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Biomedical subjects

S C Kinsky

Publications and source records attributed to S C Kinsky.

At least 19 recordsLinked to original sources
PubMed1989

Phorbol ester activation of phospholipase D in human monocytes but not peripheral blood lymphocytes.

Previous reports have shown that 12-0-tetradecanoylphorbol-13-acetate can activate phospholipase D in human peripheral blood mononuclear cells as measured by an enzyme-catalyzed transphosphatidylation reaction (phosphatidylethanol formation). In the present study, the mononuclear cells were fractionated by two procedures to identify the responsive cells. Contrary to earlier suggestions, the results indicate that phorbol ester does not stimulate phospholipase D activity in normal lymphocytes. Thus, phosphatidylethanol was not produced by T lymphocytes (isolated by sheep erythrocyte rosette formation) or by a mixture of T and B lymphocytes (isolated by centrifugal elutriation). Under the same conditions, phorbol ester was able to activate phospholipse D in fractions that contained predominately monocytes. Preliminary experiments have further shown that phorbol ester does not induce phospholipase D activity in human T cell leukemic lines (MOLT-3, CEM, JURKAT, PEER) but can do so in some, but not all, B cell lines that have been infected with Epstein-Barr virus.

B-Lymphocytes↗
PubMed1988

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine.

We have prepared liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-gamma-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects of MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N5-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N5-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.

Ammonium Chloride↗
PubMed1987

Circumvention of the methotrexate transport system by methotrexate-phosphatidylethanolamine derivatives: effect of fatty acid chain length.

Methotrexate has been conjugated (amide bond) via either the alpha or gamma, or both alpha and gamma, glutamyl carboxyl groups to the amino function of dihexanoylphosphatidylethanolamine (C6C6PE) and 1-tetradecanoyl-2-hexanoylphosphatidylethanolamine (C14C6PE). These phospholipid prodrugs (either free or incorporated into liposomes) were compared with the corresponding ditetradecanoylphosphatidylethanolamine (C14C14PE) conjugates, some of whose properties have been described previously, for their ability to inhibit the proliferation of human leukemic cells (CEM/O) or cells derived therefrom (CEM/MTX) that are resistant to methotrexate because of a defective drug transport system. Regardless of chain length, the gamma conjugates were more effective than either the alpha or the alpha, gamma conjugates, in inhibiting growth of the parent cells, confirming initial experiments with mouse cells. Chain length had, however, a pronounced influence on the capacity of the various gamma derivatives to circumvent the transport defect. For example, CEM/MTX cells were 120-fold less susceptible than CEM/O cells to inhibition by either methotrexate or methotrexate-gamma-C6C6PE, whereas both cell lines were equally sensitive to methotrexate-gamma-C14C14PE. Although less potent than either of the foregoing, methotrexate-gamma-C14C6PE could partially by-pass the defective transport system. These results suggest that methotrexate-gamma-PE derivatives with appropriate acyl residues might be useful probes to investigate the mechanism by which phospholipids in general are able to traverse cell membranes.

Biological Transport↗
PubMed1987

Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-gamma-dimyristoylphosphatidylethanolamine.

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.

Cell Division↗
PubMed1986

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability.

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.

Avidin↗
PubMed1986

Effect of liposomes sensitized with methotrexate-gamma-dimyristoylphosphatidylethanolamine on cells that are resistant to methotrexate.

This study compares the ability of methotrexate and liposomes, in which the drug is anchored to the lipid bilayers via methotrexate-gamma-dimyristoylphosphatidylethanolamine, to inhibit proliferation of human leukemic cells (CEM/O) and cells derived from this line that are resistant to methotrexate because of either a defective transport system (CEM/MTX cells) or elevated levels of dihydrofolate reductase (CEM/R1 cells). Whereas CEM/O and CEM/MTX cells show a 120-fold difference in their susceptibility to methotrexate (as measured by the incorporation of tritiated deoxyuridine into DNA), both lines are equally sensitive to the liposomes. In contrast, proliferation of CEM/MTX cells is not inhibited significantly by methotrexate-gamma-glycerophosphorylethanolamine (MTX-gamma-glyceroPE), the water-soluble analog of MTX-gamma-DMPE. Both the ability of the liposomes to circumvent the transport defect, and the inability of MTX-gamma-glyceroPE to do so, were anticipated on the basis of previous experiments which show that thiamine pyrophosphate could antagonize inhibition of mouse 3T3 and L1210 cell proliferation by methotrexate and MTX-gamma-glyceroPE, but not inhibition by liposomes. Human cells (CEM/O) behave similarly. The present experiments also suggest that liposomes prepared with MTX-gamma-DMPE can partially reverse the methotrexate resistance of CEM/R1 cells that is due to overproduction of the target enzyme.

Cell Division↗
PubMed1985

Synthesis and characterization of methotrexate-dimyristoylphosphatidylethanolamine derivatives and the glycerophosphorylethanolamine analogs.

Research in this laboratory is currently focused on the biochemical and pharmacological properties of liposomes in which an otherwise water-soluble drug is anchored to the lipid bilayers via an appropriate non-polar residue. To this end, we have synthesized three (I-III) methotrexate (MTX) derivatives of dimyristoylphosphatidylethanolamine (DMPE) by conjugation of the alpha and/or gamma glutamyl carboxyl groups of the drug with the amino function of the phospholipid. These derivatives have been characterized analytically and chromatographically as MTX-gamma-DMPE (I), MTX-alpha-DMPE (II), and MTX-alpha, gamma-diDMPE (III). The corresponding glycerophosphorylethanolamine analogs have also been prepared and identified. The biological activity of these compounds (as inhibitors of in vitro cell proliferation and dihydrofolate reductase) is described in the following paper.

Chromatography, Thin Layer↗
PubMed1985

Inhibition of cell proliferation and dihydrofolate reductase by liposomes containing methotrexate-dimyristoylphosphatidylethanolamine derivatives and by the glycerophosphorylethanolamine analogs.

Liposomes, which were prepared with the three methotrexate (MTX)-dimyristoylphosphatidylethanolamine (DMPE) derivatives described in the preceding paper, were tested for their ability to block proliferation of mouse 3T3 and L1210 cells. Tritiated deoxyuridine incorporation into DNA could be completely inhibited by liposomes sensitized with MTX-DMPE I (MTX-gamma-DMPE). Under similar conditions, liposomes containing MTX-DMPE II (MTX-alpha-DMPE) and MTX-DMPE III (MTX-alpha, gamma-diDMPE) produced partial and no inhibition, respectively. These effects on cell growth were paralleled by the capacity of liposomes, prepared with each of the DMPE derivatives, to inhibit dihydrofolate reductase isolated from L1210 cells. Analogous experiments with the three corresponding glycerophosphorylethanolamine (glyceroPE) analogs also indicated that MTX-glyceroPE I was the most effective inhibitor of both cell proliferation and enzymatic activity. However, MTX-DMPE I sensitized liposomes apparently enter target cells as a consequence of phagocytosis, and not via the ubiquitous methotrexate transport system that is employed by MTX-glyceroPE I. For example, novel use of thiamine pyrophosphate showed that this compound had no influence on inhibition of cell proliferation due to liposomes, whereas thiamine pyrophosphate could completely antagonize the inhibitory effects of methotrexate and MTX-glyceroPE I. The results are discussed with reference to possible therapeutic advantages of these liposomes.

Ammonium Chloride↗
PubMed1983

An alternative procedure for the preparation of immunogenic liposomal model membranes.

This investigation describes a new procedure for the preparation of immunogenic liposomes which circumvents the need to synthesize the N-(hapten)-substituted derivatives of phosphatidylethanolamine that were previously employed for this purpose. The method is based on the generation of liposomes containing the N-hydroxysuccinimide (NHS) esters of either palmitic acid, cholesteryl-hemisuccinate, or N-succinyl-phosphatidylethanolamine. Reaction of these preformed liposomes with a hapten that possesses a substitutable amino group (e.g., DNP-lysine) results in covalent attachment of the hapten to the lipid bilayers. As a consequence of this binding, the liposomes can elicit formation of hapten-specific plaque-forming cells in mice. The reliability of this procedure is indicated by the fact that these liposomes share the essential immunological properties of liposomes sensitized by incorporation of N-substituted phosphatidylethanolamine derivatives (e.g., DNP-Cap-PE). Thus, the magnitude of the response was found to be dependent on: (a) the presence of lipid A in the liposomes; (b) the phospholipid composition of the liposomes; (c) the distance separating the DNP determinant from the liposomal surface. Additional applications of liposomes, which contain the NHS esters, are indicated.

Animals↗
PubMed1983

A requirement for nonspecific T cell factors in antibody responses to "T cell independent" antigens.

Using murine splenic B cell preparations depleted of macrophages and rigorously depleted of T cells, we studied the role of nonspecific helper factors in in vitro antibody responses to T cell-independent (TI) type 1 and type 2 antigens. TNP-lipopolysaccharide, TNP-Brucella abortus, and DNP-liposomes containing lipid A were chosen as examples of TI type 1 antigens. DNP-Ficoll and DNP-liposomes without lipid A were chosen as TI type 2 antigens. Only the type 1 antigens were able to elicit significant, albeit very weak, responses without added helper factors. Both type 1 and 2 antigens required factors present in supernatants from concanavalin A-stimulated spleen cells (Con A SN) to stimulate optimum antibody responses. Interleukin 2- (IL 2) containing supernatant from the T cell hybridoma FS6-14.13 supported suboptimal responses to varying degrees with each TI antigen, in contrast to its lack of effect on responses to sheep red blood cells in the absence of additional factors. This activity of the FS6-14.13 supernatant was removed by absorption with the IL 2-dependent T cell line HT-2, suggesting that IL 2 was the active component. Another factor, IL-X, which is distinct from both IL 1 and IL 2 and is also found in Con A SN, was required in addition to IL 2 to achieve optimal responses with both types of TI antigens. These results clearly establish a role for factors derived from T cells in the activation of B cells by both type 1 and type 2 TI antigens.

Animals↗
PubMed1982

A role for T cell helper factors in antibody responses to "T-independent" antigens.

Plaque-forming cell responses were assessed in unprimed mouse spleen cells after stimulation in vitro with SRBC or "T-independent" type 2 (DNP-Ficoll or DNP-liposomes) or type 1 (TNP-LPS or DNP-liposomes containing lipid A) antigens. Rigorously T cell depleted, Sephadex G-10 passed spleen cells were almost completely unresponsive to all these antigens. In every case, responsiveness could be restored by adding supernates from concanavalin A stimulated spleen cells (Con A SN). Experiments in which supernates containing interleukin 1 (IL-1) from the P388D1 cell line, interleukin 2 (IL-2), and a non-IL-2 T cell replacing factor (IL-X) were mixed showed that optimal responses to all the above antigens could only be obtained if both IL-2 and IL-X were present. IL-2 was the only factor that had any effect alone. IL-2 restored responsiveness to different degrees depending on the antigen. This ranged from about 3% (for SRBC) to about 70% (for TNP-LPS) of the optimal response. The P388D1 supernate enhanced responsiveness only if IL-2 and IL-X were present, and only with the T-independent type 2 antigens. These results demonstrate that at least some examples of both type 1 and 2 "T-independent" antigens actually require nonspecific factors one of which, IL-2, is clearly T cell derived, to stimulate antibody production by B cells.

Animals↗
PubMed1982

Effect of lipid A incorporation on characterization of liposomal model membranes as thymus-independent type 1 or type 2 immunogens.

We used the following criteria to determine whether liposomal model membranes are thymus-independent type 1 (TI-1) or type 2 (TI-2) immunogens: a) in vivo response of (CBA/N X BALB/c)F1 male and female mice; b) in vitro response of spleen cells from these mice; c) ontogeny of the response in cultures of (C57BL/6 X DBA/2)F1 male cells obtained from mice of different ages; d) ability of a monoclonal anti-delta antibody to block the in vitro response of C57BL/6 spleen cells. The liposomes were sensitized with N(2,4,6-trinitrophenyl-6-N-aminocaproyl)-dipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE) and were prepared without and with incorporation of the B cell mitogen, lipid A. On the basis of the above criteria, liposomes prepared without lipid A can be characterized as a TI-2 immunogen and resemble TNP-Ficoll. In contrast, liposomes prepared with lipid A behave like a TI-1 immunogen such as TNP-BA (trinitrophenylated Brucella abortus). Conversion of liposomes from a TI-2 to a TI-1 immunogen by inclusion of lipid A in the bilayers was achieved under conditions of constant TNP-Cap-DPPE epitope density. Increasing the epitope density in liposomes made without lipid A does not result in transformation of immunogen type. These results, which bear on the features that may distinguish TI-1 from TI-2 immunogens, are discussed with regard to the involvement of macrophages and T cells in the response.

Animals↗
PubMed1979

Preparation of stable erythrocyte target cells suitable for detection of the antibody response to the haptens azobenzenearsonate, azophenyltrimethylammonium and azophenylphosphorylcholine by plaque-forming cell assay.

The preparation of sheep red blood cells (SRBC) which have been sensitized with the haptens azobenzenearsonate, phosphorylcholine and azophenyltrimethylammonium, and are suitable for use in a plaque-forming cell assay, is described. Each hapten is first coupled to a phenolic imidoester (methyl-p-hydroxybenzimidate) and the substituted imidoesters are employed to prepare target SRBC. Details are presented concerning synthesis and purification of the imidoesters, as well as conditions for coupling to SRBC. The hapten-SRBC prepared by this method are very stable and offer significant advantages over other procedures for coupling these haptens to red blood cells.

Animals↗
PubMed1979

Primary in vitro immunogenicity of liposomal model membranes in mouse spleen cell cultures.

Neither 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) nor fluoresceinthiocarbamylphosphatidylethanolamine (F1-PE) induces hapten-specific plaque-forming cells (PFC) when incubated with suspensions of spleen cells from unimmunized C57BL/6J mice. However, PFC are produced after incorporation of these synthetic lipid antigens into liposomal model membranes. The in vitro response is characterized by the following: a) it is time and dose dependent; b) the frequency of IgM PFC exceeds IgG PFC; c) both nonadherent and adherent cells are required (2-mercaptoethanol can replace the requirement for adherent cells in some experiments); d) depletion of thymus-derived cells by treatment with anti-theta antiserum plus complement does not diminish the response; e) spleen cells from nude BALB/c mice also produce PFC. Thus, the essential features of the in vivo immunogenicity of DNP-Cap-PE and F1-PE sensitized liposomes, which have been previously described, can be replicated in an in vitro cell culture system.

Aminocaproates↗