In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment.

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red-blood-cell membrane surface, namely oxidant compounds, such as ascorbate/Fe(+2) and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3-cross-linking reagents. We monitored the phagocytosis of oxidized or Band 3-cross-linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe(3+), but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross-linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS(3)) and 3,3'-dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS(3)-cross-linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short-term delivery to cells of the mononuclear phagocytic system.

Differential in vitro and in vivo behavior of mouse ascorbate/Fe3+ and diamide oxidized erythrocytes.

Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe3+ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe3+ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe3+ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used.

Influence of aerobic oxidation of mouse erythrocytes on their recognition by macrophages.

Membrane protein modification can change cell surface properties which can be correlated with altered macrophage-erythrocyte interactions. Mouse erythrocytes were incubated in phosphate buffer for different times to induce protein modification. Mouse erythrocyte membrane changes were analyzed by infrared analyses and gel electrophoresis. Proteolytic digestion of membrane proteins was observed. After 22 hours preliminary incubation, the number of erythrocytes adhering to a monolayer of macrophages reached a maximum, the majority of which had not been phagocytosed. Most of the erythrocytes incubated for 40 hours underwent phagocytosis after adhesion to the macrophages.

Delivery to macrophages of interleukin 3 loaded in mouse erythrocytes.

Mouse carrier erythrocytes containing 125I-interleukin 3 have been prepared and treated with band 3 crosslinking reagents. The incorporation of interleukin 3 by hypotonic treatment into mouse erythrocytes reached levels of about 15% of the interleukin 3 added to the medium being predominantly present in the cytosolic fraction (73%). Uptake fell to about 7.4% when using the same conditions but omitting hypotonic shock. The interaction of band 3 crosslinked interleukin 3 loaded erythrocytes with macrophages was also studied. A high level of incorporation of interleukin 3 into macrophages was observed either from band 3 crosslinked, interleukin 3-loaded erythrocytes or from interleukin 3 loaded erythrocytes. The observations encourage the view that the system may be able to deliver and target cytokines and other growth factors to macrophages.

Attempted suicide: repetition and survival--findings of a follow-up study.

OBJECTIVE: This was a prospective follow-up study of suicidal patients to assess the influence over time of different risk factors, whether on completed suicides or reattempts. Survival analysis makes it possible to weigh the influence of variables that increase or decrease a patient's life span or that make reattempts less likely. METHOD: A cohort of 150 patients admitted to a psychiatric department after a suicide attempt was followed up over 10 years. The study protocol used standardized criteria, and periodic controls were carried out in all patients. RESULTS: In total, 12% of patients completed suicide, 10% died from natural causes, 75% were still alive and 25% reattempted. In the survival analysis the risk for completed suicide or reattempting was highest during the first 2 years after the index attempt admission. Global Assessment of Functioning (GAF) was the factor that most increased survival time. The number of previous attempts decreased survival time and increased the risk of reattempts. CONCLUSION: Since suicidal risk varied over time, intensifying contact with patients during periods of psychopathological change or life events could prolong their survival.

Band-3 crosslinking-induced targeting of mouse carrier erythrocytes.

Mouse band-3 crosslinked carrier erythrocytes have been prepared. [125I]Carbonic anhydrase (CA) has been encapsulated into mouse erythrocytes. Then, loaded erythrocytes were labelled with 51Cr. Eventually, these doubly labelled cells were crosslinked with band-3 crosslinking reagents. [125I]CA was shown to have cytosolic localization in crosslinked carrier erythrocytes. Estimation of the action of band-3 crosslinkers on mouse carrier-erythrocyte membranes rendered values around 1721% of band-3 monomer reduction. Crosslinked carrier erythrocytes were in vivo targeted to liver, as shown by chromium-labelling localization. Also, encapsulated CA radioactivity was localized in vivo predominantly in liver, which is clearly in contrast with the behaviour shown by free CA injected into animals. These results support this model as a feasible system for the analysis of carrier-erythrocyte survival and targeting as well as the in vivo efficacy of release and targeting of encapsulated compounds.

In vivo behaviour of rat band 3 cross-linked carrier erythrocytes.

Rat band 3 cross-linked carrier erythrocytes have been prepared. Iodinated carbonic anhydrase has been encapsulated into rat erythrocytes. Then, carrier erythrocytes were labeled with 51chromium. Eventually, these doubly labeled rat RBCs were treated with a band 3 cross-linking reagent, namely bis(sulfosuccinimidyl)suberate (BS3). 51Chromium labeling and 125I CA showed to have cytosolic localization in cross-linked carrier erythrocytes. Estimation of the band 3 cross-linking induced by BS3 on rat carrier erythrocytes has been done rendering values around 25% of band 3 monomer reduction. BS3-cross-linked carrier erythrocytes when injected into rats are mainly targeted to liver as shown by chromium labeling localization. Also, encapsulated CA radioactivity carried by cross-linked carrier rat erythrocytes when injected into rats is localized predominantly in liver as shown by in vivo experiments. Accordingly, cross-linked carrier erythrocytes are highly recognized by peritoneal macrophages as detected by in vitro analyses of macrophage recognition. Thus, our data revealed a targeting of carrier rat erythrocytes induced by cross-linking of band 3 protein by BS3. These results support claims in favor of this animal model as a feasible system to analyze cross-linked carrier erythrocytes survival and targeting as well as the in vivo efficacy of targeting of loaded compounds to liver.

Differential induction of macrophage recognition of carrier erythrocytes by treatment with band 3 cross-linkers.

Mouse native and hypotonically loaded erythrocytes were treated with two cross-linking reagents: bis(sulphosuccinimidyl)suberate (BS3)- and 3,3'-dithiobis-(sulphosuccinimidyl propionate) (DTSP), excluding clustering agents. Microscopic analyses revealed that band 3 cross-linked native and hypotonically loaded erythrocytes are more strongly recognized by peritoneal macrophages than native and loaded erythrocytes as a result of the cross-linking of band 3 protein in accordance with studies in vivo. Macrophage-recognition analyses of 51Cr-labelled erythrocytes also demonstrated increased recognition of cross-linked and cross-linked loaded erythrocytes. This shows that the only action of these two band 3 cross-linkers on mouse erythrocytes promotes recognition by macrophages without requiring the use of clustering agents. The extent of recognition of BS3 cross-linked and cross-linked loaded erythrocytes by macrophages is dependent on the presence or absence of homologous serum or immunoglobulins. In contrast, the presence of serum factors or IgG in the incubation medium did not seem to influence the recognition of DTSP-modified erythrocytes by macrophages. These results seem to indicate a different mechanism of recognition for the erythrocytes modified with either one or the other band 3 cross-linker. In summary, the unique use of both band 3 cross-linkers procedures can be used to target carrier erythrocytes conveying active compounds to macrophages, with possible therapeutical applications. Different mechanisms of induction of macrophage recognition by these band 3 cross-linkers could reveal differential actions on erythrocytes or the involvement of different factors in the recognition process.

Cross-linking treatment of loaded erythrocytes increases delivery of encapsulated substance to macrophages.

Previous investigation has shown that osmotically loaded erythrocytes can act as drug carriers in systemic circulation, whereas chemically modified erythrocytes can be targeted to organs of the mononuclear phagocytic system because of changes introduced in the membrane that are recognized by macrophage cells. In this study we have examined the delivery of 125I-labelled carbonic anhydrase (125I-CA) carried by mouse erythrocytes, either loaded, or loaded and cross-linked with bis(sulphosuccinimidyl)suberate (BS3) and 3,3'-dithiobis-(sulphosuccinimidyl propionate), into homologous peritoneal macrophages maintained in culture. The hypotonically loaded mouse erythrocytes show a slight recognition by macrophages, similar to native erythrocytes. CA loaded into erythrocytes is thus delivered to a limited extent into macrophages. Neither the number of recognized loaded 51Cr-labelled erythrocytes nor the amount of delivered 125I-CA is affected by the presence of serum components or IgG. In contrast, cross-linking these loaded erythrocytes results in a greater phagocytosis by macrophages as assessed by microscopic observations, producing a markedly increased amount of targeted enzyme. The amount of CA delivered into macrophages, after BS3 cross-linker treatment of erythrocytes, is dependent on the presence of serum components in the incubation medium. Thus these cross-linking treatments improve the capacity of loaded mouse erythrocytes to deliver significant amounts of targeted enzyme to macrophage cells, increasing the therapeutic potential of carrier erythrocytes.

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.

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.

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.

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.

Heterogeneity of hypotonically loaded rat erythrocyte populations as detected by counter-current distribution in aqueous polymer two-phase systems.

Carrier rat erythrocytes loaded with exogenous substances ([125I] carbonic anhydrase) by hypotonic-isotonic dialysis become heterogeneous cell populations that can be fractionated using the counter-current distribution (CCD) technique. Two well-defined low- and high-partition ratio, G, subpopulations are obtained in charge-sensitive dextran-polyethylene glycol two-phase systems. The low-G subpopulation, which contains the most fragile and surface-altered cells, as deduced from their osmotic fragility curves and partition behaviour, respectively, presents a high amount of exogenous substance incorporated (134.6 cpm/10(6) cells). The high-G subpopulation, that contains cells similar to the control or isotonically dialyzed cells presents a lower amount of exogenous substance incorporated (69.8 cpm/10(6) cells). Cells in this high-G subpopulation seem to be fractionated, like the controls, according to ageing as suggested by the decline of the pyruvate kinase specific activity from the left- to the right-hand side of the CCD profile.

Effects of DNA topoisomerase II inhibitors on human bone marrow progenitor cells.

Topoisomerase II (topo II) is a target for many cytotoxic agents. Two observations, however, warrant caution in their therapeutic use: first, these agents can inhibit differentiation and second, perturbations in function render the enzyme error-prone. Illegitimate recombination events occurring at sites where topo II acts in differentiation could be particularly important in the development of secondary malignancies (relatively frequent after therapy with agents that target topo II). Topo II inhibitors are heterogeneous in mechanisms of action; in site-specificity of cleavable complex 'entrapment' (where present) and in the relative potency against the two topo II isoforms, all potentially influencing the site of maximum DNA damage. The object of this study was to examine the effect of topo II inhibitors on human haemopoietic precursor cells, to determine which have most impact on differentiation. We selected two which act via cleavable complex entrapment, but with different site preferences (m-AMSA and VP-16), and two acting via other mechanisms (merbarone and fostriecin). VP-16 and m-AMSA showed similar patterns with low dose stimulation of granulocyte-macrophage colony formation and high dose inhibition of all colony types. The stimulation was accompanied by an increase in colony size and blast content, consistent with a low dose inhibition of differentiation. Forstriecin, in contrast, stimulated predominantly mixed and erythroid colonies. Merbarone failed to increase colony formation. Neither produced substantial inhibition of colony formation. The effects on granulocyte-macrophage progenitors were confirmed using 7-day suspension cultures, using nitroblue tetrazolium (NBT) reduction and 3-4,5,dimethylthiazol 2,5-diphenyl tetrazolium bromide (MTT) assays for differentiated cells and total cell mass, respectively. These results demonstrate that the effects of topo II inhibitors on haemopoietic cell proliferation and differentiation are agent-specific and can involve lineage-restricted partial inhibition of differentiation.

Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity.

PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.

Affinity partitioning of erythrocytic phosphofructokinase in aqueous two-phase systems containing poly(ethylene glycol)-bound cibacron blue. Influence of pH, ionic strength and substrates/effectors.

Phosphofructokinase (PFK) from rat erythrocyte haemolysates has a high affinity for Cibacron Blue F3G-A covalently bound to poly(ethylene glycol) (PEG-Cb) and thus the enzyme can be extracted into the top phase of poly(ethylene glycol)-dextran aqueous two-phase systems containing PEG-Cb. The pH, ionic strength and presence of substrates/effectors affect to different extents the affinity of the enzyme for PEG-Cb and the number of PEG-Cb molecules attached per molecule of PFK (the latter probably reflecting, at saturation, the influence on the aggregation state of the enzyme) and thus influence the yield of enzyme recovered in the top phase. Increasing the pH from 6 to 7 and then to 8 leads to a higher yield of PFK in the top phase. A change in pH from 6 to 7 and 8 results in an increased number of PEG-Cb molecules attached per molecule of enzyme while the affinity of PFK for PEG-Cb shows a minimum at pH 7. The ionic strength in the range 0.017-0.164 has less influence on the partitioning of PFK. The presence of substrates or effectors of the enzyme in general reduces the recovery of PFK in the top phase. Fructose 6-phosphate increases the number of PEG-Cb molecules attached but greatly reduces the affinity of PFK for PEG-Cb. In contrast, AMP slightly reduces the number of PEG-Cb molecules attached and the affinity of PFK for PEG-Cb. ATP and ATP-Mg2+ compete with PEG-Cb for the same binding sites in PFK.(ABSTRACT TRUNCATED AT 250 WORDS)