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Biomedical subjects

T B Okarma

Publications and source records attributed to T B Okarma.

At least 37 records · Page 2Linked to original sources
PubMed1992

Rapid isolation of human CD34 hematopoietic stem cells--purging of human tumor cells.

Human CD34+ hematopoietic stem cells were purified using a new technology in which monoclonal antibodies are covalently immobilized on polystyrene surfaces. The CD34+ cell isolation scheme involved three sequential processes: (1) purification of bone marrow mononuclear cells; (2) enrichment of CD34+ cells using covalently immobilized soybean agglutinin; and (3) positive selection of CD34+ cells using polystyrene surfaces coated with the anti-CD34 monoclonal antibody ICH3. CD34+ cells purified by this process have both low-to-medium forward light scatter and low 90 degrees light-scatter properties. Moreover, the purified CD34+ cells are greater than 85% viable, express appropriate characteristic surface antigens, and are 10-50-fold enriched in short- and long-term hematopoietic activity. CD34+ cells collected in this manner from bone marrow samples contaminated with radiolabeled breast carcinoma, neuroblastoma, acute myelogenous leukemia, or small cell lung carcinoma cells were 99.9% depleted of the tumor cells. The CD34+ cell selection devices are sterile and are easily scaled-up to process clinical scale bone marrow samples.

Antibodies, Monoclonal↗
PubMed1991

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Animals↗
PubMed1991

Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor alpha-subunit.

To better define the antigenic sites on the human muscle acetylcholine receptor (AChR) that are involved in stimulating the production of pathogenic antibodies in myasthenia gravis (MG), the nucleotide sequence encoding the major extracellular domain of the AChR alpha subunit was chemically synthesized. The gene cassettes encoding amino acids (aa) 1-85 (AChR-I) and 86-210 (AChR-II), were cloned individually, and the coding sequence representing the complete major extracellular domain (aa 1-210; AChR-C) was obtained by subsequent fusion of cassettes encoding AChR-I and AChR-II. The genes were inserted into the inducible expression plasmid, pKK-223-3, and expressed in vitro and in vivo in Escherichia coli. Biological activity was demonstrated by immunoprecipitation of in vitro-synthesized AChR-C by sera from MG patients and by the alpha-bungarotoxin-binding activity of E. coli-synthesized AChR-II and AChR-C. The availability of the recombinant AChR polypeptides should facilitate studies on the molecular basis of the autoimmune response in MG.

Amino Acid Sequence↗
PubMed1991

Primary structure of bovine conglutinin, a member of the C-type animal lectin family.

We report the complete amino acid sequence of bovine conglutinin obtained by structural characterization of peptides derived from the protein by various chemical and enzymatic fragmentation methods. The protein consists of 351 amino acid residues including 55 apparent Gly-X-Y repeats with two interruptions. This 171-residue-long collagenous domain separates a short noncollagenous NH2-terminal region of 25 residues from the 155-residue-long globular COOH terminus revealing the structural relation of conglutinin with mannose-binding proteins, pulmonary surfactant-associated proteins, and a complement component C1q. Eight hydroxylysine residues were found in the collagenous domain. All of these hydroxylysine residues which occupy a Y position in a Gly-X-Y triplet are possible glycosylation sites since no phenylthiohydantoin amino acid was identified in automated Edman degradation cycles corresponding to these sites. The noncollagenous COOH domain of conglutinin, on the other hand, contains a carbohydrate recognition domain which shares substantial sequence homology with C-type animal lectins. Conglutinin has the greatest sequence similarity with mannose-binding proteins and pulmonary surfactant-associated proteins.

Amino Acid Sequence↗
PubMed1990

Enrichment of murine hematopoietic stem cells. Reconstitution of syngeneic and haplotype-mismatched mice.

A method is described to purify murine hematopoietic stem cells. The procedure involves fluorescence-activated cell sorting of nonadherent nucleated bone marrow cells for the presence of the antigen, Thy1.2, and the absence of the lineage-specific antigens, Lyt2, L3T4, Mac1, B220, and J11D.2. These Thy1.2+T-B-M-J- cells are 200-800-fold enriched in in vitro colony-forming units. Moreover, this narrow subset shows enhanced ability to form spleen colonies and engraft lethally irradiated mice. Data reported herein demonstrate that these purified pluripotent stem cell populations also have enhanced potential for the rescue of lethally irradiated haplotype-mismatched mice.

Animals↗
PubMed1990

Separation and growth of human CD4+ and CD8+ tumor-infiltrating lymphocytes and peripheral blood mononuclear cells by direct positive panning on covalently attached monoclonal antibody-coated flasks.

A direct positive panning technique was developed in order to achieve highly purified CD4+ and CD8+ cells. Fresh peripheral blood mononuclear cells or tumor-infiltrating lymphocytes derived from bulk cultures were applied to modified polystyrene surfaces to which anti-CD4 or anti-CD8 monoclonal antibodies were covalently attached. Adherent cells were allowed to grow in the original flask and were then harvested and cultured in IL-2-containing medium. This positive immunoselection technique resulted in CD4+ and CD8+ cell subsets with high cell viability and a high degree of purity. In several samples, the isolated cell subsets were subsequently subjected to a negative immunomagnetic bead selection in order to remove reciprocal cell subset contamination or double-positive CD4+/CD8+ cells. The isolated cells maintained their ability to proliferate, kept their phenotypic profiles, and remained functionally intact after long-term growth in culture without the further addition of mitogenic or allogeneic cell stimulation. This approach is a simple, rapid, and reproducible technique that might be useful on a large scale to isolate and to grow T-cell subsets for research and for clinical use.

Antibodies, Monoclonal↗
PubMed1988

Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that is dependent on adenovirus or herpesvirus for reproductive functions. We describe the construction of recombinant AAV vectors containing the chloramphenicol acetyltransferase gene or the neomycin phosphotransferase gene. These vectors carried their respective genes into a wide variety of cell types, including primary skin fibroblasts and hematopoietic cells. Infection efficiencies varied with cell type and ranged up to 3.0%. Coinfection of two different recombinant viruses was also used to introduce two different sequences simultaneously into a given cell. Finally, methods for obtaining recombinant AAV vectors with minimal contamination of wild-type virus are described. These various attributes of AAV vectors make them a viable DNA transduction system.

Animals↗
PubMed1988

Optimizing electroporation parameters for a variety of human hematopoietic cell lines.

The parameters affecting electroporation of four human hematopoietic cell lines were investigated. The optimal conditions for electroporation are described for both transient and stable expression of foreign genes. A correlation exists between the levels of transient gene expression and stable transfection frequency. In addition, linear DNA yields higher stable transfection frequencies than supercoiled DNA. The cumulative results indicate that electroporation is a simple and useful method for obtaining transient and stable expression of foreign genes in human hematopoietic cells.

Cell Line↗
PubMed1987

Inducible gene expression from multiple promoters by the tumor-promoting agent, PMA.

Phorbol ester tumor promoters affect a broad scope of changes in mammalian cells. This report describes the activation of expression of an introduced chloramphenicol acetyltransferase (CAT) reporter gene by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in a variety of fibroblast and hematopoietic cell lines. PMA-mediated activation appears to be promoter region specific, yet widespread. Enhanced gene expression is observed for four out of five promoter systems tested, and, in some cases, is dependent on the cellular environment. Further experiments indicate that PMA mediates elevated gene expression by rapidly increasing steady state levels of CAT mRNA. The broad range of promoters affected by PMA may help explain the high potency of this agent in tumor production.

Cell Line↗
PubMed1985

Isofocusing of antigenic small nuclear ribonucleoproteins. I. Compositional analysis.

This report describes the behavior of antigenic small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels. These RNA-protein complexes exhibited true isofocusing characteristics only when in the complexed form. Deproteinized snRNAs migrated to pH ranges which varied according to the pH of the application site. Immunological assays using lupus sera which recognized the La, Sm, and RNP determinants on these snRNPs established that the La and the Sm/RNP antigens segregated to pH 4.7-4.9 and 5.5-7.5, respectively. RNase digestion of these snRNPs did not alter the isofocusing migration of either the Sm or the La determinants. These antigenically active fractions contained the appropriate protein and RNA species shown by immunoprecipitation studies to associate with these antigenic determinants. The isofocusing fractions containing the uridylic acid-snRNPs were fully immunoprecipitable by anti-Sm sera, confirming their particulate integrity after isofocusing.

Animals↗
PubMed1985

Isofocusing of antigenic small nuclear ribonucleoproteins. II. Preparative isolation.

In a companion report (T.B. Okarma, W.S. Schrier, and R. Feinbaum, 1985, Anal. Biochem. 147, 27-37) the behavior of small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels was characterized. This communication extends those findings and describes a gentle procedure for the preparative isolation of snRNPs in native form from cultured murine L-5178y leukemia cells using sucrose density gradient centrifugation, preparative isofocusing, and gel filtration chromatography. Isofocusing in granulated gels separated intact uridylic acid (U)-snRNPs from tRNA and La RNPs. The U-snRNPs remained immunoprecipitable by lupus antisera throughout fractionation. The final product obtained in 2% yield contained primarily U1 and U2 snRNAs and lesser amounts of U3, U4, U5, and U6, along with the core U-snRNP polypeptides A-G. The core polypeptides displayed apparent pI's which ranged from 4.5 to 9.5 when analyzed by two-dimensional gel electrophoresis. Proteins B (28,000), D (16,000), and E (13,000) exhibited isoelectric variants. The Sm determinant proteins B' (28,000) and E (13,000) isofocused as basic peptides with apparent pI's of 9.5 and 8.5, respectively. The purity of the final fractions compared well with that of immunoprecipitates and the procedure reproducibly generated yields of native snRNPs sufficient for in vitro studies of their biological function.

Animals↗
PubMed1985

Prospective study of side effects associated with the use of silver sulfadiazine in severely burned patients.

We report the results of a prospective study of 45 burn patients in whom serum and urine levels of cream constituents (sulfadiazine and propylene glycol) were determined and correlated with clinical parameters and the presence of an immune response to sulfadiazine. Propylene glycol, measured by gas chromatography, was found in the serum of 53% of study patients. Sulfa, determined by spectroscopy, was found in the serum of all patients, with values as high as 9.1 mg/dl within 24 hours of topical application. Ninety percent of tested patients had circulating sulfadiazine antibodies, predominantly IgG. Fourteen patients died, 11 of whom had interpretable renal biopsies. Five of these biopsies showed positive immunofluorescent linear or granular staining for IgG, IgM, IgA, or C'3; none of these 5 patients had clinical evidence for the nephrotic syndrome. The association between antisulfadiazine antibodies, circulating immune complexes, immunoglobulin disposition on glomerular basement membranes, and topical application of silver sulfadiazine provides evidence for sulfadiazine sensitization in patients with severe burns.

Administration, Topical↗
PubMed1984

Partial purification and characterization of plasma DNA and its relation to disease activity in systemic lupus erythematosus.

This study reports the partial isolation and characterization of plasma DNA by phenol extraction and concentration of the extract. DNA, identified by immunologic and chemical methods, was found in plasma from both normal persons and patients with systemic lupus erythematosus in amounts varying from 0.004 to 0.4 micrograms/ml. The DNA was predominantly of low molecular weight, approximating 100 to 200 base pairs. Patients with lupus tended to have higher concentrations of plasma DNA than normal persons, with considerable overlap between the groups. Plasma DNA concentrations correlated inversely with titers of antibody to DNA but not strongly enough for prediction of either variable in individual cases. Many patients had high levels of plasma DNA and its antibody without clinical nephritis. These results indicate the ubiquity of plasma DNA and suggest the necessity of factors other than mere presence of DNA and its antibody for initiation of glomerular damage in systemic lupus erythematosus.

Autoantibodies↗
PubMed1982

Analysis of speckled fluorescent antinuclear antibody test antisera using electrofocused nuclear antigens.

Antibodies to different components of the extractable nuclear antigen (ENA) have been thought to be serological markers for clinical subsets of rheumatic diseases. However, incomplete characterization and standardization of antigenic components such as ribonucleoprotein (RNP), Sm, and SS-B (Ha), and the multiplicity of autoantibodies produced by different patients have confounded correlations between autoantibody specificity and disease subsets. This study describes the preparative separation of the antigens Sm, RNP, and Ss-B (Ha) by electrofocusing and their use in a rocket electrophoretic assay that in one step identifies and quantifies the multiple reactivities of patient sera exhibiting the speckled FANA pattern. Preparative electrofocusing generates milligram quantities of these antigens with retention of their immunologic and biochemical characteristics, facilitating further study of their biological properties and relationships to disease subsets.

Animals↗
PubMed1982

Mechanisms of proteinuria in human glomerulonephritis.

We evaluated glomerular barrier function in 28 patients with glomerulonephritis. Neutral dextrans of graded size were used to characterize the size-selective properties of the barrier. Charge selectivity was characterized by electrofocusing excreted urinary proteins. A fractional IgG clearance (relative to freely permeable inulin), smaller or greater than 100 x 10(-5) was used to distinguish patients with minor (group I, n = 13) and major (group II, n = 15) urinary IgG leakage, respectively. Fractional clearances of smaller dextrans (radii 20-50 A) were similar, but those of larger dextrans (radii 52-60 A) were elevated in group II relative to group I patients. A model of solute transport through a bimodal pore size distribution revealed the values for pore radius in the lower mode to approximate 51-55 A in both group I and group II patients. Pore radius in the upper mode, by contrast, was much larger in group II than in group I patients, approximating 87-97 vs. 72-77 A, respectively. Electrofocusing of urinary protein from group I patients revealed mostly albumin (isoelectric point 5.2). In group II patients, however, immunoglobulin excretion was copious. Moreover, the distribution of anionic, neutral, and cationic species (isoelectric points 5.5-8.5) in urinary and plasma eluates of IgG2 and IgG4 was similar. We conclude that when glomerulonephritis is associated with selective albuminuria, as in group I,, there is an isolated reduction of electrostatic retardation of relatively small anionic proteins. Major urinary IgG leakage (group II), however, appears to result from the development in the glomerular membrane of a subpopulation of enlarged pores that are highly permeable towards proteins of large size and varying charge.

Albuminuria↗