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Summary

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (pbl) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The pbl from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (lak) cells, or with phytohemagglutinin (pha) and rIL-2 to generate autologous-stimulated lymphocytes (asl). After 4 days, cytotoxicity by the asl, lak, and pbl was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The pbl cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's pbl were activated in vitro, they killed the dog's own tumor, asl more effectively than lak cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, asl generated from healthy dogs were significantly more cytolytic than lak from healthy dogs, or than asl generated from tumorbearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including pha in the assay medium with lymphocytes and Raji cells, by asl and lak was greater than cytotoxicity of Raji without pha. Because asl were more cytolytic than lak against all targets in vitro, they may be more beneficial than lak for immunotherapy of canine tumors.

Free access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)– and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses.

SAMPLE

1,440 samples of PBMCs from 6 healthy research horses.

PROCEDURES

From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA.

RESULTS

Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations.

CONCLUSIONS AND CLINICAL RELEVANCE

Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.

Full access
in American Journal of Veterinary Research

Summary

We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by infection with bovine leukemia virus (blv). The superficial cervical lymph nodes from 3 blv-infected but apparently healthy sheep and 5 sheep with fullblown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (slgM) and major histocompatibility complex (mhc) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P < 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5 B-cell lineage and to be classified as 2 types, CD5CD4CD8B2+MHC class-II+sIgM+ and CD5CD4CD8B2+mhc class-II+sIgM. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells and not in T cells. Thus, we conclude that the host's immune response may be still maintained at a lymphosarcomatous stage.

Free access
in American Journal of Veterinary Research

improved biopharmaceuticals for oncology . Pharmacotherapy 2003 ; 23 : 3S – 8S . 10.1592/phco.23.9.3S.32886 11. Molineux G . The design and development of pegfilgrastim (PEG-rmetHuG-CSF, Neulasta) . Curr Pharm Des 2004 ; 10 : 1235 – 1244 . 10

Full access
in American Journal of Veterinary Research