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Immunostimulatory effects of monoclonal antibodies (mAb) through binding to Fcγ receptors (FcγR) on immune cells are a likely cause of cytokine release syndrome. However, it is difficult to detect the potential risk of FcγR-dependent cytokine release associated with mAb in the current standard cytokine release assays (CRA), including the air-drying solid-phase method using human peripheral blood mononuclear cells (PBMC). To increase the sensitivity to detect FcγR-dependent cytokine release due to mAb, a high-density preculture (HDC) method was incorporated into the air-drying solid-phase CRA. Here, PBMC were exposed to panitumumab, trastuzumab, rituximab, or alemtuzumab at 0.1, 0.3, 1, and 3 μg/well for 24 or 48 hr under both non-HDC and HDC conditions. T-cell agonists (anti-CD3 mAb, anti-CD28 super-agonist [SA] mAb) were used as reference mAb. Panitumumab, trastuzumab, rituximab, or alemtuzumab induced cytokine release under both non-HDC and HDC conditions, and cytokine release caused by alemtuzumab was more pronounced under HDC conditions. To investigate FcγR involvement in cytokine release associated with panitumumab, trastuzumab, rituximab, and alemtuzumab, CRA of these four mAb were conducted with anti-FcγRI, -FcγRII, or -FcγRIII F(ab’)2 fragments. The results showed cytokine release caused by trastuzumab, rituximab, and alemtuzumab was significantly suppressed by anti-FcγRIII F(ab’)2 pretreatment, and slightly reduced by anti-FcγRI or anti-FcγRII pretreatment, indicating these mAb induced FcγR (especially FcγRIII)-dependent cytokine release from PBMC. Cytokine release caused by panitumumab was slightly suppressed by anti-FcγRIII F(ab’)2 pretreatment. Anti-CD3 mAb and anti-CD28 SA mAb also induced significant release of cytokines under HDC conditions compared with that under non-HDC conditions. In conclusion, CRA incorporating HDC into the air-drying solid-phase method using human PBMC could sensitively capture the FcγR-dependent cytokine release potential of mAb.

The in vivo mutagenesis from ethyl methanesulfonate (EMS) was investigated by the original Pig-a assay, which measures Pig-a mutant cells in red blood cells, and the PIGRET assay, which uses reticulocytes as a part of a collaborative study supported by the Japan Health Sciences Foundation. For the studies with the Pig-a assay, three dose levels of EMS (25, 50 and 100 mg/kg/day) were given orally to 6-week-old male F344 rats for 28 days, and peripheral blood was sampled just before the start of dosing, and after dosing for 1, 2 and 4 weeks with EMS. In the studies with the PIGRET assay, single oral dosing at two dose levels (360 and 720 mg/kg) of EMS was employed and peripheral blood was collected just before dosing, and 1 and 2 weeks after dosing of EMS. As the results, a statistically significant increase in mutant frequency of the Pig-a gene was observed at 2 and 4 weeks, but not at one week after dosing of EMS in the Pig-a assay. In the PIGRET assay, on the other hand, a statistically significant increase in mutant frequency was obtained at one week after the dosing. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier than the original Pig-a assay can by focusing on reticulocytes rather than red blood cells as the target cell population. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the role of tumor-derived angiogenic factors in solid tumor formation. We compared the angiogenic potential of tumorigenic and non-tumorigenic human tumor cell lines. All tumorigenic cell lines induced angiogenesis in vivo and their angiogenesis-inducing abilities were higher than those of the other non-tumorigenic cell lines. This in vivo angiogenic potential was well correlated with the in vitro endothelial cell growth-stimulating activity contained in the cell extract or conditioned medium of each cell line. The endothelial cell growth-stimulating activities of these cell lines were completely inhibited by neutralizing antibodies to basic fibroblast growth factor (bFGF), acidic FGF (aFGF) or vascular endothelial growth factor (VEGF). Furthermore, the levels of tumor-derived endothelial cell growth-stimulating activities depended on the amounts of angiogenic factors such as VEGF and bFGF produced by tumor cells. Although VEGF transcripts were detected in all of the cell lines by RT-PCR assay, the non-tumorigenic cell lines showed poor productivity of VEGF as well as FGFs and had less or non-potency for endothelial cell growth stimulation. These findings suggest that the increase in production of angiogenic factors by tumor cells is necessary for their in vivo angiogenic and tumorigenic potentials, and that VEGF and FGFs are the major mediators of tumor-induced angiogenesis. [ABSTRACT FROM AUTHOR]

In the view of animal welfare considerations, we investigated the suitability of modifying the rat liver micronucleus test with partial hepatectomy to include administration of an analgesic drug to minimize pain and distress as much as possible. The effects of the analgesic, buprenorphine, on the genotoxicity evaluation of structural chromosome aberration inducers (cyclophosphamide, diethylnitrosamine and 1,2-dimethylhydrazine) and numerical chromosome aberration inducers (colchicine and carbendazim) were examined. The genotoxicants were given orally to 8-week-old male F344 rats a day before or after partial hepatectomy and hepatocytes were isolated 4 days after the partial hepatectomy. Buprenorphine was injected subcutaneously twice a day with at least a 6-hr interval for 2 days from just after partial hepatectomy. As results, buprenorphine caused neither change in clinical signs (except for one animal death) nor increase in the incidence of micronucleated hepatocytes of vehicle treated animals. In the case of concomitant treatment of buprenorphine and a genotoxicant, one out of 8 animals died in each group given buprenorphine with cyclophosphamide, carbendazim or colchicine (lower dose level only). Slight changes in clinical signs were noted in the group given buprenorphine with cyclophosphamide or carbendazim. A statistically significant increase in the incidence of micronucleated hepatocytes was obtained in concomitant treatment of buprenorphine and genotoxicant compared with genotoxicant alone for 1,2-dimethylhydrazine, colchicine and carbendazim. It is concluded that use of buprenorphine as an analgesic drug to minimize pain and distress for rats that are given partial hepatectomy is not appropriate under the present experimental conditions, because it could enhance the general toxicity and genotoxicity of the test chemical. [ABSTRACT FROM AUTHOR]