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).

Serum levels of high mobility group box-1 protein (HMGB1) and soluble receptors of advanced glycation end-products (RAGE) in depressed patients treated with electroconvulsive therapy.

AIMS: High mobility group box-1 (HMGB1) is one of the damage-associated molecular patterns produced by stress and induces inflammatory responses mediated by receptors of advanced glycation end-products (RAGE) on the cell surface. Meanwhile, soluble RAGE (sRAGE) exhibits an anti-inflammatory effect by capturing HMGB1. Animal models have shown upregulation of HMGB1 and RAGE in the brain or blood, suggesting the involvement of these proteins in depression pathophysiology. However, there have been no reports using blood from depressed patients, nor ones focusing on HMGB1 and sRAGE changes associated with treatment and their relationship to depressive symptoms. METHODS: Serum HMGB1 and sRAGE concentrations were measured by enzyme-linked immunosorbent assay in a group of patients with severe major depressive disorder (MDD) (11 males and 14 females) who required treatment with electroconvulsive therapy (ECT), and also in a group of 25 age- and gender-matched healthy subjects. HMGB1 and sRAGE concentrations were also measured before and after a course of ECT. Depressive symptoms were assessed using the Hamilton Rating Scale for Depression (HAMD). RESULTS: There was no significant difference in HMGB1 and sRAGE concentrations in the MDD group compared to healthy subjects. Although ECT significantly improved depressive symptoms, there was no significant change in HMGB1 and sRAGE concentrations before and after treatment. There was also no significant correlation between HMGB1 and sRAGE concentrations and the HAMD total score or subitem scores. CONCLUSION: There were no changes in HMGB1 and sRAGE in the peripheral blood of severely depressed patients, and concentrations had no relationship with symptoms or ECT.

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.

Antidepressant amitriptyline-induced matrix metalloproteinase-9 activation is mediated by Src family tyrosine kinase, which leads to glial cell line-derived neurotrophic factor mRNA expression in rat astroglial cells.

BACKGROUND: Astrocytes have been implicated in the pathophysiology of mood disorders and in the mechanism of the pharmacological effects of antidepressant drugs by the production of neurotrophic/growth factors. Previous studies have identified astrocyte-expressed Gαi/o -coupled lysophosphatidic acid receptor 1 (LPAR1), as being involved in antidepressant-induced production of glial cell line-derived neurotrophic factor (GDNF) and matrix metalloproteinase-9 (MMP-9) activation, an important step in the production of GNDF. However, the precise mechanism of MMP-9 activation by antidepressants has yet to be identified, in particular the intracellular signaling pathway between LPAR1/Gαi/o and MMP-9. METHODS AND RESULTS: Treatment of rat C6 astroglial cells (C6 cells) with amitriptyline increased Src family tyrosine kinase phosphorylation in a time and concentration-dependent manner. Amitriptyline-induced GDNF mRNA expression was blocked by Src family tyrosine kinase inhibitors. In addition, inhibiting Src family tyrosine kinase blocked amitriptyline-induced zymographic MMP-9 activation in C6 cells. The amitriptyline-induced zymographic MMP-9 activity was completely blocked by selective inhibition of Gαi/o protein and LPAR1. Furthermore, the amitriptyline-induced Src family tyrosine kinase phosphorylation was blocked by LPAR1, but not MMP-9 inhibition, indicating that Src family tyrosine kinase involvement is downstream of LPAR1. CONCLUSIONS: The current findings suggest that the pharmacological effect of antidepressant such as amitriptyline is mediated through an intracellular signaling pathway via the LPAR1/Gαi/o /Src family tyrosine kinase, which leads to MMP-9 activation and GDNF production.

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.

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.

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.

Factors associated with relapse after a response to electroconvulsive therapy in unipolar versus bipolar depression.

BACKGROUND: While electroconvulsive therapy (ECT) treatment for depression is highly effective, the high rate of relapse is a critical problem. The current study investigated factors associated with the risk of relapse in mood disorders in patients in which ECT was initially effective. METHOD: The records of 100 patients with mood disorders (61 unipolar depression, 39 bipolar depression) who received and responded to an acute ECT course were retrospectively reviewed. Associations between clinical variables and relapse after responding to acute ECT were analyzed. The Ethics Committee of NHO Kure Medical Center approved the study protocol. RESULTS: After one year, the percentage of relapse-free patients was 48.7%. There was no significant difference between patients with either unipolar or bipolar depression who were relapse-free (unipolar: 51.1%, bipolar: 45.5%, P=0.603). Valproate maintenance pharmacotherapy in unipolar depression patients was associated with a lower risk of relapse compared to patients without valproate treatment (multivariate analysis, hazard ratio: 0.091; P=0.022). Lithium treatment, reportedly effective for unipolar depression following a course of ECT, tended to lower the risk of relapse (hazard ratio: 0.378; P=0.060). For bipolar depression, no treatment significantly reduced the risk of relapse. LIMITATIONS: The current findings were retrospective and based on a limited sample size. CONCLUSIONS: The relapse-free rate was similar between unipolar and bipolar depression. Valproate could have potential for unipolar depression patients as a maintenance therapeutic in preventing relapse after ECT.

Stimulation of spinal dorsal horn ß2-adrenergic receptor ameliorates neuropathic mechanical hypersensitivity through a reduction of phosphorylation of microglial p38 MAP kinase and astrocytic c-jun N-terminal kinase.

The noradrenaline-adrenergic system has a crucial role in controlling nociceptive transduction at the spinal level. While α-adrenergic receptors are known to regulate nociceptive neurotransmitter release at the spinal presynaptic level, it is not entirely clear whether ß-adrenergic receptors are involved in controlling pain transduction at the spinal level as well. The current study elucidated a role of ß-adrenergic receptors in neuropathic pain in mice following a partial sciatic nerve ligation (PSNL). In addition, the cellular and intracellular signaling cascade induced by ß-adrenergic receptors in neuropathic mice was elaborated. Intrathecal injection of isoproterenol (1 nmol), a nonselective ß-adrenergic receptor agonist, briefly ameliorated hind paw mechanical hypersensitivity of PSNL mice. Isoproterenol's antinociceptive effect was mediated through ß2-adrenergic receptors since pretreatment with ICI118551, a selective ß2-adrenergic receptor antagonist, but not with CGP20712A, a selective ß1-adrenergic receptor antagonist, significantly attenuated isoproterenol's effect. Furthermore, intrathecal treatment with a selective ß2-adrenergic receptor agonist, terbutaline, but not a selective ß1-adrenergic receptor agonist, dobutamine, also significantly ameliorated neuropathic pain. Fourteen days after PSNL, increased phosphorylation of both p38 Mitogen-activated protein kinase (MAPK) in microglia and c-jun N-terminal kinase (JNK) in astrocytes of ipsilateral spinal dorsal horn were observed. Phosphorylation of both microglial p38 MAPK and astrocytic JNK were downregulated by stimulation of the ß2-adrenergic receptor. Together, these results suggest that spinal ß2-adrenergic receptor have an inhibitory role in neuropathic nociceptive transduction at the spinal level through a downregulation of glial activity, perhaps through modulation of MAP kinases phosphorylation. Thus, targeting of ß2-adrenergic receptors could be an effective therapeutic strategy in treating neuropathic pain.

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 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.

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.

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.

Combination therapies with daclatasvir and asunaprevir on NS3-D168 mutated HCV in human hepatocyte chimeric mice.

BACKGROUND: Although the frequency of emergent drug-resistant strains of HCV in patients who failed to respond to simeprevir plus pegylated interferon (PEG-IFN) and ribavirin (RBV) decreased after cessation of the treatment, it is not clear whether or not the NS3-D168 variants affect the outcome of NS5A and NS3 inhibitor combination therapy. In this study, we investigated the relationship between the effect of daclatasvir plus asunaprevir treatment and the frequencies of NS3-D168 variants. METHODS: HCV genotype-1b-infected human hepatocyte chimeric mice with various frequencies of NS3-D168 amino acid substitutions were treated with asunaprevir alone or in combination with daclatasvir for 4 weeks. Frequencies of NS3-D168 substitutions at baseline were analysed by ultra-deep sequencing. Some mice with NS3-D168 substitutions were treated with PEG-IFN or telaprevir for 4 weeks. RESULTS: Mice with high frequencies of NS3-D168 showed low susceptibility to asunaprevir treatment and failed to respond to daclatasvir plus asunaprevir therapy. In contrast, mice with a low frequency (less than approximately 14%) of NS3-D168 showed a similar susceptibility to wild-type HCV-infected mice and achieved viral eradication with daclatasvir plus asunaprevir therapy. Although treatment with either telaprevir or PEG-IFN resulted in reduction of serum HCV RNA levels, no significant decrease in the frequency of NS3-D168 substitutions was achieved. CONCLUSIONS: Daclatasvir and asunaprevir treatment could eliminate NS3-D168 variant HCV if the frequency was low. It is necessary to confirm that the frequency of NS3-D168 variants has decreased sufficiently before adopting daclatasvir plus asunaprevir therapy in patients with simeprevir plus PEG-IFN/RBV treatment failure.

Elimination of HCV via a non-ISG-mediated mechanism by vaniprevir and BMS-788329 combination therapy in human hepatocyte chimeric mice.

We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C.

Effect of tenofovir disoproxil fumarate on drug-resistant HBV clones.

BACKGROUND & AIMS: Tenofovir disoproxil fumarate (TDF) has been approved for chronic hepatitis B treatment, and favorable susceptibility of hepatitis B virus (HBV) has been indicated. However, differences in TDF susceptibility among HBV genotypes and drug-resistant strains are unclear. In this study, TDF susceptibilities between genotypes A and C were evaluated in vitro and in vivo using several drug-resistant HBV clones. METHODS: HBV expression plasmids were constructed from sera of HBV carriers, and drug-resistant substitutions were introduced by site-directed mutagenesis. TDF susceptibility was evaluated by changes of core-associated HBV replication intermediates in vitro or by change of serum HBV DNA in human hepatocyte chimeric mice carrying each HBV clone in vivo. RESULTS: TDF susceptibilities of lamivudine-resistant clones (rtL180M/M204V) and lamivudine plus entecavir-resistant clones (rtL180M/S202G/M204V) were similar to wild type clones in vitro. However, lamivudine plus adefovir-resistant clones (rtA181T/N236T) acquired tolerance to TDF, and the rtN236T mutation was considered to be a causal substitution for TDF resistance. Furthermore, genotypic differences in TDF susceptibility were also observed between genotypes A and C in vitro, and the differences could be confirmed in vivo (p = 0.023). CONCLUSIONS: The present study indicates that TDF susceptibility varies among HBV genotypes and drug-resistant HBV clones.

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.

Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

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.

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.