Gilberto Berti

Binding of anti-spectrin antibodies to red blood cells and vesiculation in various in vivo and in vitro ageing conditions in the rat.

In this study, the binding of naturally occurring antibodies as well as of induced anti-spectrin antibodies to red blood cells (RBC), in relation with different ageing conditions, was investigated in the rat. RBC from aged animals, or from rats whose RBC were age-induced either by means of hypertransfusion (which blocks erythropoiesis) or by treatment with clodronate-containing liposomes (which reduces RBC removal from circulation), were used. Attainment of RBC ageing was demonstrated by MCV reduction and by an increase of both RBC density and 4.1a/4.1b RBC membrane protein ratio. The results demonstrate an augmented anti-spectrin antibody binding to RBC in relation with their ageing condition, especially when induced by hypertransfusion. The vesiculation process was also investigated and correlated with antibody binding: vesicles were found only in the plasma of clodronate-treated rats, whose RBC showed the lowest level of anti-spectrin antibody binding with respect to the other groups. In addition, RBC preserved in vitro in different media showed a binding of anti-spectrin antibody, which inversely correlated with the vesiculation process. On the whole, the latter results suggest a protective effect of vesicles towards IgG opsonization of aged RBC.

Aging of red blood cells and impaired erythropoiesis following prolonged administration of dichloromethylene diphosphonate containing liposomes in rats.

Abstract:  Objectives: To investigate whether macrophage‐depleted rats may serve as a model for studying red blood cell (RBC) aging. Methods: Rats were macrophage‐depleted by 4 weekly injections of dichloromethylene diphosphonate‐containing liposomes (Cl2MDP‐CL). The macrophage content of spleens and bone marrows (BMs) was investigated by immunohistochemistry and light microscopy and by flow cytometry, respectively, after staining with macrophage‐specific monoclonal antibodies. In addition, the ultrastructure of residual BM macrophages and their ability to phagocytose zymosan was studied. BM was also studied for apoptosis (by the TUNEL reaction) and for erythroid progenitor cell content. Furthermore, RBC indices, morphology, life span (by 51Cr labeling) and aging features (MCV, density, 4.1a/4.1b membrane protein ratio, anti‐spectrin IgG binding, microvesiculation) were investigated. Serum TNF‐α, iron, total iron‐binding capacity (TIBC) and ferritin were also determined. Results: Prolonged treatment with Cl2MDP‐CL caused an almost complete depletion of macrophages in the spleen and a 58% reduction of those in the BM; the residual BM macrophages were activated as judged by their ultrastructure and phagocytic capacity in vitro. These alterations were accompanied by an increase in RBC life span and age‐related RBC changes, as well as by mild anemia associated with a reduced reticulocyte count, reduced BM erythroid progenitors, increased numbers of apoptotic cells in the BM, low serum iron, high TIBC and increased serum TNF‐α levels. Conclusions: Rats subjected to prolonged macrophage depletion showed an increased prevalence of senescent RBC in the circulation due to their impaired clearance by macrophages. Hence, these animals provide a model system in which mechanisms of RBC aging can be delineated. They also showed impaired erythropoiesis, presumably related to a reduction in BM macrophages and increased production of proinflammatory cytokines by residual activated marrow macrophages and other cells.

Red blood cells expressing fetal antigens: their presence in adults with certain forms of anemia.

Using the IgG fraction of an antiserum against cord red blood cell (RBC) membranes (F-IgG), antigenic properties of RBC of newborns (n = 24) and patients suffering from anemia (n = 46) [either due to beta-thalassemia intermedia (n = 37) or hemorrhage (n = 9)] as compared to those of normal adults (n = 18) were examined with fluorescence microscopy, flow cytometry and radioimmunoassays (RIA). With fluorescence microscopy and flow cytometry 1.01 +/- 0.31 and 0.82 +/- 0.28% (mean +/- SD), respectively, of cord RBC and 0.79 +/- 0.31 and 0.53 +/- 0.28% of RBC from anemic patients reacted with F-IgG. RBC of normal adults showed virtually no F-IgG reactivity. In anemic patients there was a good correlation between the percent of F-IgG-reactive cells and the percent of reticulocytes, although the former were only two thirds of the latter; the ratio of F-IgG-reactive cells to reticulocytes was higher in posthemorrhagic anemia than in thalassemia. Moreover, double stainings revealed that the majority of F-IgG-reactive RBC were at the reticulocyte stage (80%), and coexpressed transferrin receptor (96%). Furthermore, the F-IgG-positive RBC correlated inversely with Hb levels. When RIA was employed, F-IgG binding to RBC of anemic patients and newborns was similar and considerably and significantly higher than that to RBC from healthy adults. The results demonstrate the reappearance in certain forms of anemia of F-IgG-reactive RBC, which are likely to represent a subpopulation of reticulocytes.

Potentiation of erythroid abnormalities following macrophage depletion in aged rats.

Objectives: The effects of prolonged macrophage depletion on haematological parameters were investigated in aged rats and compared with those in young ones. Methods: Four weekly i.v. injections of dichloromethylene diphosphonate‐containing liposomes (Cl2MDP‐CL) were employed to achieve a prolonged depletion of bone marrow (BM) and spleen macrophages. The number of BM macrophages was then assessed by flow cytometry, whereas the spleen clearance function was judged by the elimination of oxidised red blood cells (RBC). Haematological parameters and signs of RBC ageing (reduced MCV, increased density and augmented 4.1a/4.1b membrane protein ratio) were determined. Finally, the recovery from phlebotomy‐induced acute anaemia was investigated. Results: Following the Cl2MDP‐CL treatment, in comparison with young rats, the aged animals showed: (i) reduced numbers of BM macrophages; (ii) greater impairment of spleen clearance function; (iii) similar anaemic condition and signs of RBC ageing; (iv) greater increase in white blood cell (WBC) numbers (mainly neutrophils). In addition, whereas aged control rats showed a recovery from phlebotomy‐induced acute anaemia which was similar to that of the untreated young animals, in the aged‐treated rats, a significantly diminished/delayed restoration of RBC, Hb and reticulocyte to normal values was observed, accompanied by a significantly higher increase in WBC numbers than in the other groups of animals. Conclusion: Haematological abnormalities because of Cl2MDP‐CL‐induced macrophage depletion are potentiated in aged rats in which the BM regenerative potential of the erythroid lineage as well as the clearance function of the spleen appear compromised. Thus, in aged rats, macrophage dysfunction is likely to interfere with erythroid homeostasis particularly during haemopoietic stress.

Rabbit IgG Antibodies against Cord Red Blood Cell Membranes Bind to Complement Receptor 1 (CD35)

We have previously shown that a subpopulation of cord/fetal red blood cells (RBC) binds rabbit IgG antibodies raised against cord RBC and absorbed on adult RBC (F-IgG), while control IgG, raised against and absorbed on adult RBC (A-IgG), fails to do so. In the present study, F-IgG maintained its binding to cord RBC surface antigens following absorption on spectrin but not after absorption on skeleton-stripped RBC membranes. Spectrin-absorbed F-IgG- but not A-IgG-affinity-purified material from cord RBC contained polypeptides with apparent MW of complement receptor 1 (CR1) allotypes. Moreover, on immunoblotting these polypeptides reacted with 125I-F-IgG as well as with 125I-anti-CR1 mAb, and binding of 125I-anti-CR1 mAb was inhibited by unlabelled F-IgG. In addition, cord RBC incubated with F-IgG prior to reaction with anti-CR1 showed decreased fluorescence intensity on flow cytometry. Taken together the results suggest that F-IgG binds to CR1 which shows increased expression/accessibility on a subpopulation of cord/fetal RBC.

ERYTHROCYTES WITH DIFFERENT SURFACE IMMUNOLOGICAL PROPERTIES IN THE PERIPHERAL BLOOD OF β-THALASSAEMIC PATIENTS

pain as result of thrombosis of portal and splenic veins. Oral administration of polymyxin-E led to a reduction of endotoxin levels from lo4 to 1-10 ng/g faeces in the gut. The second patient had a severe haemolytic anaemia due to PNH. He was also treated with polymyxin-E which resulted in a reduction of the endotoxin level from 103-104 to 10 ng/g faeces. These low levels of endotoxin were maintained during a period of 2-3 weeks. Although in both patients the endotoxin levels in the gut were markedly reduced, the haematological parameters did not improve, while endotoxin could not be detected in the blood at any time. The lack of beneficial effects of the suppression of gut endotoxin could be due to the limited relevance of gut endotoxin in the causation of clinical problems in PNH; on the other hand the effects of more complete gut sterilization awaits study.

[A comparative immunoelectrophoretic study on extracts of LS fibroblasts and of tumors produced by inoculating them into C3H mice (author's transl)].

Aqueous extracts of LS fibroblasts (LS) and of tumors obtained by inoculating these cells into newborn C3H mice (Tu/LS) share a number of antigens detectable by means of Immunoelectrophoresis against rabbit anti-LS or anti-Tu/LS sera. Yet, at least one antigen seems to be contained only in the LS extracts; in fact, anti-LS sera, absorbed with Tu/LS extracts, reacted with the LS extracts, causing a precipitation line on Immunoelectrophoresis. This may depend on the suppression from the growing population of the inoculated cells of a line more incompatible or in any case less favored in the environment of the subcutaneous tissue of the living animal; or on the lack of in vivo production of an antigen that the LS fibroblast sinthesizes when growing in vitro.