Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA−CD8+ T cells with different CD28/CD27 phenotypes showed that CD28+CD45RA−CD8+ and CD27+CD45RA−CD8+ T cells have characteristics of memory T cells, whereas CD28−CD45RA−CD8+ and CD27−CD45RA−CD8+ T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8+ T cells into memory/effector CD8+ T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8+ T cells. Three subsets of CD45RA−CD8+ T cells were observed in both total CD8+ T cells and EBV- or HCMV-specific CD8+ T cells: CD27+CD28+, CD27+CD28−, and CD27−CD28−. A significant number of the CD27−CD28+ subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8+ T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27+CD28+CD45RA−→CD27+CD28−CD45RA−→CD27−CD28−CD45RA−. This was supported by the observation that the frequency of CCR5+ cells and CCR7+ cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27+CD28+ memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5−CCR7+, CCR5+CCR7+, and CCR5+CCR7−. These findings suggested the following differentiation pathway: CD27+CD28+CD45RA− (CCR5−CCR7+→CCR5+CCR7+→CCR5+CCR7−)→CD27+CD28−CD45RA−→CD27−CD28−CD45RA−. The presence of a CD27−CD28+ subset with a CCR5+CCR7− phenotype implies a specialized role for this subset in the differentiation of CD8+ T cells.

Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42°C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor β subunit (IRβ) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12–15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway.

tiffness of large arteries has been related to cardiovascular mortality, but the mechanisms underlying this relationship have not been established. 1 Methods are used to estimate this stiffness include carotid ultrasound (CU) and pulse-wave-velocity (PWV). 2 Carotid artery stiffness detected by CU is known to representative of systemic arteriosclerosis. The measurement of PWV is very useful for diagnosing arteriosclerosis in any part of the body 3-7 and a new method for measuring PWV has been proposed in Japan. Brachial -ankle PWV (baPWV) measures the PWV in the arm and leg by applying air pressure using the volume plethysmographic method. However, baPWV is reportedly influenced by several factors such as blood pressure (BP), autonomic nerve function etc and therefore does not reflect arteriosclerosis in some cases.The stiffness parameter is reported to be independent of BP. 8 Beta of the thoracic descending aorta (TDA) has been obtained previously only by transesophageal echocardiography (TEE), 9 but recently this problem has been solved with the advent of the cardio-ankle vascular index (CAVI). CAVI is a new parameter that is also independent of BP 10-12 and in the present study, we examined the accuracy and usefulness of CAVI and compared it with other parameters of arteriosclerosis, using CU and serum lipids measurement in patients with chest pain syndrome. Methods Principle of CAVI and Method of MeasurementCAVI was obtained by substituting the stiffness parameter in the following equation for determining vascular elasticity and PWV. The stiffness parameter indicates BPindependent patient-specific vascular stiffness measured by arterial US. The stiffness parameter is calculated as: (1) where Ps and Pd are respectively the systolic and diastolic BP in mmHg. D is the diameter of the blood vessel and ∆D is the change of D.Bramwell-Hill's formula expresses the relationship between volume elastic modulus and PWV as follows:where ∆P is pulse pressure, is blood density, V is the volume of the blood vessel and ∆V is the change of V.From equation (2), the following formula is derived:where D is the diameter of the blood vessel and ∆D is the change of D. If we substitute equation (3) for equation (1), we obtain the stiffness parameter:CAVI is measured as follows. PWV is obtained by dividing vascular length (L) by the time (T) taken for the pulse wave to propagate from the aortic valve to the ankle Circ J 2007; 71: 1710 -1714 (Received February 15, 2007 revised manuscript received June 20, 2007; accepted July 4, 2007) Division of Cardiology, Tokuyama Central Hospital, Shunan, *Division Methods and ResultsThe purpose of this study was to evaluate the accuracy and usefulness of CAVI and to compare it with other parameters of arteriosclerosis by carotid ultrasound (CU). The instantaneous dimensional change of the TDA on TEE was measured simultaneously with systemic pressure of the brachial artery in 70 patients in sinus rhythm. There were significant correlations between CAVI and age (r=0.65, p<0.01), and CAVI and...

Phenotypic classification of human CD8 + T cells using three cell surface markers, CD27, CD28 and CD45RA, was recently suggested to be useful for identification of naive, memory and effector CD8 + T cells. However, it still remains unclear whether such classification precisely reflects functional classification of CD8 + T cells. To clarify this, we characterized each CD27CD28CD45RA subset of total and human cytomegalovirus (HCMV)-specific CD8 + T cells by analyzing the expression of perforin and two chemokine receptors, CCR5 and CCR7, as well as their function. An inverse correlation between perforin and CD27 expression was found in all four CD28CD45RA subsets. Therefore, to achieve a phenotypic classification of CD8 + T cells that more precisely reflects their function, the CD27 + subset was divided into CD27 low and CD27 high subsets based on the expression level of CD27. Functional and flow cytometric analyses of CD27CD28CD45RA subsets showed that this phenotypic classification reflects functional classification of CD8 + T cells. HCMV-specific CD8 + T cells from healthy HCMV-seropositive individuals were predominantly found in effector and memory/effector subsets, indicating that HCMV-specific effector CD8 + T cells are actively induced by HCMV replication in healthy HCMV carriers. Phenotypic analyses of CD8 + T cells using this classification will enable the characterization of antigen-specific CD8 + T cells.

ObjectivesTo collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis.MethodsFifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians.ResultsThe study population comprised 26 males and 24 females aged 0–61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment.ConclusionsIn patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

Gravity acts on the circulatory system to decrease arterial blood pressure (AP) by causing blood redistribution and reduced venous return. To evaluate roles of the baroreflex and vestibulosympathetic reflex (VSR) in maintaining AP during gravitational stress, we measured AP, heart rate (HR), and renal sympathetic nerve activity (RSNA) in four groups of conscious rats, which were either intact or had vestibular lesions (VL), sinoaortic denervation (SAD), or VL plus SAD (VL + SAD). The rats were exposed to 3 G in dorsoventral axis by centrifugation for 3 min. In rats in which neither reflex was functional (VL + SAD group), RSNA did not change, but the AP showed a significant decrease (-8 +/- 1 mmHg vs. baseline). In rats with a functional baroreflex, but no VSR (VL group), the AP did not change and there was a slight increase in RSNA (25 +/- 10% vs. baseline). In rats with a functional VSR, but no baroreflex (SAD group), marked increases in both AP and RSNA were observed (AP 31 +/- 6 mmHg and RSNA 87 +/- 10% vs. baseline), showing that the VSR causes an increase in AP in response to gravitational stress; these marked increases were significantly attenuated by the baroreflex in the intact group (AP 9 +/- 2 mmHg and RSNA 38 +/- 7% vs. baseline). In conclusion, AP is controlled by the combination of the baroreflex and VSR. The VSR elicits a huge pressor response during gravitational stress, preventing hypotension due to blood redistribution. In intact rats, this AP increase is compensated by the baroreflex, resulting in only a slight increase in AP.

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domaincontaining adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.

The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.