Accelerated elimination from the circulation of homologous aged red blood cells in rats bearing anti-spectrin antibodies.

In order to analyse a possible role of anti-spectrin antibodies in the clearance of aged red blood cells (RBC), a homologous system was employed, whereby a population of aged RBC, obtained by hypertransfusion, was injected into rats bearing a high level of anti-spectrin antibodies, following immunization with spectrin. The aged RBC bound the anti-spectrin antibodies 'in vitro' and were eliminated from circulation in spectrin-treated rats at a faster rate than in control rats with naturally occurring antibodies. The analysis of the clearance curves revealed aged RBC of heterogeneous lifespans: two principal populations of short- and longer-living could be identified. In rats with anti-spectrin antibodies, the survival of the short-living population was further reduced. However, the similar kinetics of elimination of aged RBC in the two groups (with naturally-occurring and induced antibodies, respectively) suggest that anti-spectrin antibodies strengthened the intervention of the naturally-occurring ones. On the basis of these results, we assume that during their aging in circulation, RBC can accumulate surface alterations to make spectrin accessible to antibodies so that, in addition to anti-band 3 antibodies, anti-spectrin antibodies may contribute to their elimination.

[Lowered osmotic resistance of mouse LS fibroblasts grown in the presence of various types of dextran]. / Diminuzione della resistenza osmotica di fibroblasti LS di topo cresciuti in terreno addizionato di vari tipi didestrano.

Mouse fibroblasts grown in the presence of dextrans of various molecular weight at various concentrations, show a small but significant decrease of the osmotic resistance (Tab. 1,2,3; ANOVA: source of variation A and C); this decrease is related to the length of time the cells stay in the dextran containing medium (Tab. 2,3; ANOVA: source of variation B). The presence of dextran in the culture medium has no effect on the cell growth (Tab. 4).

Lowered resistance to lytic agents in sucrose vacuolation.

Vacuolation in fibroblasts cultivated in the presence of sucrose is associated with progressive accumulation of the undigestible sugar. In radioisotope experiments the process lasted several days, and when the cells were subcultured back into a medium devoid of sucrose the label was also lost after several days. This type of vacuolated cells is more fragile when it is challenged with lytic agents. 51Cr-labelled LS fibroblasts released more radioactivity when they had been growing in the presence of sucrose, whether they were suspended in media of decreasing osmolarity, in dilutions of various surfactants, exposed to high temperatures, or subjected to mechanical stress. It is concluded that these cells exhibit a lower resistance when exposed to unfavourable environments, but retain their viability in growth media despite some morphological and biochemical alterations.

[Action of high temperature on the integrity of LS fibroblasts. I. Effect of culture in the presence of sucrose]. / Azione di temperature elevate sulla integrità dei fibroblasti LS. Nota I: Effecto della coltura in presenza di saccarosio.

LS fibroblasts which have accumulated sucrose into cytoplasmic vacuoles during 4 days are more fragile than normal controls when they are exposed to temperatures between 45 degrees C and 60 degrees C. Continuing to cultivate for 6 more days in the presence of sucrose apparently improves the resistance of the vacuolated cultures to the damaging temperatures, possibly because of a selective enhancement of the more resistant cells.

[Action of high temperature on the integrity of LS fibroblasts. II. Effect of duration of presence in a medium containing sucrose]. / Azione di temperature elevate sulla integrità dei fibroblasti LS. Nota II: Effetto della diversa durata di permanenza in terreno contenente saccarosio.

The resistance to high temperatures of LS fibroblasts starts lowering quite rapidly when the cells have been suspended in a culture medium containing sucrose. On the other hand, transferring back to normal medium for increasing times cells cultivated for 4 days in the presence of sucrose seems to gradually improve their resistance.

[Polyester treatment in rats with a dorsal air pouch: chemotaxis in lavage fluid from the pouch]. / Studio del trattamento con poliestere in ratti portatori di una sacca dorsale d'aria: chemiotassi dei liquidi di lavaggio della sacca.

Minced polyester threads introduced into peritoneal cavity of rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they are released locally by cells involved in inflammation. In this paper the chemotactic effect of the peritoneal and subcutaneous air pouch fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The fluids were obtained by washing the cavity of untreated rats or rats injected with polyester, 7 days after the injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear cells from normal rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrate that a chemotactic activity is present in peritoneal and subcutaneous air pouch fluids following the inflammatory process. In conclusion the chronic inflammation determines the appearance of chemotactic factors for PMN cells, in the peritoneal cavity and in the air pouch, and the air pouch is a very convenient experimental system with the particular advantage that it permits easy repeated sampling of exudate during the course of an inflammatory response.

[Inflammatory reaction to polyester material in the guinea pig. IV. Production and liberation of beta glucuronidase and lactate dehydrogenase by peritoneal macrophages in culture]. / Il processo infiammatorio da materiale poliestere nella cavia. IV. Produzione e liberazione di beta glicuronidasi e di lattato deidrogenasi da parte di macrofagi peritoneali in coltura.

Peritoneal macrophages obtained from guinea pigs intraperitoneally injected with minced polyester threads with saline, and from control animals were cultivated and the activities of the lysosomal enzyme beta-glucuronidase and of the cytoplasmic enzyme lactate dehydrogenase were determined in the cells and in the culture media. The production and particularly the release into the medium of beta-glucuronidase increased in the cultures from the treated animals; the LDH production was also markedly increased, and the enzyme was partly lost into the medium, suggesting that the injection of Mersilene into the guinea pigs markedly induces these enzymes in the phagocytes and at the same time probably increases the leakiness of their cell membrane.

[Inflammatory reaction to polyester material in the guinea pig. V. Semiautomatic determination of peritoneal cell volume]. / Il processo infiammatorio da materiale poliestere nella cavia. V. Determinazione semiautomatica del volume delle cellule peritoneali.

The volume of the peritoneal cells of guinea pigs treated with injections of 1) minced polyester threads (Mersilene), 2) saline, and of untreated animals, has been determined utilizing a Coulter Counter coupled with a pulse height analyzer. The volumetric test on the whole cell population has emphasized a distinctly bimodal distribution, due to the presence of two cellular types picked out as first peak (P 1 degrees) and second peak (P 2 degrees), while the non-adherent cells (CNA) have shown an unimodal distribution. Statistically significant difference in the mean volumes have been found between the P 1 degrees and P 2 degrees cells and between the CNA and P 1 degrees cells. The percentage of CNA in the whole cell population significantly decreases in "Mersilene"-treated guinea pigs.

[Inflammatory reaction to polyester threads in guinea pigs. III. Reduction of Nitroblue tetrazolium by peritoneal macrophages in vitro]. / Il processo infiammatorio da materiale poliestere nella cavia. III. Riduzione in vitro del Nitroblu di tetrazolio da parte dei macrofagi peritoneali.

Peritoneal cells of guinea pigs treated or not by intraperitoneal injection of minced polyester threads have been tested by the NBT test. In phagocytic cells nitroblue tetrazolium (NBT) is reduced to formazan. This is deposited as a dark blue precipitate in the cytoplasm. Changes in reduction capacity have been quantitated by calculation of percentage of macrophages containing formazan deposits, (Tab. 1), and by extraction and photometric evaluation of formazan (Tab. 2). Peritoneal cells incubated with polyester show an increased NBT reduction, which is more apparent with the photometric evaluation. A statistically significant difference in NBT reduction has been found between the peritoneal cells of the guinea pigs treated "in vivo" with polyester and those of the untreated "in vivo" with polyester and those of the untreated animals, whether or not challenged in vitro with polyester threads. These results suggest a change in cell population following polyester treatment.

[Observations on cytolysis by cobra venom. Effect of sucrose loading]. / Osservazioni sulla citolisi da veleno di cobra. Effetto del carico con saccarosio.

LS cells cultivated for 4 days in sucrose-containing medium (Su) are more fragile than cells cultivated in medium devoid of this sugar (TN) when they are incubated with Naja-Naja venom; in fact the 51Cr radioactivity released into the medium by previously labelled cells is increased for the sucrose ones. Phospholipase does not seem to contribute to the lytic activity of the venom, which is dependent on the direct lytic factor: neither varying the temperature of incubation nor adding EDTA to the incubation medium modifies the results. Adding Ca++ decreases the 51Cr radioactivity released particularly by the cells grown in TN, suggesting a stabilizing action of this cation.

[Interference of polyester inflammation on the development of Yoshida ascites hepatoma in the rat]. / Interferenza dell'infiammazione da poliestere con lo sviluppo dell'epatoma-ascite di Yoshida nel ratto.

Minced polyester threads introduced into the peritoneal cavity cause a chronic inflammation with evidence of macrophage and lymphocyte stimulation. In this paper an interference between this kind of inflammation and the growth of Yoshida ascites hepatoma has been shown, which has been found to dependent on the time interval elapsed between the introduction of polyester (Mersilene) minces and injection of the hepatoma cells. Rats were treated intraperitoneally with Mersilene and then divided in to three groups: the first was injected intraperitoneally with hepatoma cells immediately (TM 0), the second after 7 (TM 7) and the third after 14 days (TM 14); rats untreated with polyester and implanted with the same number of hepatoma cells served as controls (NT). While in groups NT and TM 0 a rapid growth of hepatoma cells occurred, together with the accumulation of a considerable volume of ascitic fluid, no tumor growth neither ascite production occurred in groups TM 7 and TM 14; in these animals where several days were allowed to elapse after polyester introduction, the hepatoma cells which had been injected rapidly disappeared and were no more found 48 h after the intraperitoneal injection. It is suggested that the inhibition of the neoplastic growth may be dependent on the activation of macrophages (and possibly NK cells) which accompanies the development of the chronic polyester inflammation.

[Inflammatory process caused by intraperitoneal injection of polyester in the rat: chemotactic activity in lavage fluid from the cavity]. / Processo infiammatorio da inoculo intraperitoneale di poliestere nel ratto: attivita chemiotattica nei liquidi di lavaggio della cavita.

Minced polyester threads introduced into peritoneal cavity of guinea pigs or rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they may be exogenous and/or endogenous. These are released locally by the cells involved in inflammation. In this paper the chemotactic effects of the peritoneal fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The peritoneal cavity fluids were obtained by washing the cavity of untreated rats or rats intraperitoneally injected with polyester, 1, 3, 7, 14 days after the intraperitoneal injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear leukocytes from normal or treated rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrated that the peritoneal fluids taken 3 and 7 days after the intraperitoneal polyester injection, elicit an evident chemotaxis response greater than that showed by peritoneal fluids from control rats. It is suggested that chemotactic factors can be produced and released by mononuclear cells involved in the inflammatory process.

[Chemotactic activity of the peritoneal lavage fluid of guinea pigs with inflammatory processes caused by the inoculation of various types of suture thread]. / Attività chemiotattica dei liquidi di lavaggio della cavità peritoneale di cavie con processo infiammatorio da inoculo di vari tipi di fili da sutura.

Minced non adsorbable or adsorbable suture threads introduced into peritoneal cavity of guinea pigs elicit at inflammation with mononuclear and giant cells surrounding suture thread fragments. We studied the presence in the peritoneal cavity of chemotactic factors for PMN cells and we compared the results in relation to the different type of the suture threads used (Dexon, Mersilene, Gore-Tex). The peritoneal cavity was washed, the fluids collected and used as chemotactic agents. The chemotactic response was assayed by employing multiwell chemotaxis chambers (Neuro Probe) and PMNs from normal, non-treated guinea pigs. Quantification of the migration was calculate by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrate that a chemotactic activity is present in peritoneal fluids following the inflammatory process. This activity is evident at 7th day after Dexon and Mersilene inoculation; using PTFE however, it decreases at 14th d, when the inflammatory process is already developing into healing tissue. In conclusion the chronic inflammation determines the appearance of chemotactic factors for PMN cells; it is suggested that reactive, mononuclear cells, involved in the process, could be responsible for their production and release.

[Interference of inflammation caused by polyester with the growth of Yoshida ascite hepatoma. IV. Modifications of the sedimentation profile of the hepatoma cells]. / Interferenza dell 'infiammazione da poliestere con la crescita dell'epatoma ascite di Yoshida. IV. Modificazioni del profilo di sedimentazione delle cellule dell'epatoma.

It was previously shown that Yoshida's ascites-hepatoma cells (Y cells) intraperitoneally injected into rats carrying chronic inflammation caused by polyester thread rapidly disappear. In this paper the sedimentation rate in a continuous Percoll gradient at unit gravity (1 g) using the CELSEP apparatus (Sorvall), the mean diameter and volume of the Y cells harvested from the peritoneal cavity of untreated (NT) and polyester-treated (TM) animals were determined at 6-12-18-24 hours after the inoculum. At 6 hours a number of Y cells appeared enlarged both from TM and NT rats, while afterwards, and notably at 18 and 24 hours, their size was decreased and their sedimentation rate was reduced only in TM animals. It seems that the chronic inflammation causes alterations both in the size and in the density of the Y cells and probably one of the mechanisms of their death is through shrinkage and apoptosis.

[Interference of inflammation caused by polyester with the development of Yoshida ascite hepatoma in rats. V. Study of cytotoxicity in vitro]. / Interferenza della infiammazione da poliestere con lo sviluppo dell'epatoma-ascite di Yoshida nel ratto. V. Ricerca di citotossicita' in vitro.

It has been known for some years that rats developing a granulomatous inflammation following the intraperitoneal injection of fragments of a polyester suture thread (Mersilene, M) reject a graft of 10(8) Yoshida's ascites hepatoma cells, which on the contrary rapidly grow in M-uninjected animals, originating an ascites tumor and killing the rats in a few days. In this paper granulomatous (TM) and normal (control, C) small pieces of peritoneum (omentum, mesentery and parietal) have been cultivated for 15 h in Eagle-Dul becco's medium together with 10(5) 51Cr-labelled hepatoma cells: the release of the label, an index of cell lysis, was considerably higher in the TM cultures, suggesting a cytotoxic activity of some components of the granulomas. Differences have been observed in the amount of radioisotope released among the TM cultures, suggesting that the particular composition of the cell population may be relevant to the cytotoxicity versus the hepatoma 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.

Nucleotide receptors: an emerging family of regulatory molecules in blood cells.

Nucleotides are emerging as an ubiquitous family of extracellular signaling molecules. It has been known for many years that adenosine diphosphate is a potent platelet aggregating factor, but it is now clear that virtually every circulating cell is responsive to nucleotides. Effects as different as proliferation or differentiation, chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species are elicited upon stimulation of blood cells with extracellular adenosine triphosphate (ATP). These effects are mediated through a specific class of plasma membrane receptors called purinergic P2 receptors that, according to the molecular structure, are further subdivided into 2 subfamilies: P2Y and P2X. ATP and possibly other nucleotides are released from damaged cells or secreted via nonlytic mechanisms. Thus, during inflammation or vascular damage, nucleotides may provide an important mechanism involved in the activation of leukocytes and platelets. However, the cell physiology of these receptors is still at its dawn, and the precise function of the multiple P2X and P2Y receptor subtypes remains to be understood.