Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

Exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in hearts

Exercise training improves aging-induced deterioration of angiogenesis in the heart. However, the mechanisms underlying exercise-induced improvement of capillary density in the aged heart are unclear. Vascular endothelial growth factor (VEGF) is implicated in angiogenesis, which activated angiogenic signaling cascade through Akt and endothelial nitric oxide synthase (eNOS)-related pathway. We hypothesized that VEGF angiogenic signaling cascade in the heart contributes to a molecular mechanism of exercise training-induced improvement of capillary density in old age. With the use of hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and exercise-trained aged rats (23 mo old, swim training for 8 wk), the present study investigated whether VEGF and VEGF-related angiogenic molecular expression in the aged heart is affected by exercise training. Total capillary density in the heart was significantly lower in the sedentary aged rats compared with the sedentary young rats, whereas that in the exercise-trained rat was significantly higher than the sedentary aged rats. The mRNA and protein expressions of VEGF and of fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk-1), which are main VEGF receptors, in the heart were significantly lower in the sedentary aged rats compared with the sedentary young rats, whereas those in the exercise-trained rats were significantly higher than those in the sedentary aged rats. The phosphorylation of Akt protein and eNOS protein in the heart corresponded to the changes in the VEGF protein levels. These findings suggest that exercise training improves aging-induced downregulation of cardiac VEGF angiogenic signaling cascade, thereby contributing to the exercise training-induced improvement of angiogenesis in old age.     

Bloom helicase and DNA topoisomerase IIIalpha are involved in the dissolution of sister chromatids

Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.     

In vivo electrogene transfer of interleukin-12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas

BACKGROUND: Human breast cancer metastasizes mainly to lymph nodes, lungs, liver, and bone; in the majority of cases, it is the development of metastases which leads to death. In order to suppress mammary cancer metastasis, we applied in vivo electrogene transfer (non-viral method) as a means of interleukin-12 (IL-12) gene therapy on highly metastatic murine mammary cancer model. METHODS: Metastatic mammary tumors induced by inoculation in BALB/c female mice were treated by intratumoral injections of either a plasmid vector containing IL-12 or empty vector and then subjected to in vivo electrogene transfer once a week for 8 weeks. RESULTS: Treatment with IL-12 resulted in elevation of both IL-12 and IFNgamma levels in mammary tumors and in serum and intratumoral levels of CD4 and CD8 proteins were also increased. Tumor volumes and lymphatic and pulmonary metastases were significantly reduced. The histopathological changes induced by IL-12 characteristically included marked inflammation, increased apoptosis, decreased DNA synthesis, peripheral influx of significantly greater numbers of active macrophages, and reduced blood microvessel density, and apoptotic vascular endothelial cells were frequently seen. Western blotting showed decreases in VEGFR-3 of tumors exposed to IL-12 gene therapy. In adjuvant immunofluorescence studies, the CD31-positive endothelial cells of microvessels showed decreased VEGFR-3 expression in IL-12-treated tumors. However, apparent alterations in VEGFR-3 expression of podoplanin-positive lymphatic endothelial cells were not observed in IL-12-treated tumors. Although recombinant IL-12 did not inhibit tubular formation of human umbilical vein endothelial cells in a Matrigel assay, recombinant IFNgamma did completely suppress the tubular formation. CONCLUSIONS: In vivo electrogene transfer of IL-12 exerts strong anti-tumorigenic and anti-metastatic effects likely due to T-cell-mediated immune responses as well as anti-angiogenic action.     

Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.     

Differential expression of co-signal molecules and migratory properties in four distinct subsets of migratory dendritic cells from the oral mucosa

Variations in co-signal ligand expression and cytokine production greatly influence the antigen-presenting properties of migrating DCs in regional lymph nodes (RLNs). Here we investigated DCs migrating from the oral mucosa using CD326 and CD103 antigens for discriminate CD207⁺ Langerhans cells (LCs) from CD207⁺ submucosal DCs (SMDCs). Similar to DCs migrating from the skin, we identified four distinct oral mucosal DC (OMDC) subsets, CD11c^hiCD207⁻CD103⁻CD326^intCD11b^hi (F1; resident CD11b^hi SMDCs), CD11c^int/loCD207^-CD103^-CD326^loCD11b^int/hi (F2; newly recruited blood-derived SMDCs), CD11c^int/loCD207⁺CD103⁺CD326^int/hiCD11b^lo (CD103⁺ F3; resident CD207⁺ SMDCs), and CD11c^int/loCD207⁺CD103^-CD326^int/hiCD11b^lo (CD103^- F3; resident LCs). F1 DCs migrated rapidly after fluorescein isothiocyanate (FITC) painting and expressed notably high levels of CD86, CD273, and CD274 at an earlier time point. In contrast, CD103⁻ LCs expressing the highest levels of the epithelial cell adhesion molecule CD326 accounted for a minor subset at the earlier time point, but increased slowly with CD103⁺CD207⁺ SMDCs. However, their expression of CD86, CD273, and CD274 was very limited. The delayed migration and limited induction of co-signal ligands suggest that roles of OMLCs are distinct from those of the other three DC subsets. The identification of distinct subsets of OMDCs in RLNs may benefit efforts to determine the functional specialization of each subset in T cell responses against orally administrated antigens.     

The developmental process of xylem embolisms in pine wilt disease monitored by multipoint imaging using compact magnetic resonance imaging

In pine wilt disease (PWD), embolized tracheids arise after virulent pine wood nematodes (PWN), Bursaphelenchus xylophilus, invade the resin canal of pine tree; infected pine trees finally die from significant loss of xylem water conduction. We used a compact magnetic resonance imaging system with a U-shaped radio frequency (rf) probe coil to reveal the developmental process of the xylem dysfunction in PWD. Multiple cross-sectional slices along the stem axis were acquired to periodically monitor the total water distribution in each 1-year-old main stem of two 3-year-old Japanese black pines (Pinus thunbergii) after inoculation of PWN. During the development of PWD, a mass of embolized tracheids around the inoculation site rapidly enlarged in all directions. This phenomenon occurred before the significant decrease of water potential. Some patch-like embolisms were observed at all monitoring positions during the experimental period. Patchy embolisms in a cross-section did not expand, but the number of patches increased as time passed. When the significant decrease of water potential occurred, the xylem dysfunctional rate near the inoculation point exceeded 70%. Finally, almost the whole area of xylem was abruptly embolized in all cross-sections along the stem. This phenomenon occurred just after water conduction was mostly blocked in one of the cross-sections. Thus, it appears that the simultaneous expansion of embolized conduit clusters may be required to induce a large-scale embolism across the functional xylem. Consequently, xylem dysfunction in infected trees may be closely related to both the distribution and the number of PWN in the pine stem.     

Discovery of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P₁ receptor agonist with high selectivity against S1P₃ and enhanced efficacy in lowering peripheral lymphocyte counts in mice.     

Expression of the GABAA receptor/chloride channel in murine spermatogenic cells

Previous studies from our laboratory have demonstrated that γ-aminobutyric acid (GABA) and GABAB receptor subunits are expressed within the acrosome of spermatids during spermiogenesis. Furthermore, our previous study with the glutamate decarboxylase (GAD) 67-GFP knock-in mouse demonstrated that GFP-positive cells were localized to the epithelium of the caput of epididymis. In the present study, we detected GABAA subunits, including α1, α5, β1-3 and γ3, and both isoforms of GAD, GAD65 and GAD67, in mouse spermatogenic cells using RT-PCR. The expression of these proteins was subsequently confirmed by western blot analysis. Immunohistochemistry also revealed that GABA, GAD65, and α5, β1 and γ3 subunits of the GABAA receptor were localized in the membrane of spermatogenic cells, including spermatocytes and spermatids. The whole-cell patch-clamp analysis demonstrated that GABA application induced an inward chloride current in some of the large and round spermatogenic cells. Our findings show that spermatogenic cells have a GABA producing system by themselves, and that GABA may function via the ionotropic GABAA receptor. This data suggests that the GABAergic system may play important roles in the male reproductive system.