Hemocytes of Pomacea canaliculata: I. Reversible aggregation induced by Ca2+

Using a platelet aggregometer, factors involved in hemocyte aggregation of a prosobranch snail Pomacea canaliculata were investigated. Aggregation-inducing tests revealed that extracellular Ca2+ is required to provoke the cellular response which is reversible. The aggregation-dispersion response can be induced by adding Ca2+ and EDTA alternately. Aggregation was inhibited by a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in a dose-dependent manner, and by a protein kinase-C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7). Thus, calmodulin and protein kinase-C may contribute to the calcium-mediated aggregation of hemocytes, which resembles a phenomenon that occurs in vertebrate leucocytes. Unlike the aggregation of mammalian leucocytes, no stimulus other than Ca2+ is required to induce the response of Pomacea hemocytes. Thus, this system may serve as a simple model for analysing cellular aggregation responses.     

Antibacterial effect of bovine milk antibody against Escherichia coli in a mouse indigenous infection model

A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the mouse intestine. The antibacterial effect of these fractions against E. coli was examined. They contained antibody with a high affinity for E. coli strain 48, a representative strain in the mouse intestine, which is composed of a large amount of IgG and smaller amouns of IgA and IgM. Although these fractions showed no bactericidal or bacteriostatic activity against E. coli 48 directly in vitro, they exhibited strong agglutination and opsonization activities against the bacteria in vitro. The bacteria opsonized with the WPC fraction were taken up more effectively by liver macrophages in vivo, compared with unopsonized E. coli, after an intravenous injection into mice. Oral administration of the skim-milk fraction to mice significantly reduced the susceptibility to the lethal toxicity of 5-fluorouracil (5 FU). The increase in the population levels of E. coli in the intestinal tract after administration of 5 FU was inhibited by oral administration of the skimmilk fraction. These results strongly suggest that specific antibody may be effective in the prophylaxis against the indigenous infection with gram-negative bacteria such as E. coli after a period of chemotherapy in cancer patients.     

MEMBRANOUS LIPODYSTROPHY

Membranous lipodystrophy is a peculiar systemic disease almost exclusively found in Japan and Scandinavia and characterized by formation of numerous membranous structures in the adipose tissue of bone marrow and soft tissue. Light and electron microscopic study of a biopsy case of the disease was presented. The membranous structures consisted of membrane and inner space on both light and electron microscopic study. The membrane consisted of two kinds of layers, dense and amorphous. The dense layer was positive for lipid stainings. The amorphous layer did not show any definite lipld constituents and was supposed to consist of an amorphous substance derived from abnormal fat metabolism of diseased fat cells, degenerative cytoplasms of the diseased cells, and/or stromal cells. It probably also had some stromal substance alteration due to membranolipodystrophic change. Accumulation of the amorphous layer in the interstitium leads to degeneration and disappearance of adjacent fat cells. This process may play a role in further advance of membranous structures.     

Intracellular Carboxyl Esterase Activity Is a Determinant of Cellular Sensitivity to the Antineoplastic Agent KW-2189 in Cell Lines Resistant to Cisplatin and CPT-11

KW-2189, a novel antitumor antibiotic belonging to the duocarmycins, possesses marked DNA-binding activity upon activation by carboxyl esterase to its active form, DU-86. Three duocarmycins, KW-2189, DU-86 and duocarmycin SA, were active against the cisplatin (CDDP)-resistant human non-small cell lung cancer cell lines PC-9/CDDP and PC-14/CDDP, and the multidrug-resistant human small cell lung cancer cell line H69/VP. However, HAC2/0.1, a CDDP-resistant human ovarian cancer cell line which is also resistant to CPT-11 because of decreased intracellular activation of CPT-11, was about 12.8-fold more resistant to KW-2189. HAC2/0.1 was not resistant to other duocarmycins as compared to its parental cell line, HAC2. There was no difference between HAC2 and HAC2/0.1 with regard to the intracellular accumulation of KW-2189. Addition of 130 mU/ml of carboxyl esterase to the culture medium did not influence the sensitivity of HAC2 cells to KW-2189. However, the sensitivity of HAC2/0.1 cells to KW-2189 was enhanced to the level of HAC2. These results suggest that HAC2/0.1 is less potent than HAC2 in activating KW-2189. The carboxyl esterase activity of whole-cell and microsomal extracts from HAC2/0.1 was approximately 60% of that from HAC2. The cell-free experiment revealed that KW-2189 bound to DNA more efficiently in the presence of HAC2 than HAC2/0.1 cell extract. It was concluded that decreased intracellular carboxyl esterase activity in HAC2/0.1 cells caused decreased intracellular conversion of KW-2189 to its active form, thus producing resistance to KW-2189. The decreased conversion of CPT-11 to SN-38 in HAC2/0.1 cells might be explained by decreased carboxyl esterase activity.     

Protective role of metallothionein in renal toxicity of cisplatinum

To elucidate the protective role of metallothionein (MT) in the prevention of cisplatinum (cis-DDP) toxicity, we investigated the lethal and renal toxicities caused by cis-DDP in MT-null transgenic mice in comparison with wild-type control mice, and examined the effect of pretreatment with bismuth nitrate or zinc sulfate on the cis-DDP nephrotoxicity. The MT-null mice were of mixed 129 Ola and C57BL/6 genetic background. Since no differences in cis-DDP nephrotoxicity were observed between these strains, C57BL/6J mice were used as the wild-type control. The basal MT levels in the kidneys were negligible in the MT-null mice and 2.93 ± 0.77 μg/g tissue in the C57BL/6J mice. In terms of both the lethal and renal toxicities of cis-DDP, MT-null mice were far more sensitive than C57BL/6J mice. Preinduction of renal MT synthesis by administration of bismuth nitrate or zinc sulfate protected C57BL/6J mice from cis-DDP nephrotoxicity. In the case of MT-null mice, however, renal MT could not be induced by pretreatment with these metal compounds, and renal toxicity of cis-DDP was not prevented by this pretreatment. These results suggest that MT is an important factor with the potential to suppress the development of cis-DDP toxicity. Received: 17 June 1996 / Accepted: 27 November 1996     

Enhanced Topoisomerase I Activity and Increased Topoisomerase IIα Content in Cisplatin-resistant Cancer Cell Lines

Although the combined effects of cisplatin (CDDP) and DNA topoisomerase (Topo) inhibitors have been described in recent literature, little is known about the combined effects and their biological basis in CDDP-resistant cells. The aim of the present study was to elucidate the combined effect of CDDP and Topo inhibitors on CDDP-resistant cells as well as to investigate the biological factors involved in the sensitivity to these anti-cancer agents. We found synergistic actions between CDDP and SN-38 (a Topo I inhibitor) or VP-16 (a Topo II inhibitor) in KFr cells, a CDDP-resistant subline of the KF epithelial ovarian carcinoma cell line, but not in the parent KF cells. We subsequently assayed Topo protein levels and enzymatic activities in two sets of CDDP-sensitive and -resistant cell lines: KF and KFr, and HeLa and HeLa/CDDP. The levels of Topo I protein in the CDDP-resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to CDDP. Topo I enzymatic activity, however, was 2- to 4-fold higher in the CDDP-resistant cell lines than in their respective parent cell lines. In contrast, higher levels of Topo lice protein were observed both before and after CDDP exposure in the CDDP-resistant cells than in their controls. However, no difference in Topo II catalytic activity was observed between the CDDP-resistant and -sensitive cells.     

Maintenance and Characterization of an Epstein Barr Virus-infected CD56-negative T Cell Lymphoma

T cell lymphoma carrying Epstein Barr virus (EBV⁺TL) is very rare among Western countries while it is much more common among Japanese. Here we report an EBV⁺TL which has been maintained for years by the use of mice with severe combined immune deficiency (SCID) mice. Lymphoma was obtained from a 55-year-old male suffering from oculomotor nerve palsy and lymphadenopathy. A small piece of biopsied tumor was transplanted into SCID mice and the lymphoma has been maintained for over 3 years with passages every 2–3 weeks. The maintained lymphoma, termed as TMS24, and the original lymphoma cells showed identical phenotype and genotype, including diffuse medium-sized cell morphology lacking granules, suppressor/cytotoxic immunophenotype and identical T cell receptor β-chain gene rearrangement mode. Further, both were shown to carry an identical EBV clone in terms of the number of terminal repeats and the latency II-type restricted gene expression profile. Cytogenetically, TMS24 retained two characteristic chromosomal translocations of t(l;18)(q32;q21) and t(6;12)(p21;q24). Since only one cell line with such characters has been reported previously, TMS24 should be useful for detailed analysis of EBV⁺TL.     

Ileal duplication presenting as perforation: Report of a case

(Received for publication on Apr. 9, 1999; accepted on Nov. 11, 1999)     

Immunostimulating properties of intragastrically administered Acetobacter-derived soluble branched (1,4)-beta-D-glucans decrease murine susceptibility to Listeria monocytogenes

We previously found that AC-1, an extracellular polysaccharide, produced by Acetobacter xylinum and composed of (1,4)-beta-D-glucan with branches of glucosyl residues, showed a strong activity to induce production of interleukin-12 (IL-12) p40 and tumor necrosis factor alpha by macrophages in vitro via Toll-like receptor 4 (TLR-4) signaling. In the present study, we examined the effect of oral administration of AC-1 on protective immunity against Listeria monocytogenes. Mice were given AC-1 or phosphate-buffered saline (PBS) intragastrically 2 days before, on the day of, and 2 days after an intraperitoneal inoculation of L. monocytogenes. The survival rate of AC-1-treated mice was significantly improved and bacterial growth in AC-1-treated mice was severely retarded compared to those of PBS-treated mice after infection with L. monocytogenes. IL-12 p40 levels in serum and magnitudes of CD4+ Th1 and CD8+ Tc1 responses against Listeria antigen were significantly higher in AC-1-treated mice than in PBS-treated mice. The effect of AC-1 on antilisterial activity was diminished in C3H/HeJ mice carrying mutated TLR-4. Thus, AC-1, a potent IL-12 inducer through TLR-4, enhanced protective immunity against L. monocytogenes via augmentation of Th1 responses. These results suggest that infectious processes driven by intracellular microorganisms could be prevented to develop by the (1,4)-beta-D-glucan.     

Possible roles of cAMP and Ca2+ in the regulation of miracidial transformation inSchistosoma mansoni

The triggering action of physiological saline in the miracidial transformation ofSchistosoma mansoni was analyzed using various agents affecting cAMP-and Ca²⁺-dependent pathways. Potent activators of adenylate cyclase such as forskolin and serotonin, strongly inhibited the transformation provoked by saline in RPMI-1640. These inhibitory actions were diminished by the combined administration of phosphodiesterase activators such as ammonium salts or imidazole. Furthermore the exposure of miracidia to ammonium salts or imidazole in dechlorinated tap water mimicked the transformation, i.e., the cessation, of swimming and then shedding of epithelial plates. This mimic transformation was also inhibited by serotonin or forskolin. In contrast, treatment of miracidia with Ca²⁺ antagonists such as TMB-8 (an inhibitor of Ca²⁺ release), nicardipine (a Ca²⁺ channel blocker), or W-7 (a calmodulin inhibitor) in tap water produced severe vesiculation on their body surfaces and resulted in death. However, these toxic effects were abolished by a combined administration of these Ca²⁺ antagonists with saline or NH₄Cl, and the transformation was reestablished except with W-7 treatment. W-7 strongly inhibited the triggering action of saline and NH₄Cl and the worms swam slowly, whereas W-5, an inactive analogue of W-7, had no inhibitory effect on the transformation. These results suggest that the initiation of micracidial transformation to young sporocysts may be synergistically, regulated by cAMP and Ca²⁺ and that a decrease in cAMP levels and an increase in Ca²⁺ mobilization may be provoked in worms transformed by saline, ammonium salts, or imidazole.Abbreviations Nicardipine 2-(N-bentyl-N-methylamino)ethylmethyl-2,6-dimethyl-4-(m-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate - TMB-8 8-(N,N-diethyl amino)-octyl-3,4,5-trimethoxybenzoate - TPA 12-0-tetradecanoyl-phorbol-13-acetate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide