Sadayuki Sasaki

Role of milky spots as selective implantation sites for malignant cells in peritoneal dissemination in mice

We investigated the significance of milky spots for malignant cells in peritoneal dissemination using three mouse carcinomatous peritonitis models. P388 leukemia and Colon 26 cancer cells were labeled with bromodeoxyuridine (BrdU) and mice were inoculated intraperitoneally. After 24 h the greater omentum and the mesenterium were removed and stained immunohistochemically with anti-BrdU antibody. The labeled cells were found to have preferentially infiltrated into the milky spots in these specimens. Next, using B16 PC melanoma cells, which can be easily distinguished from the other cells by the intrinsic black melanin, the distribution of the melanoma cells was observed macro- and microscopically following intraperitoneal inoculation. The melanoma cells were similarly found to have selectively infiltrated into the milky spots in the omentum and mesenterium after 1 day. Moreover, the melanoma cells were growing and forming distinct metastic lesions within the milky spots 1 week later.

Enhancement of therapeutic efficacy of aclarubicin against lymph node metastases using a new dosage form: aclarubicin adsorbed on activated carbon particles.

Seven days after a subcutaneous inoculation of 5 × 105 P388 leukemia cells Into the foot pad of the left hind paw of donor mouse, aclarubicin (0.2 mg/kg body weight) was injected subcutaneously Into the hind paw of the opposite foot pad in the form of ACR-CH or aclarubicin aqueous solution. On day 10, the left popliteal and the lower para-aortic lymph nodes taken from each donor were transferred Intraperltoneally to a normal recipient mouse. The combined survival time of recipients and the viable P388 leukemia cell number In popliteal and para-aortic lymph nodes were estimated with a calibration formula. Our results showed that the survival curve of recipients given ACR-CH was statistically improved compared with that of other treatment groups.

CISPLATIN MICROCRYSTALS SUSPENDED IN OIL : PATHOLOGICAL STUDY OF ACUTE TOXICITY IN MICE

The pathological changes brought about by the i.p. administration of a new dosage format, clsplatin micro-crystals suspended in oil (CDDP-Oil), was examined in mice. CDDP-Oil decreased the absolute weight and the relative weight (organ weight/body weight) of the kidney, liver and spleen in the mice receiving the dosage form. However, the severity of the reduction of the organ weight in the CDDP-Oil administration groups was not different from that in the cisplatin aqueous solution (CDDP-Sol) administration groups. Histologically, severe degeneration and atrophy were recognized in the kidney, large intestine, small intestine, thymus, spleen, bone marrow and lymph nodes in the CDDP-Oil administration groups as well as CDDP-Sol administration groups. However, there were no additional changes in the macroscopic and microscopic findings in the CDDP-Oil groups. From these results, we concluded that this dosage form did not change the toxicity of cisplatin in terms of pathological effects.

Cisplatin microcrystals suspended in oil--toxicity in mice.

A new dosage format, cisplatin microcrystals suspended in oil (CDDP-oil), was developed for the treatment of peritoneal carcinomatoses. We studied the acute toxicity of CDDP-oil injected intraperitoneally in mice. The 50% lethal dose was 30.3 mg/kg (27.1–33.7 mg/kg at the 95% confidence level), which was 1.79 times that of a cisplatin aqueous solution (CDDP-sol) of 16.9 mg/kg (16.1–17.8 mg/kg at the 95% confidence level). There were no significant differences in the duration of the toxic effects and the toxic symptoms between these two dosage forms. However, the severity of weight loss in the group given CDDP-oil was less than the group given CDDP-sol.

Enhancement of LAK-like activity and cytokine induction in regional lymph nodes and spleen cells of mice after intralymphnodal injection of OK-432, a killed streptococcal preparation

A single dose of inactivated streptococci (OK-432) was injected into the popliteal lymph nodes of male CDF1, mice and its effects on popliteal, inguinal, and para-aortic lymph node cells and spleen cells were investigated and compared with the effects of subcutaneous injections of the same dosage of OK-432. Regional lymph node cells and spleen cells obtained from intralymphnodally injected mice lysed not only natural killer (NK)-sensitive YAC-1 cells, but also NK-resistant P-815 and meth-A cells. Lysis of target cells was inhibited when effector cells were treated with anti-Thy-1.2 or anti-Lyt-2.2 monoclonal antibody and complement, but no inhibition was apparent after treatment with anti-asialo-GM1 or anti-Lyt-1.2 antibody and complement. These results suggest that the effector cells are lymphocyte-activated killer (LAK) cells. An enhanced capacity of lymph node cells to produce cytokines, tumor necrosis factor and Interleukin 1 upon restimulation with lipopolysaccharide was found only in intralymphnodally injected mice. Thus, the induction of LAK-like cells and cytokine production in regional lymph nodes and spleen cells by the intralymphnodal administration of OK-432 should be effective for the inhibition or treatment of lymph node metastases.

Pathological study for the acute toxicity of cisplatin microcrystals suspended in oil by intraperitoneal administration in mice.

Pathological changes were compared between two dosage formulations of cisplatin microcrystals suspended in oil (CDDP-Oil), and cisplatin aqueous solution(CDDP-Sol), by intraperitoneal administration in mice. Main toxic findings such as decreasement of the organ weights and histopathological degenerations were seen in the kidney, liver, and hematologic tissues of the spleen, lymph nodes and bonemarrow. Those changes were more heavy in mice died by the toxicity than in mice surviving during the observation period. Thus, the toxic changes were principally similar in the two dosage formulations, and there were no aditional toxic effects in the CDDP-Oil.

Enhancement of various non-specific immune effector functions in mice by local injection of aclacinomycin A adsorbed onto activated carbon particles (ACR-CH)

Local injections of aclacinomycin A adsorbed onto activated carbon particles (ACR-CH) augmented the cytotoxic activities of regional lymph node cells for 7 days. In contrast NK-activity was only slightly augmented by injections of aclacinomycin A (ACR) solution or activated carbon suspension. The effects were found in lymphocytes from all regions tested. NK-activity could only be detected when both adherent and non-adherent cells were present. The cell number of L3T4+ cells in each type of lymph node tested increased, and subset analysis of the lymphocyte subpopulations revealed an increase in the ratio of L3T4+/Lyt2+ cells, suggesting that the ACR-CH selectively increased and stimulated L3T4+ cells. Enhanced capacity of lymph node cells to produce cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1) upon restimulation (with LPS) in vitro in the ACR-CH treated group was found. From these results, it appears that the new dosage form of aclacinomycin A, ACR-CH, with superior therapeutic efficacy against lymph node metastases, can also enhance the immune response of regional lymph node cells. The findings reported here will be valuable in the establishment of novel chemoimmunotherapeutic protocols using ACR-CH.