Kazuo Ohuchi

An orthotopic implantation mouse model of human malignant pleural mesothelioma for in vivo photon counting analysis and evaluation of the effect of S-1 therapy.

Human malignant pleural mesothelioma (HMPM) is an aggressive neoplasm that is highly resistant to conventional therapies. We established 3 HMPM cell lines (TCC‐MESO‐1, TCC‐MESO‐2 and TCC‐MESO‐3) from Japanese patients; the first 2 from the primary and metastatic tumors of a patient with the epithelioid type of HMPM, and the third from a patient with biphasic characteristics of the tumor (epithelioid and sarcomatous phenotypes). The 3 cell lines resembled the original HMPMs in their morphological and biological features, including the genetic alterations such as lack of p16 expression and mutation of p53. Their tumorigenicity was determined in SCID mice by orthotopic implantation (20–46%). The tumorigenicity of the HMPM cell lines, which was relatively low, was enhanced by repeated subcultures and orthotopic implantations, and 3 competent tumorigenic sublines were produced (Me1Tu, Me2Tu and Me3Tu sublines from the TCC‐MESO‐1, TCC‐MESO‐2 and TCC‐MESO‐3 cell lines, respectively). The resultant HMPM sublines efficiently generated tumors in the SCID mice (100%) following orthotopic implantation. SCID mice implanted with the competent sublines, into one of which the luciferase gene was introduced, displayed quantitative fluctuation of the bioluminescence for the tumor volume in vivo. Oral administration of S‐1, an anticancer agent, suppressed the proliferation of the luciferase gene‐expressing Me1Tu subline in the mouse models in vivo, with a treated‐to‐control ratio of the mean tumor volume of 0.2. The orthotopic implantation mouse model proved to be useful for quantitative evaluation of the efficacy of novel anticancer drugs and also for studying the biology of HMPMs in vivo.

Determination of platelet activating factor using immunoaffinity mini-columns. Stimulation of platelet activating factor production by thapsigargin and effects of dexamethasone.

Thapsigargin, a hexaoxygenated tetraacylated sesquiterpene lactone isolated from the roots of Thapsia garganica L., is one of non-TPA type tumor promoters that stimulate prostaglandin E2 production at very low concentrations and induce inflammation when applied topically. Effects of thapsigar-gin on platelet activating factor (PAF) production by rat peritoneal macrophages were examined. Levels of PAF in the conditioned medium and in the cells were determined by radioimmunoassay after extraction of PAF using immunoaffinity mini-columns. It was demonstrated that thapsigargin at concentrations of 3 to 100 ng/ml stimulated production of cell-associated PAF when measured 10 min after incubation. Levels of PAF in the conditioned medium were less than the detectable amount.The stimulative effect by thapsigargin reached a plateau at a concentration of 30 ng/ml. Time course experiments revealed that the levels of cell-associated PAF showed a peak 10 to 15 min after incubation with thapsigargin at a concentration of 30 ng/ml. The levels were then declined until 40 min after incubation. When macrophages were preincubated for 3 h with dexamethasone at concentra-tions of 0.01, 0.1 and l μg/ml, thapsigargin (30 ng/ ml) -stimulated PAF production in the cells at 10 min were decreased. However, the maximum inhibition was about 50% even at 1μg/ml of dexamethasone. These date suggest that by treatment with thapsigargin, PAF is partly produced through formation of lyso-PAF by activated phospholipase A2 since dexamethasone shows partial inhibition of thapsigargin-induced PAF production, and thap-sigargin-stimulated PAF production may partly account for proinflammatory activity of thapsigar-gin.

Histamine-production-increasing factor-II in the inflammatory exudate in the chronic phase of allergic inflammation.

Employing the air pouch-type allergic inflammation model in rats, effects of inflammatory exudate on histamine production by bone marrow cells were examined in order to clarify the mechanism of histamine production at the site of allergic inflammation. The pouch fluid collected 8 h after the induction of allergic inflammation (the acute phase) increased histamine production by bone marrow cells. On the other hand, the pouch fluid collected 5 days after the antigen challenge (the chronic phase) showed no activity by itself, but enhanced the activity by the pouch fluid collected at the acute phase. These activities were due to proteinous factors that were produced at the acute phase and at the chronic phase, named histamine-production-increasing factor (HPIF) -I and HPIF-II, respectively. HPIF-I increased histidine decarboxylase (HDC) activity in bone marrow cells but HPIF-II did not. However, HPIF-II enhanced HDC activity stimulated by HPIF-I. The cooperative effect of HPIF-I and HPIF-II on histamine production was also observed in vivo. Isoelectric point and molecular weight of HPIF-II were estimated to be 7 to 8 and about 100 kD, respectively.These results indicate that histamine production at the site of allergic inflammation might be regulated by at least two kinds of proteinous factors, HPIF-I and HPIF-II.

Anti-inflammation. Methodology and a means for the suppression of inflammation. From an aspect of arachidonic acid cascade.

Using an air pouch type allergic inflammation model in rats, roles of arachidonate metabolites in inflammatory responses were investigated in order to define characteristics of allergic inflammation in rats, and to get further insight into a methodology for suppression of inflammation from a veiw point of arachidonic acid metabolism. Biochemical and pharmacological analysis revealed following results; (1) Peptide-leukotrienes do not play any significant role in vascular permeability increase in anaphylaxis phase. (2) Leukotriene B4 also plays no role in neutrophil infiltration, suggesting that participation of lipoxygenase products in inflammatory responses is negligible in rats. (3) In contrast, antagonists of platelet activating factor (PAF) efficiently suppressed neutrophil infiltration into the inflammatory locus indicating that PAF plays a significant role for neutrophil infiltration. (4) Prostaglandin E2 participates in vascular permeability increase at a definite period, and in stimulation of neutrophil infiltraltion by suppressing histamine production at the inflammatory locus. (5) Cyclooxygenase inhibitors suppress vascular permeability and neutrophil infiltration. Suppression of neutrophil infiltration by cyclooxygenase inhibitors is mediated by stimulation of histamine production through inhibition of prostaglandin E2 biosynthesis. Histamine suppresses neutrophil infiltration by acting on histamine H2 receptors of neutrophils.Finally, present situation and point at issue for suppression of inflammation through regulation of arachidonic acid metabolism is discussed.

Regulation of histamine production of bone marrow cells stimulated by inflammatory exudate.

The mechanism for histamine release of bone marrow cells stimulated by the inflammatory exudate, the pouch fluid in air pouch type allergic inflammation in rats, was examined. When bone marrow cells were incubated in the medium containing dialyzed pouch fluid supernatant, histidine decarboxylase activity in the cells and histamine level in the medium were significantly increased when compared with those in bone marrow cells incubated in the medium alone. Addition of an RNA synthesis inhibitor, actinomycin D, or a protein synthesis inhibitor, cycloheximide, into the medium strongly reduced the dialyzed pouch fluid supernatant-induced increase in histamine level in the medium. Inhibitors of DNA synthesis, mitomycin C and cytosine arabinoside, reduced it partially. In addition, it was inhibited by retinoic acid, a differentiation-inducer, but was augmented by sodium n-butyrate, which induces differentiation of bone marrow cells to basophils. Without dialyzed pouch fluid supernatant, retinoic acid and sodium n-butyrate did not affect histamine production. These results suggest that the inflammatory exudate in allergic inflammation has an activity to stimulate histamine production by bone marrow cells by inducing de novo synthesis of histidine decarboxylase which is regulated by growth and differentiation of histamineproducing cells.

Arachidonate metabolism in macrophages and the effect of "Mao" alkaloids.

Species of Ephedra plants has been shown to have anti-inflammatory activities. Recently, four alkaloids, ephedrine, pseudoephedrine, ephedroxane, isolated from the crude drug “mao”, and chemically synthesized pseudoephedroxane have been reported to have anti-inflammatory activities. For example, these alkaloids suppressed carrageenin-induced hind-paw edema formation in mice. To elucidate the mechanism of the antiinflammatory activities, the effects of “mao” alkaloids on arachidonic acid release and its metabolism in rat peritoneal macrophages stimulated with zymosan particles were examined. When macrophages were stimulated with zymosan, arachidonic acid release and production of arachidonate metabolites such as prostaglandin (PG) E2, 6-keto-PGF1α, PGF2α, LTB4, and 12-HETE were enhanced. By treatment with dexamethasone, the releace of radioactivity was suppuressed. These four alkaloids also inhibited arachidonic acid release and PGE2 production.These results suggest that a part of the anti-inflammatory activities by “mao” alkaloids is attributable to their inhibitory effects on arachidonate metabolism.

Anti-inflammatory activities by glucocorticoids.

A review on the mechanism of anti-inflammatory actions by glucocorticoids, especially focused on the anti-inflammatory protein proposed by Tsurufuji which includes lipocortin, vasoregulin and vasocortin. A prospect concerning on these proteins is discussed.