Identification of new immunogenic peptides in conserved regions of hepatitis C virus (HCV) 1b with the potentiality to generate cytotoxic T lymphocytes in HCV1b+ HLA-A24+ patients

Aim: Hepatitis C virus (HCV) 1b is resistant to standard interferon therapy and has a high risk of developing into hepatocellular carcinoma at the late stage of infection. Therefore, new therapeutic modalities for HCV1b infection must be developed. One approach would be active specific immunotherapy with highly immunogenic HCV1b peptides. Methods: HCV1b-derived 44 synthetic peptides were selected based on their binding scores to HLA-A24. Peptide-specific IgG were measured by ELISA. Peptide-specific cytotoxic T-lymphocytes (CTLs) were induced in vitro by repeated peptide-stimulation. Results: We identified three novel candidate peptides of HCV1b proteins containing HLA-A24 binding motifs. Each of them had the ability to induce HLA-A24-restricted and peptide-specific CTL activity, and IgGs specific to each of them were detected in the plasma of HCV1b patients. Among these three peptides, a peptide NS5A 2132-2142 was recognized by both cellular and humoral immunities in the majority of blood samples of patients tested. More importantly, the peptide-stimulated peripheral blood mononuclear cells (PBMCs) showed cytotoxicity against cells cotransfected with NS5A and HLA-A2402 genes in an HLA-restricted manner. This is an additional report to our previous study. Conclusion: These findings may provide a new insight into the development of a peptide-based specific immunotherapy for HCV1b-infected patients.     

Destination after discharge from a senile dementia therapy ward before and after the implementation of long-term care insurance in Japan

Background: In Japan a new long‐term care insurance (LTCI) system, the so‐called ‘Kaigo‐Hoken’, was started in April 2000. The present study analyzes the change in the type of destination after discharge from a senile dementia therapy ward before and after the implementation of LTCI at Fukuoka Prefectural Onga Hospital, Japan. Methods: The present study examines data from 199 inpatients discharged from the Fukuoka Prefectural Onga Hospital that had been diagnosed with dementia and met the DSM IV criteria for Alzheimer's type, vascular dementia or other type of dementia. For the purposes of comparison two periods were defined, ‘the first period’ was defined as the period from 1 April 1999 to 31 March 2000, before LTCI was implemented, while ‘the second period’ was defined as the period from 1 April 2000 to 31 March 2001, after LTCI had started. Subject data was analyzed on the basis of where the subject had resided pre‐admission and their destination after discharge using the following classifications: nursing home or geriatric care facility, hospitalization, home and death. Results: While the certification rate of inpatients regarding long‐term care increased slightly in the second period, no significant change was observed based on where the subject had resided pre‐admission and their destination after discharge between the first and second periods. Conclusions: While LTCI is essential for Japan, it is necessary that people with dementia in senile dementia therapy wards are encouraged to return to their homes under the care and support of LTCI.     

A hamster temperature-sensitive G1 mutant, tsBN250 has a single point mutation in histidyl-tRNA synthetase that inhibits an accumulation of cyclin D1

Background: We have previously isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line, derived from the golden hamster. These mutants proliferate at 33.5 °C, the permissive temperature, but not at 39.5 °C the restrictive temperature. Using DNA-mediated gene transfer, human genes complementing these ts mutants were cloned.
Results: At 39.5 °C the tsBN250 cell line, a temperature-sensitive mutant of the BHK21 cell line, had a defect in the G1 phase, but not in the S phase. The human gene complementing tsBN250 cells was found to encode histidyl-tRNA synthetase. Indeed, the tsBN250 cell line had a single base change—guanine to adenine at the second position of the 362nd codon of hamster histidyl-tRNA-synthetase, converting arginine to histidine. Following release from serum starvation, cyclin E, but not cyclin D1, was accumulated, while, at 39.5 °C, the mRNA of cyclin D1 was normally expressed in tsBN250 cells. A similar inhibition of cyclin D1 accumulation was observed in another ts mutant, tsBN269, which has a single point mutation in lysyl-tRNA synthetase. Overexpression of cyclin D1 enabled tsBN250 cells to enter the S phase.
Conclusion: tsBN250 cells have a single point mutation in histidyl tRNA synthetase that causes a loss of histidyl-tRNA synthetase activity which in turn reduces the content of cyclin D1, but not of cyclin E, thereby resulting in G1 arrest.     

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein betagamma subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K(+) ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., et al. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.     

Successful endoscopic transpapillary pancreaticobiliary drainage for omental panniculitis by hepatocellular carcinoma complicated by biliary fistula and pancreatic fistula

A 78-year-old man with hepatocellular carcinoma treated by chemoembolization and percutaneous ethanol injection was admitted to our hospital because of acute abdomen. The CT scan showed biliary fistula caused by hepatocellular carcinoma protruding from S3. Endoscopic retrograde cholangiopancreatography showed disruption of an intrahepatic duct and the main pancreatic duct, and contrast agent leaked into the peritoneal cavity from each duct. Omental panniculitis with biliary fistula and pancreatic fistula was diagnosed. The symptoms improved by endoscopic nasobiliary drainage and endoscopic pancreatic stenting. On the 13th day after admission, we added endoscopic nasopancreatic drainage because his abdominal pain had been exacerbated by pancreatic juice leakage. Omental panniculitis by hepatocellular carcinoma complicated by biliary fistula and pancreatic fistula is extremely rare. Endoscopic transpapillary pancreaticobiliary drainage was effective for omental panniculitis in this case.     

Improved Orange and Red Ca2+ Indicators and Photophysical Considerations for Optogenetic Applications

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Long-Term Seizure Outcome following Resective Surgery for Epilepsy: To Be or Not to Be Completely Cured?

Surgical intervention is expected to improve the quality of life in patients with intractable epilepsy by providing adequate seizure control. Although many previous studies showed various rates of seizure freedom, definite conclusions have not yet been made regarding outcomes. In order to clarify the long-term postoperative outcome for a period up to 10 years, a retrospective review of our patients was performed longitudinally by using the survival analysis method. The postoperative state of epilepsy in 76 patients who underwent resection surgery was assessed based on Engel’s criteria. In addition, Kaplan-Meier survival analysis was used to calculate the probability of seizure freedom. In this patient group, abnormal lesion were detected by MRI in 70 out of 76 cases, and the ictal onset zone was finally identified within temporal lobe in 51 cases. The most favorable outcome, defined as Engel Class Ia, was observed in 26 (37%), 24 (40%), and 18 (41%) cases at 2, 5, and 10 years after surgery, respectively. The Kaplan-Meier survival curve in the overall group estimated the probability of seizure freedom as 75% (95% confidence interval [CI] 70–80%), 67% (62–72%), and 51% (45–57%) at 2, 5, and 10 years follow up, respectively. Half of all seizure recurrences occurred within the first 2 postoperative years. In this study, we showed that long-term favorable outcome of seizure control following resection surgery can be achieved in more than half of the patients.     

Feedback-controlled bolus plus infusion (FC-B/I) method for quantitative drug assessment in living brain with PET

We have developed a feedback-controlled bolus plus infusion (FC-B/I) method for monitoring the interaction between positron emission tomography (PET) ligands and their specific target molecules with PET. The usefulness of the FC-B/I method was evaluated by the direct interaction between [¹¹C]raclopride, a dopamine D₂ receptor (D₂R) ligand, and cold raclopride (10 and 100 μg/kg) in the brains of conscious monkeys. The present results demonstrated that the FC-B/I method could achieve the equilibrium state of [¹¹C]raclopride in the striatum of monkey brain, and also that the cold raclopride-induced reduction of [¹¹C]raclopride binding to D₂R was observed in a dose-dependent manner. Good correlations of distribution volume ratio of the striatum to cerebellum between the conventional bolus plus infusion (B/I) method and the FC-B/I method as well as between the conventional bolus injection method and the FC-B/I method were observed. These results indicated that the system could be a useful tool for the evaluation of interaction between drug candidates and their target molecules like enzymes, receptors, and transporters by using of their specific PET ligands.     

Delayed Gastric Emptying After Stapled Versus Hand-Sewn Anastomosis of Duodenojejunostomy in Pylorus-Preserving Pancreaticoduodenectomy: a Randomized Controlled trial

Background

A retrospective analysis indicated that the incidence of delayed gastric emptying (DGE) was less after using a circular stapler (CS) for duodenojejunostomy than that after hand-sewn (HS) anastomosis in pylorus-preserving pancreaticoduodenectomy (PpPD). This randomized clinical trial compared the incidence of DGE postoperative after CS duodenojejunostomy with that of conventional HS anastomosis in PpPD.

Methods

We randomly assigned 101 patients (age 20–80) undergoing PpPD to receive CS duodenojejunostomy (group CS, n?=?50) or HS duodenojejunostomy (group HS, n?=?51) in two Japanese cancer center hospitals between 2011 and 2013. The patients were stratified by institution and size of the main pancreatic duct (<3 or ≥3 mm). The primary endpoint was the incidence of grade B or C DGE according to the international definition with a non-inferiority margin of 5 %. This trial is registered with University hospital Medical Information Network (UMIN) Center: UMIN000005463.

Results

Per-protocol analysis of data on 95 patients showed that grade B or C DGE was found in 4 (8.9 %) of 45 patients who underwent CS anastomosis and in 8 (16 %) of 50 patients who underwent HS anastomosis (P?=?0.015). There were no differences in the overall incidence of DGE (P?=?0.98), passage of the contrast medium through the anastomosis (P?=?0.55), or hospital stays (P?=?0.22).

Conclusions

CS duodenojejunostomy is not inferior to HS anastomosis with respect to the incidence of clinically significant DGE, justifying its use as treatment option.

     

Effects of chlorogenic acid on carbachol-induced contraction of mouse urinary bladder

Chlorogenic acid (CGA) is a polyphenol found in coffee and medicinal herbs such as Lonicera japonica. In this study, the effect of CGA-induced relaxation on carbachol (CCh)-induced contraction of mouse urinary bladder was investigated. CGA (30–300 μg/ml) inhibited CCh- or U46619-induced contraction in a concentration-dependent manner. SQ22536 (adenylyl cyclase inhibitor) recovered CGA-induced relaxation of CCh-induced contraction; however, ODQ (guanylyl cyclase inhibitor) did not have the same effect. In addition, 3-isobutyl-1-methylxanthine (IBMX) enhanced CGA-induced relaxation; however, forskolin or sodium nitroprusside did not have the same effect. Moreover, Ro 20–1724, a selective phosphodiesterase (PDE) 4 inhibitor, enhanced CGA-induced relaxation, but vardenafil, a selective PDE5 inhibitor, did not have the same effect. In the presence of CCh, CGA increased cyclic adenosine monophosphate (cAMP) level, whereas SQ22536 inhibited the increase of cAMP levels. Moreover, higher cAMP levels were obtained with CGA plus IBMX treatment than the total cAMP levels obtained with separate CGA and IBMX treatments. In conclusion, these results suggest that CGA inhibited CCh-induced contraction of mouse urinary bladder by partly increasing cAMP levels via adenylyl cyclase activation.