Laboratorio Of Inmunohematologia

Buenos Aires, Argentina

Laboratorio Of Inmunohematologia

Buenos Aires, Argentina
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De Leon P.P.,Laboratorio Of Parasitologia | Menendez M.,Laboratorio Of Parasitologia | Bertorini G.,Laboratorio Of Parasitologia | Biondi C.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2012

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 °C. 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.


De Leon P.P.,Laboratorio Of Parasitologia | Di Vita S.,Laboratorio Of Parasitologia | Biondi C.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2012

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 that globular 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 RC C sediment with physiological solution during 120 minutes at 37 °C. Plate and Tube Agglutination Tests were performed, contrasting RC B 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.


Ponce De Leon P.,Bioquimica | Ponce De Leon P.,Laboratorio Of Parasitologia | Di Vita S.,Estudiante de Bioquimica | Di Vita S.,Laboratorio Of Parasitologia | And 4 more authors.
Acta Bioquimica Clinica Latinoamericana | Year: 2011

Erythrocyte aggregation affects microcirculation and its study is important in vascular diseases. It was communicated that Ascaris lumbricoides can capture erythrocyte sialic acid (SA) and alter the anionic charge on the red cell. The aim of this work was to study SA capture by A. lumbricoides larvae incubated in vivo with erythrocytes. Work was performed with concentrated larvae ([ALLC]) incubated in 4 tubes with phosphate buffer and antibiotics. Group O erythrocytes were added in two Tubes. The remaining were Controls. They were incubated at 37 °C (5% CO2) for 24 and 28 hours. The larvae were separated, collected, concentrated and counted microscopically (larvae/mL: [ALLC]1:1500-1700; [ALLC]3:1600-1800; [ALLC]2 and 4:200-400). Aggregation Inhibition by Polybrene (AIP) and Blue Alcian (BA) techniques were used. AIP showed a significant difference between Polybrene Titers diluted with physiological solution and with [ALLC]1 and 2, which were incubated with erythrocytes 24 and 48 hours respectively. There was no change in the Title of the corresponding Controls. BA performed in [ALLC]1 and [ALLC]3 determined CapSAC = 6.65% ±0.36 and SAC[CLAL]3 % = 0.67% ±0.36. The experience showed SA capture by larvae incubated in vivo with erythrocytes and it suggested that they have no intrinsic SA. SA kidnapping during the larval migration could be important in parasite-host interaction.


De Leon P.P.,Laboratorio Of Parasitologia | Di Vita S.,Laboratorio Of Parasitologia | Biondi C.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2011

Previous experiences have identified ABH antigens in A. lumbricoides's adult specimens. The parasites only showed epithopes expressed in their respective hosts. It was demonstrated that epithopes are acquired by absorption by means of the culture of larvae with erythrocytes. As the adult parasite lives in the small intestine, it could be present there because the fourth stage larva retains the epithopes absorbed in the migration and keeps them during the maturation process to adult, or the adult specimen would acquire them from intestinal secretions. Larvae were obtained by hatching of eggs and they were collected and cultured in RPMI medium. The aim of this work was to study the ABH soluble epithopes by larvae in culture. B soluble antigen was added into three tubes, which were incubated for 2, 3 and 6 days, and A soluble antigen was poured into two tubes, both cultured for 6 days. Each culture time had its absorption Negative Control where ABH like antigen excretion/secretion products (ES) were investigated. The Semiquantitative Inhibition Agglutination Test showed B epithopes in the larvae cultured for 3 days and A and B epithopes in the larvae incubated for 6 days. Similar A and B ES products were only evidenced in the Control of 6 days. The results conclude that A. lumbricoides may absorb ABH antigenic determiners from soluble antigens.


De Leon P.P.,Laboratorio Of Parasitologia | Foresto P.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2010

Molecular mimicry has been associated with chronicity of parasitic infections. This mechanism can be due to absorption of the host's molecules or to synthesis of similar molecules. P1 has been detected in various parasite species. Previous experiences have shown P1 epitopes in Ascaris lumbricoides's adult parasites. The aim of this study was to analyse absoption of these epitopes by nematode larvae in order to determine the mechanism by which the parasite can express these antigens on their cuticle. Larvae were hatched from the faeces with helminth eggs. Larvae were cultivated in RPMI medium with P1 red cells. The Quantitative Inhibition Agglutination Test was performed using 4 anti- P1 monoclonal antibodies. P1 fresh red cells in bromelin enzymatic medium was used as revealing system. Simil P1 excretory-secretory products were investigated using larvae cultivated in RPMI medium without P1 erythrocytes by the same technique. The larvae which were cultivated with red cells inhibited the agglutination between all anti-P1 monoclonal antibodies and P1 erythrocytes. Simil P1 excretory-secretory products were not identified. It was demonstrated that A. lumbricoides larvae absorb P1 epitopes from the culture medium. It can be concluded that the parasite can absorb this antigen during its life cycle in order to use it in molecular mimicry.


De Leon P.P.,Laboratorio Of Parasitologia | Foresto P.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2010

Hyaluronic acid (HA) participates in the immune response because it is a constituent of natural mechanical barriers, an inflammation stimulator and a ligand for receptors involved in immunity. Previous experiences have shown that adult specimens and larval concentrates of A. lumbricoides have hyaluronan binding capacity. The aim of this work was to study the kinetics of hyaluronic acid capture by this parasite. Work was carried out with an extract of adult specimen and a larval concentrate. The modified test of serum soluble CD44 Detection by Aggregation Inhibition was used. Kinetics hyaluronic acid capture by the nematode was assessed by varying the contact time between HA and parasite, from an initial time to 90 minutes, with 10-minute intervals. C expAdhEHA and IexpCPHA were calculated for all the times and they were analysed by Wilcoxon and Friedman Tests The results showed that at initial time of contact, the adult parasite and the larvae had captured a small amount of HA. The major capture was made at 10 minutes and it was maintained for 50 minutes without significant differences. From that time there was a slight increase in capture and it remained so until the end of the experiment without considerable variations. This experience demonstrates that at 10 minutes of in vitro contact, A. lumbricoides captures most of the HA, which suggests that in vivo, the parasite would be able to capture HA quickly for the purpose of modulating the immune response.


De Leon P.P.,Laboratorio Of Parasitologia | Biondi C.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2010

Sialic acids (SA) contained in glycoproteins and glycolipids participate in various biological functions; besides, its presence on the erythrocyte surface has both hemodynamical and hemorheological importance. SA are thought to intervene in the parasite-host interaction. The aim of this work was to study the effect of Ascaris lumbricoides on the erythrocyte superficial charge by using Polybrene Method. Work was carried out on 51 adult parasite extracts ([AE]) and 4 (larvae) larvae concentrates ([ALLC]: 2000 / 1000 / 500 / 250 larvae/ mL). The method was applied to non-treated (Control) and treated red cells with [AE] / all 4 [ALLC] simultaneously. Larvaé treatment was conducted over 18 experiences. Erythrocytes treated with 37. 25% of the [AE] had lower aggregation than the Control. The statistical analysis showed that this effect was not related to the protein concentration of [AE]. The aggregate decrease in the treatment with [ALLC] was dependent on larvae concentration and it was related to larvae concentrations ≥ 500 larvae/ mL. It was observed that the [AE]/ [ALLC] fixation to the erythrocyte was not strong. The results have shown that A. lumbricoides sequestrates SA erythrocyte. This capture could affect the pathology and/or participate in the escape of the host's immune response.


De Leon P.P.,Area de Parasitologia | Racca L.,Area Estadistica y Procesamiento de Datos | Menendez M.,Area de Parasitologia | Biondi C.,Laboratorio Of Inmunohematologia | Valverde J.,Laboratorio Of Inmunohematologia
Acta Bioquimica Clinica Latinoamericana | Year: 2012

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, 7PBMsaline solution, at 37°C. 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. © 2012 Federacion Bioquimica de la Provincia de Buenos Aires.


Boggione C.T.,National University of Rosario | Lujan Brajovich M.E.,National University of Rosario | Tarrago M.,Laboratorio Of Inmunohematologia | Mattaloni S.M.,National University of Rosario | And 5 more authors.
Transfusion | Year: 2014

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-Ds, 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-Ds 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. © 2014 AABB.


Brajovich M.E.L.,National University of Rosario | Brajovich M.E.L.,CONICET | Brajovich M.E.L.,Laboratorio Of Inmunohematologia | Trucco Boggione C.,National University of Rosario | And 20 more authors.
Transfusion | Year: 2012

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. © 2012 American Association of Blood Banks.

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