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V Raso

Publications and source records attributed to V Raso.

At least 19 recordsLinked to original sources
PubMed2006

Impact of acute exposure to air pollution on the cardiorespiratory performance of military firemen.

The objective of the present study was to determine the impact of acute short-term exposure to air pollution on the cardiorespiratory performance of military firemen living and working in the city of Guarujá, São Paulo, Brazil. Twenty-five healthy non-smoking firemen aged 24 to 45 years had about 1 h of exposure to low and high levels of air pollution. The tests consisted of two phases: phase A, in Bertioga, a town with low levels of air pollution, and phase B, in Cubatão, a polluted town, with a 7-day interval between phases. The volunteers remained in the cities (Bertioga/Cubatão) only for the time required to perform the tests. Cumulative load 10 +/- 2 min-long exertion tests were performed on a treadmill, consisting of a 2-min stage at a load of 7 km/h, followed by increasing exertion of 1 km h-1 min-1 until the maximum individual limit. There were statistically significant differences (P < 0.05) in anaerobic threshold (AT) between Cubatão (35.04 +/- 4.91 mL kg-1 min-1) and Bertioga (36.98 +/- 5.62 mL kg-1 min-1; P = 0.01), in the heart rate at AT (AT HR; Cubatão 152.08 +/- 14.86 bpm, Bertioga 157.44 +/- 13.64 bpm; P = 0.001), and in percent maximal oxygen consumption at AT (AT%VO2max; Cubatão 64.56 +/- 6.55%, Bertioga 67.40 +/- 5.35%; P = 0.03). However, there were no differences in VO2max, maximal heart rate or velocity at AT (ATvel) observed in firemen between towns. The acute exposure to pollutants in Cubatão, SP, caused a significant reduction in the performance at submaximal levels of physical exertion.

Adult↗
PubMed1999

Direct evidence for native CD4 oligomers in lymphoid and monocytoid cells.

CD4 is expressed by T lymphocytes and monocytes and is generally considered a monomer even though its structure was originally modelled on the REI Bence-Jones homodimer. However, native CD4 was demonstrated as both monomer and dimers of 55 and 110 kDa in lymphoid and monocytoid cells by immunoprecipitation and immunoblotting after solubilization with alkylating (iodoacetamide) or reducing (dithiothreitol, 2-mercaptoethanol) reagents. Full reduction yielded only the 55-kDa monomeric form. Purified CD4 oligomers from CEM-T4 cells were also resolved as homodimers by MALDI-Tof mass fingerprinting after tryptic digestion. Cell treatment with the membrane impermeable, free-thiol reactive, 5,5'-dithiobis-2-nitrobenzoic acid enhanced cell surface CD4 dimers and tetramers. The interaction sites producing dimerization were probably in the D4 domain as OKT4 inhibited self association of recombinant CD4 (rCD4). Oligomerization of rCD4 by glutathione and thioredoxin indicates that thiol exchange interactions were responsible. Enhanced CD4 dimer expression was also observed after PMA (20 ng/ml) activation of THP-1 cells. These findings demonstrate that different quaternary forms of CD4 such as monomers, homodimers and tetramers are expressed by T lymphocytes and monocytes/macrophages.

Amino Acid Sequence↗
PubMed1997

Antibodies capable of releasing diphtheria toxin in response to the low pH found in endosomes.

Diphtheria toxin (DT) undergoes a rapid conformational change in response to the acidity encountered within endosomes. That transition is integral to the passage of its catalytic domain into the cytosol and thus its lethal action. The importance of this translocation mechanism led us to develop several monoclonal antibodies that bind DT at neutral pH but spontaneously release the toxin when critical epitopes denature or unfold upon lowering the pH to 4.5-5.5. Hybridomas were selected using a microtiter plate assay that measured the pH-dependent detachment of antibody from immobilized toxin. The acid-sensitive epitopes involved were on the catalytic, transmembrane, and receptor binding domains of DT. This pH-induced disruption of the binding of toxin to these monoclonal antibodies was analyzed by sedimentation velocity ultracentrifugation. Antibody combining sites were fully occupied at pH 5.5, partially bound at pH 5.0, and totally empty at pH 4.5. It was estimated that the Ka for antibody-toxin binding was approximately 1000-fold lower at pH 5.0 than at neutral pH. This novel acid-triggered release mechanism provides a basis for delivery of antibody-bound toxin into cells accompanied by its immediate dissociation as the complex enters acidic vesicles.

Antibodies, Monoclonal↗
PubMed1997

Intracellular targeting with low pH-triggered bispecific antibodies.

Bispecific antibodies were designed to deliver a reversibly bound ligand into target cells and then spontaneously release it upon passage into acidified vesicles. These reagents were assembled by coupling monoclonal antibodies that recognize acid-sensitive epitopes on diphtheria toxin to cell type-specific monoclonal antibodies. The dual binding capacity of the bispecific antibodies was confirmed by delivery of 125I-diphtheria toxin to target molecules present on intact cells. Bispecific antibodies directed against transferrin receptors on human cells were loaded with toxin and tested for cytotoxicity. The mutant diphtheria toxins CRM107 and CRM45 were used since their inability to bind cell receptors renders them ordinarily nontoxic. Their full cytotoxic potential, however, was restored via bispecific antibody-mediated delivery and release within low pH intracellular vesicles. Cytotoxicity was shown to be specific by blocking receptor sites and to be acidification-dependent by protection using NH4Cl to raise endosomal pH. Kinetics for inhibition of cellular protein synthesis was identical for native diphtheria toxin and the bispecific antibody. CRM107 combination. The rate of inhibition (t1/2 = 20 min) indicated that release of CRM107 from the antibody combining site was fast, and its toxic action was unimpeded by this delivery mechanism.

Antibodies, Bispecific↗
PubMed1996

Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates.

Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfRneg) and two lines transfected with either wild-type TfR (Tfrwt) or an internalization-deficient (TfR(delta 7-58del)) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfRwt cells (ID50 < 1 nM) while both TfRneg cells and TfR(delta 7-58del) were only minimally affected (ID50 > 100 nM). Butyrate up-regulation substantially increased receptor expression on the TfRwt and TfR(delta 7-58del) cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfRwt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR(delta 7-58del) cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number.

Animals↗
PubMed1993

Potentiation of antitumor immunotoxins by liposomal monensin.

BACKGROUND: The cytotoxicity of specific ricin A-chain immunotoxins is greatly enhanced in vitro by the carboxylic ionophore monensin. However, the highly lipophilic nature of monensin, which is reflected in its poor solubility and short half-life, has restricted its use in in vivo animal studies. PURPOSE: The purpose of this study was to assess the ability of monensin incorporated in unilamellar vesicles (liposomes) to potentiate antitumor immunotoxins in vitro and in vivo. METHODS: Monensin was incorporated into liposomes and used in combination with specific immunotoxins against human tumor cell lines in vitro and in vivo. Inhibition of [3H]leucine incorporation was used to evaluate the cytotoxic action of immunotoxin with or without monensin in vitro on the following human tumor cell lines: H-MESO-1 malignant mesothelioma, LS174T colorectal carcinoma, and U373, U87, and MG-1 glioblastomas. For the in vivo studies of immunotoxins and liposomal monensin, BALB/c nu/nu mice were inoculated intraperitoneally with H-MESO-1 cells. RESULTS: Liposomal monensin potentiated the cytotoxic action of cell-specific anti-human transferrin receptor immunotoxin on H-MESO-1 target cells at a molar concentration of monensin that was 160-fold lower than the concentration of monensin in buffer that produced the same effect (0.3 nM versus 0.05 microM). Moreover, immunotoxin plus 0.1 microM liposomal monensin was fivefold more toxic for H-MESO-1 cells and 1000-fold and 2200-fold more toxic for human glioblastoma U373 and U87 cells, respectively, than immunotoxin plus 0.1 microM free monensin in buffer. Liposomal monensin produced similar effects when it was combined with different specific immunotoxins and other target cell lines (i.e., LS174T, U87, and CEM). Immunotoxin specificity was preserved with liposomal monensin, as shown by the absence of effect with non-cell-binding immunotoxins or on antigen-negative cell lines. In mice, liposomal monensin in combination with specific immunotoxin substantially prolonged survival, and three (21%) of 14 mice bearing H-MESO-1 xenografts treated with the liposomes showed no evidence of tumor at day 160 after treatment. Treatment with control immunotoxin plus liposomal monensin was ineffective. CONCLUSION: These findings suggest that encapsulation of monensin into liposomes increased the capacity of monensin to enhance the potency of cell-specific immunotoxin in vitro and in vivo.

Adjuvants, Immunologic↗
PubMed1991

Monensin in lipid emulsion for the potentiation of ricin A chain immunotoxins.

The utilization of carboxylic ionophores such as monensin for immunotoxin potentiation may be hampered by the poor solubility and short in vivo half-life of these highly lipophilic compounds. Therefore, the use of monensin formulated in a lipid/water emulsion was investigated for the in vitro and in vivo potentiation of immunotoxins. Monensin in emulsion or in buffer was equally effective for the in vitro potentiation of the cytotoxicity of both anti-human transferrin receptor and anti-carcinoembryonic antigen immunotoxins against target cells. In mice, buffer and lipid emulsion were compared as vehicles for the i.p. administration of monensin. The half-life of monensin in the peritoneal cavity of BALB/c x DBA/2 F1 (CD2F1) mice was increased 20-fold by inclusion in lipid emulsion (13 min versus 0.75 min). Treatment i.p. with anti-human transferrin receptor immunotoxin or anti-carcinoembryonic antigen immunotoxin and monensin emulsion prolonged the survival of mice with macroscopic i.p. tumor xenografts of H-Meso-1 mesothelioma and LS174T colorectal carcinoma (200-250% increased length of median survival). The in vivo antitumor effect of the cell-specific immunotoxin plus monensin emulsion was superior to immunotoxin alone or to immunotoxin plus monensin in buffer (P less than 0.03; Mann-Whitney U test). This indicates that delivery of monensin in preformed lipid emulsion may produce a reservoir effect of the ionophore in the peritoneal cavity of tumor-bearing mice. Nonspecific control immunotoxin plus monensin emulsion produced no increase in survival. Long-term tumor-free survival (greater than 150 days versus a median survival of 25 days for controls) of mice bearing microscopic LS174T xenografts was obtained by treatment with anti-human transferrin receptor immunotoxin plus monensin emulsion. Administration of either monensin in buffer or monensin in emulsion without immunotoxin had no significant effect on survival. Monensin in this pharmacologically available form significantly improved the in vivo efficacy of both anti-human transferrin receptor immunotoxin and anti-carcinoembryonic antigen immunotoxin when used as regional therapy.

Animals↗
PubMed1990

Potent cytotoxicity of an antihuman transferrin receptor-ricin A-chain immunotoxin on human glioma cells in vitro.

The cytotoxic effects of an antihuman transferrin receptor monoclonal antibody-ricin A-chain conjugate (anti-TfR-A) immunotoxin on glioma cells were assessed in vitro. Five human glioma cell lines were studied; three were derived from surgical explants (MG-1, MG-2, MG-3) and two were well characterized established glioma cells (U-87 MG, U-373 MG). The C6 rat glioma line served as a nonhuman control. One of six lines (U-373) expressed glial fibrillary acidic protein, as assessed by immunohistochemistry. All five human lines expressed human transferrin receptor, as assessed by flow cytometry; no human transferrin receptor was demonstrable on rat C6 cells. Potent inhibition of protein synthesis was found after an 18-h incubation with anti-TfR-A. Fifty % inhibitory concentration (IC50) values for human glioma cells ranged from 1.9 X 10(-9) to 1.8 X 10(-8) M. In contrast, no significant inhibition of leucine incorporation was observed when anti-TfR-A was tested on rat cells (IC50 greater than 10(-7) M) or when a control immunotoxin directed against carcinoembryonic antigen was substituted for anti-TfR-A on human glioma cells (IC50 greater than 10(-7) M). Coincubation with the carboxylic ionophore monensin (10(-7) M) decreased the IC50 of anti-TfR-A against human glioma lines from 16- to 842-fold (range, 7.0 X 10(-12) to 1.5 X 10(-10) M). In contrast, an IC50 of greater than 10(-7) M was obtained when C6 cells were incubated with anti-TfR-A and monensin. Anti-TfR-A immunotoxins potentiated by monensin are extremely potent in vitro cytotoxins for human glioma cells.

Antibodies, Monoclonal↗
PubMed1990

Immunoelectron-microscopic studies of human platelet thrombospondin, von Willebrand factor, and fibrinogen redistribution during clot formation.

The relative distributions of the human platelet alpha-granule proteins fibrinogen, thrombospondin, and von Willebrand factor were mapped by immunoelectron microscopy in thin cryosections of activated platelets, platelet aggregates, and clots during the first 24 h of in vitro clot formation. In early activated platelets, the results suggest that the canalicular system constitutes a significant component of the external platelet surface, and may act as a compartment for biochemical reactions occurring during granule release. Further, detection of coagulation proteins by various non-morphological procedures may reflect protein contained within canalicular elements. Later in the release process, von Willebrand factor was detected as a major antigen on the platelet canalicular and plasma membranes; thrombospondin, on the other hand, showed minimal binding to platelets and only limited binding to the extensive fibrin network. Comparison of radioimmunoassays of supernatants of thrombin-stimulated platelets in plasma, clotted whole blood, and Triton X-100 platelet releasates indicated that virtually all of the platelet thrombospondin appears in serum. These data confirm the immunocytochemical results indicating that very little platelet thrombospondin binds to the platelet surface, compared with von Willebrand factor, studied here under the same conditions, which binds extensively to the platelet membrane following release and clot formation.

Blood Coagulation↗
PubMed1990

Transferrin receptor in normal and neoplastic brain tissue: implications for brain-tumor immunotherapy.

The distribution of transferrin receptor (TfR) in normal human brain-tissue obtained at autopsy and in brain-tumor biopsy specimens from 27 patients was determined by immunohistochemistry using two specific murine monoclonal antibodies against human TfR. The tumors studied included 10 glioblastomas multiforme (GBM's), nine other glial tumors, and eight meningiomas. In normal brain, TfR was detected primarily in endothelial cells; rare glial cells also contained immunoreactive product. All tumors contained TfR-positive cells, although the intensity (number of cells stained) and pattern (focal vs. diffuse) of staining varied with the histopathological type of the tumor. Among gliomas, the most intense staining was seen in GBM's, especially in areas of pseudopalisading where virtually all cells were stained. A rough correlation between tumor grade, number of positively stained cells, and staining pattern was seen in the other astrocytic tumors. By contrast, all meningiomas demonstrated an identical and characteristic focal staining pattern. Considering the differential immunostaining for TfR between normal and neoplastic tissue, the authors conclude that TfR may be an appropriate target for monoclonal antibody-directed brain-tumor immunotherapy, especially in more malignant tumors such as GBM's.

Astrocytoma↗
PubMed1990

Antibodies in diagnosis and therapy. The magic bullet--nearing the century mark.

A truly successful magic bullet therapy for cancer requires the production of agents which can easily reach, accurately recognize and permanently nullify every cancer cell in the body. Moreover, these precise functions must be achieved without adversely affecting normal vital tissues. This demanding approach is examined from the standpoint of animal model systems which satisfy these criteria to various extents. The salient features which contribute to success in these models are presented to provide a basis for evaluating the performance of currently available agents, and to assist in the design of new, more highly perfected 'bullets'. Pertinent issues regarding the therapeutic impact of cellular receptors, internalization pathways, mechanisms of toxin action, kinetics of cell killing, solid tumor penetration, pharmacokinetics and relative potency on target versus non-target cells are all considered. Current strategies using advanced biotechnical approaches to construct more effective targeted agents are addressed in this context.

Animals↗
PubMed1989

Cure of experimental human malignant mesothelioma in athymic mice by diphtheria toxin.

A single ip or iv dose of 1-3 micrograms of diphtheria toxin consistently cured athymic mice of an advanced stage experimental human malignant mesothelioma. All cancer cells were killed within 18 hours and the profuse ip ascites plus large solid tumor masses associated with this model of neoplastic disease were subsequently eliminated. Treated mice appeared normal in 3 days and lived for greater than 300 days with no signs of recurrence, while control animals did not survive greater than 32 days. The complete tumoricidal effect implies that toxin readily reached, entered, and preferentially killed each human cancer cell. This outcome exemplifies the true therapeutic potential of highly selective, site-directed toxins, and offers a frame of reference for judging the performance of current as well as prospective toxin-based agents.

Animals↗
PubMed1988

Monensin is obligatory for the cytotoxic action of a disulfide linked methotrexate-anti-transferrin receptor conjugate.

Methotrexate (MTX) in the form of a gamma-cysteinylglycine derivative was disulfide linked to a monoclonal antibody reactive with the human transferrin receptor to give an antibody-MTX conjugate (anti-TfR-MTX). Antibody directed delivery of MTX to cell surface receptors was readily detected by flow cytometry using an anti-MTX antibody plus a secondary fluorescent antibody probe. Despite the presence of ample drug on the cell membrane, the conjugate alone was not cytotoxic over the course of several days. Expression of specific toxic activity, however, was obtained in conjunction with the carboxylic ionophore monensin, in whose presence anti-TfR-MTX displayed an IC50 of 8 X 10(-8) M. These results suggest that the ionophore causes antibody-drug conjugate to bypass the normal transferrin receptor cyclic pathway, allowing sufficient drug to reach, bind to, and inactivate intracellular dihydrofolate reductase.

Antibodies, Monoclonal↗
PubMed1987

Antitumor activity of intraperitoneal immunotoxins in a nude mouse model of human malignant mesothelioma.

Immunotoxins directed against human transferrin receptor have been evaluated in a nude mouse model of human malignant mesothelioma. Immunotoxins were constructed by linking ricin A chain to murine monoclonal antibodies reactive with the human transferrin receptor. A chain was obtained either by isolation from the parent toxin or by recombinant DNA techniques. These immunotoxins acted as potent in vitro cytotoxins against human malignant mesothelioma cells (H-MESO-1) (ID50, 2 X 10(-9) M). Cytotoxic potency and kinetics of cell kill were potentiated in vitro by the carboxylic ionophore monensin. For in vivo trials, nude mice were injected i.p. with 6-9 X 10(6) human malignant mesothelioma cells 24 h prior to the start of i.p. immunotoxin treatments. The survival of tumor-bearing mice was extended by 149-404%, representing a probable cell kill of 2-4 logs. Specificity of this antitransferrin receptor immunotoxin response was confirmed by the ineffectiveness of irrelevant control immunotoxins and blockade of specific immunotoxin action by excess free antibody. Monensin showed limited in vivo potentiation of immunotoxin effect, but a derivative formed by esterification of monensin with linoleic acid gave improved survival times over treatment with immunotoxin alone. Immunotoxins constructed with ricin A chain have significant tumoricidal activity in this model of regional antitumor therapy. These results may have direct relevance for treatment of i.p. malignancy in clinical settings.

Animals↗
PubMed1987

Intracellular pathways of ricin A chain cytotoxins.

Ricin A chain, a potent ribosomal poison, was disulfide linked either to the iron transport protein, transferrin, or to anti-transferrin receptor antibodies to produce highly specific derivative toxins, Tf-A and TfR-A, respectively. The ability of these agents to gain access to and damage ribosomes within the cell was accelerated in the presence of carboxylic ionophores. Their effectiveness for killing clonogenic target cells was correspondingly enhanced by 5 logs after a brief treatment with Tf-A plus ionophore. Intracellular trafficking of Tf-A and TfR-A was monitored by a variety of methods to better understand their mechanism of action. Data obtained with 125I-labeled A chain and 59Fe3+-labeled toxin probes indicated that the natural iron delivery pathway was initially followed. This was characterized by specific attachment to surface receptors, internalization, entry into low-density acidic vesicles, uncoupling of iron, an absence of lysosomal degradation, and sustained cycling. Ultrastructural studies using a colloidal gold-labeled anti-A chain probe confirmed the presence of these toxins within the structural elements associated with endocytosis. Toxic Tf-A molecules, however, diverged from this pathway (t1/2 = 88 min) to eventually kill cells as witnessed by a gradual loss in the ability to rescue cells using excess transferrin. Potentiating agents, such as carboxylic ionophores or B chain, seem to act by speeding the divergence of Tf-A and TfR-A from the normal endocytotoxic cycle.

Animals↗
PubMed1986

Elimination of clonogenic tumor cells from human bone marrow using a combination of monoclonal antibody:ricin A chain conjugates.

Effective autologous bone marrow transplantation for leukemia and lymphoma is likely to depend upon the selective removal in vitro of malignant cells from normal human bone marrow precursors. Highly specific cytotoxic conjugates formed by coupling ricin A chain to monoclonal antibodies might prove useful for the selective elimination of malignant cells. Consequently, ricin A chain conjugates have been prepared with several different murine monoclonal antibodies and tested for their ability to eliminate clonogenic Burkitt's lymphoma cells from an excess of human bone marrow. The most active reagents included an antibody:A chain conjugate which bound to the nonpolymorphic chain of the la molecule and another which reacted with the mu heavy chain of cell surface immunoglobulin. Conjugates formed with anti-common acute lymphoblastic leukemia antigen, anti-Mr 26,000 glycoprotein, and anti-B1 were much less active on these Burkitt's cells, contrasting with results of complement-dependent tumor cell lysis. Tumor cell kill was partially inhibited by the addition of greater than 2 X 10(6) human bone marrow cells/ml but could be potentiated by increasing the concentration of conjugate or by the addition of 10 mM ammonium chloride. In the presence of ammonium chloride, at least 4 logs of clonogenic tumor cells could be eliminated within 24 h from a 20-fold excess of bone marrow using 10(-7) M ricin A chain linked to one or two different antibodies. Similar treatment of normal human bone marrow temporarily inhibited granulocyte-macrophage colony-forming units (cell) formation but did not compromise establishment of continuous bone marrow cultures. The degree of selective elimination of tumor cells with A chain antibody conjugates was comparable to that achieved with 4-hydroperoxycyclophosphamide or with multiple monoclonal antibodies and complement.

Ammonium Chloride↗