Safety of nivolumab monotherapy in five cancer types: pooled analysis of post-marketing surveillance in Japan.

BACKGROUND: Nivolumab has been approved for treating ≥ 10 cancer types. However, there is limited information on the incidence of rare, but potentially serious, treatment-related adverse events (TRAEs), as well as notable TRAEs in patients with certain medical disorders or older patients in Japan. METHODS: We performed pooled analyses of data from published post-marketing surveillance in Japan of nivolumab monotherapy for patients with malignant melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck cancer, and gastric cancer to determine the frequencies of 20 categories of TRAEs of special interest overall and in patient groups with higher perceived safety risks (history of autoimmune disease, interstitial lung disease, tuberculosis, or hepatitis B/C; patients vaccinated during nivolumab treatment; and older patients [≥ 75 years]). RESULTS: The overall population comprised 7421 patients treated with nivolumab. TRAEs were reported in 49.1% of patients, with grade ≥ 3 TRAEs in 16.7%. Endocrine disorders (14.4%), hepatobiliary disorders (10.9%), and interstitial lung disease (7.0%) were the three most common categories (any grade). The incidences of rare TRAEs with high risk of becoming serious, which occurred in < 1% of patients, were consistent with those in previous reports. The frequencies of TRAEs were not markedly increased in the specified patient groups relative to the overall population. CONCLUSION: To our knowledge, this is the largest study examining the safety of nivolumab-treated patients in real-world clinical practice including rare but potentially serious TRAEs. We found no new signals in the safety of nivolumab among the patient groups relative to the overall population, and no additional safety measures are required in these groups. Trial registration UMIN000048892 (overall analysis), JapicCTI-163272 (melanoma), Japic-163271 (non-small cell lung cancer), JapicCTI-184071 (head and neck cancer), JapicCTI-184070 (gastric cancer), and JapicCTI-184069 (renal cell cancer).

Reduced Serum and Cerebrospinal Fluid Levels of Autotaxin in Major Depressive Disorder.

BACKGROUND: The autotaxin/lysophosphatidic acid axis is involved in diverse biological processes including neurodevelopment, inflammation, and immunological functioning. The lysophosphatidic acid 1 receptor has been implicated in the pathophysiology of major depressive disorder and in the mechanism of action of antidepressants. However, it is unclear whether central or peripheral autotaxin levels are altered in patients with major depressive disorder. METHODS: Serum autotaxin levels were measured by an enzyme-linked immunosorbent assay in 37 patients with major depressive disorder diagnosed using DSM-IV-TR who underwent electroconvulsive therapy and were compared with those of 47 nondepressed controls matched for age and sex between January 2011 and December 2015. Patient serum levels of autotaxin before and after electroconvulsive therapy were also compared. In a separate sample set, cerebrospinal fluid autotaxin levels were compared between 26 patients with major depressive disorder and 27 nondepressed controls between December 2010 and December 2015. A potential association was examined between autotaxin levels and clinical symptoms assessed with the Hamilton Depression Rating Scale. RESULTS: Before electroconvulsive therapy, both serum and cerebrospinal fluidautotaxin levels were significantly lower in major depressive disorder patients than in controls (serum: P = .001, cerebrospinal fluid: P = .038). A significantly negative correlation between serum, but not cerebrospinal fluid, autotaxin levels and depressive symptoms was observed (P = .032). After electroconvulsive therapy, a parallel increase in serum autotaxin levels and depressive symptoms improvement was observed (P = .005). CONCLUSION: The current results suggest that serum autotaxin levels are reduced in a state-dependent manner. The reduction of cerebrospinal fluidautotaxin levels suggests a dysfunction in the autotaxin/lysophosphatidic acid axis in the brains of patients with major depressive disorder.

Shared preventive factors associated with relapse after a response to electroconvulsive therapy in four major psychiatric disorders.

AIM: The efficacy of electroconvulsive therapy (ECT) has been established in psychiatric disorders but the high rate of relapse is a critical problem. The current study sought preventative factors associated with relapse after a response to ECT in a continuum of four major psychiatric disorders. METHODS: The records of 255 patients with four psychiatric disorders (83 unipolar depression, 60 bipolar depression, 91 schizophrenia, 21 schizoaffective disorder) were retrospectively reviewed. RESULTS: The relapse-free rate of all patients at 1 year was 56.3% in the four psychiatric disorders without a difference. As a result of univariate analysis, three items could be considered as preventative factors associated with relapse: a small number of psychiatric symptom episodes before an acute course of ECT, the use of mood stabilizers, and the use of maintenance ECT. Multivariate analysis was performed, keeping age, sex, and diagnosis constant in addition to the three items, and small number of psychiatric symptom episodes before an acute course of ECT (P = 0.003), the use of lithium (P = 0.025), the use of valproate (P = 0.027), and the use of maintenance ECT (P = 0.001) were found to be significant preventative measures against relapse. CONCLUSION: The use of mood stabilizers, such as lithium and valproate, and maintenance ECT could be shared preventive factors associated with relapse after a response to ECT in four major psychiatric disorders.

Possible Association between Serum Matrix Metalloproteinase-9 (MMP-9) Levels and Relapse in Depressed Patients following Electroconvulsive Therapy (ECT).

Background: Matrix metalloproteinases are involved in neuroinflammatory processes, which could underlie depression. Serum levels of MMP-9 and MMP-2 in depressed patients are significantly altered following electroconvulsive therapy, but an association between altered matrix metalloproteinases after successful ECT and possible relapse has yet to be investigated. Methods: Serum was obtained twice, before and immediately after a course of electroconvulsive therapy, from 38 depressed patients. Serum was also collected, once, from two groups of age- and gender-matched healthy controls, 40 volunteers in each group. Possible associations between levels of matrix metalloproteinases and relapse during a 1-year follow-up period were analyzed. Results: Excluding patients who did not respond to electroconvulsive therapy and patients lost to follow-up, data from 28 patients were evaluated. Eighteen of the patients (64.3%) relapsed within 1 year. In the group that did not relapse, serum levels of MMP-9 were significantly decreased after a course of electroconvulsive therapy, but not in the group that relapsed. No association between MMP-2 and relapse was observed. Conclusion: The degree of change in serum MMP-9 change could be associated with relapse following electroconvulsive therapy in depressed patients.

Identification of Lysophosphatidic Acid Receptor 1 in Astroglial Cells as a Target for Glial Cell Line-derived Neurotrophic Factor Expression Induced by Antidepressants.

Preclinical and clinical evidence suggests that glial cell line-derived neurotrophic factor (GDNF) is important in the therapeutic effect of antidepressants. A previous study demonstrated that the tricyclic antidepressant amitriptyline induces Gαi/o activation, which leads to GDNF expression in astrocytes. However, the specific target expressed in astrocytes that mediates antidepressant-evoked Gαi/o activation has yet to be identified. Thus, the current study explored the possibility that antidepressant-induced Gαi/o activation depends on lysophosphatidic acid receptor 1 (LPAR1), a Gαi/o-coupled receptor. GDNF mRNA expression was examined using real-time PCR and Gαi/o activation was examined using the cell-based receptor assay system CellKeyTM in rat C6 astroglial cells and rat primary cultured astrocytes. LPAR1 antagonists blocked GDNF mRNA expression and Gαi/o activation evoked by various classes of antidepressants (amitriptyline, nortriptyline, mianserin, and fluoxetine). In addition, deletion of LPAR1 by RNAi suppressed amitriptyline-evoked GDNF mRNA expression. Treatment of astroglial cells with the endogenous LPAR agonist LPA increased GDNF mRNA expression through LPAR1, whereas treatment of primary cultured neurons with LPA failed to affect GDNF mRNA expression. Astrocytic GDNF expression evoked by either amitriptyline or LPA utilized, in part, transactivation of fibroblast growth factor receptor and a subsequent ERK cascade. The current results suggest that LPAR1 is a novel, specific target of antidepressants that leads to GDNF expression in astrocytes.

The glycosylation stoichiometry of EWS species in neuronal cells.

Although Ewing sarcoma protein (EWS) is known to be glycosylated by O-linked ß-N-acetylglucosamine (O-GlcNAc), the dynamics and stoichiometry of its glycosylation remain obscure. Here, we report a dynamic change in the glycosylation stoichiometry of EWS species during neuronal differentiation of embryonic carcinoma P19 cells. Our findings suggest that O-GlcNAc glycosylation participates in the regulation of EWS functions in neuronal cells.

Protease Inhibitor Resistance Remains Even After Mutant Strains Become Undetectable by Deep Sequencing.

BACKGROUND: Although treatment-emergent NS3/4A protease inhibitor (PI)-resistant variants typically decrease in frequency after cessation of PI therapy in patients with chronic hepatitis C virus (HCV) infection, HCV susceptibility to PIs in patients who have not responded to previous PI therapy has not been addressed. METHODS: Patients with chronic HCV genotype 1 infection were treated either with simeprevir plus interferon or with daclatasvir plus asunaprevir. Frequencies of drug-resistant mutations among patients with treatment failure were analyzed by deep sequencing. Human hepatocyte chimeric mice were injected with serum samples obtained from either treatment-naive patients or nonresponders to treatment with daclatasvir plus asunaprevir and then were treated with simeprevir and sofosbuvir. RESULTS: Virological response to daclatasvir plus asunaprevir treatment was significantly lower in patients with simeprevir treatment failure as compared to those without previous treatment. Deep-sequencing analysis showed that the frequency of PI treatment-emergent NS3-D168 mutations gradually decreased and were completely replaced by wild-type genes after cessation of therapy. However, mice injected with serum obtained from a patient with PI treatment failure rapidly developed NS3-D168 mutations at significantly higher frequencies following either simeprevir or sofosbuvir treatment. CONCLUSIONS: The virological response to daclatasvir plus asunaprevir treatment was low in patients with simeprevir treatment failure. PI resistance remains even after disappearance of mutant strains by deep sequencing.

Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

The expression of glial cell line-derived neurotrophic factor mRNA by antidepressants involves matrix metalloproteinase-9 activation in rat astroglial cells.

Neurotrophic/growth factors derived from glial cells, especially astrocytes, have been implicated in mood disorders and the pharmacological effects of antidepressant drugs. Previous studies demonstrated that the release of glial cell line-derived neurotrophic factor (GDNF) induced by the tricyclic antidepressant amitriptyline was significantly inhibited by a broad-spectrum matrix metalloproteinase (MMP) inhibitor in rat C6 astroglial cells (C6 cells). However, it is unknown whether amitriptyline affects MMP enzymatic activity or expression, and the MMP subtype has yet to be identified. The current study measured the effect of antidepressants on MMP activity with gelatin zymography, an in vitro assay for enzymatic activity, in C6 cells and primary cultured rat astrocytes (primary astrocytes). Treatment with amitriptyline increased zymographic MMP-9 activity without changing MMP-9 mRNA expression in C6 cells. Several different classes of antidepressants significantly increased zymographic MMP-9 activity in C6 cells and primary astrocytes, whereas antipsychotic drugs without antidepressant pharmacological activity did not. The amitriptyline-induced expression of GDNF mRNA was completely blocked by selective inhibition of MMP-9 in C6 cells. Treatment of C6 cells and primary astrocytes with exogenous recombinant MMP-9 increased GDNF mRNA expression, similar to that observed with amitriptyline. Inhibiting MMP-3 blocked amitriptyline-induced zymographic MMP-9 activation in C6 cells and primary astrocytes, indicating that MMP-3 is necessary for MMP-9 activity. The current study suggests that MMP-9 activation is indispensable in the amitriptyline-induced expression of GDNF mRNA in astrocytes and further supports a role of astrocytic neurotrophic/growth factors in the pharmacological effect of antidepressants.

Human Cytotoxic T Lymphocyte-Mediated Acute Liver Failure and Rescue by Immunoglobulin in Human Hepatocyte Transplant TK-NOG Mice.

UNLABELLED: Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are critical in eliminating infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by HBV-specific CTLs. After HBV inoculation in human hepatocyte-transplanted herpes simplex virus type-1 thymidine kinase-NOG mice, human peripheral blood mononuclear cells (PBMCs) were administered, and albumin, HBV DNA, alanine aminotransferase (ALT), and cytokine levels were analyzed. Histopathological and flow-cytometric analysis of infiltrating human immune cells were performed, and the efficacy of CTL-associated antigen-4 immunoglobulin (CTLA4Ig) against liver damage was evaluated. PBMC treatment resulted in massive hepatocyte damage with elevation of ALT, granzyme A, and gamma interferon and decrease in albumin and HBV DNA. The number of liver-infiltrating human lymphocytes and CD8-positive cells was significantly higher in HBV-infected mice. HBV-specific CTLs were detected by core and polymerase peptide-major histocompatibility complex-tetramer, and the population of regulatory T cells was significantly decreased in HBV-infected mice. Serum hepatitis B surface (HBs) antigen became negative, and HBs antibody appeared. CTLA4Ig treatment strongly inhibited infiltration of mononuclear cells. CTLA4Ig treatment will be used to treat patients who develop severe acute hepatitis B to prevent liver transplantation or lethality. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B. IMPORTANCE: Without liver transplantation, some HBV-infected patients will die from severe liver damage due to acute overreaction of the immune system. No effective treatment exists, due in part to the lack of a suitable animal model. An animal model is necessary to investigate the mechanism of hepatitis and to develop therapeutic strategies to prevent acute liver failure in HBV infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by human HBV-specific CTLs. In this model, HBV-infected human hepatocytes were transplanted into severely immunodeficient NOG mice in order to reconstruct elements of the human immune system. Using this model, we found that CTL-associated antigen-4 immunoglobulin was able to suppress damage to HBV-infected hepatocytes, suggesting an approach to treatment. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B.

Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice.

The molecular mechanisms underlying the hepatitis B virus (HBV) life cycle are poorly understood because of the lack of appropriate in vitro infection models. Herein, we report a highly effective in vitro HBV infection system using fresh human hepatocytes (HHs) isolated from chimeric mice with humanized livers. After the inoculation of sera collected from HBV-infected chimeric mice or patients to HHs, we measured levels of HBV DNA, mRNA, covalently closed circular DNA, and viral protein expression in HHs. We investigated the neutralization activity of hepatitis B immune globulin and the effects of siRNA against sodium taurocholate-cotransporting polypeptide and clathrin heavy chain on HBV infection. We confirmed the expression of viral antigens in HHs and the presence of extracellular HBV DNA and hepatitis B surface antigen. The maximum infection rate was approximately 80%. Lamivudine and hepatitis B immune globulin treatment reduced HBV DNA levels in a dose-dependent manner. Knockdown of sodium taurocholate-cotransporting polypeptide and clathrin heavy chain significantly reduced the levels of hepatitis B surface antigen. Infection was successfully established using different donor HHs and inocula. Elevation of extracellular HBV DNA levels and the increase of HBV-positive HHs were blocked by continuous hepatitis B immune globulin treatment, indicating virus spread in this model. Chimeric mouse-derived HHs provide a robust in vitro infection model that can completely support the HBV life cycle.

Altered Serum Levels of Matrix Metalloproteinase-2, -9 in Response to Electroconvulsive Therapy for Mood Disorders.

BACKGROUND: Inflammatory processes could underlie mood disorders. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMP) are inflammation-related molecules. The current study sought an association between mood disorders and systemic levels of MMPs and TIMPs. METHODS: Serum was obtained from patients with mood disorders (n=21) and patients with schizophrenia (n=13) scheduled to undergo electroconvulsive therapy. Serum was also obtained from healthy controls (n=40). Clinical symptoms were assessed by the Hamilton Rating Score for Depression and the Brief Psychiatric Rating Scale. Serum levels of MMPs and TIMPs were quantified by ELISA. RESULTS: The serum levels of MMP-2 in mood disorder patients, but not in schizophrenia patients, prior to the first electroconvulsive therapy session (baseline) was significantly lower than that of healthy controls. At baseline, levels of MMP-9 and TIMP-2, -1 were not different between patients with mood disorder and schizophrenia and healthy controls. After a course of electroconvulsive therapy, MMP-2 levels were significantly increased in mood disorder patients, but MMP-9 levels were significantly decreased in both mood disorder and schizophrenia patients. In mood disorder patients, there was a significant negative correlation between depressive symptoms and serum levels of MMP-2 and a positive correlation between depressive symptoms and MMP-9. In addition, alterations of serum levels of MMP-2 and MMP-9 were significantly correlated each other and were associated with certain depressive symptoms. CONCLUSION: A change in inflammatory homeostasis, as indicated by MMP-2 and MMP-9, could be related to mood disorders, and these markers appear to be sensitive to electroconvulsive therapy.

Association between variants in the interferon lambda 4 locus and substitutions in the hepatitis C virus non-structural protein 5A.

BACKGROUND & AIMS: Single nucleotide polymorphisms within the interferon lambda 4 (IFNL4) locus are strongly associated with spontaneous clearance of hepatitis C virus (HCV) infection and early viral response to interferon therapy. Interaction between host genotype and amino acid substitutions might also influence the risk of antiviral resistance in interferon-free direct acting antiviral (DAA) therapies. METHODS: The relationship between IFNL4 genotype and HCV substitutions was analyzed in 929 patients with chronic HCV genotype 1b infection. Ultra-deep sequencing and quasispecies reconstruction was performed on the N-terminal region of NS5A in 57 patients. RESULTS: IFNL4 genotype was strongly associated with HCV NS5A Y93 and core protein substitutions, and the number and diversity of predicted quasispecies was marginally greater in IFNL4 TT/TT patients compared to TT/ΔG, ΔG/ΔG patients. RNA secondary structure prediction of the NS5A region suggests that variable sites are more likely to occupy unpaired, high entropy positions. CONCLUSIONS: HCV infection is proposed to induce a more efficient antiviral response in individuals with the IFNL4 TT/TT genotype that results either in viral clearance or selection for viral adaptations. The association between IFNL4 TT/TT genotype and Y93 substitutions may impact the risk of antiviral resistance in NS5A inhibitors in DAA therapy.

Long term persistence of NS5A inhibitor-resistant hepatitis C virus in patients who failed daclatasvir and asunaprevir therapy.

Although interferon-free antiviral treatment is expected to improve treatment of hepatitis C, it is unclear to what extent pre-existing drug-resistant amino acid substitutions influence response to therapy. The impact of pre-existing drug-resistant substitutions on virological response to daclatasvir and asunaprevir combination therapy was studied in genotype 1b hepatitis C virus (HCV)-infected patients. Thirty-one patients were treated with daclatasvir and asunaprevir for 24 weeks. Twenty-six patients achieved sustained virological response (SVR), three patients experienced viral breakthrough, and two patients relapsed. Direct sequencing analysis of HCV showed the existence of daclatasvir-resistant NS5A-L31M or -Y93H/F variants in nine out of 30 patients (30%) prior to treatment, while asunaprevir-resistant NS3-D168 mutations were not detected in any patient. All 21 patients with wild-type NS5A-L31 and -Y93 achieved SVR, whereas only four out of nine patients (44%) with L31M or Y93F/H substitutions achieved SVR (P = 0.001). Ultra-deep sequencing analysis showed that treatment failure was associated with the emergence of both NS5A-L31/Y93 and NS3-D168 variants. NS5A-L31/Y93 variants remained at high frequency through post-treatment weeks 103 through 170, while NS3-D168 variants were replaced by wild-type in all patients. In conclusion, pre-existence of NS5A inhibitor-resistant substitutions compromised the response to daclatasvir and asunaprevir combination therapy, and treatment failure was associated with the emergence of both NS5A-L31/Y93 and NS3-D168 variants. While asunaprevir-resistant variants that emerged during therapy returned to wild-type, daclatasvir-resistant variants tended to persist in the absence of the drug.

Aryl hydrocarbon receptor protects against bacterial infection by promoting macrophage survival and reactive oxygen species production.

Aryl hydrocarbon receptor (AhR) is crucial for various immune responses. The relationship between AhR and infection with the intracellular bacteria Listeria monocytogenes (LM) is poorly understood. Here, we show that in response to LM infection, AhR is required for bacterial clearance by promoting macrophage survival and reactive oxygen species (ROS) production. AhR-deficient mice were more susceptible to listeriosis, and AhR deficiency enhances bacterial growth in vivo and in vitro. On the other hand, pro-inflammatory cytokines were increased in AhR-deficient macrophages infected with LM despite enhanced susceptibility to LM infection in AhR-deficient mice. Subsequent studies demonstrate that AhR protects against macrophage cell death induced by LM infection through the induction of the antiapoptotic factor, the apoptosis inhibitor of macrophages, which promotes macrophage survival in the setting of LM infection. Furthermore, AhR promotes ROS production for bacterial clearance. Our results demonstrate that AhR is essential to the resistance against LM infection as it promotes macrophage survival and ROS production. This suggests that the activation of AhR by its ligands may be an effective strategy against listeriosis.

Aryl hydrocarbon receptor plays protective roles in ConA-induced hepatic injury by both suppressing IFN-γ expression and inducing IL-22.

The aryl hydrocarbon receptor (AhR), a ligand-activated nuclear transcription factor, is known to mediate the toxic and carcinogenic effects of various environmental pollutants, while AhR has been shown to protect animals from various types of tissue injury. ConA-induced hepatitis is known as a mouse model of acute liver injury. Here, we found a protective role of AhR in ConA-induced hepatitis. AhR is induced in the liver during ConA-induced hepatitis, and Ahr (-/-) mice were highly sensitive to this model. Bone marrow chimera experiments indicate that Ahr (-/-) hematopoietic cells are responsible for hypersensitivity to ConA-induced hepatitis. We found that IFN-γ from invariant NKT cells was up-regulated and IL-22 from innate lymphoid cells (ILCs) was abolished in Ahr (-/-) mice. In addition, IL-22 production was still observed in Rag2 (-/-) mice but it was severely reduced in Ahr (-/-) Rag2 (-/-) mice. ConA-induced IL-22 production was also dependent on retinoic acid-related orphan receptor γt. These results show that AhR has crucial protective roles in ConA-induced liver injury via promoting IL-22 production from ILCs and suppressing IFN-γ expression from NKT cells.

Effects of bisphosphonate zoledronic acid in hepatocellular carcinoma, depending on mevalonate pathway.

BACKGROUND AND AIM: Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate and is used to reduce cancer-induced osteolysis. We reported previously that ZOL delayed both the growth and pain progression of bone metastases from hepatocellular carcinoma. The present study was designed to evaluate the effects of ZOL on hepatoma cell lines and the molecular mechanisms of such effects. METHODS: Cell viability assay, scratch assay, immunohistochemistry, Western blotting, and flow cytometry analysis were performed using Huh7 and HepG2 cells treated with and without ZOL. RESULTS: ZOL reduced cell growth in a dose-dependent manner and prevented cell migration when used at a concentration exceeding 10 µM. Immunohistochemistry showed that the inhibitory effects of ZOL on hepatoma cell progression was not due to the suppression of Ras and RhoA expression but due to inhibition of their translocation from the cytosol to the cell membrane, which terminates mevalonate pathway. Immunoblotting and flow cytometry showed that ZOL inhibited the mitogen-activated protein kinase pathway (MAPK) and induced apoptosis of hepatoma cells. CONCLUSIONS: Our results indicated that ZOL prevented cell growth and metastasis based on direct antitumor effects in hepatoma cells. The use of ZOL could not only suppress the progression to bone metastatic lesions but also prevented growth of primary hepatocellular carcinoma.

Clock gene Per1 regulates the production of CCL2 and interleukin-6 through p38, JNK1 and NF-κB activation in spinal astrocytes.

It has been previously reported that spinal clock genes controlled under circadian rhythm contribute to the regulation of astrocytic function, which in turn is involved in diverse processes such as nociceptive transduction and the induction of inflammation. However, how clock genes expressed in spinal cord astrocytes are associated with the modulation of the inflammatory response is poorly understood. In the current study, the role of Period1 (Per1), one of clock genes, in the expression of chemokine (C-C motif) ligand 2 (CCL2) and interleukin-6 (IL-6), which are typical pro-inflammatory mediators produced by spinal astrocytes, was investigated. It was found that the knockdown of Per1 by using RNA interference led to a significant increase of the expression of CCL2 and IL-6 in cultured rat spinal astrocytes. Moreover, the silencing of the Per1 gene also increased the phosphorylation of p38, c-Jun N-terminal kinase (JNK) 1 and IκBα, and led to the translocation of p65 from the cytosol to the nucleus. The induction of CCL2 and IL-6 was significantly inhibited by treatment with the inhibitors of p38, JNK, and NF-κB. By contrast, the overexpression of PER1 by transfection vector significantly blocked the expression of CCL2 and IL-6, and the activation of p38, JNK, and NF-κB. Together, these results suggest that down-regulation of Per1 induced the phosphorylation of p38 and JNK1 and the subsequent activation of NF-κB, and that these events contribute to neuroinflammatory state in the spinal cord via the induction of the release of inflammatory mediators.

A ß1/2 adrenergic receptor-sensitive intracellular signaling pathway modulates CCL2 production in cultured spinal astrocytes.

The phosphorylation of c-jun N-terminal kinase (JNK) and the subsequent production of C-C chemokine CCL2 (monocyte chemoattractant protein; MCP-1) in spinal astrocytes contribute to the initiation of neurological disorders including chronic pain. Astrocytes express neurotransmitter receptors which could be targeted to ameliorate neurological disorders. In the current study, the involvement of the ß-adrenergic system in the regulation of JNK activity and CCL2 production after stimulation with tumor necrosis factor (TNF)-α, one of many initiators of neuroinflammation, was elucidated. Treatment of cultured spinal astrocytes with isoproterenol (a ß-adrenergic receptor agonist; 1 µM) reduced both TNF-α-induced JNK1 phosphorylation, as observed by Western blotting, and the subsequent increase of both CCL2 mRNA expression and CCL2 production, which were measured by real time-PCR and ELISA, respectively. The effects of isoproterenol were completely blocked by pretreatment with either propranolol (a ß-adrenoceptor antagonist) or H89 (a protein kinase A [PKA] inhibitor). The current study revealed that the regulation of glycogen synthase kinase-3ß (GSK-3ß) activity is a crucial factor in the inhibitory action of isoproterenol. The TNF-α-induced JNK1 phosphorylation was significantly blocked by treatment with GSK-3ß inhibitors (either LiCl or TWS119), and stimulation of ß-adrenergic receptors induced the inhibition of GSK-3ß through the phosphorylation of Ser(9) . Moreover, treatment with isoproterenol markedly suppressed the TNF-α-induced increase of CCL2 mRNA expression and CCL2 production through a ß-adrenergic receptor-PKA pathway mediated by GSK-3ß regulation. Thus, activation of ß1/2 adrenergic receptors expressed in spinal astrocytes could be a novel method of moderating neurological disorders with endogenous catecholamines or selective agonists.

Ultradeep sequencing study of chronic hepatitis C virus genotype 1 infection in patients treated with daclatasvir, peginterferon, and ribavirin.

Direct-acting antivirals (DAAs) are either part of the current standard of care or are in advanced clinical development for the treatment of patients chronically infected with hepatitis C virus (HCV) genotype 1, but concern exists with respect to the patients who fail these regimens with emergent drug-resistant variants. In the present study, ultradeep sequencing was performed to analyze resistance to daclatasvir (DCV), which is a highly selective nonstructural protein 5A (NS5A) inhibitor. Eight patients with HCV genotype 1b, who were either treatment naive or prior nonresponders to pegylated interferon plus ribavirin (Rebetol; Schering-Plough) (PEG-IFN/RBV) therapy, were treated with DCV combined with PEG-IFN alpha-2b (Pegintron; Schering-Plough, Kenilworth, NJ) and RBV. To identify the cause of viral breakthrough, the preexistence and emergence of DCV-resistant variants at NS5A amino acids were analyzed by ultradeep sequencing. Sustained virological response (SVR) was achieved in 6 of 8 patients (75%), with viral breakthrough occurring in the other 2 patients (25%). DCV-resistant variant Y93H preexisted as a minor population at higher frequencies (0.1% to 0.5%) in patients who achieved SVR. In patients with viral breakthrough, DCV-resistant variant mixtures emerged at NS5A-31 over time that persisted posttreatment with Y93H. Although enrichment of DCV-resistant variants was detected, the preexistence of a minor population of the variant did not appear to be associated with virologic response in patients treated with DCV/PEG-IFN/RBV. Ultradeep sequencing results shed light on the complexity of DCV-resistant quasispecies emerging over time, suggesting that multiple resistance pathways are possible within a patient who does not rapidly respond to a DCV-containing regimen. (This study has been registered at ClinicalTrials.gov under registration no. NCT01016912.).