Claudia Biondi

SENESCENT ERYTHROCYTES: FACTORS AFFECTING THE AGING OF RED BLOOD CELLS

Human red blood cells (RBC) have a well-defined lifespan of 120 days affected by many cellular parameters. The aim of the present study was to investigate through a functional assay the effect of some factors in the interaction of erythrocytes with monocytes: heat rigidification, equilibration at different pH and desialyzation. We also studied the interaction between stored RBC and peripheral blood monocytes with this functional erythrophagocytosis assay. Blood samples from 30 volunteer donors were investigated. 1) Senescent (Se) and Young (Y) RBC were obtained by differential centrifugation. 2) Erythrocyte suspensions: Aliquots of each sample were subjected to the following treatments: a) Rigidification by heat (RRBC), b) Equilibration at different pH (5.34, 6.30, 7.33, 9.20) and c) Desialyzation with neuraminidase and trypsin. The functional assay was performed incubating monocytes obtained by glass adherence with these suspensions of RBC. Whole blood samples (n=20) were stored during different periods of time (0, 7, 14, 21, 28, 35 and 42 days). The erythrophagocytosis assay was performed during six weeks incubating isologous monocytes with RBC from every unit. Negative and positive controls were performed using non sensitized (NSRBC) and sensitized with IgG anti-RhD (SRBC) red cells. The percentage of active monocytes (AM) obtained were: 1) YRBC: 2.8±0.9 and SeRBC: 17.5±2.1; 2a) RRBC: 3.0±0.9; 2b) 10.9±0.9, 15.5±0.8, 3.1±1.0, 4.0±1.1; 2c) 11.1±1.4 and 3.9±1.0; SRBC 32.1±1.7 and NSRBC: 2.8±1.5. The % of AM with SeRBC was higher (p<0.001) than those obtained with NSRBC. The data of AM with RRBC were significantly lower (p<0.001) than those obtained with SeRBC and SBRC, indicating that heat rigidification of RBC does not increase phagocytosis by monocytes. The values of AM obtained from the suspensions of erythrocytes equilibrated at different pH indicate that the acidification of RBC increases the interaction with monocytes. The % AM with neuraminidase treated RBC was higher than those observed with YRBC and NSRBC (p<0.001). No modifications were observed with trypsin treated RBC. These results suggest that the loss of sialic acid may be involved in the physiological phagocytosis. The values of AM of stored whole blood were: 2.3±1.3, 2.7±1.3, 4.4±1.6, 6.7±1.2, 9.6±1.0, 11.7±0.8 and 13.0±1.2. The results showed a significant increase in the %of AM as a function of the preservation time from 2,3±1,3 for the first day to 13,0±1,2 for the 42nd day (p<0.001). The data obtained in this ex vivo model show a significant increase (p<0.001) in the phagocytosis of RBC equilibrated at low pH, desialinized (greater than 80%) with neuraminidase and stored for over 28 days. These factors would be involved in erythrocyte removal via phagocytosis during tissular homeostasis.

Early detection of RhD status in pregnancies at risk of hemolytic disease of the newborn

Abstract The aim of this work was to investigate the presence of the RHD gene in fetal cells obtained from amniotic fluid. We studied 65 samples of amniotic fluid, 11 from RhD-negative mothers sensitized with anit-D alloantibodies. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from amniotic fluid and maternal blood. The RHD genotyping was performed in non-contaminated samples (n=62) using a multiplex polymerase chain reaction strategy that yields three amplification products from RhD-positive phenotypes (intron 4 of both RHCE and RHD genes and exon 10 of the RHD gene) and 1 DNA fragment from RhD-negative phenotypes (intron 4 of the RHCE gene). We genotyped 54 RhD-positive fetuses (8 from RhD-negative sensitized mothers) and 8 RhD-negative fetuses (3 from RhD-negative sensitized mothers). The fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD-negative invasive procedures can be avoided.

Association of the 'secretor state' with the presence and recurrence of urinary infections in pregnant women.

Urinary tract infection (UTI) is the most common medical complication of pregnancy. The prevalence increases with age, low socioeconomic status, sexual activity, in multiparous women and in cases of untreated pathologies. Most of the patients referred to our service belong to these groups and have an incidence of UTI of 17-20%. The binding of bacteria to urinary tract epithelial cells is an important step in the development of UTI. The receptors for uropathogenic organisms, particularly Escherichia coli, are carbohydrate residues located on surface glycolipids and glycoproteins of urothelial cells. I Blood group antigens (A, B, H, Lea, LeO) are also a group of carbohydrate determinants, found in erythrocytes and certain epithelial tissues, including urothelium. The presence or absence of these antigens on the urothelial cell surface may influence individual susceptibility to UTI. ABH and Lewis antigens are present in bodily fluids of individuals expressing the secretor state.v It is possible that if the secretion of blood group antigens is a protective factor in hostparasite interactions then there will be an increased incidence of non-secretors among pregnant women with UTI. The aim of this work was to investigate the association of erythrocyte antigens with the presence and recurrence of UTI in pregnant patients.

A Dc- phenotype encoded by an RHCE–D(5–7/8)–CE hybrid allele

Background and Objectives The Rh system is genetically controlled by the homologous RHD and RHCE genes that encode the RhD and RhCcEe polypeptides, respectively. Deletions, point mutations and rearrangements between both genes are responsible for the great polymorphism of this system. The aim of this work was to analyse the genetic basis of a Dc‐ phenotype.

Study of phagocytosis of senescent erythrocytes in young and elderly individuals

Abstract. There is a decrease in the percentage contribution of a heavy density fraction of red blood cells to whole blood with increasing age. The aim of this study was to investigate in the young and elderly the interaction between monocytes and different erythrocyte suspensions: senescent red blood cells, erythrocytes stored with or without serum, and desialylated red blood cells. The results obtained with senescent red blood cells and erythrocytes stored with serum show the involvement of autologous IgG in the selective removal of erythrocytes. These values were higher in elderly individuals, indicating that this process increases with age. Our observation suggest that desialylation is not involved in the increased removal of erythrocytes observed in elderly individuals.

Blood Groups and Oral Lesions Diagnostics

Cancer incidence in humans has gradually increased over the last century. Surgical, radio, chemotherapeutic and biological treatments have experienced important advances, with concomitant reduction in the morbidity associated with the radical surgical practices of the past. The term “oral cancer” includes a diverse group of tumors arising from the oral cavity (Khalili, 2008). Usually included are cancers of the lip, tongue, pharynx, and oral cavity. The World Health Organization (WHO) reported oral cancer as having one of the highest mortality ratios amongst all malignancies (Parkin et al., 2000). Although oral cancer is rare and attracts little attention, it is more common than Hodgkin’s disease tumours of brain, liver, bone, thyroid gland, stomach, ovaries, or cancer of the cervix. It ranks 12th among all cancers (Jemal et al., 2002). The vast majority of malignant neoplasms in the mouth are squamous cell carcinomas. Oral cancer incidence and mortality rates vary widely across the world. Mortality rate is an important tool that provides implicit information about incidence, diagnosis stage, solving capacity of health services, available technology and health programs to be applied. Although globally oral cancer represents an incidence of 3% (males) and 2% (females) of all malignant neoplasm, it has one of the lowest survival rates — 50 percent, within a five-year period (Greenlee et al., 2001).