0,20). Conclusiones: Ascaris lumbricoides es capaz de captar ácido siálico del eritrocito, lo que contribuiría a explicar la trombosis atribuida al parásito, pero también sugeriría que el nematodo lo podría utilizar en sus rutas metabólicas o en sus estrategias de evasión inmunológica.

Introduction: the study of the host-parasite interactions is a new challenge to understanding some aspects of the parasitic metabolism and the mechanisms of invasion, immunological evasion and damage. Ascaris lumbricoides may cause anemia and thrombosis. It was previously shown that Ascaris lumbricoides modified the superficial charge of erythrocytes, which means that the parasite can capture sialic acid from the red blood cell. Objective: to study the effect of adult parasite extracts on the erythrocyte charge using the Alcian Blue method and to compare its sensitivity with the Polybrene method. Methods: fifty five adult parasite extracts and Group O erythrocyte suspensions were used. The erythrocytes were treated by incubating the sediment with an equal volume of parasite extracts for one hour at 37 ºC. The control group (erythrocytes without any contact with the parasite extracts) was incubated with pH 7.4phosphate buffer solution. Alcian Blue method was applied and the percentage erythrocyte anionic charge was determined in the control group and in the treated red cells. The experimental coefficient of erythrocyte anionic charge was defined as the quotient between the initial and the final percentage erythrocyte anionic charge. Results: it was shown that 27 out of 55 parasite extracts (49.1 percent) modified the charge of the red blood cells, being their experimental coefficient of the erythrocyte anionic charge 0.75 ± 0.1144 whereas the same coefficient amounted to 0,94 ± 0.0445 for those which did not show any charge variation. The statistical analysis concluded that the Polybrene and Alcian Blue Methods had comparable sensitivities (p>0.20). Conclusions: A. lumbricoides is able to capture sialic acid from the erythrocyte, which would not only explain the thrombosis attributed to the parasite, but also suggest that the nematode could use this acid either in its metabolic routes or for its strategies of immunological evasion.