Decreased expression of interferon-stimulated genes in B cells of patients with chronic hepatitis C during interferon-free therapy potentially suggests the eradication of hepatitis C virus in the B cells: A cohort study.

AIMS: Hepatitis C virus (HCV) infection is monitored by the host innate immunity that includes the endogenous interferon (IFN), which up-regulates IFN-stimulated genes (ISGs). HCV is both hepatotropic and lymphotropic, but HCV replication in lymphoid cells is a controversial issue. Here, we analyzed the mRNA levels of the ISGs in B cells of HCV-infected patients during antiviral therapy and investigated the effects of viral eradication. METHODS: One hundred and eighty-one patients with chronic hepatitis C and 26 healthy volunteers were enrolled in this study. Levels of HCV RNA and mRNA of ISGs in B cells isolated from the patients were monitored before, during, and after antiviral therapy. RESULTS: HCV RNA was detected in B cells of 133/175 (76.0%) patients who achieved sustained virologic response (SVR) before therapy was started. The positive ratio of HCV RNA in B cells was higher in patients with genotype 1 and the non-major genotype of interleukin 28B. HCV RNA in B cells of most patients disappeared 1 week after antiviral therapy was started. The baseline expression of ISG mRNA was significantly higher in the patients than in the healthy volunteers. Levels of ISG mRNA were increased and remained high throughout the IFN-based therapy. In contrast, levels of ISG mRNA in patients who achieved SVR were significantly decreased 1 week after the IFN-free therapy was started and remained low during the therapy. CONCLUSIONS: These results suggested that IFN-free therapy potentially eradicated HCV in the B cells, leading to the down-regulation of endogenous ISGs. The level of ISG mRNA could be used as a marker for viral eradication in B cells.

Candidatus Mycoplasma haemohominis in Human, Japan.

Hemotropic mycoplasmas are common pathogens in animals, but it remains unclear what role these pathogens play in human infections. We report clinical and biologic characterization of Candidatus Mycoplasma haemohominis infection in a 42-year-old man in Japan. The patient had severe hemophagocytic syndrome 1 month after an accidental needlestick injury. Metagenomic deep sequencing identified Candidatus M. haemohominis and determined its draft genome for an isolate from serum of the patient. A high copy number of the Candidatus M. haemohominis genome was detected in serum and bone marrow samples. Electron microscopy examination showed morphologic characteristics of Candidatus M. haemohominis. Levofloxacin monotherapy induced resistance caused by a gyrase A gene mutation in the quinolone resistance-determining region, but a combination treatment with moxifloxacin and minocycline was effective. We identified Candidatus M. haemohominis in a patient who had life-threatening symptoms related to multiple organ infection. Human infection with this mycoplasma might occur more frequently than has been generally recognized.

Single-incision cholecystectomy in a patient with situs inversus totalis presenting with cholelithiasis: A case report.

Laparoscopic cholecystectomy has become the gold standard for the treatment of cholelithiasis, and many reports of single-incision laparoscopic cholecystectomy have been published in the past few years. Situs inversus totalis is a very rare condition, but the variant anatomy should not preclude a minimally invasive approach to surgery. We report a case of successful single-port laparoscopic cholecystectomy in a patient with situs inversus totalis, describe the technical advantages, and review the literature.

Natural human interferon beta plus ribavirin combination therapy in Japanese patients infected with hepatitis C virus and a high viral load.

OBJECTIVE: The aim of this pilot study was to determine the safety and efficacy of natural human interferon beta (nIFNbeta) plus ribavirin (RBV) in patients with chronic hepatitis C who did not respond to pegylated interferon alpha (PEG-IFN), with special emphasis on the incidence of mental disorders or refusal for fear of adverse effects. METHODS: We studied 19 patients with HCV genotype 1b, 2a or 2b and a high viral load, including 8 patients with mental disorders. They were treated with nIFNbeta-RBV. Eleven patients with HCV genotype 1b of these patients were treated with nIFNbeta-RBV for 48 weeks (group A), and compared with 22 matched controls treated with PEG-IFN plus RBV for 48 weeks (group B). The other 8 patients with HCV genotype 2 were treated with nIFNbeta-RBV for 24 weeks. RESULTS: Six of 8 patients with mental disorders and 9 of 11 patients without mental disorders completed nIFNbeta-RBV therapy; 1 patient with mental disorder dropped out due to exacerbation of depression, and 3 patients suspended the therapy due to insufficient response. The sustained virological response (SVR) was 27% (3/11) in group A and 41% (9/22) in group B (p = 0.70). During treatment, platelet count increased in group A but not in group B. SVR was 88% (7/8) in patients of genotype 2 and high viral load treated with nIFNbeta plus RBV. CONCLUSION: nIFNbeta-RBV therapy offers sufficient safety and efficacy for patients with mental disorders, and thus could represent an excellent second-line therapy for subpopulations that are not suitable for PEG-IFN-RBV.

The N-terminal 15-48 region of cyclin kinase inhibitor p21 is a determinant of basal expression.

The cyclin kinase inhibitor, p21, inhibits or arrests cell cycle progression in response to DNA damage and regulates the progression of apoptosis, either negatively or positively depending on the situation. The stability of p21 is regulated by its phosphorylation or through binding with partner molecules, and, when cells grow without DNA damage, the level of p21 is regulated by proteasome degradation. In this study, we analyzed the mechanism by which the basal expression level of p21 is stabilized. The transient expression of various p21 deletion mutants revealed a specific mutant with a deletion of 15-48 aa (Delta15-48C) that was extremely unstable. This mutant was stabilized by the proteasome inhibitor, lactacystin. Since the cysteine in the region of the alanine mutant did not destabilize p21, possible disulfide bonds formed by cysteines in the region are not responsible for the stabilization. The Delta15-48C was unstable in the cells stably expressing the 1-60 aa region, indicating that the 1-60 aa region did not function in trans. Fusion of the 1-60 aa fragment to the N-terminal of Delta15-48C stabilized the product, indicating that the 1-60 aa region in the molecule is effective for the stabilization. We constructed cells that stably expressed Delta15-48C. The Delta15-48C was unstable, but was stabilized by lactacystin. Irradiation (5 Gy) enhanced the expression of Delta15-48C without elevation of mRNA levels and increased the binding with cyclin A or CDK2. Taken together, the 15-48 aa region of p21 is essential for basal expression by preventing degradation by the proteasome, which is distinct from the mechanism induced by DNA damage.

Cytoplasmic Localization of Cyclin Kinase Inhibitor p21 Delays the Progression of Apoptosis.

The different functions of the cyclin kinase inhibitor, p21, rely on its localization to either the cytoplasm or nucleus. Phosphorylation at Thr-145 and/or Ser-146 was reported to target p21 to the cytoplasm. To clarify the function of cytoplasmic p21, we constructed non-phosphorylatable mutants, Thr-145 to Ala (T145A) and Ser-146 to Ala (S146A), and phosphorylation mimic mutants, Thr-145 to Asp (T145D) and Ser-146 to Asp (S146D), and the cells stably expressing those mutants were identified. The association of all four mutants with either CyclinA or CDK2 was increased by Á-irradiation, indicating that the mutants functioned as cyclin kinase inhibitors. PCNA binding was detected in T145A and S146A, but not in T145D and S146D. In the stably-expressing cells, T145D and S146D binding was observed in the cytoplasm, while T145A and S146A in the nucleus. Further, lactacystin treatment enhanced T145A and S146A, but not T145D and S146D, which is consistent with the degradation of p21 by proteasome in the nucleus. Apoptosis induced by Á-irradiation was delayed in the cells expressing either T145D or S146D. The activities of caspase 3 were not reduced in mutant-expressing cells. These results suggest that the PCNA-unbound form of the full length p21 in the cytoplasm delays apoptosis through the interaction with caspase 3 or downstream components.