23 results on '"Zivin RA"'
Search Results
2. Fluorescence imaging of electrically stimulated cells.
- Author
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Burnett P, Robertson JK, Palmer JM, Ryan RR, Dubin AE, and Zivin RA
- Subjects
- Calcium pharmacology, Cell Line, Tumor, Cell Survival drug effects, Electric Conductivity, Electric Stimulation, Humans, Indium pharmacology, Inhibitory Concentration 50, Ion Channel Gating drug effects, Membrane Potentials drug effects, Patch-Clamp Techniques, Sodium Channels metabolism, Tetrodotoxin pharmacology, Time Factors, Veratridine pharmacology, Cell Physiological Phenomena drug effects, Fluorescence
- Abstract
Designing high-throughput screens for voltage-gated ion channels has been a tremendous challenge for the pharmaceutical industry because channel activity is dependent on the transmembrane voltage gradient, a stimulus unlike ligand binding to G-protein-coupled receptors or ligand-gated ion channels. To achieve an acceptable throughput, assays to screen for voltage-gated ion channel modulators that are employed today rely on pharmacological intervention to activate these channels. These interventions can introduce artifacts. Ideally, a high-throughput screen should not compromise physiological relevance. Hence, a more appropriate method would activate voltage-gated ion channels by altering plasma membrane potential directly, via electrical stimulation, while simultaneously recording the operation of the channel in populations of cells. The authors present preliminary results obtained from a device that is designed to supply precise and reproducible electrical stimuli to populations of cells. Changes in voltage-gated ion channel activity were monitored using a digital fluorescent microscope. The prototype electric field stimulation (EFS) device provided real-time analysis of cellular responsiveness to physiological and pharmacological stimuli. Voltage stimuli applied to SK-N-SH neuroblastoma cells cultured on the EFS device evoked membrane potential changes that were dependent on activation of voltage-gated sodium channels. Data obtained using digital fluorescence microscopy suggests suitability of this system for HTS.
- Published
- 2003
- Full Text
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3. Apolipoprotein E protects against NMDA excitotoxicity.
- Author
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Aono M, Lee Y, Grant ER, Zivin RA, Pearlstein RD, Warner DS, Bennett ER, and Laskowitz DT
- Subjects
- Animals, Calcium metabolism, Cell Death drug effects, Cells, Cultured, Neuroglia cytology, Neurons cytology, Neurons metabolism, Neurotoxins, Rats, Rats, Sprague-Dawley, Apolipoproteins E pharmacology, Excitatory Amino Acid Agonists toxicity, N-Methylaspartate toxicity, Neurons drug effects, Neuroprotective Agents pharmacology
- Abstract
Preclinical and clinical evidence implicates a role for endogenous apolipoprotein E in modifying the response of the brain to focal and global ischemia. To investigate whether apoE modulates the neuronal response to glutamate excitotoxicity, we exposed primary neuronal glial cultures and a neuronal cell line to biologically relevant concentrations of apolipoprotein E prior to NMDA exposure. In both of these paradigms, apolipoprotein E exerted partial protective effects. At neuroprotective concentrations, however, apolipoprotein E failed to block NMDA-induced calcium influx to the same magnitude as the NMDA receptor antagonist MK-801. These results suggest that one mechanism by which apolipoprotein E modifies the central nervous system response to ischemia may be by reducing glutamate-induced excitotoxicity.
- Published
- 2002
- Full Text
- View/download PDF
4. Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II clinical trial.
- Author
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Utset TO, Auger JA, Peace D, Zivin RA, Xu D, Jolliffe L, Alegre ML, Bluestone JA, and Clark MR
- Subjects
- Adolescent, Adult, Aged, Arthritis, Psoriatic diagnosis, Dose-Response Relationship, Drug, Drug Administration Schedule, Female, Follow-Up Studies, Humans, Male, Middle Aged, Pain Measurement, Severity of Illness Index, Treatment Outcome, Arthritis, Psoriatic drug therapy, Maximum Tolerated Dose, Muromonab-CD3 therapeutic use
- Abstract
Objective: Treatment of autoimmune diseases with therapies that tolerize pathogenic lymphocytes may obviate the need for longterm global immunosuppression. In vitro, non-Fc receptor binding derivatives of anti-murine CD3 monoclonal antibodies tolerize type 1 T cells and stimulate type 2 T cells. Recently, a humanized non-FcR binding derivative of the anti-human CD3 Mab OKT3, huOKT3gamma1(ala-ala), has been described. We hypothesized that this Mab may be safe and efficacious in the treatment of type 1 T lymphocyte mediated chronic autoimmune diseases such as psoriatic arthritis (PsA)., Methods: In a Phase I/II trial, 7 patients with PsA were treated with escalating daily doses of huOKT3gamma1(ala-ala) for 12 to 14 days. Number of tender and swollen joints and a visual analog pain scale were used to rate disease activity at entry and Day 30 and Day 90 after treatment., Results: At Day 30, 6 of 7 patients had > or = 75% improvement in the number of inflamed joints and an average 63% improvement on the patient pain scale. Two of 6 responders had sustained improvement at Day 90. No patient treated with an initial dose < or = 1 mg had significant side effects, nor did they have detectable increases in serum cytokines. One patient treated with 4 mg without escalation developed mild cytokine release symptoms associated with elevation of interleukin 10. Transient T cell depletion occurred following treatment with the maximum dose of 4 mg, which resolved by Day 30. Antiidiotypic antibodies developed in 2 patients; however, there was no concurrent decrease in efficacy., Conclusion: These data indicate that huOKT3gamma1(ala-ala) may be useful in treating PsA.
- Published
- 2002
5. Simultaneous intracellular calcium and sodium flux imaging in human vanilloid receptor 1 (VR1)-transfected human embryonic kidney cells: a method to resolve ionic dependence of VR1-mediated cell death.
- Author
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Grant ER, Dubin AE, Zhang SP, Zivin RA, and Zhong Z
- Subjects
- Benzofurans toxicity, Capsaicin toxicity, Cell Death physiology, Cell Line, Cells, Cultured, Cloning, Molecular, Diterpenes metabolism, Ethers, Cyclic toxicity, Fluorescent Dyes, Fura-2, Humans, Hydrogen-Ion Concentration, Neurotoxins metabolism, Reactive Oxygen Species, Receptors, Drug genetics, Receptors, Drug physiology, Transfection, Calcium metabolism, Kidney metabolism, Receptors, Drug metabolism, Sodium metabolism
- Abstract
The vanilloid receptor 1 (VR1) is a ligand-gated, nonselective cation channel important for the sensory processing of painful stimuli. Activation of VR1 leads to increases in intracellular concentrations of calcium and sodium. Prolonged activation of VR1 in mammalian expression systems leads to cell death. The mechanism of VR1-mediated toxicity may have relevance to pathophysiological processes that can occur in neurons. Therefore, we have evaluated the relative contributions of intracellular calcium and sodium changes to VR1-mediated toxicity in human embryonic kidney 293 cells stably transfected with the human VR1 channel. The data demonstrate that VR1 receptor agonists capsaicin and resiniferatoxin lead to a sustained increase in intracellular calcium and sodium in a concentration-dependent manner, followed by cell death. Pretreatment with VR1 receptor antagonists capsazepine or ruthenium red block both the calcium and sodium responses to agonists, and block agonist-induced cell death in a concentration-dependent manner. However, addition of antagonists several minutes after agonists selectively reverses the agonist-induced increase in intracellular calcium, but does not reverse the elevated intracellular sodium concentration. Nonetheless, antagonists retain protective efficacy against capsaicin toxicity when added several minutes after capsaicin, conditions in which the cells still manifest elevated intracellular sodium, but not elevated intracellular calcium. In addition, a transient VR1-mediated increase in intracellular calcium that returns to baseline within minutes, induced by a rapid drop in pH, from pH 7.5 to pH 6.3, also does not lead to cell death. Collectively, these data demonstrate that the most important intracellular ionic change for mediating VR1-dependent toxicity is a sustained increase of calcium.
- Published
- 2002
- Full Text
- View/download PDF
6. Kinetics of small molecule inhibitor binding to p38 kinase.
- Author
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Thurmond RL, Wadsworth SA, Schafer PH, Zivin RA, and Siekierka JJ
- Subjects
- Animals, Drosophila, Enzyme Inhibitors pharmacology, Humans, Kinetics, Mitogen-Activated Protein Kinases antagonists & inhibitors, Protein Binding, Surface Plasmon Resonance, p38 Mitogen-Activated Protein Kinases, Enzyme Inhibitors metabolism, Mitogen-Activated Protein Kinases metabolism
- Abstract
p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.
- Published
- 2001
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7. Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.
- Author
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Kraft PJ, Haynes-Johnson DE, Patel L, Lenhart JA, Zivin RA, and Palmer SS
- Subjects
- Animals, Animals, Newborn, Cricetinae, Electrophoresis, Polyacrylamide Gel, Fluorescence Polarization, Humans, Substrate Specificity, Collagen Type IV metabolism, Fibronectins metabolism, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 7 metabolism
- Abstract
Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.
- Published
- 2001
- Full Text
- View/download PDF
8. Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis.
- Author
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McMillian MK, Grant ER, Zhong Z, Parker JB, Li L, Zivin RA, Burczynski ME, and Johnson MD
- Subjects
- Animals, Cell Survival, Dogs, Fatty Liver metabolism, Fatty Liver pathology, Hepatocytes drug effects, Hepatocytes metabolism, Hepatocytes pathology, Humans, In Vitro Techniques, Indicators and Reagents pharmacokinetics, Microscopy, Fluorescence, Oxidation-Reduction, Rats, Tumor Cells, Cultured, Fatty Liver chemically induced, Fluorescent Dyes, Lipids analysis, Oxazines pharmacokinetics, Steroids toxicity, Xanthenes
- Abstract
Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation in vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.
- Published
- 2001
- Full Text
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9. Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay.
- Author
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Burczynski ME, McMillian M, Parker JB, Bryant S, Leone A, Grant ER, Thorne JM, Zhong Z, Zivin RA, and Johnson MD
- Subjects
- Animals, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System genetics, Dexamethasone pharmacology, Enzyme Induction, Male, Mass Spectrometry, Protein Folding, Rats, Rats, Sprague-Dawley, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System biosynthesis, Hepatocytes enzymology, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction
- Abstract
The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the numbers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug candidates will induce various members of the drug-metabolizing cytochrome P450 (CYP) enzyme superfamily is currently of great interest to the pharmaceutical industry. In the present study, we describe the rapid and reliable analysis of CYP induction in a readily obtained model system (cultured rat hepatocytes) using both real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary inducible rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in untreated and induced (beta-naphthoflavone, phenobarbital, and hydrocortisone) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, various culture conditions were examined for optimizing induction of the three CYP subfamily members. Both real-time RT-PCR and the RNA invasive cleavage assay prove to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes.
- Published
- 2001
10. Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells.
- Author
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Grant ER, Errico MA, Emanuel SL, Benjamin D, McMillian MK, Wadsworth SA, Zivin RA, and Zhong Z
- Subjects
- Animals, Apoptosis, Butadienes pharmacology, Cell Differentiation, Drug Interactions, MAP Kinase Kinase 1, MAP Kinase Kinase 2, Mice, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinases metabolism, N-Methylaspartate pharmacology, Neurons enzymology, Nitriles pharmacology, Phosphorylation, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases antagonists & inhibitors, Receptors, N-Methyl-D-Aspartate biosynthesis, Tumor Cells, Cultured, Enzyme Inhibitors pharmacology, Glutamic Acid pharmacology, Mitogen-Activated Protein Kinase 1 antagonists & inhibitors, Mitogen-Activated Protein Kinases antagonists & inhibitors, Neurons drug effects, Neuroprotective Agents pharmacology
- Abstract
Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.
- Published
- 2001
- Full Text
- View/download PDF
11. In vitro characterization of five humanized OKT3 effector function variant antibodies.
- Author
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Xu D, Alegre ML, Varga SS, Rothermel AL, Collins AM, Pulito VL, Hanna LS, Dolan KP, Parren PW, Bluestone JA, Jolliffe LK, and Zivin RA
- Subjects
- Animals, Antibody Affinity, Complement Activation, Complement C1q metabolism, Dose-Response Relationship, Drug, Genetic Variation, Graft Rejection drug therapy, Humans, Immunoglobulin Constant Regions genetics, Immunoglobulin Heavy Chains genetics, Immunoglobulin Variable Region genetics, Immunosuppressive Agents isolation & purification, Immunosuppressive Agents pharmacology, Kidney Transplantation immunology, Lymphocyte Activation, Mice, Muromonab-CD3 genetics, Muromonab-CD3 isolation & purification, Muromonab-CD3 pharmacology, Mutagenesis, Protein Binding, Protein Engineering methods, Receptors, IgG metabolism, Recombinant Proteins isolation & purification, T-Lymphocytes immunology, CD3 Complex immunology, Immunosuppressive Agents immunology, Muromonab-CD3 immunology
- Abstract
Orthoclone OKT 3 (mOKT3) is a highly effective agent for the reversal of steroid-resistant renal allograft rejection. However, its wider use has been limited by the development of a human anti-mouse antibody response (HAMA) and by the "cytokine release syndrome" (CRS). CRS has been associated with T cell/monocyte activation and, secondarily, with activation of the complement cascade. These processes are mediated through Abs' Fc regions by their abilities to cross-link T cells and mononuclear cells and to activate complements. To alleviate these problems, a group of five huIgG1- and huIgG4-based OKT3 wild-type antibodies and their corresponding Fc mutants with altered residues at amino acids 234, 235, and 318, reported to be required for FcgammaRI and FcgammaRII binding and complement fixation, were constructed. Characterization of these humanized OKT3 Abs, denoted huOKT3gamma1, huOKT3gamma4, huOKT3gamma1(A(234), A(235)), huOKT3gamma4(A(234), A(235)), and huOKT3gamma1(A(318)), has demonstrated that huOKT3gamma1(A(234), A(235)) and huOKT3gamma4(A(234), A(235)), and have at least a 100-fold reduced binding to FcgammaRI and FcgammaRII. As expected, they are much less potent in the induction of T cell activation and cytokine release, yet retain in vitro immunosuppressive effects as potent as those of mOKT3. Unexpectedly, while huOKT3gamma1(A(318)) did not show any reduction in its ability to bind C1q and to fix a complement, huOKT3gamma1(A(234), A(235)) was completely inactive. The in vitro characteristics of huOKT3gamma1(A(234), A(235)) are consistent with recent in vivo studies, in which this Ab showed greatly reduced HAMA and CRS with the retention of its ability to reverse ongoing graft rejection in man., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
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12. Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3gamma1(Ala-Ala) in the treatment of acute renal allograft rejection.
- Author
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Woodle ES, Xu D, Zivin RA, Auger J, Charette J, O'Laughlin R, Peace D, Jollife LK, Haverty T, Bluestone JA, and Thistlethwaite JR Jr
- Subjects
- Acute Disease, Adult, Antigens, CD blood, Cytokines blood, Dose-Response Relationship, Drug, Female, Half-Life, Humans, Male, Middle Aged, Muromonab-CD3 administration & dosage, Muromonab-CD3 adverse effects, Treatment Outcome, Graft Rejection therapy, Immunosuppressive Agents therapeutic use, Kidney Transplantation, Muromonab-CD3 immunology, Muromonab-CD3 therapeutic use, Receptors, Fc immunology
- Abstract
Background: HuOKT3gamma1(Ala-Ala) is a genetically-engineered derivative of the parental murine OKT3 monoclonal antibody, in which the six complementarity-determining regions have been grafted within a human IgG1 mAb, and whose C(H)2 region has been altered by site-directed mutagenesis to alter FcR-binding activity, thereby eliminating T cell activation properties. This report describes the results of a phase I trial of huOKT3gamma1(Ala-Ala) treatment of acute renal allograft rejection., Methods: Acute renal allograft rejection in kidney and kidney-pancreas transplant recipients was treated with huOKT3gamma1(Ala-Ala). huOKT3gamma1(Ala-Ala) dosing consisted of daily 5- or 10-mg doses adjusted initially to achieve target levels of 1000 ng/ml., Results: A total of seven patients, five kidney transplant and two kidney-pancreas transplant recipients, were treated with the monoclonal antibody for first rejection episodes. Corticosteroids (500 mg i.v. Solumedrol) were given 2 hr before the first huOKT3gamma1(Ala-Ala) dose only. Banff classification of treated rejections were the following: grade I, 1 patient, grade IIA, 1 patient, grade IIB, 4 patients, and grade III, 1 patient. Median time from transplant to rejection was 15 days, and median follow up 12 months (range 10-17 months). HuOKT3gamma1(Ala-Ala) therapy was given for 10.1+/-2.5 days, and mean total dose was 76+/-27 mg. Rejection was reversed in five of seven patients, and recurrent rejection was observed in one patient. Serum creatinine values peaked on day 1 of huOKT3gamma1(Ala-Ala) therapy, and thereafter demonstrated a progressive decline. Rejection reversal (return of creatinine to baseline) occurred at a median of 4 days and a mean of 4.1+/-2 days. Renal allograft biopsies obtained during huOKT3gamma1(Ala-Ala) therapy provided evidence of rapid rejection reversal. Patient and graft survival were both 100%. First dose reactions were minimal, and anti-OKT3 antibodies were not detected. Elevations in serum IL-10, but not IL-2 levels were observed after the first huOKT3gamma1(Ala-Ala) dose. Marked reductions in circulating CD2+, CD4+, and CD8+ T cells were observed after the first huOKT3gamma1(Ala-Ala) dose, followed by a slow progressive return of cell counts toward pretreatment values. Pharmacokinetic analysis revealed a half-life of 142+/-32 hr., Conclusions: HuOKT3gamma1(Ala-Ala) possesses the ability to reverse vigorous rejection episodes in kidney and kidney-pancreas transplant recipients, and in comparison to murine OKT3, possesses minimal first dose reactions and does not seem to induce antibodies that bind the OKT3 idiotype. These results support the conduct of additional clinical trials with the huOKT3gamma1(Ala-Ala) antibody.
- Published
- 1999
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13. Humanized, nonmitogenic OKT3 antibody, huOKT3 gamma(Ala-Ala): initial clinical experience.
- Author
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Woodle ES, Bluestone JA, Zivin RA, Jolliffe LK, Auger J, Xu D, and Thistlethwaite JR
- Subjects
- Female, Follow-Up Studies, Humans, Immunosuppressive Agents pharmacokinetics, Male, Muromonab-CD3 pharmacokinetics, Time Factors, Graft Rejection therapy, Immunosuppressive Agents therapeutic use, Kidney Transplantation immunology, Muromonab-CD3 adverse effects, Muromonab-CD3 therapeutic use, Pancreas Transplantation immunology
- Published
- 1998
- Full Text
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14. Amino-terminal dimerization of an erythropoietin mimetic peptide results in increased erythropoietic activity.
- Author
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Johnson DL, Farrell FX, Barbone FP, McMahon FJ, Tullai J, Kroon D, Freedy J, Zivin RA, Mulcahy LS, and Jolliffe LK
- Subjects
- Animals, Binding, Competitive drug effects, Cell Division physiology, Chromatography, High Pressure Liquid, Cross-Linking Reagents, Erythrocyte Membrane metabolism, Erythropoietin isolation & purification, Extracellular Space metabolism, Humans, In Vitro Techniques, Iron Radioisotopes, Mass Spectrometry, Mice, Molecular Weight, Polycythemia blood, Polyethylene Glycols metabolism, Receptors, Erythropoietin agonists, Receptors, Erythropoietin biosynthesis, Recombinant Proteins, Erythrocytes metabolism, Erythropoiesis physiology, Erythropoietin metabolism
- Abstract
Background: Erythropoietin (EPO), the hormone involved in red blood cell production, activates its receptor by binding to the receptor's extracellular domain and presumably dimerizing two receptor monomers to initiate signal transduction. EPO-mimetic peptides, such as EMP1, also bind and activate the receptor by dimerization. These mimetic peptides are not as potent as EPO, however. The crystal structure of the EPO receptor (EBP) bound to EMP1 reveals the formation of a complex consisting of two peptides bound to two receptors, so we sought to improve the biological activity of EPO-mimetic peptides by constructing covalent dimers of EMP1 and other peptide mimetics linked by polyethylene glycol (PEG)., Results: The potency of the PEG-dimerized EPO peptide mimetics both in vitro and in vivo was improved up to 1,000-fold compared to the corresponding peptide monomers. The dimers were constructed using peptide monomers which have only one reactive amine per molecule, allowing us to conclude that the increase in potency can be attributed to a structure in which two peptides are linked through their respective amino termini to the difunctional PEG molecule. In addition, an inactive peptide was converted into a weak agonist by PEG-induced dimerization., Conclusions: The potency of previously isolated peptides that are modest agonists of the EPO receptor was dramatically increased by PEG-induced dimerization. The EPO receptor is thought to be dimerized during activation, so our results are consistent with the proposed 2:2 receptor : peptide stoichiometry. The conversion of an inactive peptide into an agonist further supports the idea that dimerization can mediate receptor activation.
- Published
- 1997
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15. Humanization and molecular modeling of the anti-CD4 monoclonal antibody, OKT4A.
- Author
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Pulito VL, Roberts VA, Adair JR, Rothermel AL, Collins AM, Varga SS, Martocello C, Bodmer M, Jolliffe LK, and Zivin RA
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal isolation & purification, Antibody Affinity genetics, Cloning, Molecular, Computer Simulation, Humans, Immunoglobulin Heavy Chains genetics, Immunoglobulin Heavy Chains isolation & purification, Immunoglobulin Light Chains genetics, Immunoglobulin Light Chains isolation & purification, Lymphocyte Culture Test, Mixed, Mice, Molecular Sequence Data, Recombinant Fusion Proteins isolation & purification, Species Specificity, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, CD4 Antigens immunology, Models, Molecular, Recombinant Fusion Proteins chemistry
- Abstract
OKT4A, a murine mAb that recognizes an epitope on the CD4 receptor, is a potent immunosuppressive agent in vitro and in a variety of nonhuman primate models of graft rejection and autoimmune disease. Initial human cardiac transplant trials suggest that OKT4A does not cause either cytokine release syndrome or CD4+ cell depletion, but does induce a human anti-mouse Ab (HAMA) response despite strong concurrent immunosuppression. To further investigate the potential of OKT4A as an immunomodulator, it was necessary to decrease its immunogenicity. Therefore, we developed a humanized version of this Ab (gOKT4A-4), which has the same binding affinity and in vitro immunosuppressive properties of OKT4A, but retains only three murine sequence-derived amino acid residues outside of the complementarity-determining regions (CDRs). Detailed computer modeling of both OKT4A and gOKT4A-4 provided a computational rationale for the changes necessary to regain activity after humanization. This has also provided a plausible representation of the Ag binding site. Preliminary clinical results with gOKT4A-4 suggest that we have eliminated the immunogenicity observed in the parent murine Ab.
- Published
- 1996
16. A non-activating "humanized" anti-CD3 monoclonal antibody retains immunosuppressive properties in vivo.
- Author
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Alegre ML, Peterson LJ, Xu D, Sattar HA, Jeyarajah DR, Kowalkowski K, Thistlethwaite JR, Zivin RA, Jolliffe L, and Bluestone JA
- Subjects
- Animals, Graft Survival, Humans, Interleukin-2 biosynthesis, Lymphocyte Activation, Lymphocyte Depletion, Mice, Mice, SCID, Muromonab-CD3 immunology, Immunosuppressive Agents therapeutic use, Muromonab-CD3 therapeutic use
- Abstract
OKT3, a mouse anti-human CD3 mAb, is a potent immunosuppressive agent used in clinical transplantation to prevent or treat allograft rejection. Associated with this therapy is the systemic release of several cytokines that result in a series of adverse side effects. This release of cytokines is dependent on the cross-linking mediated by OKT3 between T cells and the Fc gamma R-bearing cells. To generate an anti-human CD3 mAb with reduced activating properties as compared with OKT3, we have transferred the complementary determining regions of OKT3 onto human IgG frameworks and then performed point mutations that reduce the affinity of the "humanized" anti-CD3 mAbs for Fc gamma Rs. Initial, in vitro, studies showed that whereas OKT3 and the parental humanized anti-CD3 mAbs activated T cells similarly, a humanized Fc variant failed to do so. Both the Fc variant and the activating anti-CD3 mAbs induced comparable modulation of the TCR and suppression of cytolytic T cell activity, in vitro. In the current study, we exploited an experimental model in which human splenocytes from cadaveric organ donors were inoculated into severe combined immunodeficient mice (hu-SPL-SCID mice) to test the activating and immunosuppressive properties of these anti-human CD3 mAbs in vivo. Unlike injection of OKT3 or of the parental humanized mAb, administration of the Fc variant did not result in T cell activation in vivo, as evidenced by the lack of induction of surface markers of activation, and of systemic human cytokines, including IL-2. Importantly, similar prolongation of human allograft survival was achieved with all anti-CD3 mAbs, indicating that the nonactivating anti-CD3 mAbs retained significant immunosuppressive properties in vivo. Thus, the use of an Fc variant in clinical transplantation should result in fewer side effects than observed with OKT3, while maintaining its clinical efficacy.
- Published
- 1994
17. Effect of a single amino acid mutation on the activating and immunosuppressive properties of a "humanized" OKT3 monoclonal antibody.
- Author
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Alegre ML, Collins AM, Pulito VL, Brosius RA, Olson WC, Zivin RA, Knowles R, Thistlethwaite JR, Jolliffe LK, and Bluestone JA
- Subjects
- Amino Acid Sequence, Animals, Antibody Affinity, Antigens, Differentiation metabolism, Binding Sites, Antibody, CD3 Complex, Granulocyte-Macrophage Colony-Stimulating Factor biosynthesis, Humans, Immunosuppression Therapy, In Vitro Techniques, Interferon-gamma biosynthesis, Lymphocyte Activation, Mice, Molecular Sequence Data, Muromonab-CD3 metabolism, Receptors, Fc metabolism, Receptors, IgG, Recombinant Fusion Proteins, T-Lymphocytes, Cytotoxic immunology, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha biosynthesis, Antigens, Differentiation, T-Lymphocyte immunology, Muromonab-CD3 chemistry, Receptors, Antigen, T-Cell immunology
- Abstract
The binding specificity of the murine OKT3 has been transferred into a human antibody framework to reduce its immunogenicity. This "humanized" anti-CD3 mAb (gOKT3-5) was previously shown to retain, in vitro, all the properties of native OKT3, including T cell activation, which has been correlated, in vivo, with the severe side effects observed in transplant recipients after the first administration of the mAb. T cell activation is thought to be triggered by the cross-linking mediated by the antibodies between T cells and Fc receptor-bearing cells. In this study, we introduced a single amino acid mutation from a leucine to a glutamic acid at position 235 in the Fc receptor binding segment of the gOKT3-5 mAb to produce Glu-235 mAb. This mutation generated a 100-fold decrease in the affinity of the antibody for the Fc receptor on U937 cells, without affecting Ag binding. In parallel, we observed a marked reduction in the T cell activation triggered by the mAb (proliferation, cell surface expression of early activation markers including Leu 23 and IL-2R, and release of TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF). In contrast, the mutated mAb retained suppressive properties similar to the gOKT3-5 mAb, as assessed by significant modulation of the T cell receptor complex and suppression of Ag-specific CTL activity. We conclude that this anti-CD3 mAb bearing a single amino acid mutation in its Fc portion retains important immunosuppressive properties, while exhibiting significantly less T cell activation than OKT3 in vitro. This drug might achieve potent immunosuppression while minimizing acute toxicity in vivo and thus be useful in transplantation as well as in autoimmune diseases.
- Published
- 1992
18. Humanized OKT3 antibodies: successful transfer of immune modulating properties and idiotype expression.
- Author
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Woodle ES, Thistlethwaite JR, Jolliffe LK, Zivin RA, Collins A, Adair JR, Bodmer M, Athwal D, Alegre ML, and Bluestone JA
- Subjects
- Amino Acid Sequence, Antigen-Antibody Reactions, Binding, Competitive, Cytotoxicity Tests, Immunologic, Dose-Response Relationship, Immunologic, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Humans, Immunoglobulin Heavy Chains genetics, Immunoglobulin Light Chains genetics, In Vitro Techniques, Interferon-gamma biosynthesis, Lymphocyte Activation immunology, Lymphocyte Culture Test, Mixed, Molecular Sequence Data, Muromonab-CD3 genetics, Receptors, Antigen, T-Cell immunology, Receptors, Interleukin-2 biosynthesis, Sequence Homology, Nucleic Acid, T-Lymphocytes immunology, T-Lymphocytes metabolism, Tumor Necrosis Factor-alpha biosynthesis, Immunoglobulin Idiotypes analysis, Muromonab-CD3 immunology
- Abstract
Antibodies that possess the Ag-binding regions of OKT3 within the context of a human framework (Hu-OKT3 Ab) offer distinct advantages for optimizing anti-CD3 mAb therapy. First, manipulation of Ab genes to produce humanized Ab that retain Ag-binding activity may circumvent antigenicity problems. Second, Ab gene engineering provides a means for modifying functional properties, including T cell activation and immune suppression. The purpose of this study was to determine the functional properties of Hu-OKT3 Ab and to compare the functional properties and idiotypes of Hu-OKT3 Ab to those of murine OKT3. Three Hu-OKT3 IgG4 Ab, a chimeric OKT3 antibody (cOKT3-1) (grafted sequences comprising all OKT3 VH and VL regions) and two complementarity determining region (CDR)-grafted antibodies, gOKT3-5 and gOKT3-6 (grafted sequences comprising only OKT3 VH and VL CDR and some framework amino acids, were analyzed. Initial studies demonstrated that the cOKT3 and gOKT3-5 Ab bound selectively to T cells and competitively inhibited OKT3-FITC binding with avidities similar to that of murine OKT3. Binding avidity of the gOKT3-6 Ab was markedly less than that of the other two Hu-OKT3 Ab. Serologic analysis suggested that cOKT3 and gOKT3-5 Ab possess idiotypes (combining sites) similar to murine OKT3. T cell activation potency of all three Hu-OKT3 Ab was assessed by proliferation, induction of activation marker expression (IL-2R and Leu 23), and lymphokine production (TNF-alpha and IFN-gamma). The cOKT3 and gOKT3-5 Ab demonstrated T cell activation potencies similar to murine OKT3 as assessed by each parameter. CD3 coating and modulation by these two Ab was effective but somewhat less potent than that observed with OKT3. Finally, cOKT3 and gOKT3-5 Ab both inhibited CTL activity comparably to murine OKT3. In conclusion, these studies indicate that gOKT3-5 and cOKT3 Ab possess immune modulating properties similar to murine OKT3 and thus offer attractive alternatives to murine OKT3 for in vivo therapy.
- Published
- 1992
19. Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC.
- Author
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Ellis RW, Keller PM, Lowe RS, and Zivin RA
- Subjects
- Cloning, Molecular, DNA, Recombinant metabolism, Nucleic Acid Hybridization, Plasmids, Protein Biosynthesis, RNA, Messenger genetics, Serotyping, Escherichia coli genetics, Genes, Genes, Viral, Genetic Vectors, Herpesvirus 3, Human genetics, Viral Proteins genetics
- Abstract
The genome of varicella-zoster virus (VZV) encodes at least three major glycoprotein genes. Among viral gene products, the gC gene products are the most abundant glycoproteins and induce a substantial humoral immune response (Keller et al., J. Virol. 52:293-297, 1984). We utilized two independent approaches to map the gC gene. Small fragments of randomly digested VZV DNA were inserted into a bacterial expression vector. Bacterial colonies transformed by this vector library were screened serologically for antigen expression with monoclonal antibodies to gC. Hybridization of the plasmid DNA from a gC antigen-positive clone revealed homology to the 3' end of the VZV Us segment. In addition, mRNA from VZV-infected cells was hybrid selected by a set of VZV DNA recombinant plasmids and translated in vitro, and polypeptide products were immunoprecipitated by convalescent zoster serum or by monoclonal antibodies to gC. This analysis revealed that the mRNA encoding a 70,000-dalton polypeptide precipitable by anti-gC antibodies mapped to the HindIII C fragment, which circumscribes the entire Us region. We conclude that the VZV gC glycoprotein gene maps to the 3' end of the Us region and is expressed as a 70,000-dalton primary translational product. These results are consistent with the recently reported DNA sequence of Us (A.J. Davison, EMBO J. 2:2203-2209, 1983). Furthermore, glycosylation appears not to be required for a predominant portion of the antigenicity of gC glycoproteins. We also report the tentative map assignments for eight other VZV primary translational products.
- Published
- 1985
- Full Text
- View/download PDF
20. Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase.
- Author
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Benfield PA, Zivin RA, Miller LS, Sowder R, Smythers GW, Henderson L, Oroszlan S, and Pearson ML
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, DNA Restriction Enzymes, Genes, Poly A genetics, Protein Biosynthesis, RNA genetics, RNA, Messenger genetics, Rats, Cloning, Molecular, Creatine Kinase genetics, DNA isolation & purification, Muscles enzymology
- Abstract
A series of cDNA clones corresponding to 1494 bases of rat muscle creatine kinase mRNA has been isolated and characterized. The identity of these clones has been confirmed by DNA sequence analysis and by comparison of the predicted amino acid sequence with that determined for the purified protein. The cDNA sequence accounts for the entire coding sequence of the creatine kinase protein in addition to the complete 3' untranslated region and 68 bases of 5' noncoding region. Sequences corresponding to the active site region of the protein, the initiation codon, the termination codon, and poly(A) addition signal have been identified.
- Published
- 1984
21. Molecular cloning and characterization of DNA sequences encoding rat and human atrial natriuretic factors.
- Author
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Zivin RA, Condra JH, Dixon RA, Seidah NG, Chrétien M, Nemer M, Chamberland M, and Drouin J
- Subjects
- Amino Acid Sequence, Animals, Atrial Natriuretic Factor, Base Sequence, Cloning, Molecular, DNA genetics, Genes, Humans, Natriuretic Agents, Protein Precursors genetics, RNA, Messenger genetics, Rats, Muscle Proteins genetics, Natriuresis, Proteins genetics
- Abstract
A cDNA copy of the message encoding rat atrial natriuretic factor (ANF) has been cloned in Escherichia coli, and its nucleotide sequence was determined. ANF appears to be synthesized as a larger precursor, atrial pronatriodilatin. The cDNA has an open reading frame potentially encoding a protein of 152 amino acids, of which the first 24 amino acids strongly resemble a signal sequence. This is followed by a sequence with 80% homology to a second vasoactive protein, porcine cardiodilatin. The ANF peptide is contained in the COOH-terminal portion of the protein. The DNA sequence corresponding to human ANF is also presented and displays a high degree of homology to its rat counterpart. These data provide further evidence for the expression in cardiac atria of a multifactor system that may contribute to the regulation of blood pressure and extracellular fluid volume.
- Published
- 1984
- Full Text
- View/download PDF
22. High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin.
- Author
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Gan ZR, Condra JH, Gould RJ, Zivin RA, Bennett CD, Jacobs JW, Friedman PA, and Polokoff MA
- Subjects
- Amino Acid Sequence, Base Sequence, Chromatography, High Pressure Liquid, DNA genetics, Electrophoresis, Polyacrylamide Gel, Intercellular Signaling Peptides and Proteins, Leucine genetics, Molecular Sequence Data, Plasmids, Platelet Aggregation, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transformation, Genetic, Viper Venoms biosynthesis, Viper Venoms metabolism, Cloning, Molecular, Escherichia coli genetics, Genes, Synthetic, Peptides, Viper Venoms genetics
- Abstract
A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.
- Published
- 1989
- Full Text
- View/download PDF
23. Gene structure of human cardiac hormone precursor, pronatriodilatin.
- Author
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Nemer M, Chamberland M, Sirois D, Argentin S, Drouin J, Dixon RA, Zivin RA, and Condra JH
- Subjects
- Alleles, Amino Acid Sequence, Atrial Natriuretic Factor, Base Sequence, DNA isolation & purification, DNA Restriction Enzymes, Genetic Variation, Heart Atria metabolism, Humans, Transcription, Genetic, Genes, Muscle Proteins genetics, Myocardium metabolism, Protein Precursors genetics
- Abstract
Atrial cardiocytes contain granules typical of protein-secreting cells, and atrial extracts are known to contain a powerful natriuretic and diuretic activity and to possess smooth muscle relaxant activity. A variety of active atrial peptides have been isolated, including a family of related peptides showing natriuretic, diuretic and smooth muscle relaxant activities in rat and human atria; these peptides were named atrial natriuretic factor (ANF). Another unrelated peptide from pig atria, cardiodilatin, is thought to possess only smooth muscle relaxant activity. Its partial amino acid sequence shows no homology with ANF sequences. The sequence analysis of a large form (106 amino acids) of ANF and of ANF complementary DNA clones indicates that cardiodilatin and ANF peptides are synthesized from a common precursor. This precursor also contains a signal peptide sequence expected of a secretory protein. We now describe the complete structure and sequence of the human gene for this novel hormone precursor that we call pronatriodilatin.
- Published
- 1984
- Full Text
- View/download PDF
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