Essential role of Rap signal in pre-TCR-mediated beta-selection checkpoint in alphabeta T-cell development

We demonstrate that lck promoter-driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of alphabeta T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting gammadelta T-cell development. The effect was specific to the DN stage, because CD4 promoter-driven SPA-1 expression hardly affected T-cell development. Rap1A17, a dominant-negative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2(-/-) fetal thymocytes in vitro in the presence of anti-CD3epsilon antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3epsilon chimera), which substituted autonomous pre-T-cell receptor (TCR) signal, inducing CD69 expression and CD25 down-regulation. Reciprocally, expression of C3G, a Rap guanine nucleotide exchange factor, in both normal and Rag2(-/-) DN cells markedly enhanced Notch-dependent generation and expansion of DP cells without additional anti-CD3epsilon antibody, thus bypassing pre-TCR. Defective alphabeta T-cell development in the conditional SPA-1-transgenic mice was restored completely by introducing a p53(-/-) mutation. These results suggest that endogenous Rap GTPases downstream of pre-TCR play an essential role in rescuing pre-T cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation.     

Development of the Home Care Quality Assessment Index (HCQAI)

AIM: To develop a Home Care Quality Assessment Index (HCQAI) that may be used for overall assessment of home care in three areas: 1) conditions of the impaired elderly (outcome); 2) caregiver and caregiving situation (process); and 3) the home care environment (input). METHODS: To develop the HCQAI, a list of items for assessment was drawn up, and the reliability of each item was verified. Reliability was investigated by a) test-retest reliability, and b) inter-rater reliability. Impaired elderly and their family caregivers who used the visiting nurse station of the Okazaki Medical Association were surveyed. A kappa coefficient of 0.4 or greater generally served as the inclusion criteria for test-retest and inter-rater reliability of each item. A factor analysis was conducted for items satisfying the above criteria, using 10 scales. RESULTS: Cronbach's alpha showing internal consistency (reliability) for these scales was 0.6-0.9. Two scales corresponded to care within the home: the "barrier-free" and "improvement of water facilities"; three to the caregiver situation: "dressing appropriate for the season," "mistreatment by the elderly," and "hygiene and assistance"; and five involved conditions of the impaired elderly: "cognition," "paralysis," "vision and hearing," "ADL," and "gross motor." CONCLUSION: The HCQAI developed in the present study, consisting of 41 items, can assess quality of home care both objectively and comprehensively, based on professional staff observation. Few indexes of this kind exist worldwide to scientifically assess input, process and outcome in the delivery of quality home care for the impaired elderly.     

Combined primary aldosteronism and Cushing's syndrome due to a single adrenocortical adenoma complicated by Hashimoto's thyroiditis

A 43-year-old Japanese woman presented hypertension, hypokalemia and typical Cushingoid signs. Autonomous secretion of both aldosterone and cortisol was shown. Abdominal computed tomography demonstrated a single tumor in the right adrenal gland, which established the diagnosis of combined primary aldosteronism and Cushing's syndrome. The resected tumor was a golden yellow-colored adenoma (diameter 4.3 cm) which expressed P450(aldo) and P450(11beta), causing oversecretion of both hormones from this adenoma. After tumor resection, overproduction of both hormones disappeared and she developed adrenal insufficiency, suggesting the strong suppression of normal adrenal function. This case was complicated by Hashimoto's thyroiditis.     

Risk factors for heavy burden among family caregivers in a rural town in Hokkaido

OBJECTIVE: The present study was conducted in order to investigate risk factors for heavy burden of family caregivers. STUDY DESIGN: Cross-sectional study. PARTICIPANTS: 51 pairs of the frail elderly and their family caregivers in one town in Hokkaido. RESULTS: Compared to those with a lighter burden, family caregivers with heavier burden looked after the frail elderly with more behavior disturbances due to dementia. They cared for the elderly longer and had less time to go out without accompanying their charges than less burdened caregivers. On the other hand, the elderly had similar activities of daily living and degree of need of care between the two groups. In addition, physical caring time did not differ between the two groups. These findings suggest that the psychological burden may be more important than the physical burden. In addition, caregivers used only 30-40% of services they had the right to use with long-term care insurance. These findings suggest that more convenient services for users should be provided.     

Effects of local administrations of tempol and diethyldithio-carbamic on peripheral nerve activity

We have recently shown that systemic administration of a superoxide dismutase mimetic, tempol, resulted in decreases in mean arterial pressure and heart rate along with a reduction in renal sympathetic nerve activity (RSNA). It has also been shown that these parameters are significantly increased by systemic administration of a superoxide dismutase inhibitor, diethyldithio-carbamic (DETC), indicating a potential role of reactive oxygen species in the regulation of RSNA. In this study, we examined the effects of local administrations of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) and DETC on RSNA in anesthetized rats. Either tempol or DETC was directly administered onto the renal sympathetic nerves located between the electrode and ganglion. Local application of tempol (10 microL, 0.17 to 1.7 mol/L, n=6) resulted in dose-dependent decreases in integrated RSNA (by -81+/-6% at 1.7 mol/L) without alterations in mean arterial pressure and heart rate. In contrast, DETC (10 microL, 0.17 to 1.7 mol/L, n=6) increased RSNA dose-dependently. The responses of RSNA to tempol and DETC were significantly greater in spontaneously hypertensive rats than in normotensive rats (n=6, respectively). Local application of sodium nitroprusside (1 mmol/L) or N(G)-nitro-L-arginine methyl ester (0.11 mol/L) altered neither basal RSNA nor tempol-induced reductions in RSNA (n=6 and 5, respectively). A voltage-gated potassium channel blocker, 4-aminopyridine (0.1 mol/L), significantly decreased basal RSNA (by -81+/-1%) and completely prevented DETC-induced increases in RSNA (n=5). These results suggest that reactive oxygen species play a role in the regulation of peripheral sympathetic nerve activity, and that at least part of this mechanism is mediated through voltage-gated potassium channels.     

Functional engraftment of human peripheral T and B cells and sustained production of autoantibodies in NOD/LtSzscid/IL‐2Rγ−/− mice

NOD/LtSzscid/IL‐2Rγ^?/? (NSG) mice have advantages in establishing humanized mouse models. However, transferring human PBMCs into these mice often causes lethal GVH disease. In this study, we discovered an improved method for the engraftment of normal or pathological human PBMCs into NSG mice and examined the subsequent induction of specific immune responses. We sequentially transferred human CD4⁺ memory T (Tm) and B cells obtained from PBMCs of healthy adults or patients with autoimmune diseases into NSG mice. Removing naïve CD4⁺ T cells from the transferred PBMCs allowed successful engraftment without lethal GVH disease. The transferred Tm cells were found to reside mainly in the spleen and the lymphoid nodules, where they expressed MHC class II molecules and produced cytokines, including IL‐21. Surprisingly, the transferred B cells were also well maintained in the lymphoid organs, underwent de novo class‐switch recombination, and secreted all isotypes of human Igs at significant levels. Moreover, transferring patient‐derived Tm and B cells resulted in sustained production of IgM‐rheumatoid factor and antiaminoacyl transfer RNA synthetase Abs in these mice. These results suggest that transfer of Tm and B cells derived from human PBMCs into NSG mice could be a useful method for the study of human autoimmune mechanisms.     

Optimization and pharmacological characterization of receptor‐mediated Gi/o activation in postmortem human prefrontal cortex

The biochemical abnormalities in transmembrane signal transduction mediated through G protein‐coupled receptors (GPCRs) have been postulated as underlying pathophysiology of psychiatric diseases such as schizophrenia and mood disorders. In the present study, the experimental conditions of agonist‐induced [³⁵S]GTPγS binding in postmortem human brain membranes were optimized, and the responses induced by a series of agonists were pharmacologically characterized. The [³⁵S]GTPγS binding assay was performed in postmortem human prefrontal cortical membranes by means of filtration techniques, and standardized as to GDP concentration, membrane protein content, MgCl₂ and NaCl concentrations in assay buffer, incubation period and effect of white matter contamination. Under the standard assay conditions, the specific [³⁵S]GTPγS binding was stimulated by the addition of 15 compounds in a concentration‐dependent manner. Of these agonists, R(+)‐8‐OH‐DPAT, UK‐14,304, DAMGO and DPDPE showed apparently biphasic concentration‐response curves. As for these four responses, only higher‐potency site was pharmacologically characterized. The receptors involved in the responses investigated were 5‐HT_1A receptor (probed with R(+)‐8‐OH‐DPAT or 5‐HT), α_2A‐adrenoceptor (UK‐14,304 or (?)‐epinephrine), M₂/M₄ mAChRs (carbachol), adenosine A₁ receptor (adenosine), histamine H₃ receptor (histamine), group II mGlu (l ‐glutamate), GABA_B receptor (baclofen), μ‐opioid receptor (DAMGO or endomophin‐1), δ‐opioid receptor (DPDPE or SNC‐80) and NOP (nociceptin). Although dopamine also activated specific [³⁵S]GTPγS binding, this response was likely mediated via α_2A‐adrenoceptor, but not dopamine receptor subtypes. The present study provides us with fundamental aspects of the strategy for elucidation of probable abnormalities of neural signalling mediated by G proteins activated through multiple GPCRs in the brain of psychiatric patients.     

Clinical relevance of frozen diagnosis of ductal margins in surgery of bile duct cancer

It is anticipated that free surgical margin is crucial for curative resection of bile duct cancer. However, the clinical relevance of the ductal margin is somewhat controversial. A role of frozen section diagnosis used for evaluation of the ductal margin during surgery is also ambiguous. We reviewed the current knowledge about frozen section diagnosis and the clinical relevance of the margin status in surgery of the bile duct cancer. Frozen section diagnosis of the ductal margin of bile duct cancer is necessary to ensure free margins; however, it is quite challenging even for experienced pathologists because the bile duct involved with bile duct cancer is often inflamed severely due to obstruction and/or insertion of a draining tube, which induces epithelial regeneration with atypia. Also accessory ducts/peribiliary glands and their conduits in ductal wall can mimic invasive ductal components, which requires careful examination to evaluate regenerative change, carcinoma in situ, or invasive carcinoma. Published studies assessing an association between the ductal margin state and prognosis in relatively large cohorts of patients undergoing surgery for bile duct cancer indicate that the ductal margin status is an independent prognostic factor; and the ductal margin with carcinoma in situ is comparable to free margin; however, the margin with invasive carcinoma is significantly adverse for patients' prognoses.