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E Zocchi

Publications and source records attributed to E Zocchi.

53 records · Page 3Linked to original sources
PubMed1989

Encapsulation of doxorubicin in liver-targeted erythrocytes increases the therapeutic index of the drug in a murine metastatic model.

Doxorubicin-loaded, glutaraldehyde-treated murine erythrocytes, once reinjected into circulation, are rapidly taken up by liver and lungs and behave as an organ-targeted, slow delivery system for the encapsulated drug. The antitumor activity of encapsulated doxorubicin (former generic name, adriamycin) was compared with that of the free drug in a murine hepatic and pulmonary tumor model. This was obtained by intrasplenic injection of L1210 lymphoma cells followed by splenectomy. Different schedules of treatment of tumor-bearing mice with erythrocyte-encapsulated or free doxorubicin were investigated. The optimal schedule of treatment for free doxorubicin proved to be i.v. bolus administration on the day of splenectomy. Under these conditions, the dose producing 50% inhibition of metastatic growth in the liver, as measured by inhibition of 5-[125I]iodo-2'-deoxyuridine uptake 9 days after tumor induction, was 6.3 mg/kg for free doxorubicin and 0.48 mg/kg for the encapsulated drug. In these conditions pulmonary tumor development was even more efficiently prevented by encapsulated doxorubicin as compared with the free drug. The values of the therapeutic index (TI), defined as the ratio between the maximal tolerated dose (LD10) and the minimal effective dose (ED90, producing 90% inhibition of liver metastatic growth), were 4.2 and 1.8 for encapsulated and free doxorubicin, respectively.

Alanine Transaminase↗
PubMed1989

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes.

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.

2,3-Diphosphoglycerate↗
PubMed1988

Improved metabolic properties of hexokinase-overloaded human erythrocytes.

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Adenine Nucleotides↗
PubMed1988

Conversion of encapsulated 5-fluoro-2'-deoxyuridine 5'-monophosphate to the antineoplastic drug 5-fluoro-2'-deoxyuridine in human erythrocytes.

The fluoropyrimidine deoxyribonucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was encapsulated in human erythrocytes by a procedure based on hypotonic hemolysis and isotonic resealing. Encapsulated FdUMP (up to 9 mumol/ml of packed erythrocytes) did not affect erythrocyte metabolism or morphology. Hemolysates were found to catalyze efficient dephosphorylation of FdUMP to yield nearly stoichiometric amounts of the corresponding deoxyribonucleoside 5-fluoro-2'-deoxyuridine (FdUrd), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas. The dephosphorylation reaction had an apparent Km of 7.7 +/- 1.2 mM FdUMP at pH 7.4 and was remarkably slower at pH 8.2. ATP, GTP, and UTP inhibited both the disappearance of FdUMP and the formation of FdUrd in hemolysates. The enzyme responsible for the FdUMP-to-FdUrd conversion was identified with the deoxyribonucleotide-specific isozyme of erythrocyte pyrimidine 5'-nucleotidase (EC 3.1.3.5). Intracellular formation and subsequent release of FdUrd were observed in intact erythrocytes loaded with FdUMP. Inhibition of FdUrd release from these erythrocytes was obtained by raising the pH intracellularly and, alternatively, by coencapsulation of ATP. Autologous FdUMP-loaded erythrocytes might be used as endogenous bioreactors designed for time-programmed and liver-targeted delivery of FdUrd.

Antineoplastic Agents↗
PubMed1988

In vivo liver and lung targeting of adriamycin encapsulated in glutaraldehyde-treated murine erythrocytes.

Treatment of adriamycin-loaded erythrocytes from B6D2F1 mice with 0.1% glutaraldehyde produced the following effects: a considerable decrease in the in vitro leakage of the unmodified drug and a selective liver (and, to a lesser extent, lung) uptake of the encapsulated drug (70% of the injected dose) compared to drug leakage from, and tissue distribution of, carrier erythrocytes not treated with glutaraldehyde. The liver vascular bed was not saturated by five daily intravenous injections of 20 microliters of glutaraldehyde-treated erythrocytes, which allows a total dosage of 200 micrograms of the drug (half the LD50 value) to be administered. No appreciable liver damage results from extensive and prolonged uptake of glutaraldehyde-treated carrier erythrocytes. Entrapment of adriamycin within erythrocytes along with glutaraldehyde treatment of the carrier cells seems to be a promising therapeutic strategy against liver (and lung) tumors.

Animals↗
PubMed1987

Favism: impairment of proteolytic systems in red blood cells.

Red blood cells (RBC) from favic patients are characterized by (a) severe oxidative damage (contributed by autoxidation of divicine and isouramil, two pyrimidine aglycones present in fava beans) and (b) greatly increased calcium levels. In vitro, both autoxidation of divicine and calcium loading produced marked alterations of proteolytic systems in intact RBC. Specifically, autoxidizing divicine inactivated procalpain, the proenzyme species of calcium-activated cytosolic neutral proteinase, or calpain. Inactivation was much greater with glucose-6-phosphate dehydrogenase (G6PD)-deficient RBC than with normal RBC. On the other hand, loading of normal and G6PD-deficient RBC with calcium resulted in conversion of procalpain to calpain and eventual autoproteolytic inactivation of calpain itself, and extensive release of acid endopeptidase activity from the membranes into the cytosol. Damaged RBC from favic patients had significantly lowered procalpain activity and an abnormal subcellular distribution of acid proteinase activity that was found mostly in the cytosol. When purified calpain was incubated with membranes from acetylphenylhydrazine (APH)-treated RBC, significant proteolysis was observed affecting mostly band 3 and hemoglobin chains, ie, the two proteins involved in the onset of aggregation of Heinz bodies. Moreover, exposure of intact RBC to 20 mmol/L APH induced depletion of procalpain activity for which the time course was inversely related to formation of Heinz bodies. These findings support the role of procalpain in protecting G6PD-deficient RBC from oxidant-induced Heinz body formation and imply that exhaustion of the procalpain-calpain system is an important step in the mechanisms of RBC damage and destruction in favism.

Calcium↗
PubMed1987

Hepatic or splenic targeting of carrier erythrocytes: a murine model.

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H2O2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved.

Animals↗
PubMed1986

Encapsulation of adriamycin in human erythrocytes.

Adriamycin (doxorubicin) was encapsulated in human erythrocytes by means of a dialysis technique involving transient hypotonic hemolysis followed by isotonic resealing. Up to 1.6 mg of the drug was entrapped per ml of packed erythrocytes, with the efficiency of encapsulation being 60-80%. In vitro incubation of the Adriamycin-loaded erythrocytes in autologous plasma was accompanied by progressive release of unaltered Adriamycin in the medium. The efflux was still evident after 50 hr. The metabolism of encapsulated Adriamycin was restricted to limited formation of the C-13 hydroxylated metabolite, adriamycinol, in the normal erythrocytes but not in erythrocytes from individuals deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) activity. Reductive bioactivation of encapsulated Adriamycin to yield the corresponding aglycones was not observed in a variety of conditions. However, when NADPH ferredoxin reductase and ferredoxin, both purified from spinach leaves, were co-entrapped within erythrocytes and allowed to catalyze electron transfer to Adriamycin intracellularly under N2, a quantitative conversion to 7-deoxyadriamycin aglycone was obtained. Adriamycin-loaded erythrocytes did not show any significant oxidative damage, except for a variable increase of methemoglobin, suggesting some redox cycling between native Adriamycin and its semiquinone radical. Encapsulation of Adriamycin in autologous human erythrocytes may represent a therapeutic strategy for the slow release in circulation of this antineoplastic drug in order to reduce or prevent its adverse effects and especially the delayed cardiotoxicity that limits its use in patients with neoplastic disease.

Biotransformation↗
PubMed1986

Alveolar macrophage stimulation of T-cell proliferation in autologous mixed lymphocyte reactions. Role of HLA-DR antigens.

Alveolar macrophages act as accessory cells in lymphocyte response to mitogens or alloantigens. Because the autologous mixed lymphocyte reaction (MLR), in which HLA-DR-positive non-T cells stimulate the proliferation of autologous T lymphocytes, represents a good model to study macrophage-T cell interaction, we examined and compared the ability of human alveolar macrophages and peripheral blood-derived monocytes to induce T-cell proliferation in autologous MLR. Maximal T lymphocyte proliferation was observed in both alveolar-macrophage- and blood-monocyte-stimulated autologous MLR at a T cell to alveolar macrophage or blood monocyte ratio of 4:1, but the ability to stimulate T-cell proliferation was lower for alveolar macrophages than for blood monocytes (p less than 0.01). Because HLA-DR antigens modulate monocyte-T cell interaction, we quantified the proportions of HLA-DR-positive cells in alveolar macrophage and blood monocyte suspensions and determined the inhibitory effects on T-cell proliferation of masking HLA-DR antigens on stimulator cells with monoclonal antibodies. The proportions of HLA-DR-positive cells were higher in alveolar macrophage than in blood suspensions (p less than 0.01); interestingly, however, the preincubation of the stimulator cells with anti-HLA-DR monoclonal antibodies inhibited to a similar extent both alveolar-macrophage- and blood-monocyte-stimulated autologous MLR (p greater than 0.2). These studies indicate that alveolar macrophages are less effective than blood monocytes are as stimulator cells in autologous MLR and that, although the masking of HLA-DR molecules results in inhibition of autologous MLR, T-cell proliferation is not dependent on the numbers of stimulator cells bearing HLA-DR antigens. The autologous MLR may represent a good model to study the functions of alveolar macrophages during their interaction with autologous T cells in health and disease.

Adult↗
PubMed1986

Effect of a monoclonal antibody against GPs IIb-IIIa on platelet aggregation and ATP secretion.

A murine monoclonal antibody against glycoproteins IIb-IIIa of platelet membrane completely abolished platelet aggregation induced by epinephrine, arachidonic acid, and low concentration of collagen and thrombin, but it had only minor inhibitory effects on aggregation induced by ADP, higher amounts of collagen and thrombin, and these agents combined in pairs. The simultaneous studies of aggregation and ATP secretion demonstrated that aggregation plays an essential role in stimulating the platelet-release reaction, except for the thrombin-induced ATP secretion that seems to be largely dependent on fibrinogen binding to platelet surface.

Adenosine Triphosphate↗
PubMed1985

Establishment and characterization of a new human cloned myelomonocytic cell line (ZC-1.6).

A human cell line, ZC, derived from the blood of a patient with acute myelomonocytic leukemia, was established and cloned. One of the clones, ZC-1.6, was subsequently characterized. As for its morphology and cytochemistry, ZC-1.6 clone shares a number of features with immature cells of the monocytic or myelocytic lineages. Surface marker analysis shows positivity for 4F2 (100% of the cells), and OKM1 (38%) monoclonal antibodies, and presence of surface HLA-D/DR antigens (100%) and Fc (11%) and complement (C3b) receptors (100%). Functional capabilities of ZC-1.6 cells include adherence, spreading, and phagocytosis of latex and opsonized zymosan particles. Despite its morphological immaturity, the ZC-1.6 clone produces relevant amounts of O2- in the presence of different stimuli (phorbol myristate acetate, opsonized zymosan, or latex particles). The production of O2- by ZC-1.6 cells is the first evidence that reactive oxygen intermediate production may represent an early feature of cells of the myelomonocytic lineage.

Adult↗
PubMed1984

Progesterone enhances reactive oxygen intermediates production by cultured human monocytes.

Progesterone at placental tissue concentrations (5-20 micrograms/ml) markedly increases in vitro PMA-stimulated O-2 and H2O2 production by human cultured monocytes. This appears to contrast the well-known suppressive action of the hormone on the other cell mediated defence mechanisms. We suggest that these findings could cast a new light on progesterone's multiple and differentiated functions in the placental environment.

Cells, Cultured↗
PubMed1983

A monoclonal antibody to platelet glycoproteins IIb and IIIa complex: its use in purifying human megakaryocytes from sternal bone marrow aspirates for immunofluorescence studies of Ia-like antigens.

A monoclonal antibody (PBM 6.4) to platelet and megakaryocyte glycoproteins IIb and IIIa has been obtained and used to purify human megakaryocytes from sternal bone marrow aspirates by a simple method, consisting of a Percoll gradient centrifugation followed by affinity adherence on PBM 6.4-coated plastic surface, "panning." Megakaryocytes, 80-90% pure and morphologically well preserved, were attached to poly-L-ornithine-coated multi-well microscope slides and immunofluorescence was done using monoclonal antibodies to human Ia-like antigens (DR, DC1). A higher proportion of DR-positive megakaryocytes was found, in comparison to the values reported by others, while DC1 antigen was detected on about 20% of megakaryocytes. The method described has the unique feature of enriching cells of the human megakaryocytic lineage from simple diagnostic sternal aspirates in an amount adequate for immunofluorescent and morphological analysis.

Animals↗
PubMedDate not supplied

Enhanced antitumor activity of adriamycin by encapsulation in mouse erythrocytes targeted to liver and lungs.

Adriamycin was encapsulated within human and murine (B6D2F1 female mice) erythrocytes using a procedure based on hypotonic hemolysis followed by isotonic resealing and reannealing. Following drug encapsulation the murine erythrocytes were treated with glutaraldehyde to obtain: a) control of Adriamycin efflux from loaded erythrocytes, b) appropriate hepatic and pulmonary targeting of the in vivo re-infused cells. The antitumor effect of equivalent amounts of bolus (i.v.) administered Adriamycin, 1) free, 2) encapsulated within erythrocytes, 3) encapsulated within glutaraldehyde-treated erythrocytes, was compared using an in vivo model of metastasis based on selective hepatic and pulmonary dissemination of intrasplenically injected L1210 cells in B6D2F1 mice. The therapeutic index (TI) of Adriamycin encapsulated within glutaraldehyde-treated erythrocytes increased by more than two-fold over that of the free drug.

Animals↗
PubMedDate not supplied

The immunological events leading to the "in vitro" response to PPD.

In normal individuals the 5/9 monoclonal antibody (5/9 MAb) recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5/9+ and 5/9- T lymphocytes were isolated from tuberculim skin-positive subjects and the proliferative response induced by PPD was investigated. The results show that the total PPD-induced lymphocyte DNA synthesis is confined to the 5/9+ T cell fraction. PPD was unable to induce a direct or indirect (through the 5/9+ cells) proliferation of T8+ cells. Whether 5/9 antigen, expressed on the PPD responsive T-cell subset, is involved in PPD-induced cell proliferation, was also analyzed. No significant effect was observed adding 5/9 MAb, whereas complete inhibition of antigen-induced blastogenesis was observed upon addition of a MAb (D1.12) directed to common determinants of Ia antigens. The supernatant fluids of 5/9+ and 5/9- cells stimulated with PPD were studied for the presence of IL-2 and IFN-gamma. Production of both these lymphokines was detected in the 5/9+ T-cell supernatants, whereas neither IL-2 or IFN-gamma were found in supernatants of 5/9- cells. In addition, preliminary experiments suggested that only the supernatants of PPD-stimulated 5/9+ T cells were able to activate a macrophage cell line, inducing a remarkable release of hydrogen peroxide. These results indicate that the lymphocyte responsiveness to PPD is confined in a small T cell fraction, expressing the 5/9 antigen; only these T cells are able to release soluble factors (i.e., IL-2 and IFN-gamma); those or other soluble factor(s) produced in PPD-stimulated cultures seem able to activate the macrophages.

Adult↗
PubMedDate not supplied

Construction of glucose oxidase-loaded human erythrocytes: a model of oxidative cytotoxicity.

Human red blood cells were loaded with Glucose oxidase from Aspergillus niger by a standardized procedure of encapsulation involving transient hypotonic hemolysis followed by isotonic resealing. The amount of loaded enzyme activity, as evaluated by O2 consumption at 5 mM glucose, ranged from 40 to 75 mumoles O2/hr/ml of packed red cells at 37 degrees C. The red cells loaded with Glucose oxidase were found to behave as efficient glucose-consuming bioreactors. Moreover, at 5 mM glucose, no clear mechanism of H2O2-induced damage was apparent, with the exception of a significantly increased formation of methemoglobin (10% approximately) and of a several-fold stimulated intracellular rate of hexose monophosphate shunt activity, indicating glutathione peroxidase-mediated draining of reduced glutathione for removal of bursts of H2O2. The Glucose oxidase-loaded red cells represent a convenient model system for cytotoxicity studies aiming at clarifying the effects of intracellularly formed H2O2.

Aspergillus niger↗