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Biomedical subjects

E Mihich

Publications and source records attributed to E Mihich.

At least 37 records · Page 2Linked to original sources
PubMed1998

Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-alpha therapy: phenotypic and functional characterization up to 20 months after initial tumor inoculation.

As reported previously, cyclophosphamide plus tumor necrosis factor-alpha treatment of C57BL/6 mice bearing advanced EL4 lymphoma induced approx. 60% long-term (i.e., >60 days) survivors. These mice developed protective immunity, as evidenced by 1) rejection (100% survival) of EL4 tumor re-implanted on day 60 (day 0 = initial tumor implantation); and 2) development of significant levels of specific EL4 tumor cell killing activity by both splenocytes and thymocytes. Using this model, age-related changes in functionally and phenotypically definable thymocyte subsets were assessed. In thymocytes from 90 to 308 day survivors, specific immune memory was long term; both CD4+ and CD8+ cells were required for the ex vivo stimulation of lytic activity, but the specific anti-EL4 cytotoxic effector was CD4-CD8+. On day 520, the surviving mice were randomized into 2 groups. One group received a second re-challenge with EL4 tumor cells and all survived. The other group was sacrificed on day 520. Their thymocytes, exposed to X-irradiated EL4, developed anti-EL4 lytic activity and, in comparison with thymocytes of young and age-matched control mice, were markedly enriched in CD4-CD8+CD44+ cells. On day 625, thymocytes from the survivors of the day 520 re-challenge were evaluated and were found to have developed specific anti-EL4 lytic activity. Phenotypically, they had returned toward the pattern seen in age-matched control mice although CD4-CD8+CD44+ cells remained increased. These mice were > or = 2 years old, the median life span of C57BL/6 mice. Thus, mice cured of tumor by an immuno-modulating regimen rejected re-implanted primary tumor and maintained specific thymic anti-tumor immune memory for life.

Animals↗
PubMed1998

Doxorubicin plus tumor necrosis factor alpha combination treatments in EL4-lymphoma-bearing C57BL/6 mice.

The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor alpha (TNFalpha) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFalpha administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFalpha, i.v., at either 16000 U (7 microg)/injection, on days 1 and 8 or 4000 U (1.7 microg)/injection, on days 11-15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFalpha-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFalpha-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFalpha in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFalpha Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established.

Animals↗
PubMed1996

Evidence for the involvement of ecto-5'-nucleotidase (CD73) in drug resistance.

Increased ecto-5'-nucleotidase (ecto-5'NT) protein expression in several multidrug-resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto-5'NT protein with its mRNA expression. An inhibitor of ecto-5'NT enzymatic activity, alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP-CP (0.3 mM) reversed the resistance of ecto-5'NT-positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto-5'NT-negative parental cell lines or that of 2 ecto-5'NT-negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP-CP increased rhodamine uptake and inhibited rhodamine efflux from ecto-5'NT-positive MDR cells without affecting ecto-5'NT-negative MDR cells. The presence of exogenous adenosine (0.5 microM) circumvented AMP-CP-induced inhibition of rhodamine efflux from EL4/ADM cells. AMP-CP inhibited the growth of the ecto-5'NT-positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto-5'NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto-5'NT serves as a required accessory molecule in resistance mediated by ATP-dependent mechanisms and that growth-sustaining nucleosides are provided by this salvage pathway.

5'-Nucleotidase↗
PubMed1996

Specific anti-EL4-lymphoma immunity in mice cured 2 years earlier with doxorubicin and interleukin-2.

This laboratory has reported the conditions for an effective, non-toxic, chemoimmunotherapy utilizing doxorubicin in combination with prolonged administration of interleukin-2 and the identification of the critical role of activated CD8+ T cells in the therapeutic effect. Mice (C57BL/6) cured in those studies have been followed for the remainder of their life spans. These mice, approximately 2 months of age when initially inoculated with syngeneic EL4 lymphoma, survived for more than 2 years, the normal life span of C57BL/6 mice. Mice 4 months old reinoculated with the EL4 cells all survived. At about 1 year of age mice were sacrificed and the ability of their thymocytes and splenocytes to develop specific CD8+ anti-EL4 activity was as high as it had been at the time of tumor rejection. At about 2 years of age EL4 was reimplanted into mice; all of them survived. These surviving mice, at 2 years 2 months of age, as well as a group of 2-year-old mice not rechallenged, were killed and functional antitumor activity and phenotype characteristics of various lymphocyte populations were determined in comparison to those of young and age-matched control mice. The phenotyping of the lymphocytes from the cured mice indicated very notable differences in subset distribution and increased CD44 expression. Functionally they developed high levels of anti-EL4 activity, which was ablated by combined treatment with monoclonal antibodies against CD8 and CD44, indicating the role of memory cells. Consistent with cells from aged mice, these same cell populations had a very reduced allogeneic responsiveness. It appears that cured mice have developed an immune memory specific for EL4.

Animals↗
PubMed1995

Cyclophosphamide plus tumor necrosis factor-alpha chemoimmunotherapy cured mice: life-long immunity and rejection of re-implanted primary lymphoma.

Changes in functionally and phenotypically definable splenocyte subsets in aging mice which had been rendered tumor-free in early life by immunochemotherapy (cyclophosphamide plus tumor necrosis factor-alpha) were studied in the syngeneic EL4 lymphoma-C57BL/6 murine model. Treatment-induced long-term survivors (LTS) surviving rechallenge are termed "immune-LTS". On day 120 (day 0, initial tumor inoculation), splenocytes from day 60 rechallenged immune-LTS developed significantly greater specific anti-EL4 cytolytic activity in an ex vitro assay than those from non-rechallenged LTS. Splenocytes from combination-treated groups developed significantly higher activity than those from cyclophosphamide-induced immune-LTS. The splenic effector precursor was a CD8+ T cell. The specific anti-EL4 effector cell from the cyclophosphamide-induced immune-LTS was CD4- CD8+; however, approximately 50% of those from combination-treated immune-LTS appeared to be CD4+CD8+. On day 520 immune-LTS were randomized into 2 groups. One group was re-implanted with EL4 tumor; all mice survived. The other group was killed and, even though their splenocytes developed considerable anti-EL4 activity, their allogeneic responsiveness was as reduced as that of age-matched controls. Phenotypic analysis, compared with splenocytes from young and age-matched controls, revealed changes in the makeup of each T-cell subset, except the CD4+CD8+, and all subsets, except the CD4-CD8-, had increases in CD44 positivity. On day 625, the age of these mice was equivalent to the median life-span of C57BL/6 mice; nevertheless, their splenocytes developed high anti-EL4 activity. Phenotypic analysis indicated that, compared to day 520, there was a major decrease in CD4-CD8+ splenocytes; we suggest that these cells had migrated to the site of tumor eradication.

Animals↗
PubMed1995

Murine splenic macrophage tumoricidal activation by cytokines.

Interleukin-2 (IL-2), IL-1 beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), each alone and in all possible combinations, were studied for their capacity to activate murine resident splenic macrophages to a tumoricidal state. Two approaches were used in these studies. The first approach was to add cytokine directly to the adherent macrophages that had been washed free of nonadherent spleen cells. The only agent effective alone was IL-2, inducing significant tumoricidal activity in macrophages obtained after culturing whole spleen cell suspensions for 4, but not 1 to 3, days. Nonadherent splenic populations were required during this 4-day macrophage "culture conditioning." Only combinations of cytokines containing IL-2 were effective, but none more than IL-2 alone. The second approach was to add cytokine to the whole spleen cell suspensions for an activation period before isolation of adherent macrophages. Again, the only agent effective alone was IL-2. Macrophage tumoricidal activity was highest when IL-2 was added to the whole spleen cell suspensions at the initiation of the 4-day activation culture. In addition, TNF-alpha, but none of the other cytokines, significantly augmented the IL-2-induced effect. The tumoricidal activity was not a consequence of lipopolysaccharide contamination or of lymphokine-activated killer cells. Based on the utilization of neutralizing antibodies, IL-1 alpha, IL-1 beta, and IFN-gamma were not involved as soluble mediators during the activation of tumoricidal splenic macrophages by IL-2 with or without TNF-alpha.

Animals↗
PubMed1995

Doxorubicin and cyclosporin A affect murine lymphoid cells expressing different antigenic determinants.

Cyclosporin A (CsA) enhances the antitumor activity of doxorubicin (Dox) as well as that of some other cytotoxic drugs against drug-resistant tumor variants. In some cases, however, such combination treatments result in severe unexplained toxicities. In this study the possibility was explored that the effects of CsA and Dox on lymphoid cells may be instrumental, at least in part, in determining their toxicological effects. Subsets of spleen and thymus lymphoid cells, from mice with or without Dox or CsA treatment, were identified by flow cytometry based upon their plasma membrane antigenic determinants. The results indicate that there is essentially no cross-sensitivity/resistance between the two agents. The most Dox-susceptible cells were immature (non-proliferating) CD4+CD8+ thymocytes and CD3-B220- as well as CD3-B220+ splenocytes. These populations were intact following CsA treatment, but the numbers of mature CD4+CD8- and CD4-CD8+ cells were substantially reduced. Similar "mirror image" differences were found for other populations examined. When considered together, these findings suggest that in combination Dox and CsA would affect nearly all subsets of lymphoid cells, providing one possible explanation of why increased leukopenia, toxicity and immunosuppression are found after their combined administration. Since leukemias, lymphomas and, to a more limited extent, certain solid tumors express these same phenotypic markers, similar analyses should be considered for monitoring and perhaps even predicting neoplastic cell sensitivity to treatment with such agents.

Animals↗
PubMed1994

Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin.

Cytochemical screening for a panel of enzymes revealed increased 5' nucleotidase (5'NT) expression in 3 of 3 P-glycoprotein 170 (Pgp170)-positive multidrug-resistant (MDR) variants of the murine EL4 T-lymphoma cell line (EL4/ADM, ER2 and ER13). Electron microscopic localization established the presence of the membrane-bound ecto-form of the enzyme. Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto-5'NT. Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line L1210A) showed increased expression of ecto-5'NT, when compared with their parental cell lines. The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto-5'NT. All MDR cell lines with elevated ecto-5'NT expression were generated by doxorubicin treatment. These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5-3.0 mM, confirming that the expressed ecto-5'NT was biologically active. The parental and MDR cells did not differ, in general, in their sensitivity to adenosine. An inhibitor of ecto-5'NT, alpha,beta-methyleneadenosine 5'-diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin. The possibility exists of a functional relationship between the ecto-5'NT molecule and the members of the ATP-binding cassette transporter superfamily, important components of MDR, in some cell types.

5'-Nucleotidase↗