The effect of cholesterol in a liposomal Muc1 vaccine.

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.

Interleukin-2-induced small unilamellar vesicle coalescence.

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (

In situ vaccination against a non-immunogenic tumour using intratumoural injections of liposomal interleukin 2.

Cancers appear to escape surveillance by the immune system at least in part because they fail to induce a protective immune response. Therapeutic vaccines based on specific tumour antigens and tumour cells modified ex vivo by genetic techniques are but two strategies being used to circumvent this problem. In this report, we describe a simple, yet effective alternative in which tumour-specific responses are induced by in situ administration of a well-characterized liposomal formulation of the cytokine interleukin 2 (IL-2). Using the non-immunogenic B16 melanoma model, intratumoural injections of liposomal IL-2 L(IL2), were shown to induce a long-lived immune response specific for the injected tumour. In conjunction with subsequent removal of the primary tumours by surgery, the injections increased mean survival to 57 days from a control value of 32 days and partially protected surviving mice against re-challenge with B16. L(IL2) induced an early infiltration of inflammatory cells within the tumours which was followed several days later by an influx of CD3+ T cells. The cellular influx and a coincident decrease in tumour growth were noted in both injected tumours and a second non-injected tumour on the same animal, thereby demonstrating the systemic nature of the immune response. Intratumoural injections of soluble IL-2 at the same dose failed to induce B16-specific cellular immunity or to prolong survival of the mice. Thus, liposomal formulation of the cytokine was fundamental to successful induction of immunity by this in situ vaccination regimen.

Biopharmaceutics of liposomal interleukin 2, oncolipin.

Oncolipin is a multilamellar liposomal (dimyristoyl phosphatidylcholine) formulation of interleukin 2 (IL-2) and human serum albumin (HSA) with distinct surface characteristics which may influence its biological activities. IL-2 and HSA were detected on the surface of the liposomes using specific antibody staining. Surface expression of IL-2 was also demonstrated by the observation that Oncolipin bound to cells expressing IL-2 receptors (IL-2R) containing alphabetagamma or betagamma subunits. Binding and internalization of Oncolipin by cells expressing alphabetagamma or betagamma receptor subunits was blocked by excess free IL-2 or a neutralizing antibody against the beta chain. The display of surface IL-2 on Oncolipin's liposomes was maintained in vivo after intravenous injection into mice. IL-2 was also present between the lipid bilayers of the multilamellar liposomes based on the unique physical characteristics detected by freeze fracture electron microscopy. The bulk of the liposome-associated IL-2 was released from the liposomes upon incubation at 37 degrees C in medium containing serum, indicating that the IL-2 was not irreversibly entrapped on or in the liposome structure. Thus, Oncolipin is receptor-targeted to activated T and NK cells by virtue of its surface expression of IL-2 and has the potential to release IL-2 following deposition within lymphoid organs. These properties may confer distinct advantages over soluble IL-2 for immunotherapy of cancer and viral diseases.

A comparison of biodistribution of liposomal and soluble IL-2 by a new method based on time-resolved fluorometry of europium.

A novel method was developed to determine the pharmacokinetics and biodistribution of cytokines and lymphokines based on time-resolved fluorometry (TRF) of europium (Eu). The comparison of two formulations of IL-2 was used to illustrate the sensitivity and applicability of this method as well as to extend the information on the pharmacokinetics of liposomal IL-2 and soluble IL-2. The blood kinetics and biodistribution of liposomal and soluble IL-2 in lymphoid organs and kidneys as measured by TRF were similar to those determined by the radioisotopic method. In both instances, the formulation of IL-2 into liposomes increased its serum half-life and accumulation in reticuloendothelial and lymphoid organs. The increased sensitivity of the Eu/TRF method permitted the extension of observational time points and the analysis of biodistribution in organs such as lymph nodes and bone marrow. These results suggest that Eu-labelled proteins in conjunction with TRF offer a suitable alternative to radiolabelled proteins for pharmacokinetics and tissue distribution studies in animals. This method offers distinct advantages over traditional techniques employing radioistopes since it has greater sensitivity, no half-life limitations and no radioactive or hazardous waste disposal.

Anti-tumour effects of interleukin 1 beta: in vivo induction of immunity to B16 melanoma, a non-immunogenic tumour.

The anti-tumour properties of interleukin 1 beta (IL-1 beta) were examined using an intradermal B16 murine melanoma surgical model. B16 cells were injected intradermally on the right ventral side and surgery was performed on days 10-20 to remove the primary tumours. IL-1 beta or vehicle was administered prior to surgery for 5-7 consecutive days. In mice which received only injections of vehicle, survival ranged between 0 and 30% when measured on day 120 after implantation of B16 cells. Mice died of metastases and growth of B16 cells in the thoracic lymph nodes. When mice without metastases were rechallenged with viable B16 cells, only one out of 22 mice (5%) failed to develop tumours. No significant immunity to B16 cells was detected in this group of mice. In contrast, in mice which received injections of IL-1 beta, survival ranged between 70-100% on day 120 after implantation of B16 cells. When IL-1 beta treated mice were rechallenged with viable B16 cells on day 120, 20 out of 32 (63%) mice failed to develop B16 tumours suggesting that some of these mice had immunity to B16 melanoma cells. Moreover, mice with immunity to B16 cells did develop tumours when injected with another syngeneic tumour, MCA 105. In vitro specific immune responses were also demonstrated in spleen cells and sera from mice treated with IL-1 beta, but not in the spleen cells or sera of mice that received only vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Suramin, an anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation, and induction of urokinase-type plasminogen activator.

Suramin, an anticancer agent in current clinical trials, is a prototype of a pharmacological antagonist of growth factors, including basic fibroblast growth factor (bFGF). Suramin inhibited angiogenesis in the chick chorioallantoic membrane assay in a dose-dependent fashion. Suramin, 200 mg/kg i.v., inhibited rat corneal angiogenesis induced by bFGF-impregnated polymers; addition of heparin stimulated angiogenesis and counteracted the inhibition of suramin. The half-maximal inhibitory concentration (IC50) of suramin was determined for key cellular mechanisms that regulate angiogenesis: (a) low and high affinity cellular binding of bFGF to bovine capillary endothelial (BCE) cells with IC50s, respectively, of 24.3 and 71.5 micrograms/ml; (b) spontaneous migration of bovine pulmonary artery endothelial and normal AG 7680 fetal bovine aortic endothelial cells; bFGF-stimulated migration of BCE and transformed GM 7373 fetal bovine aortic endothelial cells with IC50s of 200-320 micrograms/ml; (c) proliferation of bovine pulmonary artery endothelial cells at > 100 micrograms/ml and of BCE cells at > 250 micrograms/ml; and (d) urokinase-type plasminogen activator activity of GM 7373 endothelial cells stimulated by bFGF with an IC50 of 211 micrograms/ml and of BCE cells stimulated by bFGF at > 100 micrograms/ml, but not plasminogen activator activity induced by phorbol 12-myristate 13-acetate. Suramin inhibited multiple control points of angiogenesis, including those stimulated by bFGF. Because tumor growth is angiogenesis dependent, the clinical efficacy of suramin may relate, in part, to angiosuppression.

In vivo inhibition of tumor growth of B16 melanoma by recombinant interleukin 1 beta. II. Mechanism of inhibition: the role of polymorphonuclear leukocytes.

Recombinant human interleukin 1 beta (IL 1 beta) inhibits growth of B16 melanoma in syngeneic C57BL/6 mice in a dose-dependent manner when given intratumorally, intradermally, or intramuscularly over a period of 5 to 7 days. Inhibition of tumor growth was rapid and measurable within 3 days after the initial injection and occurred regardless of the route of injection. However, only intratumoral (ITU) injections of IL 1 beta resulted in greater than 90% inhibition in tumor growth. This enhanced inhibition of tumor growth was not dependent on T or NK cells since inhibition of tumor growth occurred in nude and Beige mice. Also, a profound lymphopenia occurred in mice receiving IL 1 beta. Inhibition of tumor growth did correlate with an increase in the number of polymorphonuclear leukocytes (PMN's) in the circulation. However, only ITU injections of IL 1 beta increased the number of PMN's within the tumors. IM injections of IL 1 beta, while increasing the number of PMN's in the circulation, did not increase the influx of PMN's into the tumors. Furthermore, the transfer of PMN's directly into B16 tumors caused a 49% reduction in tumor growth without the presence of IL 1 beta. These results suggest that in vivo, PMN's may effectively control the growth of tumors and that IL 1 beta may increase this effectiveness by increasing the number of PMN's in the circulation and by locally stimulating the production of chemotactic factors for PMN's within the tumor.

In vivo inhibition of tumor growth of B16 melanoma by recombinant interleukin 1 beta. I. Tumor inhibition parallels lymphocyte-activating factor activity of interleukin 1 beta proteins.

Seven daily intratumoral injections of human recombinant interleukin 1 beta (rHu-IL 1 beta) inhibit the growth of B16 melanoma in syngeneic female C57BL/6 mice. Inhibition was dose dependent and ranged from 36% to 93%. Other routes of injection of rHu-IL 1 beta (intramuscular, intraperitoneal, intradermal) inhibited tumor growth but to a lesser degree (27% to 50%). Two different rIL 1 beta s, one a mutein of rHu-IL 1 beta (Glu-4) and the other one murine IL 1 beta (rM-IL 1 beta), were tested in the tumor inhibition model. rM-IL 1 beta inhibited tumor growth at lower concentrations than did rHu-IL 1 beta and also had enhanced IL 1 activity in the thymocyte assay in vitro. The mutein of rHu-IL 1 beta (Glu-4) had significantly reduced in vitro IL1 activity and did not inhibit tumor growth. No cytotoxic or cytostatic effects of rHu-IL 1 beta were observed in in vitro assays. These results suggest that rHu-IL 1 beta has antitumor activity in vivo that is probably not due to its direct effects on B16 cells but rather is mediated by secondary effects of IL 1 beta.

51Cr-uptake assay. A sensitive and reliable method to quantitate cell viability and cell death.

This report describes a sensitive and reproducible cytotoxicity assay which is ideal for screening toxic compounds or soluble lytic factors from leukocytes. The assay relies on the ability of sodium [51Cr]chromate to be taken up by viable but not dead cells. This assay is the reverse of the conventional 51Cr-release assay. The target cells are 51Cr-labeled after incubation with cytotoxic factors. 51Cr-uptake does not depend on DNA, RNA or protein synthesis and the only parameters which influence its uptake are temperature, viability, number and cell volume.

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes.

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.

Activation of Fc receptor-bearing lymphocytes by immune complexes. I. Stimulation of lymphokine production by nonadherent human peripheral blood lymphocytes.

Activation of human peripheral blood lymphocytes by incubation with particulate immune complexes or aggregated human gamma-globulin was studied by measuring the release of leukocyte migration inhibitory factor (LIF) activity. LIF-active supernatants were consistently produced when nonadherent lymphocytes containing less than 1% surface immunoglobulin-bearing cells and less than 0.2% nonspecific esterase-positive monocytes were incubated in the presence of RBC sensitized with rabbit or human antibodies or with pooled heat-aggregated human gamma-globulin. This immune complex-induced lymphokine production (ICLP) was dependent on the presence of cells bearing receptors for the Fc portion of IgG (Fc gamma). ICLP could not be demonstrated with lymphocyte preparations enriched for B cells even though the latter showed vigorous LIF production in the presence of complement-sensitized erythrocytes. ICLP was dependent on the concentration of lymphocytes and of stimulant as well as on the duration of coincubation, and it required active metabolic processes and RNA and protein synthesis but not DNA synthesis. Ca++ but not Mg++ was obligatory. ICLP by non-B Fc gamma receptor-bearing lymphocytes may play a role in antibody-dependent protective inflammation and immunologic injury phenomena, which is similar to that of lymphokine release by antigen-activated T cells in delayed hypersensitivity responses.

Activation of Fc receptor-bearing lymphocytes by immune complexes. II. Killer lymphocytes mediate Fc ligand-induced lymphokine production.

Cells that participate in immune complex-induced production of leukocyte migration inhibitory factor (LIF) activity can be concentrated in a population making up 2-4% of human peripheral blood lymphocytes in which greater than 90% of the cells are active in a single cell antibody-dependent cellular cytotoxicity assay. When so concentrated, such killer (K) cell preparations are as efficient in producing LIF activity as mitogen activated T lymphocytes. Other Fc receptor (FcR)-bearing lymphocytes, including natural killer (NK) cells, do not produce measurable LIF activity when incubated with immune complexes (additional evidence that the K and NK cells among ligands to the FcR of the appropriate lymphocytes, possibly without need for exogenous receptor bridging, is the only requirement for their activation to immune complex-induced lymphokine production (ICLP). It is probable that ICLP by K cells palays a role in antibody-mediated effector functions in vivo.

Human killer cells and natural killer cells: distinct subpopulations of Fc receptor-bearing lymphocytes.

Unstimulated human peripheral blood lymphocytes were depleted of K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC) without removing NK cells, which mediate natural killing (NK). K cell depletion was achieved by buoyant centrifugation removal of lymphocytes that bound to glutaraldehyde-treated P815-AB cells at high lymphocyte-to-target ratios. Likewise, NK cells were removed with glutaraldehyde-treated K562 cells without removing K cells. Furthermore, both cytotoxic cell populations were observed directly in one agarose single-cell cytotoxic assay (ASCA) using P815-AB and K562 cells simultaneously as target cells. Moreover, the percentage of total cytotoxic cells was equal to the sum of the percentage of K and NK cells observed in separate ASCA. Collectively, these results indicate that K cells and NK cells are distinct subsets of FcR-bearing lymphocytes. One subset, K cells, has more avid Fc receptors (fcR) than NK cells and are 'activated' via thier FcR to kill antibody-coated target cells. The second subset, NK cells, have less avid FcR and are not 'activated' through their FcR to kill antibody-coated target cells.

Frequency determination of K cells by a single cell cytotoxic assay.

This report describes a highly reproducible single cell cytotoxic assay which can measure the frequency of K cells in human peripheral blood lymphocytes. This assay is based on the ability of FcR-bearing lymphocytes to bind to antibody sensitized target cells via their FcR. Approximately 30% of lymphocytes which bound antibody sensitized target cells via their FcR. Approximately 30% of lymphocytes which bound antibody sensitized P815 cells (P815-AB) were K cells. Cytolysis of the conjugated P815-AB fulfilled all the criteria for ADCC. Furthermore, an excellent correlation was observed between the frequency of K cells and % cytotoxicity as measured by the ADCC 51Cr-release assay. The mean frequency of human k cells in peripheral blood lymphocytes from 20 volunteers was measured to be 3.2% of the total lymphocyte populations.

Differential biosynthesis of polyunsaturated fatty acids by Tetrahymena supplemented with ergosterol.

Tetrahymena grown with foreign sterols such as ergosterol incorporate them into cellular membranes at the expense of the native compound, tetrahymanol. It is shown that cells grown with ergosterol have a lessened capacity to produce the polyunsaturated linoleic and gamma-linolenic acids from [14C]oleic acid. However, the same cells have normal capacities to introduce double bonds at C-6 into linoleate, alpha-linolenate, or cis-vaccenate. Thus, a presumed 12-desaturase is inhibited in the presence of ergosterol, while desaturation at C-6 is unaffected.

Comparison of three procedures for isolating DNA from bacteria.

Three methods employing chloroform-isoamyl alcohol (CI), phenol, or enzymes, were evaluated for isolating DNA from Escherichia coli, Bacillus subtilis, and Arthrobacter globiformis. For the amounts of reagents employed at optimum conditions in the CI and phenol procedures, 0.4-0.9 mg of DNA/g wet weight of cells was isolated. Using the enzymatic procedure, approximately twice as much DNA was isolated. DNA isolated by the CI procedure contained 0.03-0.09% protein and 0.08-0.12% RNA. DNA isolated by the phenol procedure contained 0.02-0.05% protein and 2.2-2.6% RNA. DNA isolated by an enzymatic procedure, which is described in detail, contained 32.2-45.7% protein and 0.3-0.6% RNA. DNA isolated by all three procedures are double-stranded and at least 10(6) in molecular weight, as suggested by data from thermal transition analyses and transformations. These data emphasize that the desired characteristics of DNA for experimental purposes must be considered in selecting an isolation procedure.

Reliability of thermal transitions for characterizing DNA from micro-organisms.

Data from thermal transitions were evaluated for their usefulness in detecting similarities in the deoxyribonucleic acid (DNA) of micro-organisms. Levels of reproducibility were determined for thermal transition analysis; methods of purification of DNA and the solvents, pH, and temperature intervals used during thermal transitions did not greatly affect the reproducibility of the values obtained. The shape of the thermal transition curve is a stable property of a given kind of DNA, and this property can be described quantitatively and with satisfactory precision.

Evaluation of alternate coupling reagents to replace alpha-naphthyl amine for the detection of nitrate reduction.

Four naphthyl compounds, N,N,dimethyl-1-naphthylamine, N-1-naphthyl-ethylene-diamine dihydrochloride, 8-amino-1-naphthol-5-sulphonic acid, and 1-dimethylamino naphthalene-5-sulphonic acid, were evaluated as replacements for alpha-naphthyl amine in the bacteriological nitrate reduction test. These compounds were observed for their ability to detect standardized nitrite concentrations and nitrate reduction in eight bacterial cultures. The results indicated both N,N,dimethyl-1-naphthylamine and N-1-naphthyl-ethylene-diamine dihydrochloride, at concentrations of 0.035 M, were satisfactory alternatives. The working concentration of the latter might be reduced, as indicated by the results of sensitivity tests.