Thea Pugatsch

Induction of graft vs. tumor effect in a murine model of mammary adenocarcinoma

We have attempted to induce immune‐mediated graft‐vs.‐tumor (GVT) effects against solid tumors, using a murine model of mammary adenocarcinoma derived from BALB/c(H‐2d) mice. A cell line (4TI) isolated from this tumor model was highly tumorigenic in syngeneic (BALB/c) or haplo‐identical (BALB/c × C57B1/6)F1 mice (F1), was only partially tumorigenic in an H‐2d congenic strain of mice (DBA/2) and was non‐tumorigenic in a major histocompatible (MHC)‐unrelated (H‐2b) strain of mice (C57B1/6). 4T1 cells express class I MHC antigens and adhesion molecules but do not express MHC class II antigens or B7‐I co‐stimulatory molecules. Female BALB/c (H‐2d) or F1 (H‐2d/b) mice were reconstituted with male bone marrow (BM) cells derived from minor histocompatible (MiHC)‐mismatched DBA (H‐2d) donors or with MHC‐mismatched C57B1/6 (H‐2b) BM cells, respectively, 24 hr following lethal total body irradiation. Recipient mice carrying MiHC‐ or MHC‐mismatched donor cells were inoculated with 4T1 cells 2–3 months following BM reconstitution. Chimeras reconstituted with allogeneic donor cells that were MiHC‐ or MHC‐incompatible with tumor cells were able to down‐regulate the development of the primary tumour expressing host‐type MHC alloantigens. Tumor size in BM chimeras across MiHC or MHC antigens was significantly smaller than tumor size observed in normal BALB/c or F1 controls. The GVT effect might be of help in improving immunotherapy for solid tumors in humans. Int. J. Cancer, 71:59–63, 1997.

Volatile anesthetic preconditioning attenuates myocardial apoptosis in rabbits after regional ischemia and reperfusion via Akt signaling and modulation of Bcl-2 family proteins

We tested whether isoflurane preconditioning inhibits cardiomyocyte apoptosis and evaluated the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic preconditioning and determined whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in anesthetic preconditioning. Six-month-old New Zealand rabbits subjected to 40 min of myocardial ischemia followed by 180 min of reperfusion were assigned to the following groups: ischemia-reperfusion (I/R), isoflurane preconditioning and isoflurane plus PI3K inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002) (0.6 and 0.3 mg/kg i.v., respectively). Sham-operated, wortmannin + I/R, wortmannin + sham, LY294002 + I/R, and LY294002 + sham groups were also included. Infarct size was assessed by triphenyltetrazolium chloride staining. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and activated caspase-3 assays. Akt phosphorylation, Bax, Bcl-2, Bad, and phosphorylated Bad (phospho-Bad) expression was assessed by immunoblotting. Isoflurane preconditioning reduced infarct size compared with the I/R group: 22 ± 4 versus 41 ± 5% (p < 0.05). The percentage of apoptotic cells decreased in the isoflurane group (3.8 ± 1.2%) compared with the I/R group (12.4 ± 1.6%; p < 0.05). These results were also confirmed by the activated caspase-3 assay. Wortmannin and LY294002 inhibited the effects of isoflurane. Myocardial infarction increased to 44 ± 3 and 45 ± 2% and the percentage of apoptotic cells was 11.9 ± 2.1 and 11.7 ± 3.3%, respectively. Akt phosphorylation and Bcl-2 and phospho-Bad expression increased after isoflurane preconditioning, whereas Bax expression decreased. These effects were inhibited by wortmannin and LY294002. The data indicate that isoflurane preconditioning reduces infarct size and myocardial apoptosis after I/R. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play a role in isoflurane-induced myocardial protection.

Mitral Regurgitation Augments Post-Myocardial Infarction Remodeling: Failure of Hypertrophic Compensation

OBJECTIVES We examined whether mitral regurgitation (MR) augments post-myocardial infarction (MI) remodeling. BACKGROUND MR doubles mortality after MI, but its additive contribution to left ventricular (LV) remodeling is debated and has not been addressed in a controlled fashion. METHODS Apical MIs were created in 12 sheep, and 6 had an LV-to-left atrial shunt implanted, consistently producing regurgitant fractions of approximately 30%. The groups were compared at baseline, 1, and 3 months. RESULTS Left ventricular end-systolic volume progressively increased by 190% with MR versus 90% without MR (p < 0.02). Pre-load-recruitable stroke work declined by 82 +/- 13% versus 25 +/- 16% (p < 0.01) with MR, with decreased remote-zone sarcoplasmic reticulum Ca(2+)-ATPase levels (0.56 +/- 0.03 vs. 0.76 +/- 0.02, p < 0.001), and decreased isolated myocyte contractility. In remote zones, pro-hypertrophic Akt and gp130 were upregulated in both groups at 1 month, but significantly lower and below baseline in the MR group at 3 months. Pro-apoptotic caspase 3 remained high in both groups. Matrix metalloproteinase (MMP)-13 and membrane-type MMP-1 were increased in remote zones of MR versus infarct-only animals at 1 month, then fell below baseline. The MMP tissue inhibitors rose from baseline to 3 months in all animals, rising higher in the MI + MR-group border zone. CONCLUSIONS In this controlled model, moderate MR worsens post-MI remodeling, with reduced contractility. Pro-hypertrophic pathways are initially upregulated but subsequently fall below infarct-only levels and baseline; with sustained caspase 3 elevation, transformation to a failure phenotype occurs. Extracellular matrix turnover increases in MR animals. Therefore, MR can precipitate an earlier onset of dilated heart failure.

Eimeria maxima: identification of gametocyte protein antigens.

The antigenicity of Eimeria maxima gametocyte proteins during the course of an infection and when injected into mice and rabbits was demonstrated using the Western blotting technique. Serum taken from chickens at various times postinfection reacted to a few gametocyte proteins, with the strongest reactivity seen with serum taken 14-days postinfection. Two major antigens having molecular weights of 56,000 and 82,000 were consistently detected by these sera. Using immune rabbit or mouse sera to whole gametocyte detergent extracts, the 56,000 and 82,000 molecular weight proteins were again the immunodominant antigens, despite their representing only a small proportion of the extract which was used to immunize the animals. These results, together with those obtained by Rose (1971) using recovered chicken serum to passively immunize chickens, indicate that these two gametocyte antigens may play a role in protective immunity to E. maxima.

Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment

Successful engraftment of hematopoietic stem cells requires a supportive hematopoietic stromal microenvironment (HSM). Defects in the HSM associated with aplastic anemia, myelofibrosis, or caused by intensive ionizing radiation and chemotherapy generally result in failure of bone marrow (BM) engraftment. Transplantation of donor BM within donor HSM may therefore provide optimal conditions for allogeneic BM transplantation. We have transplanted donor hematopoietic cells together with their own HSM to improve acceptance of allogeneic or xenogeneic BM. The non-myeloablative treatment used induced tolerance to murine allografts and provided conditions for the life-long acceptance of allogeneic HSM. Allogeneic BM transplanted within its own HSM under the kidney capsule caused less graft-versus-host disease than BM transplanted i.v. Tolerance in mice to xenogeneic (rat) HSM was less complete. Ectopic ossicles were small and contained fewer hematopoietic cells. However, simultaneous transplantation of rat BM and HSM to preconditioned mice improved engraftment of rat BM compared with transplantation of BM alone. Donor hematopoietic cells survived longer on their own HSM than on HSM of recipients.

Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic bone marrow transplantation for β-thalassemia major

PURPOSE Donor lymphocyte infusion (DLI) was used to reverse relapse after allogeneic bone marrow transplantation (BMT) in a patient with beta-thalassemia major. PATIENTS AND METHODS The patient with unstable mixed chimerism after BMT was treated with graded increments of donor lymphocytes (10(5) T cells/kg to 5 x 10(7) T cells/kg) to displace residual hematopoietic host cells. RESULTS DLI resulted in complete donor-derived reconstitution of the hematopoietic compartment. The patient developed mild graft-versus-host disease (GVHD) that could be controlled by steroid treatment. CONCLUSIONS This case report shows that DLI can effectively eradicate host stem cells in mixed chimeras after BMT in nonmalignant hematopoietic diseases.

Anti-erbB2 treatment induces cardiotoxicity by interfering with cell survival pathways

IntroductionCardiac dysfunction is among the serious side effects of therapy with recombinant humanized anti-erbB2 monoclonal antibody. The antibody blocks ErbB-2, a receptor tyrosine kinase and co-receptor for other members of the ErbB and epidermal growth factor families, which is over-expressed on the surface of many malignant cells. ErbB-2 and its ligands neuregulin and ErbB-3/ErbB-4 are involved in survival and growth of cardiomyocytes in both postnatal and adult hearts, and therefore the drug may interrupt the correct functioning of the ErbB-2 pathway.MethodsThe effect of the rat-anti-erbB2 monoclonal antibody B-10 was studied in spontaneously beating primary myocyte cultures from rat neonatal hearts. Gene expression was determined by RT-PCR (reverse transcription polymerase chain reaction) and by rat stress-specific microarray analysis, protein levels by Western blot, cell contractility by video motion analysis, calcium transients by the FURA fluorescent method, and apoptosis using the TUNEL (terminal uridine nick-end labelling) assay.ResultsB-10 treatment induces significant changes in expression of 24 out of 207 stress genes analyzed using the microarray technique. Protein levels of ErbB-2, ErbB-3, ErbB-4 and neuregulin decreased after 1 day. However, both transcription and protein levels of ErbB-4 and gp130 increased several fold. Calreticulin and calsequestrin were overexpressed after three days, inducing a decrease in calcium transients, thereby influencing cell contractility. Apoptosis was induced in 20% cells after 24 hours.ConclusionBlocking ErbB-2 in cultured rat cardiomyocytes leads to changes that may influence the cell cycle and affects genes involved in heart functions. B-10 inhibits pro-survival pathways and reduces cellular contractility. Thus, it is conceivable that this process may impair the stress response of the heart.

Allogeneic cell-mediated and cytokine-activated immunotherapy for malignant lymphoma at the stage of minimal residual disease after autologous stem cell transplantation

Immunocompetent donor-derived T lymphocytes play a crucial role in the elimination of residual leukemic cells post allogeneic bone marrow transplantation. Because this graft versus leukemia (GVL) effect is absent after autologous stem cell transplantation (ASCT), a high rate of relapse ensues. We introduced cell-mediated immunotherapy at the stage of minimal residual disease in lymphoma patients to help effect a GVL-like reaction by adoptive transfer of immunocompetent human leukocyte antigen-matched donor peripheral blood lymphocytes (PBL). Thirteen consecutive patients with high-risk lymphoma were treated with allogeneic cell therapy (AlloCT) after having undergone ASCT. In the absence of graft-versus-host disease, cell therapy-induced graft-versus-lymphoma reaction was amplified by human recombinant interleukin 2 (rlL-2) during 3 days to activate donor PBL in vivo, followed by infusion of in vitro rlL-2 activated donor lymphocytes combined with 3-day rlL-2 therapy. Nine of the patients underwent the treatment protocol well. In the four other patients, in whom the AlloCT resulted in marrow aplasia due to elimination of host hematopoietic cells, treatment with donor marrow cell infusion without further conditioning was performed. Adoptive cell therapy in the form of AlloCT may turn out to be an effective therapeutic modality for the treatment of resistant residual disease in lymphoma patients.

Eimeria maxima: Isolation of gametocytes and their immunogenicity in mice, rabbits, and chickens

Eimeria maxima gametocytes were isolated from infected chicken intestinal tissue by treatment with hyaluronidase and subsequent filtration through polymon filters. The isolated gametocytes were analyzed by microscopical and biochemical methods and shown to be highly enriched. The antigenicity of the gametocytes was analyzed in mice, rabbits, and chickens by ELISA and indirect immunofluorescence. Contrary to published results, we have found gametocytes to be highly immunogenic in all animals tested.

Tumor-cell vaccination induces tumor dormancy in a murine model of B-cell leukemia/lymphoma (BCL1)

Immunity to murine B‐cell leukemia/lymphoma (BCL1) induced by multiple injections with irradiated tumor cells, prevented leukemia development in primary and adoptive transfer recipients despite long‐lasting persistence of residual tumor cells. Detection of dormant BCL1 cells was carried out by PCR analysis using the VH‐rearranged DNA sequence as a BCL1 clonal marker. Dormant tumor cells were detected >250 days following immunity induction in 40% of spleens from healthy immune mice having no detectable symptoms of disease. Tumor dormancy was not abrogated by adoptive transfer of BCL1‐containing splenocytes into syngeneic recipients, indicating that cell‐mediated anti‐tumor immunity contributes to maintenance of the tumor dormant state and prevents renewed tumor‐cell growth. Splenocytes but not sera from immune mice conferred specific radiosensitive protection from a lethal dose of BCL1 cells included in cell mixtures transferred to secondary recipients. A therapeutic effect of transferred immune splenocytes was shown in BCL1‐bearing mice, which remained disease‐free for >200 days after inoculation; nevertheless, dormant BCL1 cells were detected by PCR analysis in some of the surviving mice. Our results suggest that an efficient tumor‐cell vaccine can lead to induction of tumor dormancy that can be maintained by a cell‐derived mechanism for a long period of time.