Biomedical subjects

A Herráez

Publications and source records attributed to A Herráez.

10 recordsLinked to original sources

PubMed1998

Hypotonically loaded rat erythrocytes deliver encapsulated substances into peritoneal macrophages.

Previous work has shown increased uptake of hypotonically loaded rat RBCs by the spleen and liver "in vivo," suggesting that the cells of MPS are involved in their elimination from the circulation. In order to elucidate the mechanism of such elimination, we have undertaken studies on the interaction of such loaded RBCs, in comparison with native RBCs, with peritoneal macrophages. Erythrophagocytosis assays were performed in well plates to which thioglycollate-induced peritoneal macrophages had adhered. Native or loaded 51Cr-RBCs were added under different opsonization conditions to monolayer adherent macrophages, and then the amount of RBCs that were recognized was determined, with separation into adhesion and phagocytosis fractions. Native RBCs are slightly recognized by peritoneal macrophages, about one RBC per macrophage (Mphi). Osmotic treatment of rat RBCs used for encapsulation (independently of the encapsulated substance, 125I-CA or FITC-dextran) produces some modification in the erythrocyte membrane that induces higher recognition of these cells, about three loaded RBCs per macrophage. Consequently, both fluorescent (FITC-Dx) and radioactive (125I-CA) substances previously encapsulated in RBCs were transferred to M(phi)s. The fluorescence microscopic observations confirmed these results. Moreover, in the case of carrier 51Cr-cells loaded with 125I-CA, the amount of 125I-radioactivity delivered into M(phi)s was relatively higher than that of 51Cr. The highest ratio, 125I-CA (encapsulated substance)/51Cr-RBCs (carrier cells), present in M(phi)s means there was a stronger interaction with macrophages of RBCs that carry a higher amount of encapsulated CA, as a function of the heterogeneity of the loaded rat RBCs population previously reported. Finally, the adhesion and phagocytosis of loaded RBCs seem not to involve complement receptors or Fc receptors on the macrophages.

Animals↗

PubMed1997

In vitro properties and organ uptake of rat band 3 cross-linked erythrocytes.

Chemical conditions for cross-linking rat erythrocytes with bis(sulfosuccinimidyl)suberate (BS3) and 3,3' dithiobis(sulfosuccinimidyl propionate) (DTSSP) have been studied. These two cross-linking reagents seem to react with band 3 proteins in rat erythrocyte membrane. Two different conditions have been assayed using different cell/cross-linker concentration ratios. Similar cell volumes were observed in cross-linked rat erythrocytes compared to rat control erythrocytes. Cell yields after cross-linking account for about 65% when a high ratio of cell-to-cross-linker was used. Slightly lower cell yields (about 62%) were obtained when this ratio was reduced. Estimation of band 3 cross-linking by gel electrophoresis revealed a level of cross-linking of around 45% and 50% at the different conditions used. In vivo behavior of these modified rat erythrocytes revealed that they do not circulate, showing a predominant localization in the liver. This effect is evident from the concentration (5 mM BS3 or DTSSP) used. Based on these results, BS3 and DTSSP can be considered as useful tools to cross-link rat erythrocyte band 3 and to target rat erythrocytes to the liver.

Animals↗

PubMed1996

Fluorescence analysis of carrier rat and human erythrocytes loaded with FITC-dextran.

Rat and human erythrocytes are inherently different with respect to slow dialysis encapsulation used in preparing carrier erythrocytes. The incorporation process, commonly measured with radioactive tracers, is always larger in human erythrocytes, mainly because the rat carrier cells are more fragile. When FITC-Dextran (Dx) is used in the encapsulation process, and loaded rat and human RBCs are studied by fluorescence intensity, some additional events are evident. Not all cells of each population appear with a fluorescence signal, and not all show similar fluorescence intensity. Human RBCs show a higher percentage of marked cells and a higher fluorescence intensity than rat RBCs. Two populations, of high and low fluorescence, appear in FITC-Dx loaded rat erythrocytes. The human loaded RBCs show a similar peak distribution together with another peak in the middle scale of fluorescence. Therefore, a heterogeneity in the cell population as a result of the encapsulation process is manifested for both species. The fractionation of RBCs, loaded with either FITC-Dx or 125I-CA, by centrifugation on Ficoll-Paque reveals that the low density cells have much more substance incorporation than the counterpart cell subpopulation in the pellet. Therefore, the cell modifications produced by the encapsulation process are independent of the substance being incorporated. On the other hand, FITC-Dx, but not 125I-CA, shows a certain degree of association to RBCs membranes, especially in humans.

Animals↗

PubMed1996

In vivo survival and organ uptake of loaded carrier rat erythrocytes.

Rat RBCs loaded with 125I-CA by hypotonic dialysis and isotonic resealing were evaluated as a carrier system. Loaded RBCs stored at 4 degrees C remained unlysed (90% survival) allowing release of encapsulated 125I-CA for up to 4 days. Thereafter, cellular lysis increased significantly. IP-injected loaded RBCs reached the maximum level (50%) in circulation at 24 h post-injection. Circulating loaded RBCs showed a half-life of 8-10 days, which was advantageous for carrier function. In contrast to IP-injected free CA, which remained in circulation for only a short time, encapsulated CA showed significant levels in circulation up to 10 days post-injection. The profile of organ uptake with time is essentially not altered for loaded with respect to native cells, being higher the removal of loaded cells and mainly localized in spleen. Nevertheless, liver is the organ with highest elimination capacity for both native and loaded cells, showing its maximum at 24 h post-injection. Concomitantly, the concentration of 125I-CA in all organs studied was highest at this time. These data demonstrate that rat loaded RBCs can potentially be used as a carrier system for long-term dissemination of drug into the organism, with specially increased delivery to the spleen. They also support the use of the rat as an experimental model for biochemical and pharmacological studies in these therapeutic systems.

Animals↗

PubMed1996

Behaviour of isolated rat and human red blood cells upon hypotonic-dialysis encapsulation of carbonic anhydrase and dextran.

Rat and human carrier red blood cells (RBCs) were prepared by hypotonic-dialysis encapsulation. The standard conditions used for encapsulation were 80 mOsm/kg for 60 min. The encapsulation behaviour of rat and human RBCs was studied using radiolabelled carbonic anhydrase and fluorescently labelled dextran. Both markers are incorporated to slightly greater extents by human than by rat RBCs by hypotonic treatment. Cell recovery of rat and human RBCs loaded with either carbonic anhydrase or fluorescent dextran accounted for 49% and 80% respectively. The cellular integrity of the loaded cells was revealed by the presence of fluorescence labelling in rat and human RBCs. Fluorescence studies showed an increase of size dispersion in loaded rat and human RBCs, giving cellular volume variations in both types of cells resulting from the encapsulation procedure. Two loaded cell populations were evident in both species, one with high fluorescence content and another with background staining. Apparently the proportion of high fluorescently labelled loaded cells was higher in the case of the human RBCs. A reduced level of fluorescence labelling was observed in rat and human RBC membranes, which indicates a process of adsorption of dextran to the membranes.

Adsorption↗

PubMed1993

ELEASY: a program for processing experimental enzymoimmunoassay data.

A computer program is described that automates processing of data from enzyme immunoassays. This includes statistics, data editing, graphic display and fitting of data to both a straight line and a sigmoidal curve. The program can work either without the need for the user to enter choices, initial values, etc., or else with user-controlled choices for adaptation to special data sets.

Enzyme-Linked Immunosorbent Assay↗

PubMed1993

Immunoanalysis and quantitation of membrane sialoglycoproteins (glycophorins) in rat erythrocytes and reticulocytes.

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of rat erythrocyte membrane sialoglycoproteins (glycophorins) has been developed. Samples of erythrocytes and reticulocytes were analysed using this assay, and response compared among them and purified glycophorins samples. A broadly homologous behaviour of glycophorins was found in both cell types, suggesting the presence in reticulocytes of glycophorin molecules closely similar to those on the erythrocyte. A quantitative evaluation of glycophorins on both cell types yielded comparable levels of these glycoproteins, but with significantly higher values (1.7-fold) for reticulocytes. It is suggested that the lower number of epitopes present on the erythrocyte membrane might be due to the disappearance of some glycophorin-associated antigenic determinants during the maturation of reticulocyte to erythrocyte.

Animals↗

PubMed1993

Immunological relationship among rat erythrocyte membrane sialoglycoproteins and with human glycophorins.

1. Specific antibodies against a whole preparation of glycophorins from rat erythrocyte membrane and against its most prominent component (32 kDa) were prepared. 2. Both antisera and the respective affinity-purified antibodies recognize the 74, 32 and 25 kDa components of rat glycophorins; therefore, a close antigenic relationship between them can be inferred. 3. On the other hand, a cross-reaction with human glycophorin A (both MM- and NN-type) is observed for both antisera. 4. This suggests the existence of epitopes common to human and rat glycophorins.

Animals↗

PubMed1992

Rat erythrocyte glycophorins can be isolated by the lithium diiodosalicylate method used for other glycophorins.

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff's reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.

Animals↗

PubMed1989

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes.

A decrease in the number of binding sites for 125I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.

Anemia↗