Further characterization of memory T cells existing in a case of CD8 deficiency

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4+ T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from "naive" to "memory." In this study, we further characterized CD45RO+ T cells in a CD8 deficient patient. We showed that the patient's CD45RO+ T cell population had a wide variety of T cell receptor Vbeta-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient's T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient's CD45RO+CD4+ T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.     

Epithelioid leiomyosarcoma of the external deep soft tissue

Epithelioid leiomyosarcoma in the external deep soft tissue is extremely rare. Most epithelioid leiomyosarcomas occur in the uterus. We present a case of epithelioid leiomyosarcoma occurring in the muscle of the thigh of a 78-year-old man. Histologically, the tumor predominantly consisted of round or polygonal cells arranged in sheets with a focal spindle cell component. Immunohistochemical analysis revealed that the tumor cells expressed vimentin, alpha-smooth muscle actin, and alpha-sarcomeric actin. The tumor was negative for desmin, S100 protein, glial fibrillary acidic protein, pan-keratin, epithelial membrane antigen, CAM 5.2, HMB-45, leukocyte common antigen, factor VIII-associated antigen, and CD34. Electron microscopically, some tumor cells contained abundant actin-type filaments in their cytoplasm.     

Anin vitro invasion model for human renal cell carcinoma cell lines mimicking their metastatic abilities

We developed a modifiedin vitro invasion assay system using monolayers of vascular endothelial cells. A type I collagen gel was formed in plastic dishes, and overlaid with type IV collagen. Calf pulmonary arterial endothelial (CPAE) cells were seeded onto these plates, and incubated until they reached confluence. Five human renal cell carcinoma cell lines with various metastatic potentialsin vivo were then seeded on the monolayer CPAE cells, and their colony formation and invasion activities were examined for 9 days. At day 4, the highly metastatic cell lines increased the number of colony foci on monolayer CPAE cells several fold higher than their poorly metastatic counterpart. The horizontal spreading patterns were also different between poorly and highly metastatic cell lines. On day 9, the number of carcinoma foci that penetrated the monolayer of CPAE cells and type IV collagen sheets into type I collagen gels in highly metastatic cell lines greatly increased as compared with that of poorly metastatic cell lines. Ourin vitro invasion assay using monolayer CPAE cells would be useful to evaluate protease activities and colony formation during invasion.     

Effects of dipyridamole on Plasmodium falciparum-infected erythrocytes

This study assessed the antimalarial activity of dipyridamole, a well-known vasodilator and inhibitor of platelet aggregation. Dipyridamole was effective against all of the erythrocytic stages such as rings, trophozoites and schizonts, and induced ultrastructural changes during the transition from trophozoite to schizont in vitro. Merozoites were also inhibited from invading dipyridamole-treated erythrocytes. It seems that dipyridamole binds to the erythrocyte membrane blocking the receptors for the merozoite. The 50% inhibitory concentration (IC(50)) of dipyridamole against Plasmodium falciparum infection was 30 nM. The IC(50) of chloroquine decreased from 97.0 nM to 13.7 nM when combined with dipyridamole (0.1 nM). Therefore, we suggest that dipyridamole has antiplasmodial activity due to its ability to arrest parasite development and by inhibiting merozoite invasion of the erythrocytes. Chloroquine activity against P. falciparum is also enhanced by the addition of dipyridamole. Treatment with a combination of chloroquine and dipyridamole may lead to a more effective treatment for chloroquine-resistant strains of P. falciparum.     

Non-Polarized Secretion of Mouse Interferon-β from Gene-Transferred Human Intestinal Caco-2 Cells

Purpose. The intestinal epithelium is considered to be a feasible target for somatic gene therapy. To this end, Caco-2 cells derived from human colon carcinoma were transfected with a mouse interferon- (IFN-) expression vector and several stable sublines were established; this hetero-specific cytokine allows unexpected cellular effects to be avoided. Using the highest mouse IFN--producing sublines, the mode of IFN secretion was examined. Methods. The secretion polarity of mouse IFN- in its gene-transduced Caco-2 sublines was studied in a bicameral culture system in which the chambers were separated by microporous filters. Results. Mouse IFN- was secreted to the same extent from both apical and basolateral surfaces of the transduced cells regardless of cell aging. Conclusions. These results suggest that in the intestinal epithelium exogenous gene products such as IFNs can be delivered to both the luminal and blood sides in vivo. Thus, the intestinal epithelium may be suitable for systemic and local delivery of therapeutic proteins by gene transfer.     

Activation of trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene to genotoxic metabolites by rat and human cytochromes P450

In order to address the hypothesis that 6-aminochrysene (6-AC)is converted to genotoxic products by cytochrome P450 enzymesvia two activation pathways (N-hydroxylation and epoxidation),the activation of 6-AC and trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene(6-AC-diol) to genotoxic metabolites was examined in rat andhuman liver microsomal cytochrome P450 enzymes using Salmonellatyphimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009)as tester strains. The latter bacteria, an O-acetyl-transferase-overexpressingstrain, was highly sensitive to metabolites derived from activationof 6-AC, but not those from 6-AC-diol, using liver microsomesfrom phenobarbital-treated rats or a reconstituted monooxygenasesystem containing P4502B1 or -2B2, thus suggesting the rolesof P450 and acetyltransferase systems in the activation process.6-AC-diol, on the other hand, was activated very efficientlyby liver microsomes prepared from ß-naphthoflavone-treatedrats or a reconstituted system containing P4501A1 or -1A2; theactivation reaction is considered to proceed through diol-epoxideformation. The contribution of rat P4501A enzymes towards activationof 6-AC-diol was confirmed by the inhibitory effects on theactivation process of -naphthoflavone, a specific inhibitorof P4501 A-related activities, and antibodies raised againstpurified P4501A1 and -1A2. In humans, P4501A2 was found to bethe major enzyme involved in the activation of 6-AC-diol togenotoxic metabolites while the parent compound 6-AC was activatedmainly by P4503A4. Experiments using recombinant P450 proteinsexpressed in human lymphoblastoid cells lines showed that humanP4501A1 could also activate 6-AC-diol to reactive metabolitesat almost the same rate measured with P4501A2. In addition,P4502B6 was found to efficiently catalyze the activation of6-AC to genotoxic metabolites, and P4503A4 was active in theactivation of 6-AC-diol as well as 6-AC. Addition of purifiedrat epoxide hydrolase to the incubation mixture containing purifiedrat P4501A1 or microsomes expressing human P4501A1 caused inhibitionof activation of 6-AC-diol. These results suggest the existenceof different enzymatic activation pathways for 6-AC and 6-AC-diol.The former carcinogen may be N-hydroxylated principally by P4502Benzymes in rats and P4503A4 and -2B6 in humans and activationto its ultimate metabolites may proceed through esterificationof the N-hydroxy metabolites by an N-acetyltransferase. The6-AC-diol is metabolized to its ultimate diolepoxide productby P4501A enzymes in rat and human liver microsomes. P4503A4(humans) and P4503A2 (rats) may also contribute to some extentin the activation of 6-AC-diol, albeit at lower rates than thoseof P4501A enzymes.     

Epidural fentanyl improves the onset and spread of epidural mepivacaine analgesia

Purpose

To determine the extent of enhanced blockade by the combined use of epidural fentanyl and mepivacaine. We compared the onset of hypoalgesia, analgesia and the threshold of pressure pain.

Methods

Thirty patients were randomly divided into three groups. The fentanyl group received 10 ml saline containing 0.1 mg fentanyl, mepivacaine group received 10 ml mepivacaine 1% and a mixed group received 10 ml mepivacaine 1% with 0.1 mg fentanyl. All solutions, without epinephrine, were injected through an epidural catheter at T_5–6 to T_6–7. The change in sensation, loss of pin-prick and pain threshold sensation, measured by pressure algometer, were assessed at 2.5-min intervals for 15 min at the T₄ dermatome. Spread of analgesia was determined at 15 min.

Results

Loss of pinprick was more rapid in the mixed, 11.0 ± 2.7 (SD) min, than in the mepivacaine group, 15.0 ± 2.9 min, (P < 0.05), although there was no difference in change of sensation. Pressure pain threshold increased with time in the mepivacaine (P < 0.05) and mixed (P < 0.05) groups. It was higher in the mixed than in the fentanyl and mepivacaine groups at 5, 7.5 and 10 min (P < 0.05). The lower level of analgesia was lower in the mixed than in the mepivacaine groups (P < 0.05). Blood pressure was unchanged in the three groups, but heart rate decreased at 7.5, 10, 12.5, and 15 min in the mepivacaine and mixed groups (P < 0.05).

Conclusions

The addition of fentanyl to mepivacaine accelerates the onset of analgesia and enhances the analgesic effect of epidural block.     

In vitro metabolism of fenthion and fenthion sulfoxide by liver preparations of sea bream, goldfish, and rats.

The in vitro metabolism of fenthion and its sulfoxide (fenthion sulfoxide) in sea bream (Pagrus major) and goldfish (Carassius auratus) was investigated and compared with that in rats. Fenthion was oxidized to fenthion sulfoxide and the oxon derivative, but not to its sulfone, in the presence of NADPH by liver microsomes of sea bream, goldfish, and rats. These liver microsomal activities of the fish were lower than those of rats but were of the same order of magnitude. The NADPH-linked oxon- and sulfoxide-forming activities of liver microsomes of the fish and rats were inhibited by SKF 525-A, metyrapone, alpha-naphthoflavone, and carbon monoxide. The oxidizing activity to fenthion sulfoxide was also inhibited by alpha-naphthylthiourea. Several cytochrome P450 isoforms and flavin-containing monooxygenase 1 exhibited these oxidase activities. Fenthion sulfoxide was reduced to fenthion with liver cytosol of the fish and rats upon addition of 2-hydroxypyrimidine, N(1)-methylnicotinamide, or butyraldehyde, each of which is an electron donor of aldehyde oxidase, under anaerobic conditions. The activity was inhibited by menadione, beta-estradiol, and chlorpromazine, which are inhibitors of aldehyde oxidase. The activities in the fish livers were similar to those of rat liver. Aldehyde oxidase purified from the livers of sea bream and rats exhibited the reducing activity. Thus, fenthion and fenthion sulfoxide are interconvertible in fish and rats through the activities of cytochrome P450, flavin-containing monooxygenase, and aldehyde oxidase.