ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder associated with depletion, multiple deletions and site-specific point mutations of mitochondrial DNA (mtDNA). MNGIE is caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP; endothelial cell growth factor 1). Deficiency of TP leads to dramatically elevated levels of circulating thymidine and deoxyuridine. The alterations of pyrimidine nucleoside metabolism are hypothesized to cause imbalances of mitochondrial nucleotide pools that, in turn, may cause somatic alterations of mtDNA. We have now identified five major forms of mtDNA deletions in the skeletal muscle of MNGIE patients. While direct repeats and imperfectly homologous sequences appear to mediate the formation of mtDNA deletions, the nicotinamide adenine dinucleotide dehydrogenase 5 gene is a hot-spot for these rearrangements. A novel aspect of the mtDNA deletions in MNGIE is the presence of microdeletions at the imperfectly homologous breakpoints.     

Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.     

Dynamic changes in intranuclear and subcellular localizations of mouse Prrp/DAZAP1 during spermatogenesis: the necessity of the C-terminal proline-rich region for nuclear import and localization

Mouse Prrp (mPrrp)/DAZAP1 is a mouse ortholog of Xenopus Prrp, which is involved in vegetal pole localization of Vg1 mRNA in oocytes and is highly expressed in the testis. The mouse protein has been reported to be a shuttling protein which localizes in the nucleus of pre-meiotic spermatogenic cells and round spermatids, and shifts its location into the cytoplasm in elongating spermatids, suggesting that mPrrp may be involved in mRNA transport as well as that of the Xenopus ortholog. We reexamined immunohistochemical analyses of mPrrp/DAZAP1 during spermatogenesis utilizing a newly established monoclonal antibody and reconfirmed it to be a shuttling protein. We also carried out new observations that included remarkable intranuclear movement during spermatogenesis. In addition, we found that a long amino acid stretch which spanned over the C-terminal half of the protein was required for the nuclear import. These observations demonstrated dynamic changes in subnuclear and subcellular localization which might reflect specific functions during spermatogenesis.     

Multiple bone fractures found in a young sarcoidosis patient with long stable disease

A 22-year-old Japanese man was found to have bilateral hilar lymphadenopathy (BHL), and was diagnosed with sarcoidosis in 1995. He was followed without treatment until 2002, when a bone fracture due to osseous sarcoidosis was found in his left thumb. Despite systemic treatment with corticosteroid and methotrexate, a new bone lesion developed in his right foot and his right middle finger was fractured. The patient also suffered multiple organ involvements including brain and muscle lesions. This is the first report of a sarcoidosis patient who presented with BHL, and developed bone fractures after a long stable period of more than 5 years.     

Respiratory function after esophagectomy for patients with esophageal cancer

BACKGROUND/AIMS: Postoperative respiratory hypofunction sometime ruins quality of life of patients with esophageal cancer. From 1993, we introduced transhiatal esophagectomy without thoracotomy as a less invasive surgery to prevent postoperative respiratory complications for patients who have relatively early stage of esophageal cancer and have preoperative respiratory complication, or who are older in age. In this study, postoperative long-term evaluation of respiratory functions of patients with esophageal cancer who underwent esophagectomy was performed. METHODOLOGY: Among the patients with esophageal cancer who underwent esophagectomy in our hospital between 1993 and 1995, we selected 13 patients who underwent transhiatal esophagectomy (transhiatal group) and 9 patients who underwent transthoracic esophagectomy (transthoracic group). Conventional respiratory function tests (VC, vital capacity; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 second; FEV1/FVC, ratio of FEV1 to FVC; PEF, peak expiratory flow) were compared between the two groups at 3, 6, and 12 months after operation. RESULTS: In the transhiatal group, postoperative average values of VC, FVC, and FEV1 recovered 92%, 98%, and 93% of preoperative average values at 6 months after operation, while in the transthoracic group, the average values of VC, FVC, and FEV1 were still 78%, 78%, and 72% of preoperative average values at 6 months after operation. Postoperative respiratory complications were detected in 4 patients (transhiatal: 2 and transthoracic: 2). The recovery rates of VC, FVC, FEV1, FEV1/FVC, and PEF at 6 months after operation of these 4 patients were not different from those of 18 patients without postoperative respiratory complications. CONCLUSIONS: In patients treated with transthoracic esophagectomy, postoperative respiratory hypofunctions continued over 6 months after surgery. However, postoperative respiratory complications may not be related with the long-term postoperative respiratory hypofunction in patients with esophageal cancer.     

Growth mechanism of human myeloma cells by interleukin-6

Human myeloma cells are heterogenous morphologically and phenotypically. Myeloma cells can be classified into at least 5 subpopulations; MPC-1-CD45+CD49e-, MPC-1-CD45-CD49e- immature myeloma cells, MPC-1+CD45-CD49e-, MPC-1+CD45+CD49e- intermediate myeloma cells and MPC-1+CD45+CD49e+ mature myeloma cells. Interleukin-6(IL-6) is a major growth factor for human myeloma cells, but only MPC-1-CD45+CD49e- immature myeloma cells can response directly to IL-6 to proliferate. In the U-266 cell lines, IL-6 can lead to the induction of CD45 expression and CD45+ U-266 cells can proliferate in response to IL-6. In primary myeloma cells, MPC-1-CD45-CD49e- immature myeloma cells sorted from bone marrow samples can be changed to CD45+ cells by addition of IL-6 in vitro. In both CD45- and CD45+ U-266 cells, STAT3 and MAPK(ERK1/2) can be activated in response to IL-6 equally between them, but src family kinases such as Lyn, Fyn can be activated only in CD45+ U-266 cells. Thus, the activation of the src family kinases associated with CD45 expression is a prerequisite for the proliferation of myeloma cells. In the bone marrow of myeloma patients, most myeloma cells do not express CD45, and CD45+ immature myeloma cells are only 1 approximately 2%. In order to clarify the difference of cellular context between CD45- and CD45+ myeloma cells, PCR-based cDNA subtraction was performed from CD45+ U-266 cells to CD45-U-266 cells. The series of this subtraction selected several genes. Furthermore, sensitivity to stress stimuli between CD45+ and CD45- U-266 cells was also compared. CD45-U-266 cells were markedly more resistant to stress conditions such as serum-free condition. Therefore, we can speculate that in the bone marrow of human myelomas IL-6 can induce proliferation of CD45+ immature cells, but the amount of IL-6 is too low to support CD45+ myeloma cells and loss of CD45 results in no direct response to IL-6 to proliferate but confers resistance to stress condition leading to the longer survival at the limited amount of IL-6.     

25-hydroxyvitamin D(3) suppresses hepatitis C virus production

Because the current interferon (IFN)-based treatment for hepatitis C virus (HCV) infection has a therapeutic limitation and side effects, a more efficient therapeutic strategy is desired. Recent studies show that supplementation of vitamin D significantly improves sustained viral response via IFN-based therapy. However, mechanisms and an active molecular form of vitamin D for its anti-HCV effects have not been fully clarified. To address these questions, we infected HuH-7 cells with cell culture-generated HCV in the presence or absence of vitamin D(3) or its metabolites. To our surprise, 25-hydroxyvitamin D(3) [25(OH)D(3) ], but not vitamin D(3) or 1,25-dihydroxyvitamin D(3) , reduced the extra- and intracellular levels of HCV core antigen in a concentration-dependent manner. Single-cycle virus production assay with a CD81-negative cell line reveals that the inhibitory effect of 25(OH)D(3) is at the level of infectious virus assembly but not entry or replication. Long-term 25(OH)D(3) treatment generates a HCV mutant with acquired resistance to 25(OH)D(3) , and this mutation resulting in a N1279Y substitution in the nonstructural region 3 helicase domain is responsible for the resistance. Conclusion: 25(OH)D(3) is a novel anti-HCV agent that targets an infectious viral particle assembly step. This finding provides insight into the improved efficacy of anti-HCV treatment via the combination of vitamin D(3) and IFN. Our results also suggest that 25(OH)D(3) , not vitamin D(3) , is a better therapeutic option in patients with hepatic dysfunction and reduced enzymatic activity for generation of 25(OH)D(3) . (HEPATOLOGY 2012).     

Comparison of complete sequences of hepatitis B virus genotype C between inactive carriers and hepatocellular carcinoma patients before and after seroconversion

Background Most patients who acquire chronic hepatitis B virus (HBV) infection by perinatal transmission become inactive carriers (IC) after hepatitis B e (HBe) antigen seroconversion, whereas some patients have persistent abnormal serum transaminase levels and develop hepatocellular carcinoma (HCC) in the anti-HBe-positive phase. The aim of this study was to investigate the HCC-related mutations of HBV. Methods Complete sequences of HBV were examined among eight IC and eight HCC patients infected with HBV genotype C before and after seroconversion. Results The frequency of the T1653 mutation tended to be higher among HCC patients after seroconversion (16.7% vs. 62.5%; P = 0.086). The prevalence of a basal core promoter double mutation (T1762/A1764) was high among both IC and HCC patients after seroconversion (83.3% vs. 87.5%; P = 0.825). Among the HCC patients, a pre-S deletion mutant was detected in 62.5% patients before seroconversion, and in 37.5% patients after seroconversion. The core deletion mutant was also detected in 50% of HCC patients only before seroconversion. Deletion mutants of the pre-S or core region before seroconversion were significantly associated with HCC patients (0% vs. 62.5%; P = 0.007, 0% vs. 50%; P = 0.021, respectively). Conclusions Our data showed a significant association of pre-S and core deletion mutants before seroconversion with HCC development. The T1653 mutation after seroconversion was frequently found in HCC patients infected with HBV genotype C. These results suggest that mutations may be predictive factor for development of HCC.     

Leukocytapheresis for the treatment of active pouchitis: a pilot study

BACKGROUND: Pouchitis is a major long-term complication of ileal pouch-anal anastomosis for ulcerative colitis. The aim of this study is to investigate the efficacy of leukocytapheresis for the treatment of active pouchitis. METHODS: Eight patients with active pouchitis received leukocytapheresis weekly for 5 weeks in an open-label treatment protocol together with baseline therapy. RESULTS: Patients showed significant improvement in their pouchitis disease activity index scores, from 9.5 (range, 8-10) to 4.0 (range, 2-8) (P < 0.05). Six (75%) of the 8 treated patients achieved remission. No adverse events were observed. CONCLUSIONS: Leukocytapheresis therapy could be a new therapeutic strategy for patients with pouchitis after ileal pouch-anal anastomosis for ulcerative colitis. These encouraging results lead us to propose a randomized controlled trial.     

Two patients treated with pegylated interferon/ribavirin/telaprevir triple therapy for recurrent hepatitis C after living donor liver transplantation

It is difficult to use protease inhibitors in patients with recurrent hepatitis C virus (HCV) infection after liver transplantation (LT) due to interaction with immunosuppressive drugs. We report our experience with two patients treated with telaprevir (TVR) combined with pegylated interferon/ribavirin (PEG IFN/RBV) for recurrent HCV genotype 1 infection after LT. The first was a 63‐year‐old man with HCV‐related liver cirrhosis, who failed to respond to IFN‐β plus RBV after LT. Treatment was switched to PEG IFN‐α‐2b plus RBV and TVR was started. The donor had TT genotype of interleukin (IL)‐28 single nucleotide polymorphisms (SNP) (rs8099917). The recipient had TT genotype of IL‐28 SNP (rs8099917). Completion of 12‐week triple therapy was followed by PEG IFN‐α‐2b plus RBV for 36 weeks. Finally, he had sustained viral response. The second was a 70‐year‐old woman with HCV‐related liver cirrhosis and hepatocellular carcinoma. She failed to respond to PEG IFN‐α‐2b plus RBV after LT, and was subsequently switched to PEG IFN‐α‐2b/RBV/TVR. Genotype analysis showed TG genotype of IL‐28 SNP for the donor, and TT genotype of IL‐28 SNP for the recipient. Serum HCV RNA titer decreased below the detection limit at 5 weeks. However, triple therapy was withdrawn at 11 weeks due to general fatigue, which resulted in HCV RNA rebound 4 weeks later. Both patients were treated with cyclosporin, starting with a small dose to avoid interactions with TVR. TVR is a potentially suitable agent for LT recipients who do not respond to PEG IFN‐α‐2b plus RBV after LT.