Appeals Court rejects "apples/oranges" comparison in pharmacy cost case.
Explore the source record for details and available documents.
Insurance, Health, Reimbursement↗
Biomedical subjects
R A Klein
Publications and source records attributed to R A Klein.
MS and NMR analysis of the cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 variant specific glycoprotein.
The cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 is known to contain galactose, mannose and non-acetylated glucosamine. The structural elucidation of this oligosaccharide has been impeded by an unusual non-glycosidic linkage to the peptide chain and a glycosidic linkage to inositol phosphate on either side of the oligosaccharide. Using two different approaches for the isolation of the glycan, namely hydrolysis to give the oligosaccharide directly or pronase digestion to yield the glycan-containing C-terminal glycophosphopeptide, the structure of this glycan was elucidated by mass spectrometry and 1H-NMR spectroscopy. There was evidence of heterogeneity in the glycan residue.
Animals↗
Occult (non-surface expression) of T, B and monocyte markers in human large granular lymphocytes.
Human large granular lymphocytes were examined for non-surface expression with a panel of monoclonal antibodies to T cell, B-cell and monocyte markers. T101, antibody to the T65 antigen, showed binding to crude fractions containing intracellular membranes but not to immobilized whole cells. Non-surface expression of T65 was also demonstrated by flow cytometry using lysolecithin to transiently permeabilize cells. With the latter technique nonsurface expression was also demonstrated with monoclonal antibodies B2 and MO-2. T65 was shown to be synthesized by large granular lymphocytes by metabolic labeling, indirect immunoprecipitation and SDS-PAGE. T65 from large granular lymphocytes was the same mol. wt as antigen for T cells derived from the same donor. These results indicate that human large granular lymphocytes synthesize, but do not express on the surface, certain monoclonal antibody-derived markers heretofore considered specific for other cell lineages.
Antibodies, Monoclonal↗
Induction of early atherosclerosis in rats using parenterally-administered lipid emulsions.
It has been demonstrated by us and other workers that rats receiving I.V. infusions of Lipofundin-S will develop aortic changes indicative of early atherosclerosis. However, different lipid emulsions which are used in the clinical setting for parenteral nutrition vary substantially in chylomicron size and fatty acid composition. Therefore, in an attempt to better understand the mechanism by which a lipid emulsion might induce vessel lesions, we compared the nature of potential aortic changes resulting from infusions of Liposyn, Intralipid, or Lipofundin-S into the tail veins of Sprague-Dawley rats. Three groups of animals received either Liposyn (N = 10), Intralipid (N = 5), or Lipofundin-S (N = 9) at the rate of 6 g fat/kg body wt/day for 10 consecutive days. A fourth group (N = 5) received saline in equivalent dose to evaluate the effect of injection volume on vessel lesion formation. The other controls (N = 6) received no injections. Rats were sacrificed 24 hrs after the last infusion, and 1 mm rings from the top of the aortic arch and proximal third of the thoracic aorta were prepared for transmission electron microscopy (TEM). Examination by TEM allows two main conclusions to be drawn for both segments of the aorta. First, all three emulsions are capable of inducing early vessel changes which include endothelial damage, platelet adherence to damaged endothelium or subendothelial collagen, intimal phagocytic cells, and intimal smooth muscle cells surrounded by collagen bundles and elastin plates. Saline-infused rats only show occasional subendothelial swelling. None of the above-described changes are seen in any of the uninjected controls. Second, Lipofundin-S induces smooth muscle penetration of the intima in 7 of 9 rats, while Liposyn causes such changes in 2 of 10 animals. This difference in the efficiency with which the two emulsions induce the most advanced changes is statistically significantly by Chi Square (p less than 0.05). Intralipid produces smooth muscle penetration of the intima in 2 of 5 rats. The composition of the three emulsions suggests that the lower percent of linoleic acid and larger chylomicron size in Lipofundin-S may account for these differences, at least in part.
Animals↗
Lectin interactions with the variant surface glycoprotein from Trypanosoma brucei brucei incorporated into liposomes.
The variant specific surface glycoprotein from Trypanosoma brucei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed.
Animals↗
Detection of the intracellular ras p21 oncogene product by flow cytometry.
Rapid identification of the expression of oncogene products in specific cell types could potentially be useful in the diagnosis and treatment of human malignancy. We have now observed that through the use of lysolecithin permeabilization and fluorescence-activated flow cytometry, cells expressing high levels of the v-Ha-ras oncogene product, p21, can readily be distinguished from the nontransformed parent cells in a rapid and quantitative manner.
Animals↗
Hydrogen peroxide metabolism in Trypanosoma brucei.
Contrary to previous reports in the literature, bloodstream forms of the haemoflagellate protozoan Trypanosoma brucei brucei are not deficient in their ability to metabolize hydrogen peroxide, although they either lack or only possess the normal enzymes for H2O2 detoxification, catalase (EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.9), at extremely low levels. The hydrogen peroxide which is consumed appears to be reduced by NADPH derived from glucose via the pentose phosphate pathway. This process requires the newly discovered cofactor trypanothione.
Animals↗
A study of the membrane attachment site of the membrane-form variant surface glycoprotein from Trypanosoma brucei brucei using lipid vesicles as a model of the plasma membrane.
The incorporation of the membrane form and the soluble form of variant surface glycoprotein (mfVSG and sVSG) from Trypanosoma brucei brucei into liposomes has been investigated. It was found that selective incorporation of mfVSG into liposomes was possible in the presence of 8 M urea as a denaturing agent; sVSG, by contrast, was incorporated only poorly into liposomes. After proteolysis of mfVSG incorporated into liposomes, a compound was isolated from the liposomes which carries with it constituents of the glyophospholipid membrane anchor: ethanolamine, di-14:0-diglyceride and phosphatidylinositol were identified by electron impact and fast atom bombardment mass spectrometry, thus providing direct confirmation of the earlier findings of Holder [Holder, A.A. (1983) Biochem. J. 209, 261-262] and Ferguson et al. [Ferguson, M.A.J., Haldar, K. and Cross, G.A.M. (1985) J. Biol. Chem. 260, 4963-4968; Ferguson, M.A.J., Low, M.G. and Cross, G.A.M. (1985) J. Biol. Chem. 260, 14547-14555].
Animals↗
Spectroscopic investigations on the binding site of bovine hepatic fatty acid binding protein. Evidence for the existence of a single binding site for two fatty acid molecules.
The hydrophobic region of the binding site of a bovine fatty acid binding protein (pI 7.0-FABP) has been characterized using fluorescence and circular dichroism (CD) spectroscopy. Blue-shifts of fluorescence emission maxima and increased lifetimes of naphthylamine dyes, anthroyloxy-fatty acids, pyrene nonanoic acid and trans-parinaric acid indicated a hydrophobic interaction with FABP. The fluorescence quenching of various anthroyloxy-fatty acids by iodide and acrylamide showed lower accessibility to the fluorophore linked to the carbon adjacent to the carbonyl group and towards the methyl end of the fatty acid. Binding stoichiometries were different for fatty acids and their bulky fluorescent analogues. trans-Parinaric acid when bound to FABP showed a complex induced CD-spectrum, which is explained by a close proximity of two ligands in the same binding site. Fluorescent derivatives of phosphatidylcholine with trans-parinaric acid and cholesteryl trans-parinarate did not bind to FABP. Thus, the binding site appears to be constructed for high affinity binding of long chain fatty acids.
Animals↗
Bleeding after liver biopsy in a patient with systemic mastocytosis and malabsorption.
Systemic mastocytosis, with its diffuse infiltration of mast cells into various organs, has resulted in intestinal malabsorption and bleeding diatheses. The pathophysiology underlying these phenomena is unclear, but may be related to the release of histamine and heparin containing mast cell granules. A patient with systemic mastocytosis had malabsorption and developed massive bleeding after percutaneous liver biopsy. Histologic involvement of skin, duodenum, rectum, liver, and bone marrow was documented. Mastocytosis should be considered in the differential diagnosis of malabsorption.
Aged↗
Induction and enhancement by monocytes of antibody-induced modulation of a variety of human lymphoid cell surface antigens.
We have previously reported that the addition of monocytes results in enhanced modulation of the T65 antigen when normal or leukemic lymphoid cells were cultured in vitro with the T101 monoclonal antibody. In the present investigation, we extend these findings to demonstrate that monocyte-enhanced modulation is a phenomenon that occurs with a variety of T and B lymphoid antigens identified by murine monoclonal antibodies. Two patterns of monocyte-enhanced modulation were observed: (1) augmentation by monocytes of existing antigen modulation by the T101 and anti-Leu-4 antibodies, and (2) induction by monocytes of previously unrecognized modulation with the anti-Leu-2 and anti-Leu-9 antibodies. Enhancement of modulation by monocytes was also detected with antibodies to surface IgM and HLA-DR antigens. Antigen modulation on lymphoid cell lines appeared to be more variable than on fresh cells, with or without monocytes. Monocyte-enhanced antigen modulation was not demonstrated with two monoclonal antibodies against solid tumors. Monocyte-enhanced modulation was shown to be dependent upon the Fc portion of the antibody, but independent of proteolytic or oxidative compounds released by monocytes. These findings indicate that the results obtained during in vitro studies of antigen modulation may vary with the source of cells and the extent to which monocytic cells are present. In addition, these findings suggest an enhanced role for Fc receptor-bearing cells of monocytic origin in antigen modulation following in vivo administration of monoclonal antibodies.
Adult↗
Intratumor localization of monoclonal antibody in patients with melanoma treated with antibody to a 250,000-dalton melanoma-associated antigen.
Antibody localization at the tumor site was assessed in melanoma patients who received the murine monoclonal antibody 9.2.27. Antibody was administered twice weekly in escalating doses from 1 to 500 mg. Localization was assessed by biopsies of cutaneous and lymph node lesions obtained 24-96 hours following therapy. The percentage of tumor cells that bound the antibody in vivo was dose dependent, with similar findings obtained by either flow cytometry or immunoperoxidase staining techniques. Little or no in vivo binding of the 9.2.27 antibody to tumor cells was found following 1- and 10-mg doses, whereas all specimens demonstrated in vivo binding of the antibody following 200- and 500-mg doses. Fluorescence staining intensity, as quantitated by flow cytometry, was employed to determine the degree of in vivo saturation of antibody binding sites following therapy. The degree of saturation was found to vary substantially among patients: Some patients demonstrated nearly 100% saturation after 200-mg doses of 9.2.27 antibody, whereas others demonstrated only half maximal saturation after doses of 500 mg. Although immunoperoxidase staining provided important qualitative information regarding the distribution of antigen and antibody within the tumor, these studies demonstrated the usefulness of immunofluorescent flow cytometry for quantitative assessment of antibody localization in solid tumors and provided information necessary for the design of further trials of monoclonal antibodies and immunoconjugates.
Antibodies, Monoclonal↗
Detection of intracytoplasmic antigens by flow cytometry.
A new technique is described for the detection of intracellular antigens by immunofluorescence and flow cytometry. The technique utilizes lysolecithin (lysophosphatidylcholine), a naturally occurring phospholipid, to permeabilize cell membranes and allow antibodies to reach intracellular antigens. The technique is rapid and sensitive, and retains sufficient integrity of the cells being treated to enable differentiation of cell types on the basis of light scatter (e.g., lymphocytes from monocytes). Permeabilization of cells following lysolecithin was assessed using standard techniques including trypan blue exclusion, propidium iodide staining, and hydrolysis of fluorescein diacetate. Lysolecithin treatment was accompanied by only minimal increases in non-specific background fluorescence, and no increase in autofluorescence. Our studies have demonstrated that lysolecithin treatment of human mononuclear cell populations permits flow cytometric analysis of cytoskeletal structures, including intermediate filaments, as well as cytoplasmic immunoglobulin. Studies currently in progress in our laboratory have demonstrated broad intracellular reactivity with some monoclonal antibodies identifying leukocyte differentiation and tumor-associated antigens. These findings contrast with the more restricted expression of these antigens on the cell surface and demonstrate not only the value of the lysolecithin technique but also the importance of the study of intracellular antigens in our overall understanding of the specificity, distribution, synthesis, and function of cellular antigens.
Antigens↗
Threonine uptake in Trypanosoma brucei.
Threonine uptake by the parasitic protozoan Trypanosoma brucei has been assessed using an oil-phase separation technique for measuring rapid amino acid fluxes. It was shown that the storage conditions for the organism were critical for the production of reproducible data. Using this method it has been shown that threonine uptake occurs rapidly, being linear for less than 30 s at 37 degrees C in contrast with previous reported results. Kinetic analysis of threonine uptake at 25 degrees C and at physiological plasma threonine levels (20-700 microM) gave a Km of 250 microM and a Vmax of 8 nmol mg-1 cell protein min-1. At these concentrations threonine uptake takes place against a concentration gradient.
Alanine↗
Pyruvate kinase: a carnitine-regulated site of ATP production in Trypanosoma brucei brucei.
Pyruvate kinase activity in Trypanosoma brucei brucei is stimulated in the presence of L-carnitine and is inhibited by acetyl CoA, ATP or the ATP-Mg2+ complex. Increased pyruvate kinase activity is associated with stimulation of ATP synthesis in the presence of L-carnitine. There is evidence that carnitine stimulates pyruvate kinase activity indirectly by removing the inhibitory modulator acetyl CoA as a result of the carnitine acetyl transferase (CAT) also present in the trypanosomes.
Acetyl Coenzyme A↗
The measurement of motility in micro-organisms and its biochemical significance.
Explore the source record for details and available documents.
Acetylcarnitine↗
Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen.
Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.
Antibodies, Monoclonal↗
T65 antigen modulation in a phase I monoclonal antibody trial with chronic lymphocytic leukemia patients.
Antigenic modulation of the T65 cell surface antigen was assessed in chronic lymphocytic leukemia patients (CLL) receiving therapy with the murine monoclonal antibody T101 in a phase I clinical trial. A total of 12 patients received 1, 10, or 40 mg doses administered over 2 hr, or 50 or 100 mg doses administered over 50 hr. Decreases in T65 antigen expression (up to 90%) coincided with decreases in the circulating leukemic cell count in those patients who received T101 over a 2-hr period, indicating that clearance of circulating T65 antigen-positive cells could account for most of the observed decreases in T65 antigen expression. In contrast, analysis of bone marrow specimens from these patients indicated that decreases in T65 antigen density in this relatively stationary population resulted not from a cell decrement but rather from antigenic modulation. Pulmonary toxicity prevented administration of doses greater than 40 mg over a 2-hr period; therefore, higher doses (50 and 100 mg) were administered over a 50-hr period. This treatment schedule resulted in greater than 90% reduction in T65 antigen density of both circulating and bone marrow leukemic cells without dramatic drops in circulating leukemic cell counts, indicating that antigenic modulation accounted for most of the observed decreases in T65 antigen density under these conditions. Reexpression of T65 antigen by modulated cells was observed both in vitro and in vivo within 2 to 4 days. Immunoperoxidase staining of in vivo-modulated specimens and in vitro modulation studies with 125I-T101 suggested that T65 antigen-T101 antibody complexes were internalized during modulation. Although antigenic modulation inhibits the potential therapeutic effectiveness of unconjugated T101 antibody in CLL patients, treatment of CLL with T101 drug or toxin immunoconjugates under conditions that bring about rapid and extensive internalization of the T65 antigen may provide an effective means of therapy.
Antibodies, Monoclonal↗