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Biomedical subjects

E Mihich

Publications and source records attributed to E Mihich.

At least 55 records · Page 3Linked to original sources
PubMed1994

Lipopolysaccharide and splenic tumoricidal macrophage activation.

Splenic macrophage tumoricidal activity was examined and a splenic macrophage tumoricidal assay was established. Initially, mixtures of lipopolysaccharide (LPS) and spleen single cell suspensions (SSCS) were cultured for 1-4 days. Adherent macrophages, washed free of nonadherent cells and LPS, were then examined and were found to lack tumoricidal activity in a standard 18-h 51Cr release assay. However, tumoricidal activity was generated if LPS was added to the SSCS cultures at later time points during the 4-day incubation period; maximal activity was seen when LPS was added on day 3. In parallel, significant changes in macrophage autofluorescence and morphology, but not phenotype, were observed. Next, SSCS were cultured for 1-4 days without stimulating agents. Adherent macrophages were then washed free of nonadherent cells and LPS was added. Significant tumoricidal activity developed in time- and LPS concentration-dependent fashions. The presence of nonadherent spleen cells in physical contact with the macrophages during the SSCS culture was essential for the macrophages in the resultant monolayer to be responsive to LPS. Activated splenic macrophage-mediated lysis of tumor cells was shown to depend on the contact between the two cells.

Animals↗
PubMed1994

Effect of perioperative chemoimmunotherapy with cyclophosphamide and autologous tumor vaccine in murine MBT-2 bladder cancer.

The in vitro cytotoxic activity of splenocytes from C3H/He mice implanted subcutaneously with 10(6) syngeneic MBT-2 tumor cells on day 0 was significantly enhanced after cyclophosphamide (100 mg./kg., intraperitoneally) given 2 days before tumor resection on day 17, with or without active specific immunization with BCG plus autologous irradiated tumor cells (vaccine) 1 week after tumor resection. Furthermore, a significantly lower tumor incidence was seen in mice challenged with 10(5), but not 10(6), tumor cells per mouse 24 hours after tumor resection on day 17 and treated with cyclophosphamide on day 15 and postoperatively with vaccine than was found in nontreated tumor resected mice. Phenotypic analysis of cells from spleen showed that cyclophosphamide pretreatment and postoperative vaccine, either singly or in combination, induced a significant increase of both CD44+ memory T cells and CD11b+ myeloid/macrophage cells. Thus, in addition to a specific antitumor immune response, a nonspecific cytolytic mechanism may also play a role in the observed antitumor effect.

Animals↗
PubMed1994

Intracellular doxorubicin kinetics in lymphoma cells and lymphocytes infiltrating the tumor area in vivo: a flow cytometric study.

Recently we have reported the development of a safe and effective chemoimmunotherapy protocol involving doxorubicin (Dox) in combination with interleukin 2 which, in C57BL/6 mice, boosts local T cell responses, and, in 50 to 80% of the cases, this resulted in the complete eradication of established syngeneic EL4 lymphoma or its Dox-resistant variant, EL4/A. Accumulation of host-derived leukocytes in the peritoneal cavity was increased up to 8-fold after tumor inoculation, but, in absolute numbers, did not increase further following Dox administration. The cellular pharmacokinetic studies undertaken to clarify the role of Dox following a single IV injection indicate that 4 h later, lymphocytes found in the peritoneal cavity have detectable levels of Dox; but the lymphoma cells (both EL4 and EL4/A) have, in proportion to their larger size, taken up more drug as judged by flow cytometry. The estimated drug "concentration" (i.e., intracellular amount divided by estimated cell size) at the 4-h time point, however, was found to be essentially equivalent in both the lymphoma cells and the lymphocytes. Thereafter, the drug content and intracellular "concentration" in the EL4/A cells rapidly declined while their numbers progressively increased. In contrast, the EL4 lymphoma cells and the lymphocytes found in the peritoneal cavity in the presence of either lymphoma consistently exhibited higher levels of drug 24-48 h than at 4 h. Splenic and tumor-infiltrating mature T (CD3+) cells were completely insensitive to Dox cytotoxicity and actually showed increased CTL activity when examined ex vivo. Although EL4 cells had identical Dox uptake patterns to those of CD3+ cells, they were sensitive to the drug and their numbers decreased, resulting in increased host/tumor cell ratios in these mice. The pharmacokinetic parameters of the drug and the insensitivity of the mature T cells to the drug determined in this study can explain, in part, the efficacy of a chemoimmunotherapy protocol boosting local T-cell responses.

Animals↗
PubMed1994

Doxorubicin-induced DNA degradation in murine thymocytes.

Exposure of murine thymocytes to doxorubicin (Dox) (0.5-1.0 microM, 24 hr) triggered rapid DNA degradation, as indicated by the appearance of a major subdiploid population demonstrated by DNA flow cytometry. Electron microscopic comparison of samples with large subdiploid populations versus those with little or no such subset revealed significantly more cells with the characteristic features of apoptosis, the morphologically definable stage of programmed cell death. These features include unipolar condensed chromatin, zeiosis, and electron-dense cytoplasm. Dox-induced apoptosis occurred without prior S or G2/M phase arrest or cell size increase. The subset most susceptible to Dox-induced apoptosis in vitro and in vivo was CD3-CD4+CD8+. The same subset is affected by dexamethasone (Dex); as reported for Dex-induced apoptosis, actinomycin D and cycloheximide also blocked Dox-induced apoptosis. Thymocytes exposed to higher Dox concentrations (2-10 microM) did not have a subdiploid population. Although at 2-10 microM Dox significantly reduced cell numbers (probably as a result of necrosis), at least 5-10% of the population was viable at 24 hr. Thymocytes exposed to low concentrations of Dox (0.001-1.0 microM) plus Dex (0.1 microM) exhibited additive induction of apoptosis, whereas those exposed to high concentrations of Dox (2-10 microM) plus Dex were completely devoid of any evidence of apoptosis. These results indicate that the Dox-induced killing in thymocytes (mostly noncycling cells) occurs via different mechanisms depending upon the Dox concentration.

Animals↗
PubMed1994

TNF-alpha potentiation of the lymphokine-activated killer response of murine thymus cells.

The role of tumor necrosis factor (TNF-alpha) on in vitro generation of lymphokine-activated killer (LAK) cells from murine thymocytes was investigated and compared to that on generation of LAK from splenocytes. TNF-alpha increased the potential of interleukin-2 (IL-2) at suboptimal concentrations to generate LAK activity in thymocytes even more than in splenocytes. In parallel, augmented [3H]thymidine uptake by thymocytes and splenocytes was seen. However, no net increase in viable cell number was observed. LAK effector cells from TNF-alpha plus IL-2 cultures responded with an increased [3H]thymidine uptake to restimulation by IL-2 alone. These results suggest that TNF-alpha + IL-2 may be inducing the expansion of a small subset of cells. NK1.1+ cells are a very minor subset of thymocytes, nevertheless phenotype analysis showed that in thymocytes, IL-2 + TNF-alpha generates NK1.1+ CD8- LAK effectors in contrast to NK1.1- CD8+ cells found with IL-2 alone. This result is consistent with the finding in the proliferation studies. The fact that thymocytes are stimulated by the TNF-alpha + IL-2 combination to proliferate as well as to develop a phenotypically distinct effector supports the role of TNF-alpha in intra- and extrathymic regulation.

Animals↗
PubMed1993

Polymyxin B-mediated lysis of tumor cells.

Polymyxin B (PmB) in the concentration range of 10-50 micrograms/ml is used routinely in immunological studies to neutralize low levels of contaminating lipopolysaccharide (LPS) in media or reagents. While using PmB for such a purpose unexpected results were obtained, which led to the finding that low levels of PmB are cytotoxic to certain tumor cells. Further examination of a panel of 10 tumor cell lines revealed that in an 18-hour 51Cr release assay, EL4 cells and EL4/ADM cells were very sensitive (lysed by > or = 10 micrograms PmB/ml), C1498 cells and REH cells were moderately sensitive (lysed by > or = 20 micrograms PmB/ml) and cells of the remaining 6 lines were resistant (lysed only by 100 micrograms/ml) to PmB. A similar pattern of sensitivity was observed when 3H-thymidine incorporation was used as a measure of PmB effects in cell proliferation. The PmB concentration needed to kill 50% of the tumor cells in a suspension differed greatly among lines; thus for cells of a resistant line 8-fold more PmB was required for 50% killing than for those of a sensitive line. PmB toxicity toward EL4 cells was shown to increase to a plateau level with increasing time of exposure; however, the higher the concentration the earlier the plateau was reached. LPS may prevent PmB toxic effects since PmB binds to the lipid A portion of the LPS molecule, but 100 micrograms LPS/ml was only able to reduce the toxicity of 10 and 20 micrograms PmB/ml, and not that of 50 or 100 micrograms PmB/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Immunological responses critical to the therapeutic effects of adriamycin plus interleukin 2 in C57BL/6 mice bearing syngeneic EL4 lymphoma.

A safe and effective therapeutic combination of moderate doses of Adriamycin (doxorubicin, 4 mg/kg, IV, Days 1 and 8 or only Day 8) plus prolonged administration of moderate doses of interleukin 2 (2 micrograms, b.i.d., Days 9-40) was developed in the syngeneic EL4 (5 x 10(4) cells, IP, Day 0) lymphoma--C57B1/6 mouse model and has been reported in the companion paper. The studies described herein demonstrate that the effectiveness of this combination treatment against EL4 lymphoma growing intraperitoneally in C57B1/6 mice was dependent upon the presence of CD8+ cells. Thus, the induction of long-term survivors (60-80%) by Adriamycin plus interleukin 2 was completely ablated by pretreatment of mice with anti-CD8 monoclonal antibody (MAb), whereas pretreatment with anti-CD4 MAb only partially inhibited the therapeutic effects and anti-NK1.1 MAb had no effect. A close association between survival, an increase in phenotypically identified CD8+ cells, and an increase in specific anti-EL4 cytolytic activity was demonstrated with cells from the tumor site (peritoneum) but not consistently with cells from the spleen. No association was observed between survival and modulations in natural killer (NK), lymphokine-activated killer (LAK), or tumoricidal macrophage activity of spleen or peritoneal cells. Taken together the results indicate that, in this model, the most relevant correlate of a therapeutically effective host antitumor response is the level of specific EL4 tumor killing by cells present at the tumor site. Based on the findings reported herein, it can be predicted that weakly immunogenic tumors may be eradicated by immunologic mechanisms elicited in conjunction with properly designed therapeutic regimens.

Adjuvants, Immunologic↗
PubMed1993

Development of a safe and effective adriamycin plus interleukin 2 therapy against both adriamycin-sensitive and -resistant lymphomas.

This laboratory has extensively studied Adriamycin (doxorubicin)-induced immunomodulation. Despite demonstration of favorable effects, little therapeutic advantage was seen, and it was decided to test Adriamycin in combination with interleukin 2 (IL2). Considerable toxicity was seen with either high-dose IL2 or high-dose Adriamycin alone, using the syngeneic C57B1/6-EL4 T cell lymphoma model. When the doses of either agent were reduced to decrease toxicity, little therapeutic effect was seen. In contrast, an effective protocol without apparent toxicity was developed by combining a moderate dose of Adriamycin (4 mg/kg, IV, Days 1 and 8 or only Day 8) with prolonged administration of a moderate dose of IL2 (2 micrograms, b.i.d., i.p., Days 9 to 40). This protocol resulted in up to 80% long-term survivors among mice inoculated on Day 0 with EL4 lymphoma (5 x 10(4) cells). It should be noted that under these conditions, neither agent, when administered singly, induced long term survivors, and that following the inoculation of only 10-100 EL4 tumor cells all animals died in the absence of treatment. The survivors developed protective immunity as demonstrated by their ability to resist reimplantation with EL4 tumor. Furthermore, this resistance to tumor reimplantation could be transferred into naive hosts with spleen cells from tumor-bearing mice receiving the combination protocol; exposure of mice to sublethal whole body irradiation prior to tumor implantation completely abrogated the efficacy of this combination treatment. Finally, it was shown that this combination protocol was equally effective against an Adriamycin-resistant subline of EL4 that expresses the multidrug resistance phenotype.

Animals↗
PubMed1992

Tumoricidal activation of murine resident peritoneal macrophages by interleukin 2 and tumor necrosis factor alpha.

The capacity of recombinant human interleukin 2 (rH-IL2), alone or in combination with recombinant tumor necrosis factor (r-TNF alpha), to activate murine resident peritoneal macrophages to a tumoricidal state was examined. Resident peritoneal exudate cells from C57BL/6 mice were cultured for 18 h with activating agents and washed and the adherent cells (macrophages) were assessed for cytolytic activity against radiolabeled target tumor cells (EL4, P815). Under these conditions, rH-IL2 alone activated macrophages to a tumoricidal state in a concentration dependent fashion. Neither murine nor human r-TNF alpha alone had any activating effect but, when combined with rH-IL2, further stimulated rH-IL2-inducible responses. Using polymyxin B, it was shown that macrophage activation was not due to an inadvertent lipopolysaccharide contamination of the r-TNF alpha or rH-IL2 preparations. It was also unlikely that target cell lysis was a direct result of increased TNF alpha production by rH-IL2 stimulated macrophages since P815 is totally resistant to lysis by r-TNF alpha. Although the lytic effector function was mediated by adherent cells, nonadherent peritoneal exudate cells were required for activation to occur. Furthermore, antisera against murine gamma-interferon, when added to activation cultures, reduced the level of cytolytic activity which developed. These data suggest that rH-IL2-induced peritoneal macrophage activation requires stimulation of nonadherent cells and is dependent upon gamma-interferon mediated mechanisms.

Animals↗
PubMed1992

Adriamycin-induced modulation of host defenses in tumor-bearing mice.

Using the C57BL/6/EL4 tumor model, studies were carried out to demonstrate the feasibility of administering Adriamycin (ADM) in therapeutic doses and schedules such that the host antitumor defenses would not be suppressed and in some cases might be stimulated by treatment. ADM treatment caused prolongation of survival and, in general, either stimulated host cytolytic activities above untreated control levels or had no effect. These effects by ADM were observed with the ADM-sensitive parent EL4 line as well as with an ADM-resistant subline, indicating that the effects did not result entirely from direct antitumor activity. The cytolytic activities examined were those of cytolytic T-lymphocytes, lymphokine-activated killer cells, and splenic and peritoneal macrophages. All activities were assessed against the syngeneic EL4 target line. The information obtained in this investigation provides a rational basis for the future development of curative protocols with ADM plus biological response modifiers, which would depend on a functional immune system for optimum efficacy and would also exploit synergistic immunomodulating effects of the agents used in combination.

Animals↗
PubMed1990

The relationship between multidrug resistance and tumor necrosis factor resistance in an EL4 cell line model.

A number of recent studies have implied that a relationship exists between cellular sensitivities to tumor necrosis factor (TNF) and expression of the classic multidrug resistance (MDR) phenotype. However, different conclusions have been reported concerning whether TNF sensitivity is positively or negatively correlated with MDR (Hong, W.-S.; Sijo, N.; Sasaki, Y.; Shinkai, T.; Eguchi, K.; Sakurai, M.; Takamashi, H.; Nakano, H.; Nakagawa, K.; Twentyman, P. R. Jpn. J. Can. Res. (Gann) 78:1274-1280; 1987 and Dollbaum, C.; Creasey, A. A.; Dairkee, S. H.; Hiller, A. J.; Rudolph, A. R.; Lin, L.; Vitt, C.; Smith, H. S. Proc. Natl. Acad. Sci. USA 85:4740-4755; 1988). An apparent relationship of TNF sensitivity to P-glycoprotein (P-170gp) mediated MDR was investigated in EL4 murine T-lymphoma cell lines sensitive and resistant to Adriamycin (ADM). No consistent association was found between MDR and TNF responses when the lines were subcloned. Whereas the MDR phenotype of subclones (as assessed by ADM resistance and P-170gp expression) reflected that of the cell line from which they were derived, the TNF sensitivity of subclones varied widely. Also consistent with independence of P-170gp mediated MDR and TNF response, the P388/ADM cell line (exhibiting P-170gp mediated MDR) remained as resistant to TNF as the P388 parental line. In addition, no evidence was found of modified recognition of MDR EL4 cell lines by host defense effector cells, and gamma-interferon failed to enhance the susceptibility of either parental or MDR cell line to TNF. These results may be of value in considering therapeutic studies using the ADM/TNF combination treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗