Elevated receptor for activated C kinase 1 expression is involved in intracellular Ca2+ influx and potentially associated with compromised regulatory T cell function in patients with asthma.

BACKGROUND: Regulatory T cells (T(regs)) are activated during anergy in response to T cell receptor (TCR) activation and functional immune suppression. Anergy of paediatric T(regs) is partially dependent on intracellular calcium mobility; following TCR activation, T(regs) do not exhibit increased intracellular Ca(2+) concentration ([Ca(2+) ](i)). OBJECTIVE: We determined whether [Ca(2+) ](i) in adult T(regs) defined their anergy, if intracellular Ca(2+) movement was linked to regulatory functions, whether [Ca(2+)](i) was indicative of asthma pathology, and the potential molecular mechanism responsible for Ca(2+) movement in T(regs). METHODS: T(regs) were purified by the magnetic bead method, and their regulatory functions were assessed by monitoring carboxyfluorescein succinimidyl ester-labelled responder T cell proliferation. The Ca(2+) response of Fura-2-labelled cells was measured using a video image analysis system. To analyse the functions of T(regs) at the molecular level, we generated Jurkat Tet-On(®) clones with doxycycline (Dox)-induced forkhead box P3 (FOXP3) protein expression. RESULTS: CD4(+) CD25(+) CD127(-/low) T(regs) from participants without asthma did not elicit Ca(2+) influx in response to TCR activation, exhibited little proliferation and suppressed proliferation of CD4(+) CD25(-) T cells. In contrast, under similar conditions, T(regs) from patients with asthma exhibited increased [Ca(2+)](i) and robust proliferation with partial loss of regulatory functions. FOXP3 protein levels in Tet-On(®) clones were high after both 2- and 5-day Dox treatment; however, 5-day cells were comparable with T(regs) from patients with asthma, whereas 2-day cells were similar to T(regs) from participants without asthma. Increasing [Ca(2+)](i) induced a high level of receptor for activated C kinase 1 (RACK1) expression in 5-day cells. CONCLUSIONS AND CLINICAL RELEVANCE: We confirmed that T(regs) in patients with asthma are functionally impaired and that the abnormal regulatory functions of these cells can be analysed by [Ca(2+)](i) following TCR engagement. Furthermore, the impaired functioning of T(regs) evident in patients with asthma may be due to a high level of RACK1.

Randomized comparison of pitavastatin and pravastatin treatment on the reduction of urinary albumin in patients with type 2 diabetic nephropathy.

The effect of pitavastatin and pravastatin treatment on renal function was compared in type 2 diabetic patients with nephropathy in a randomized, controlled, open-label, parallel and multi-centre study. Type 2 diabetic patients with modest renal impairment (serum creatinine level <1.4 mg/dl) accompanied by albuminuria (30-600 mg/g creatinine) were randomly assigned to receive 2 mg of pitavastatin (n = 44) or 10 mg of pravastatin (n = 43) for 12 months. At 12 months, pitavastatin significantly reduced urinary albumin-to-creatinine ratio than pravastatin in subjects with macroalbuminuria (-67.2% vs. +14.5%, p = 0.0040), but not in subjects with microalbuminuria. There was no significant difference in the change in estimated glomerular filtration rate between the two groups. Pitavastatin is more effective than pravastatin for the reduction of albuminuria in type 2 diabetic patients with early stage of diabetic nephropathy.

Biliverdin protects against the deterioration of glucose tolerance in db/db mice.

AIMS/HYPOTHESIS: We have previously shown a negative correlation between serum bilirubin levels and prevalence of type 2 diabetes, suggesting that bilirubin inhibits development of this disease. To confirm this hypothesis, we investigated whether administration of biliverdin, the precursor of bilirubin, protects against the deterioration of glucose tolerance in db/db mice, a rodent model of type 2 diabetes. METHODS: Biliverdin (20 mg/kg daily) was orally administered to 5-week-old db/db mice for 4 weeks. After 4 weeks of treatment, i.p. glucose tolerance and insulin tolerance tests were performed. Insulin content was evaluated by immunostaining and ELISA. Oxidative stress markers (8-hydroxy-2'-deoxyguansosine and dihydroethidium staining) and expression of NADPH oxidase components Pdx1 and Bax were also evaluated in isolated islets. RESULTS: Treatment with biliverdin partially prevented worsening of hyperglycaemia and glucose intolerance in db/db mice. This effect was accompanied by a significant increase in insulin content and Pdx1 expression, and a significant decrease of apoptosis and Bax expression in pancreatic islets from db/db mice. At the same time, levels of oxidative stress markers and NADPH oxidase component production in islets were normalised. Biliverdin had little effect on HOMA of insulin resistance or insulin resistance evaluated by insulin tolerance tests. CONCLUSIONS/INTERPRETATION: Biliverdin may protect against progressive worsening of glucose tolerance in db/db mice, mainly via inhibition of oxidative stress-induced beta cell damage.

Association of genetic variants for susceptibility to obesity with type 2 diabetes in Japanese individuals.

AIMS/HYPOTHESIS: In populations of East Asian descent, we performed a replication study of loci previously identified in populations of European descent as being associated with obesity measures such as BMI and type 2 diabetes. METHODS: We genotyped 14 single nucleotide polymorphisms (SNPs) from 13 candidate loci that had previously been identified by genome-wide association meta-analyses for obesity measures in Europeans. Genotyping was done in 18,264 participants from two general Japanese populations. For SNPs showing an obesity association in Japanese individuals, we further examined diabetes associations in up to 6,781 cases and 7,307 controls from a subset of the original, as well as from additional populations. RESULTS: Significant obesity associations (p < 0.1 two-tailed, concordant direction with previous reports) were replicated for 11 SNPs from the following ten loci in Japanese participants: SEC16B, TMEM18, GNPDA2, BDNF, MTCH2, BCDIN3D-FAIM2, SH2B1-ATP2A1, FTO, MC4R and KCTD15. The strongest effect was observed at TMEM18 rs4854344 (p = 7.1 × 10(-7) for BMI). Among the 11 SNPs showing significant obesity association, six were also associated with diabetes (OR 1.05-1.17; p = 0.04-2.4 × 10(-7)) after adjustment for BMI in the Japanese. When meta-analysed with data from the previous reports, the BMI-adjusted diabetes association was found to be highly significant for the FTO locus in East Asians (OR 1.13; 95% CI 1.09-1.18; p = 7.8 × 10(-10)) with substantial inter-ethnic heterogeneity (p = 0.003). CONCLUSIONS/INTERPRETATION: We confirmed that ten candidate loci are associated with obesity measures in the general Japanese populations. Six (of ten) loci exert diabetogenic effects in the Japanese, although relatively modest in size, and independently of increased adiposity.

Multiple ulcers in the small and large intestines occurred during tocilizumab therapy for rheumatoid arthritis.

Tocilizumab is a monoclonal antibody against human interleukin-6 receptor which blocks the binding of interleukin-6 to its receptor. Tocilizumab is effective for the treatment of inflammatory disorders including rheumatoid arthritis. We report a case of multiple ulcers in the small and large intestines, which occurred during tocilizumab therapy. A 57-year-old woman started to use tocilizumab for rheumatoid arthritis. Three months later, she complained of hematochezia. Double-balloon endoscopy revealed multiple small aphthoid ulcers in the small and large intestines. One month after the woman had recovered, she was given tocilizumab again. The woman had hematochezia and abdominal pain again 2 weeks later. Colonoscopy revealed multiple round, discrete punched-out ulcers in the terminal ileum, and vast deep ulcers from the cecum to the descending colon. Bioptic histopathology and cultivation showed non-specific findings. Six weeks after discontinuation of tocilizumab, ulcers in the small and large intestine dramatically improved, leaving ulcer scars. This disease course and the results of examination made us strongly suspect that tocilizumab induced multiple ulcers in the small and large intestines. Interleukin-6 is a pleiotropic cytokine and involved in intestinal mucosal wound healing as well as in inflammatory processes. It is possible that tocilizumab inhibited tissue repair of the intestine and caused intestinal ulcers.

Initial analysis of relationship between plasma platinum concentration and hematological adverse reaction associated with weekly chemotherapy using nedaplatin in combination with radiotherapy for cervical carcinoma.

PURPOSE: Established therapeutic guidelines for cervical carcinoma recommend concurrent chemo- and radiotherapy as standard treatment for locally advanced cervical carcinoma. Nedaplatin (CDGP) is a platinum agent developed in Japan that is less nephrotoxic than cisplatin (CDDP), but with equivalent antitumor potency. In the standard dosage regimen for cervical carcinoma, CDGP is administered once every four weeks (monthly regimen). We investigated the efficacy and safety of a new dosage regimen, in which CDGP was administered once weekly for five weeks (weekly regimen). METHODS: We measured plasma platinum concentration of patients after administration of CDGP, and analyzed the relationship between plasma platinum concentration and hematological adverse reactions such as thrombocytopenia and leucopenia. RESULTS: The relative rates of change in platelet and white blood cell counts tended to increase as the plasma concentration of platinum increased. Furthermore, the rate of change in platelet counts in relation to the area under the curve was greater for the monthly regimen as compared to weekly. On the other hand, the relative rates of change in WBC were nearly the same between the regimens. CONCLUSIONS: These findings indicate that when using chemotherapy with CDGP for a patient with a cervical carcinoma, a weekly regimen might reduce the severity of thrombocytopenia, while still exhibiting the same therapeutic efficacy as the monthly regimen.

Common variants at the GCK, GCKR, G6PC2-ABCB11 and MTNR1B loci are associated with fasting glucose in two Asian populations.

AIMS/HYPOTHESIS: To test fasting glucose association at four loci recently identified or verified by genome-wide association (GWA) studies of European populations, we performed a replication study in two Asian populations. METHODS: We genotyped five common variants previously reported in Europeans: rs1799884 (GCK), rs780094 (GCKR), rs560887 (G6PC2-ABCB11) and both rs1387153 and rs10830963 (MTNR1B) in the general Japanese (n = 4,813) and Sri Lankan (n = 2,319) populations. To identify novel variants, we further examined genetic associations near each locus by using GWA scan data on 776 non-diabetic Japanese samples. RESULTS: Fasting glucose association was replicated for the five single nucleotide polymorphisms (SNPs) at p < 0.05 (one-tailed test) in South Asians (Sri Lankan) as well as in East Asians (Japanese). In fine-mapping by GWA scan data, we identified in the G6PC2-ABCB11 region a novel SNP, rs3755157, with significant association in Japanese (p = 2.6 x 10(-8)) and Sri Lankan (p = 0.001) populations. The strength of association was more prominent at rs3755157 than that of the original SNP rs560887, with allelic heterogeneity detected between the SNPs. On analysing the cumulative effect of associated SNPs, we found the per-allele gradients (beta = 0.055 and 0.069 mmol/l in Japanese and Sri Lankans, respectively) to be almost equivalent to those reported in Europeans. CONCLUSIONS/INTERPRETATION: Fasting glucose association at four tested loci was proven to be replicable across ethnic groups. Despite this overall consistency, ethnic diversity in the pattern and strength of linkage disequilibrium certainly exists and can help to appreciably reduce potential causal variants after GWA studies.

Persistent gut motor dysfunction in a murine model of T-cell-induced enteropathy.

BACKGROUND: Inflammatory bowel disease (IBD) patients in remission often experience irritable bowel syndrome (IBS)-like symptoms. We investigated the mechanism for intestinal muscle hypercontractility seen in T-cell-induced enteropathy in recovery phase. METHODS: BALB/c mice were treated with an anti-CD3 antibody (100 microg per mouse) and euthanized at varying days post-treatment to investigate the histological changes, longitudinal smooth muscle cell contraction, cytokines (Th1, Th2 cytokines, TNF-alpha) and serotonin (5-HT)-expressing enterochromaffin cell numbers in the small intestine. The role of 5-HT in anti-CD3 antibody-induced intestinal muscle function in recovery phase was assessed by inhibiting 5-HT synthesis using 4-chloro-DL-phenylalanine (PCPA). KEY RESULTS: Small intestinal tissue damage was observed from 24 h after the anti-CD3 antibody injection, but had resolved by day 5. Carbachol-induced smooth muscle cell contractility was significantly increased from 4 h after injection, and this muscle hypercontractility was evident in recovery phase (at day 7). Th2 cytokines (IL-4, IL-13) were significantly increased from 4 h to day 7. 5-HT-expressing cells in the intestine were increased from day 1 to day 7. The 5-HT synthesis inhibitor PCPA decreased the anti-CD3 antibody-induced muscle hypercontractility in recovery phase. CONCLUSIONS & INFERENCES: Intestinal muscle hypercontractility in remission is maintained at the smooth muscle cell level. Th2 cytokines and 5-HT in the small intestine contribute to the maintenance of the altered muscle function in recovery phase.

Differential diagnosis of cystic tumors of the pancreas by endoscopic ultrasonography.

BACKGROUND AND STUDY AIMS: Generally, cystic tumors are divided into two categories: neoplastic cystic tumors and non-neoplastic cystic (NNC) tumors. Neoplastic cystic tumors include mucinous cystic neoplasm (MCN), intraductal papillary-mucinous neoplasm (IPMN), and serous cystic neoplasm (SCN). MCNs and IPMNs have the potential to progress to a malignant state, whereas SCNs are known for their almost benign behavior. Thus, in order to make management decisions, it is important to distinguish between potentially malignant (MCN and IPMN), and benign (SCN and NNC) tumors. The aim of this study was to retrospectively investigate the value of endoscopic ultrasonography (EUS) for the differential diagnosis of cystic tumors of the pancreas. PATIENTS AND METHODS: A total of 76 patients with cystic tumors of the pancreas were preoperatively examined by EUS. Eight cases were MCNs, 45 were IPMNs, 13 were SCNs, and 10 were NNC tumors. The EUS findings relevant to distinguishing between potentially malignant and benign were analyzed statistically. RESULTS: All patients with MCNs were female and all these tumors were located in the pancreatic body/tail. IPMN, however, occurred predominantly in men, and in the pancreatic head. Eight of 11 monolocular cystic tumors were NNC in nature. Eleven of 13 SCNs included microcystic areas within the tumors. All MCNs were round in appearance, whereas 93 % of IPMNs were not round in appearance. Mural nodules were present in 25 % of MCN and 38 % of IPMN cases. In univariate analysis, age, tumor size, locularity, the number of cystic formation, cystic component, and appearance were significant variables. In multivariate analysis, locularity and cystic component were important for differential diagnosis of potentially malignant cystic tumors. CONCLUSIONS: The characteristics of cystic tumors of the pancreas revealed by EUS are useful for their differential diagnosis.

Impact of double-balloon endoscopy on the diagnosis of jejunoileal involvement in primary intestinal follicular lymphomas: a case series.

In recent years, primary gastrointestinal follicular lymphoma has been increasingly detected in the duodenum on esophagogastroduodenoscopy (EGD). Primary gastrointestinal follicular lymphomas are frequently distributed to multiple sites in the gastrointestinal tract. Therefore, investigation into the spread of follicular lymphomas in the small bowel is important in order to determine the most appropriate treatment strategy. The performance of double-balloon endoscopy (DBE) in the diagnosis of jejunoileal follicular lymphoma lesions has not been fully evaluated. We aimed to investigate the value of DBE in addition to computed tomography (CT) and (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG-PET) in the diagnosis of jejunoileal follicular lymphoma. DBE with biopsy was performed in seven patients with primary duodenal follicular lymphoma diagnosed by EGD, in order to investigate jejunoileal involvement. Jejunoileal follicular lymphoma lesions were detected by DBE in six out of the seven patients (three in the jejunum and three in the jejunum and ileum), whereas CT and (18)F-FDG-PET failed to detect the existence of these lesions. Endoscopic findings of the jejunoileal lesions revealed multiple white nodules and white villi, which were similar to those of duodenal lesions. DBE was more useful for the diagnosis of jejunoileal involvement in primary intestinal follicular lymphoma than CT and (18)F-FDG-PET. The use of DBE will become important for determining the most appropriate treatment for gastrointestinal follicular lymphoma.

Preparation of functionally preserved CD4+ CD25high regulatory T cells from leukapheresis products from ulcerative colitis patients, applicable to regulatory T-cell transfer therapy.

BACKGROUND: Ulcerative colitis (UC) is an intractable disease; therefore new therapies need to be developed. CD4(+) CD25(high) regulatory T cells (Treg) significantly ameliorate colitis in animal models. In active UC patients, although Treg are functionally preserved, their proportion in peripheral blood decreases. Thus Treg transfer therapy is expected to be efficacious for UC. During leukapheresis for UC, Treg are depleted, as well as colitogenic effector leukocytes. We therefore designed a leukapheresis/Treg transfer therapy in which Treg are isolated from leukapheresis products and transfused to patients, and studied large-scale germ-free methods of Treg preparation. METHODS: Using the CliniMACS cell selection system, we conducted Treg isolation experiments from leukapheresis products in which B and CD8(+) T cells were depleted, followed by positive selection of CD25(+) cells. In some experiments, isolated Treg or non-Treg were expanded with interleukin-2 (IL-2) +/- transforming growth factor (TGF)-beta1. Expression of a Treg-specific marker, FOXP3, and gut-homing receptors, and suppressor activity of isolated or cultured cells, were analyzed. RESULTS: CD4(+) CD25(high) T cells were collected and efficiently enriched with a good recovery rate. Isolated cells preferentially expressed FOXP3 and significantly suppressed T-cell proliferation in vitro. In addition, isolated Treg could be efficiently expanded, and Treg could be induced from non-Treg with TGF-beta1 in vitro. TGF-beta1 significantly up-regulated alphaEbeta7 and alpha4beta7 integrins. DISCUSSION: We have established a method of Treg isolation from leukapheresis products that can be used clinically; therefore, Treg transfer therapy is feasible in combination with leukapheresis for UC. Expansion or induction of Treg in vitro may be another approach to Treg-based immunotherapy.

Pitavastatin ameliorates albuminuria and renal mesangial expansion by downregulating NOX4 in db/db mice.

Recent studies have uncovered various pleiotrophic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase-inhibiting drugs (statins). Several studies have identified a beneficial effect of statins on diabetic nephropathy; however, the molecular mechanisms are unclear. In this study, we show that statin ameliorates nephropathy in db/db mice, a rodent model of type 2 diabetes, via downregulation of NAD(P)H oxidase NOX4, which is a major source of oxidative stress in the kidney. Pitavastatin treatment for 2 weeks starting at 12 weeks of age significantly reduced albuminuria in the db/db mice concomitant with a reduction of urinary 8-hydroxy-2'-deoxyguanosine and 8-epi-prostaglandin F(2alpha). Immunohistochemical analysis found increased amounts of 8-hydroxy-2'-deoxyguanosine and NOX4 protein in the kidney of db/db mice. Quantitative reverse transcription-polymerase chain reaction also showed increased levels of NOX4 mRNA. Pitavastatin normalized all of these changes in the kidneys of diabetic animals. Additionally, 12-week treatment with the statin completely normalized the levels of transforming growth factor-beta1 and fibronectin mRNA as well as the mesangial expansion characteristic of diabetic nephropathy. Our study demonstrates that pitavastatin ameliorates diabetic nephropathy in db/db mice by minimizing oxidative stress by downregulating NOX4 expression. These findings may provide insight into the mechanisms of statin therapy in early stages of diabetic nephropathy.

An increase in the serum amylase level in patients after peroral double-balloon enteroscopy: an association with the development of pancreatitis.

BACKGROUND AND STUDY AIMS: Double-balloon enteroscopy (DBE) is a novel technique that allows the enteroscope to be inserted deep into the small intestine. The procedure has been thought to be safe, but cases of acute pancreatitis after peroral DBE have recently been observed. The aim of this study was to confirm the occurrence of hyperamylasemia after peroral DBE. PATIENTS AND METHODS: Peroral DBE was carried out in 13 patients from July 2005 to February 2006. Blood samples were taken before and 3 h after the procedure, and serum pancreatic amylase levels were measured. The patients were also evaluated for pancreatic-type abdominal pain after the procedure. Hyperamylasemia after peroral DBE was defined as an elevation of the serum pancreatic amylase level to more than the upper normal limit and twice the level before the procedure. Pancreatitis was diagnosed on the basis of both pancreatic-type abdominal pain and hyperamylasemia. RESULTS: Hyperamylasemia after peroral DBE occurred in six patients (46.2 %). One of the six patients with hyperamylasemia had pancreatic-type abdominal pain after the procedure and developed acute pancreatitis. The average procedure time was 105 min (range 65 - 155 min) in the patients with hyperamylasemia, and did not significantly differ from that in the group without hyperamylasemia (99 min). CONCLUSIONS: Hyperamylasemia after peroral DBE occurs frequently and may be associated with development of pancreatitis.

[Analysis of 5-HT3 receptor antagonist, ramosetron hydrochloride, based on receptor occupancy considering its active metabolite].

Severe nausea and vomiting induced by antineoplastics diminish the patient's quality of life and the ability to tolerate further chemotherapy. Ramosetron hydrochloride is a 5-HT3 receptor antagonist, which has an active metabolite (M-1), expected to be useful in the inhibition of chemotherapy-induced nausea and vomiting. In the present study, in order to analyze the pharmacological effect of ramosetron hydrochloride in a comprehensive manner, we estimated the 5-HT3 receptor occupancy after intravenous administration of ramosetron hydrochloride using pharmacokinetic parameters and the dissociation constants for the 5-HT3 receptor. The average total receptor occupancy after intravenous administration of 0.3 mg of ramosetron hydrochloride to human was calculated to be 82.9% (ramosetron, 77.8%; M-1, 5.1%), thus exhibiting a significant antiemetic activity. Furthermore, the estimated time course of 5-HT3 receptor occupancies after intravenous administration of 0.3 mg of ramosetron hydrochloride suggested a substantial impact of the active metabolite (M-1). It suggested that M-1 contributed to the long duration of binding on the 5-HT3 receptor. The present analysis method should be useful for designing the rational dosage regimen of ramosetron hydrochloride and predicting the duration of its antiemetic activity in a quantitative manner.

Tributyltin or triphenyltin inhibits aromatase activity in the human granulosa-like tumor cell line KGN.

The superimposition of male sex organs (penis and vas deferens) in a female gastropod, called imposex, is widely attributed to the exposure to tributyltin (TBT) compounds, used world-wide in antifouling paints for ships. It has been hypothesized that the TBT-induced imposex is mediated by an increasing androgen level relative to the estrogen level, namely a decreased conversion of androgens to estrogens (i.e., aromatization). In the present study, we tested this hypothesis by examining the effects of TBT or triphenyltin (TPT) on the aromatase activity in a cultured human granulosa-like tumor cell line, KGN, which was recently established by our group. Treatment with more than 1000 ng/ml TBT compounds was very toxic to the cells and caused immediate cell death within 24 h, while 200 ng/ml was found to cause apoptosis of the cells. Treatment of the KGN cells for more than 48 h with 20 ng/ml TBT or TPT, which is a concentration level reported to cause imposex in marine species, did not affect cell proliferation but significantly suppressed the aromatase activity determined by a [(3)H]H(2)O release assay. Treatment with 20 ng/ml TBT compounds for 7 days also resulted in a reduction of the E2 production from Delta 4-androstenedione stimulated by db-cAMP. The changes in the aromatase activity by TBT compounds were associated with comparable changes in P450arom mRNA assessed by RT-PCR. The luciferase activity of the P450arom promoter II (1 kb) decreased after the addition of 20 ng/ml TBT compounds in transfected KGN cells either in a basic state or in states stimulated by db-cAMP. The Ad4BP-dependent increase in the luciferase activity of P450arom promoter II was also downregulated by such treatments. These results indicate that TBT compounds inhibited the aromatase activity and also decreased the P450arom mRNA level at the transcriptional level in KGN cells. The direct inhibitory effect of TBT compounds on the aromatase activity may therefore partly explain the induction of imposex by these compounds in female species.