Dale D. Isaak

Inhibitory Effects of Progesterone on Plasma Membrane Fluidity and Tumorigenic Potential of Ovarian Epithelial Cancer Cells

The lethality of common (surface) epithelial ovarian cancer is contingent on its metastatic capacity. Dissemination of the neoplasia throughout the abdominal cavity has been associated with secretion of proteolytic enzymes from vesicles shed by ovarian cancer cells. We report that the lipophilic steroid hormone progesterone decreases the fluid dynamics of plasma membranes of human SKOV-3 adenocarcinoma cells. The decrease in membrane fluidity was related to an inhibition in vitro of exocytotic vesicle release, cellular invasiveness into Matrigel, and colony formation in three-dimensional collagen matrix. Tumorigenesis was suppressed by progesterone in immunocompromised nude mice inoculated intraperitoneally with SKOV-3 cells. Progestins could therefore be of benefit in the prevention and(or) treatment of early-stage ovarian carcinomatosis.

Degradable Cisplatin-Releasing Core-Shell Nanogels from Zwitterionic Poly(β -Aminoester)-Graft-PEG for Cancer Chemotherapy

Cisplatin conjugated onto macromolecules or loaded in micelles can be preferentially delivered to tumors to minimize its toxicity to healthy tissues and increase its drug efficacy. Herein, we report cisplatin-containing nanogels possibly useful for targeted delivery of cisplatin. Carboxylic acid-functionalized poly(β -aminoester)graft-poly(ethylene glycol) copolymers were synthesized by cocondensation polymerization of piperazine with 2,2-bis(acryloxymethyl)propionic acid, PEG 2,2-bis(acryloxymethyl)propionate macromonomer (mPEG). The graft copolymers formed 100–200 nm nanogels with low size-distribution by the complexation of their carboxylic groups with cisplatin. The nanogels were negatively charged and had a PEG outer layer. Thus, they had “stealth properties” suitable for in vivo applications. The nanogels had significantly lower in vitro cytotoxicity to SKOV-3 ovarian cancer cells than free cisplatin, but similar anticancer activity toward SKOV-3 tumors xenografted to immunocompromised mice.

Progesterone facilitates cisplatin toxicity in epithelial ovarian cancer cells and xenografts.

OBJECTIVES The underlying premise of these investigations was that the lipophilic hormone progesterone, which partitions into and (at relatively high concentrations) impedes the fluid mechanics of the plasmalemma, would perturb integral associations between membrane lipids and exporter pumps that otherwise confer drug resistance. That progesterone can affect susceptibility of ovarian adenocarcinoma cells and xenografts to cisplatin was tested. METHODS The cisplatin-resistant human cell lines SKOV-3 and OVCAR-3 were treated for 24 hours with cisplatin (0.1 microg/ml)+/-progesterone (0.01, 0.1 microg/ml). Cytotoxicity and platinum were measured by MTT assay and inductively coupled plasma mass spectrometry, respectively. Athymic mice were inoculated intraperitoneal (ip) with SKOV-3 cells. Cisplatin (2 mg/kg/week)+/-progesterone (25 mg sustained-release pellet) regimens were initiated ip at one week (when micrometastases were present) and continued to six weeks post-xenograft. Tumor burdens, histopathology, and platinum concentrations were assessed upon necropsy at 24 hours after the final injection of cisplatin. RESULTS There were no significant in vitro/vivo anticancer effects of cisplatin alone. High-dose progesterone enhanced platinum accretion and induced drug toxicity in both cell lines. Tumorigenesis was suppressed by cisplatin+progesterone. The treatment synergy was related to elevated tumor platinum and morphological evidence of apoptosis. CONCLUSION It appears that the addition of progesterone to ovarian cancer therapeutic modalities represents a step in improving responses.

Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5×104 CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of protective immunity.

Inhibition of Antigen- and Mitogen-Induced Activation of Mouse Splenocytes by Uterine Secretions From Pregnant and Progesterone-Treated Ewes

ABSTRACT: Pregnant and progesterone‐treated, ovariectomized ewes accumulate secretory products in the uterus that are immunosuppressive in mitogen‐stimulated and mixed lymphocyte sheep cell cultures. In this study, uterine secretions from pregnant (Preg‐UTM) and progesterone‐treated, ovariectomized (P‐UTM) ewes were equally effective in suppressing 3H‐Tdr incorporation in mouse spleen cells stimulated with PHA. P‐UTM inhibited PHA‐stim‐ulated, purified T‐cells and separated L3T4+ and Lyt2+ T‐cell subpopulations more than Preg‐UTM, however. Both fluids were slightly inhibitory to conA‐stimulated mouse spleen cells, enriched T‐cells, and Lyt2+ T‐cells but neither inhibited L3T4+ T‐cells. For LPS‐stimulated cells, P‐UTM caused more suppression than Preg‐UTM of enriched B‐cells; however, suppression was similar for the two fluids on unseparated splenic cells. In antigen‐stimulated mouse spleen cell cultures, both fluids inhibited antibody‐forming cell responses to sheep erythrocytes, a thymus‐dependent antigen, but neither suppressed antibody‐forming cell responses to TNP‐Ficoll, a thymus‐independent antigen. These data indicate that uterine secretions in the ewe produced under the influence of progesterone or pregnancy contain immunoregulatory molecule(s) which modulate the activity of both homologous sheep and unrelated mouse lymphocytes. These studies establish the mouse as a useful in vivo model for studying the biological actions of these molecules.

T-cells inhibit Friend murine leukemia virus infection of B-cells in vitro

The ability of splenic T-cells to regulate Friend murine leukemia virus replication in lipopolysaccharide-activated target B-cells infected in vitro was investigated. Removal of the T-cell fraction from spleen cells resulted in an 8- to 10-fold enhancement in the number of productively infected cells in the remaining B-cell-enriched fraction, as compared with unseparated spleen cells, and the addition of increasing numbers of purified T-cells to isolated B-cells prior to infection resulted in a directly proportional reduction in the number of B-cells releasing infectious progeny virus. Separation of splenic T-cells into Lyt 2- and Lyt 2+ T-cells before addition to infected B-cell cultures resulted in inhibition of infection only with the Lyt 2- T-cells; Lyt 2+ T-cells did not inhibit infection, even at high 1:1 ratios. Similarly, separation of splenic T-cells into L3T4+ and L3T4- T-cells before addition resulted in inhibition by L3T4+ but not L3T4- T-cells. Also, cytotoxic treatment of splenic T-cells with monoclonal anti-L3T4 antibody and complement before addition to B-cell cultures destroyed the regulatory effects. Finally, depletion of macrophages from both T-cells and B-cells before infection and coculture had no effect on the ability of T-cells to regulate B-cell infection. Collectively these results demonstrate that L3T4+ T-cells can inhibit Friend murine leukemia virus replication in target B-cells. Culture of isolated splenic T-cells with Friend murine leukemia virus in vitro resulted in the induction of alpha/beta but not interferon-gamma synthesis and in some experiments interferon-containing supernatants from T-cell-virus cultures were able to mediate suppression of B-cell infection with Friend helper virus; the addition of antibody specific for interferon-alpha/beta to cultures inhibited the ability of T-cells to regulate B-cell infection.

Mapping of HIV-1 Determinants of Apoptosis in Infected T Cells

HIV-1 infection leads to death of CD4(+) T cells in vivo and in vitro, although the mechanisms of this cell death are not well defined. We used flow cytometry to concurrently analyze infection and apoptosis of the CD4(+) CEM T cell line and human peripheral blood mononuclear cells (PBMC). Surprisingly, T cells productively infected with HIV-1 IIIB showed less apoptosis than control, uninfected T cells. This relative paucity of apoptosis was a characteristic of IIIB, since a large number of cells infected with the viral clone, HIV-1 NL4-3, were apoptotic. The nef, vpr, and vpu gene products were not responsible for apoptosis of NL4-3-infected cells, since NL4-3DeltaVprDeltaVpuDeltaNef and HXB-2 (a nef, vpr, and vpu triple mutant derived from IIIB) also killed infected cells. Moreover, only IIIB-infected cells showed a resistance to background levels of apoptosis. Thus, the apoptotic (and antiapoptotic) properties of HIV-1 do not map solely to mutations in nef, vpr, or vpu. We postulate that, in vivo, HIV variants that do not induce rapid apoptosis in the cells they infect may have a selective advantage.