A nonsynonymous polymorphism in the human fatty acid amide hydrolase gene did not associate with either methamphetamine dependence or schizophrenia

Genetic contributions to the etiology of substance abuse and dependence are topics of major interest. Acute and chronic cannabis use can produce drug-induced psychosis resembling schizophrenia and worsen positive symptoms of schizophrenia. The endocannabinoid system is one of the most important neural signaling pathways implicated in substance abuse and dependence. The fatty acid amide hydrolase (FAAH) is a primary catabolic enzyme of endocannabinoids. To clarify a possible involvement of FAAH in the etiology of methamphetamine dependence/psychosis or schizophrenia, we examined the genetic association of a nonsynonymous polymorphism of the FAAH gene (Pro129Thr) by a case-control study. We found no significant association in allele and genotype frequencies of the polymorphism with either disorder. Because the Pro129Thr polymorphism reduces enzyme instability, it is unlikely that dysfunction of FAAH and enhanced endocannabinoid system induce susceptibility to either methamphetamine dependence/psychosis or schizophrenia.     

IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-gamma increase in normal pregnancy but decrease in spontaneous abortion

Recent data demonstrated that CD4+CD25+ regulatory T (Treg) cells and an enzyme called indoleamine 2,3-dioxygenase (IDO) mediate maternal tolerance to the fetus. Interestingly, Treg cells express the CTLA-4 molecule on their surface, and B7 (CD80/86) ligation by CTLA-4 enhanced IDO activity of dendritic cells (DCs) and monocytes by the induction of interferon gamma (IFN-gamma) production. In this study, we studied the IDO expression on peripheral blood monocytes and decidual monocytes or DCs after treatment with CTLA-4/Fc fusion protein or IFN-gamma using flow cytometry. IDO expressions on both peripheral blood DC and decidual DC and monocytes were up-regulated during normal pregnancy. On the other hand, both IDO expression on DC and monocytes after IFN-gamma treatment or CTLA-4 treatment were decreased in spontaneous abortion cases. The expression of CD86 on peripheral blood and decidual monocytes and DC in spontaneous abortion cases was lower compared with those in normal pregnancy subjects. Also, IFN-gamma production by decidual and peripheral blood mononuclear cells after CTLA-4/Fc treatment in spontaneous abortion cases was significantly lower than those in normal pregnancy subjects. These data suggest that CTLA-4 on Treg cells up-regulates IDO expression on decidual and peripheral blood DC and monocytes by the induction of IFN-gamma production.     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP

To elucidate the frequency of mutations of the β/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val → Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.     

Surface properties of LDL-binding lymphocytes in human peripheral blood.

Surface properties of low density lipoprotein (LDL)-binding lymphocytes were evaluated to determine whether LDL binds with a subpopulation of human peripheral blood lymphocytes (PBL). B- and T-cell rich fractions were prepared from PBL using E-rosette formation or nylon reticulum columns. Binding of FITC-labelled LDL with these cell fractions was determined with a fluorescent microscope and a fluorescence-activated cell sorter (FACS II). The specificity of the binding was evaluated by a dose-dependent inhibition of LDL binding with the addition of unlabelled lipoproteins. In parallel studies, surface properties including E-rosette formation, surface immunoglobulins, and receptors for IgG-Fc, as well as human and mouse C3 were examined. LDL binding lymphocytes were enriched in the B-cell rich fraction, and depleted in the T-cell rich fraction. In addition, FITC-LDL binding lymphocytes were selectively collected by the FACS II. These LDL binding cells restored surface immunoglobulins after incubation in serum-free medium following trypsinization. The majority of lymphocytes stimulated by PHA and PWM in vitro bound with LDL. It is concluded that LDL binds with B cells in fresh human PBL, while it binds with B and T cells in mitogen-stimulated lymphocytes. It is suggested that the selective collection of LDL binding lymphocytes by the FACS II can be applied to the evaluation of cellular interaction of these cells in various immunological reactions.     

Mesenteric lymphadenitis of swine caused by Rhodococcus sputi.

Rhodococcus sputi caused tuberculosislike lymphadenitis of mesenteric lymph nodes in swine. This is the first study reporting that R. sputi can be a pathogen in swine.     

Fatal necrotic enteritis associated with Clostridium perfringens in wild crows (Corvus macrorhynchos)

Sporadic outbreaks of fatal enteritis occurred among free-living wild crows (‘large billed’ or ‘wok’ crow; Corvus macrorhynchos) in an open-air park in Japan in 2002. Eight crows were found dead during February, followed by two more in September, and five of the eight were examined histopathologically. At necropsy, all cases showed a markedly dilated small intestine, especially the jejunum and ileum, with large amounts of gas, and dark red to greenish–brown soft content. The necrotic intestinal wall was markedly thickened with multifocal haemorrhages. All cases had multifocal white foci in the liver, and four cases showed marked splenomegaly. Histologically, there was severe necrotic enteritis characterized by extensive mucosal necrosis and multifocal haemorrhages, as well as inflammatory cell infiltrations. A prominent pseudo-membrane formation was noted in the affected intestine. Severe adhesive peritonitis was also observed in three cases. Gram-positive bacilli were present in large numbers in the lumen, and in and around necrotic lesions in the affected intestine. The bacilli were positive for Clostridium perfringens enterotoxin type A by immunohistochemistry, and were also positive for C. perfringens type A using the immunofluorescence method. C. perfringens was isolated by anaerobic culture from the intestinal contents. The present enteritis was thought to be induced by proliferated C. perfringens in the intestine, and to be the cause of death.     

Dissociation of interleukin-2 production from the cell activation in response to the mitogenic lectin in peripheral CD4+ T cells of LEC mutant rats.

We have recently shown that an exogenous gradient of interleukin-8 (IL-8) induces the transendothelial migration of neutrophils. Treatment of endothelium with the cytokines IL-1 or tumour necrosis factor (TNF) also causes neutrophil transmigration, and recent evidence suggests that this may be due to endogenous IL-8 produced by the endothelium. We have used specific chemotactic desensitization of neutrophils to investigate the role of IL-8 in transmigration through cytokine-activated endothelium. Preincubation of neutrophils with IL-8 reduced their chemotactic transmigration response to an IL-8 gradient by 81%, demonstrating desensitization. Transmigration in response to cytokine-activated endothelium was inhibited by 104% after IL-8 preincubation, thus tending to support the role of IL-8. However, preincubation with another neutrophil chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP), which did not affect the IL-8, response, also inhibited transmigration, by 74%. This suggests that FMLP preincubation acts to inhibit a non-IL-8-dependent mechanism of transmigration through cytokine-activated endothelium. Chemotactic factor pretreatment of neutrophils did not reduce their adhesion to activated endothelium, but specifically blocked the transmigration step. We have therefore shown that chemotactic transmigration can be subjected to factor-specific desensitization, and have used this to provide evidence supporting a role for IL-8 in transmigration through cytokine-activated endothelium, as well as suggesting a further IL-8-independent mechanism. These data also provide a mechanism for the observed defect in accumulation of neutrophils at inflammatory sites when chemotactic factors are infused intravenously.     

Pituitary Folliculo-Stellate-Like Cells Stimulate Somatotroic Pituitary Tumor Growth in Nude Mice

A new cell line (TtT/GF) established from a murine pituitary thyrotropic tumor having characteristics similar to those of pituitary folliculo-stellate cell (FS cell) was implanted into nude mice together with cells from a rat pituitary somatotrophic tumor cell line (MtT/S) to determine whether the former enhances pituitary tumor growth. For as long as 2-3 mo after implantation, MtT/S cells implanted either alone or together with fibroblasts formed either no tumors or only very small tumors in the nude mice. In contrast all of the nude mice that had received MtT/S cells implanted together with TtT/GF cells developed large tumors. Furthermore, the mice bearing the MtT/S and TtT/GF implants showed a significantly higher body weight and serum growth hormone level than those bearing only MtT/S cells or a combination of MtT/S cells and fibroblasts. The TtT/GF cell line itself had no tumorigenicity during the experimental period. Therefore, the TtT/GF cell line as a model of FS cells enhanced pituitary endocrine cell tumor formation. Additionally, immunocytochemistry showed that TtT/GF cells positive for glial fibrillary acidic protein (GFAP) or S-100 protein were present in the parenchymatous tissue elements or connective tissue surrounding the tumor nests. In the parenchymatous tissue, the TtT/GF cells exhibited a stellate appearance and surrounded neighboring tumor cells with their long cell processes. These results suggest that TtT/GF cells can serve as a model for pituitary FS cells, and are capable of stimulating pituitary tumor growth either by modifying the microenvironment or producing growth factors.