Mary E. Neville

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.

In vivo inhibition of tumor growth of B16 melanoma by recombinant interleukin 1β: 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 (PMNs) in the circulation. However, only ITU injections of IL 1 beta increased the number of PMNs within the tumors. IM injections of IL 1 beta, while increasing the number of PMNs in the circulation, did not increase the influx of PMNs into the tumors. Furthermore, the transfer of PMNs directly into B16 tumors caused a 49% reduction in tumor growth without the presence of IL 1 beta. These results suggest that in vivo, PMNs may effectively control the growth of tumors and that IL 1 beta may increase this effectiveness by increasing the number of PMNs in the circulation and by locally stimulating the production of chemotactic factors for PMNs within the tumor.

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

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 (<or=25 nm). Intermediates (partially coalesced vesicles) within the forming multilamellar structures were identified by freeze-fracture electron microscopy and their presence was corroborated by differential scanning calorimetry. Several distinct steps were identified in the coalescence process. In the initial step, rhIL-2 rapidly bound to the DMPC SUVs. This was followed by a pH-dependent conformational change in the protein, as evidenced by an increase in tryptophan fluorescence intensity. The SUVs then aggregated in large clusters that eventually annealed to form closed MLVs. In this process over 90% of the rhIL-2 was bound to and incorporated within the multilamellar structures.

Anti-tumour effects of interleukin-1β : 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)

Angiosuppressive and Antiproliferative Actions of Suramin: A Growth Factor Antagonist

Suramin is a novel anticancer agent1 that appears to be effective against advanced adrenocortical carcinoma2,3, prostatic cancer4,5, ovarian cancer6, renal cell carcinome and certain refractory lymphomas8. The antiproliferative action is possibly related to the ability of suramin to block the binding of autocrine growth factors to their receptors9-18, to inhibit a variety of cytoplasmic and intranuclear enzymes critical for cell maintenance and proliferation19,20, and to disrupt cellular respiration and energy balance21. Cell migration and adhesion of B16 melanoma cells to the extracellular matrix is inhibited following suramin exposure 22,23 suggesting a mechanism for suramin to inhibit tumor invasion.