Gallbladder microbiota composition is associated with pancreaticobiliary and gallbladder cancer prognosis.

BACKGROUND: The microbial population of the intestinal tract and its relationship to specific diseases has been extensively studied during the past decade. However, reports characterizing the bile microbiota are rare. This study aims to investigate the microbiota composition in patients with pancreaticobiliary cancers and benign diseases by 16S rRNA gene amplicon sequencing and to evaluate its potential value as a biomarker for the cancer of the bile duct, pancreas, and gallbladder. RESULTS: We enrolled patients who were diagnosed with cancer, cystic lesions, and inflammation of the pancreaticobiliary tract. The study cohort comprised 244 patients. We extracted microbiome-derived DNA from the bile juice in surgically resected gallbladders. The microbiome composition was not significantly different according to lesion position and cancer type in terms of alpha and beta diversity. We found a significant difference in the relative abundance of Campylobacter, Citrobacter, Leptotrichia, Enterobacter, Hungatella, Mycolicibacterium, Phyllobacterium and Sphingomonas between patients without and with lymph node metastasis. CONCLUSIONS: There was a significant association between the relative abundance of certain microbes and overall survival prognosis. These microbes showed association with good prognosis in cholangiocarcinoma, but with poor prognosis in pancreatic adenocarcinoma, and vice versa. Our findings suggest that pancreaticobiliary tract cancer patients have an altered microbiome composition, which might be a biomarker for distinguishing malignancy.

Anti-AQP4 autoantibodies promote ATP release from astrocytes and induce mechanical pain in rats.

BACKGROUND: Intractable neuropathic pain is a common symptom of neuromyelitis optica spectrum disorder (NMOSD). However, the underlying mechanism of NMOSD pain remains to be elucidated. In this study, we focused on ATP, which is one of the damage-associated molecular patterns, and also a well-recognized molecule involved in peripheral neuropathic pain. METHODS: We assessed the development of pain symptoms by injecting anti-AQP4 recombinant autoantibodies (rAQP4 IgG) into rat spinal cords. We incubated HEK293 cells expressing AQP4 (HEK-AQP4) and rat astrocytes with rAQP4 IgG and assessed the level of ATP in the supernatant. We performed transcriptome analysis of the spinal cords injected with rAQP4 IgG. Pharmacological inhibition was also applied to investigate the involvement of ATP in the development of neuropathic pain in our rat model. The ATP concentration within the cerebrospinal fluid was examined in patients with NMOSD and other neurological diseases. RESULTS: Development of mechanical allodynia was confirmed in rAQP4 IgG-treated rats. AQP4-Ab-mediated extracellular ATP release from astrocytes was observed in vitro, and pharmacological inhibition of ATP receptor reversed mechanical allodynia in the rAQP4 IgG-treated rats. Furthermore, transcriptome analysis revealed elevation of gene expressions related to several ATP receptors including P2rx4 and IL1B in the spinal cord of rAQP4 IgG-treated rats. In patients, CSF ATP concentration was significantly higher in the acute and remission phase of NMOSD than in multiple sclerosis or other neurological disorders. CONCLUSION: Anti-AQP4 antibody was shown to induce the release of extracellular ATP from astrocytes. The ATP-mediated development of mechanical allodynia was also suggested in rats treated with anti-AQP4 antibody. Our study indicates the pivotal role of ATP in the pain mechanism of NMOSD.

Increased cerebrospinal fluid adenosine 5'-triphosphate in patients with amyotrophic lateral sclerosis.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) has been suggested to cause neuroinflammation and motor neuron degeneration by activating microglia and astrocytes in amyotrophic lateral sclerosis (ALS). Since we have developed a highly sensitive ATP assay system, we examined cerebrospinal fluid (CSF) ATP levels in patients with ALS whether it can be a useful biomarker in ALS. METHODS: Forty-eight CSF samples from 44 patients with ALS were assayed for ATP with a newly established, highly sensitive assay system using luciferase luminous reaction. CSF samples from patients with idiopathic normal pressure hydrocephalus (iNPH) were assayed as a control. Patients were divided into two groups depending on their disease severity, as evaluated using the Medical Research Council (MRC) sum score. Correlations between the CSF ATP levels and other factors, including clinical data and serum creatinine levels, were evaluated. RESULTS: CSF ATP levels were significantly higher in patients with ALS than in the iNPH (716 ± 411 vs. 3635 ± 5465 pmol/L, p < 0.01). CSF ATP levels were significantly higher in the more severe group than in the iNPH group (6860 ± 8312 vs. 716 ± 411 pmol/L, p < 0.05) and mild group (6860 ± 8312 vs. 2676 ± 3959 pmol/L, p < 0.05) respectively. ALS functional rating scale-revised (ALSFRS-R) (37.9 ± 5.7 vs. 42.4 ± 2.8, p < 0.01) and serum creatinine levels (0.51 ± 0.13 vs. 0.68 ± 0.23 mg/dL, p < 0.05) were significantly lower in the severe group than in the mild group respectively. A negative correlation of CSF ATP levels with MRC sum score was demonstrated in the correlation analysis adjusted for age and sex (r = -0.3, p = 0.08). CONCLUSIONS: Extracellular ATP is particularly increased in the CSF of patients with advanced ALS. CSF ATP levels may be a useful biomarker for evaluating disease severity in patients with ALS.

Sema4A is implicated in the acceleration of Th17 cell-mediated neuroinflammation in the effector phase.

BACKGROUND: Sema4A is a regulator of helper T cell (Th) activation and differentiation in the priming phase, which plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). However, the role of Sema4A in the effector phase remains elusive. We aimed to investigate the role of Sema4A at the effector phase in adoptively transferred EAE model. Clinical features and cytokine profiles of MS patients with high Sema4A levels were also examined in detail to clarify the correlation between Sema4A levels and disease activity of patients with MS. METHODS: We adoptively transferred encephalitogenic Th1 or Th17 cells to wild type (WT) or Sema4A-deficient (Sema4A KO) mice and assessed severity of symptoms and cellular infiltration within the central nervous system (CNS). In addition, we analyzed clinical and radiological features (n = 201), levels of serum IFN-γ and IL-17A (n = 86), complete remission ratio by IFN-ß (n = 38) in all of relapsing-remitting multiple sclerosis (RRMS) patients enrolled in this study. RESULTS: Sema4A KO recipient mice receiving Th17-skewed WT myelin oligodendrocyte glycoprotein (MOG)-specific encephalitogenic T cells showed a significant reduction in the clinical score compared to the WT recipient mice. However, Sema4A KO recipient mice showed similar disease activity to the WT recipient mice when transferred with Th1-skewed encephalitogenic T cells. Bone marrow chimeric study indicated that Sema4A expressed on hematopoietic cells, but not the CNS resident cells, are responsible for augmenting Th17-mediated neuroinflammation. Additionally, in contrast to comparable IFN-γ levels, IL-17A is significantly higher in RRMS patients with high Sema4A level than those with low Sema4A patients with high Sema4A levels showed earlier disease onset, more severe disease activity and IFN-ß unresponsiveness than those with low Sema4A levels. CONCLUSIONS: Sema4A is involved not only in the Th cell priming but also in the acceleration of Th17 cell-mediated neuroinflammation in the effector phase, which could contribute to the higher disease activity observed in RRMS patients with high serum Sema4A levels.

Group-sequential logrank methods for trial designs using bivariate non-competing event-time outcomes.

We discuss the multivariate (2L-variate) correlation structure and the asymptotic distribution for the group-sequential weighted logrank statistics formulated when monitoring two correlated event-time outcomes in clinical trials. The asymptotic distribution and the variance-covariance for the 2L-variate weighted logrank statistic are derived as available in various group-sequential trial designs. These methods are used to determine a group-sequential testing procedure based on calendar times or information fractions. We apply the theoretical results to a group-sequential method for monitoring a clinical trial with early stopping for efficacy when the trial is designed to evaluate the joint effect on two correlated event-time outcomes. We illustrate the method with application to a clinical trial and describe how to calculate the required sample sizes and numbers of events.

Cerebrospinal fluid mitochondrial DNA in neuromyelitis optica spectrum disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disease of the central nervous system. Although complement-dependent astrocyte damage mediated by anti-aquaporin 4 autoantibody (AQP4-Ab) is well acknowledged to be the core of NMOSD pathogenesis, additional inflammatory cascades may contribute to the establishment of lesion formation. Thus, in this study, we investigated the possible pathogenic role of immune-reactive mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) of NMOSD patients. METHODS: Using quantitative polymerase chain reaction, we measured extracellular mtDNA levels in CSF of NMOSD patients positive for AQP4-Ab. Patients with multiple sclerosis or other neurological diseases were examined as controls. Pre- and post-treatment extracellular mtDNA levels were also compared in the NMOSD group. Extracellular mtDNA release from human astrocytes was analyzed in vitro utilizing NMOSD sera, and interleukin (IL)-1ß production was measured in supernatants of mixed glial cells stimulated with DNA fraction of CSF derived from NMOSD patients. Furthermore, specific innate immune pathways mediating the IL-1ß production by mtDNA were investigated in peripheral blood mononuclear cells with selective inhibitors of Toll-like receptor 9 (TLR9) and NOD-like receptor protein 3 (NLRP3) inflammasomes. RESULTS: Extracellular mtDNA level was specifically elevated in acute phase of NMOSD CSF. In vitro studies provided the evidence that mtDNA is released from human astrocytes by NMOSD sera. In addition, DNA fraction isolated from NMOSD CSF promoted secretion of IL-1ß from mixed glial cells. Selective inhibition of TLR9 and NLRP3 inflammasomes revealed that mtDNA-mediated IL-1ß production depends on specific innate immune pathways. CONCLUSION: Extracellular mtDNA is specifically elevated in the CSF of patients with acute phase NMOSD, and mtDNA released by AQP4-Ab-mediated cellular damage elicits the innate immune cascades via TLR9 and NLRP3 inflammasomes pathways. Our study highlights mtDNA-mediated innate immune pathways as a novel therapeutic target for future treatment of NMOSD patients.

Sizing clinical trials when comparing bivariate time-to-event outcomes.

Clinical trials with multiple primary time-to-event outcomes are common. Use of multiple endpoints creates challenges in the evaluation of power and the calculation of sample size during trial design particularly for time-to-event outcomes. We present methods for calculating the power and sample size for randomized superiority clinical trials with two correlated time-to-event outcomes. We do this for independent and dependent censoring for three censoring scenarios: (i) the two events are non-fatal; (ii) one event is fatal (semi-competing risk); and (iii) both are fatal (competing risk). We derive the bivariate log-rank test in all three censoring scenarios and investigate the behavior of power and the required sample sizes. Separate evaluations are conducted for two inferential goals, evaluation of whether the test intervention is superior to the control on: (1) all of the endpoints (multiple co-primary) or (2) at least one endpoint (multiple primary). Copyright © 2017 John Wiley & Sons, Ltd.

Reducing unnecessary measurements in clinical trials with multiple primary endpoints.

Clinical trials often involve two or more primary endpoints. However, observing or measuring high-cost endpoints often reduces the efficiency of the study because of high medical costs, highly invasive measurements, or long-term follow-up. Further, the individual powers to demonstrate the overall efficacy of a new intervention for the multiple endpoints often differ under a given sample size. We propose an efficient clinical trial design in which the sample size for each of the endpoints is individually determined, taking into consideration both the cost and the individual power for each endpoint. We compared the efficiency of the proposed design with that of the conventional design using three variables: (1) the number of participants in the study, (2) the total number of measurements for all endpoints, and (3) the cost of enrolling the participants and obtaining the measurements for all endpoints. We extended the proposed design to a group-sequential design. Numerical examples show that the proposed design can reduce unnecessary measurements and adjust the individual powers for the endpoints, especially when the individual power for one endpoint is relatively higher than that for other endpoints in a study with multiple co-primary endpoints.

A logrank test-based method for sizing clinical trials with two co-primary time-to-event endpoints.

We discuss sample size determination for clinical trials evaluating the joint effects of an intervention on two potentially correlated co-primary time-to-event endpoints. For illustration, we consider the most common case, a comparison of two randomized groups, and use typical copula families to model the bivariate endpoints. A correlation structure of the bivariate logrank statistic is specified to account for the correlation among the endpoints, although the between-group comparison is performed using the univariate logrank statistic. We propose methods to calculate the required sample size to compare the two groups and evaluate the performance of the methods and the behavior of required sample sizes via simulation.

Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis.

BACKGROUND: Accumulating evidence has shown that the inflammatory process participates in the pathogenesis of amyotrophic lateral sclerosis (ALS), suggesting a therapeutic potential of anti-inflammatory agents. Janus kinase 2 (JAK2), one of the key molecules in inflammation, transduces signals downstream of various inflammatory cytokines, and some Janus kinase inhibitors have already been clinically applied to the treatment of inflammatory diseases. However, the efficacy of JAK2 inhibitors in treatment of ALS remains to be demonstrated. In this study, we examined the role of JAK2 in ALS by administering a selective JAK2 inhibitor, R723, to an animal model of ALS (mSOD1G93A mice). FINDINGS: Orally administered R723 had sufficient access to spinal cord tissue of mSOD1G93A mice and significantly reduced the number of Ly6c positive blood monocytes, as well as the expression levels of IFN-γ and nitric oxide synthase 2, inducible (iNOS) in the spinal cord tissue. R723 treatment did not alter the expression levels of Il-1ß, Il-6, TNF, and NADPH oxidase 2 (NOX2), and suppressed the expression of Retnla, which is one of the markers of neuroprotective M2 microglia. As a result, R723 did not alter disease progression or survival of mSOD1G93A mice. CONCLUSIONS: JAK2 inhibitor was not effective against ALS symptoms in mSOD1G93A mice, irrespective of suppression in several inflammatory molecules. Simultaneous suppression of anti-inflammatory microglia with a failure to inhibit critical other inflammatory molecules might explain this result.

Sample size determination in group-sequential clinical trials with two co-primary endpoints.

We discuss sample size determination in group-sequential designs with two endpoints as co-primary. We derive the power and sample size within two decision-making frameworks. One is to claim the test intervention's benefit relative to control when superiority is achieved for the two endpoints at the same interim timepoint of the trial. The other is when superiority is achieved for the two endpoints at any interim timepoint, not necessarily simultaneously. We evaluate the behaviors of sample size and power with varying design elements and provide a real example to illustrate the proposed sample size methods. In addition, we discuss sample size recalculation based on observed data and evaluate the impact on the power and Type I error rate.

Elevation of Sema4A implicates Th cell skewing and the efficacy of IFN-ß therapy in multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the CNS and a leading cause of lasting neurologic disabilities in young adults. Although the precise mechanism remains incompletely understood, Ag presentation and subsequent myelin-reactive CD4(+) T cell activation/differentiation are essential for the pathogenesis of MS. Although semaphorins were initially identified as axon guidance cues during neural development, several semaphorins are crucially involved in various phases of immune responses. Sema4A is one of the membrane-type class IV semaphorins, which we originally identified from the cDNA library of dendritic cell (DC). Sema4A plays critical roles in T cell activation and Th1 differentiation during the course of experimental autoimmune encephalomyelitis, an animal model of MS; however, its pathological involvement in human MS has not been determined. In this study, we report that Sema4A is increased in the sera of patients with MS. The expression of Sema4A is increased on DCs in MS patients and shed from these cells in a metalloproteinase-dependent manner. DC-derived Sema4A is not only critical for Th1 but also for Th17 cell differentiation, and MS patients with high Sema4A levels exhibit Th17 skewing. Furthermore, patients with high Sema4A levels have more severe disabilities and are unresponsive to IFN-ß treatment. Taken together, our results suggest that Sema4A is involved in the pathogenesis of MS by promoting Th17 skewing.

Elevated Serum Xanthine Oxidase and Its Correlation with Antioxidant Status in Patients with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative movement disorder associated with a loss of dopamine neurons in the substantia nigra. The diagnosis of PD is sensitive since it shows clinical features that are common with other neurodegenerative diseases. In addition, most symptoms arise at the late stage of the disease, where most dopaminergic neurons are already damaged. Several studies reported that oxidative stress is a key modulator in the development of PD. This condition occurs due to excess reactive oxygen species (ROS) production in the cellular system and the incapability of antioxidants to neutralize it. In this study, we focused on the pathology of PD by measuring serum xanthine oxidase (XO) activity, which is an enzyme that generates ROS. Interestingly, the serum XO activity of patients with PD was markedly upregulated compared to patients with other neurological diseases (ONDs) as a control. Moreover, serum XO activity in patients with PD showed a significant correlation with the disease severity based on the Hoehn and Yahr (HY) stages. The investigation of antioxidant status also revealed that serum uric acid levels were significantly lower in the severe group (HY ≥ 3) than in the ONDs group. Together, these results suggest that XO activity may contribute to the development of PD and might potentially be a biomarker for determining disease severity in patients with PD.

Switching disease-modifying therapies from sphingosine-1-phosphate receptor modulators to natalizumab or dimethyl fumarate restores immune responses after SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis.

OBJECTIVES: This study aimed to evaluate whether switching disease-modifying therapies (DMTs) from sphingosine-1 phosphate (S1P) receptor modulators to either natalizumab (NTZ) or dimethyl fumarate (DMF) could restore the effectiveness of SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis (MS). METHODS: This study included 9 controls and 33 patients with MS: 7 patients treated with DMF, 7 patients treated with NTZ, 9 patients treated with S1P receptor modulators, and 10 patients who had switched DMTs from S1P receptor modulators to DMF or NTZ by the second vaccine dose. The patients who had switched DMTs were classified into two groups, based on whether their lymphocyte counts were above or below 1000/µL at the time of vaccination. In addition, relapses within 6 months after switching DMTs were also evaluated in these patients. Six months after the second dose of the vaccination, anti-SARS-CoV-2 spike antibodies were evaluated in all participants, and spike specific CD4+ T cells were also assessed in patients who had switched DMTs from S1P receptor modulators. RESULTS: Patients treated with S1P receptor modulators had lower levels of anti-SARS-CoV-2 spike antibodies than the controls and patients treated with DMF and NTZ. On the other hand, in patients who had switched DMTs from S1P receptor modulators, a recovery of lymphocyte counts above 1000/µL resulted in restored humoral and cellular immune responses to the vaccination. There were no neurological relapses in patients who had switched DMTs from S1P receptor modulators to NTZ. CONCLUSION: SARS-CoV-2 mRNA vaccination is expected to be effective in patients whose lymphocyte counts have recovered due to switching DMTs from S1P receptor modulators. Switching DMTs from S1P receptor modulators to NTZ before vaccination may be beneficial in achieving efficacy for SARS-CoV-2 mRNA vaccination, with a reduced risk of relapse.

Sample size determination for clinical trials with co-primary outcomes: exponential event times.

Clinical trials with event-time outcomes as co-primary contrasts are common in many areas such as infectious disease, oncology, and cardiovascular disease. We discuss methods for calculating the sample size for randomized superiority clinical trials with two correlated time-to-event outcomes as co-primary contrasts when the time-to-event outcomes are exponentially distributed. The approach is simple and easily applied in practice.

Identification of double-stranded DNA in the cerebrospinal fluid of patients with acute neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disease of the central nervous system (CNS) characterized by severe myelitis and optic neuritis. Double-stranded DNA (dsDNA) is involved in the pathogenesis of various autoimmune diseases, such as systemic lupus erythematosus. However, its role in NMOSD remains unclear. In this study, the concentration of dsDNA in the cerebrospinal fluid (CSF) was quantified in 23 patients with NMOSD and 16 patients with other neurological diseases (ONDs). CSF dsDNA levels in patients with NMOSD (median: 0.03 ng/µL) were significantly higher than those in patients with ONDs (median: 0.01 ng/µl). CSF dsDNA levels showed no significant difference before and after treatment. Elevation of CSF dsDNA levels may suggest its essential role in the augmentation of CNS inflammation in patients with NMOSD.

RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS.

Repulsive guidance molecule A (RGMa) was originally identified as a neuronal growth cone-collapsing factor. Previous reports have demonstrated the multifunctional roles of RGMa mediated by neogenin1. However, the pathogenic involvement of RGMa in amyotrophic lateral sclerosis (ALS) remains unclear. Here, we demonstrated that RGMa concentration was elevated in the cerebrospinal fluid of both patients with ALS and transgenic mice overexpressing the mutant human superoxide dismutase1 (mSOD1 mice). Treatment with humanized anti-RGMa monoclonal antibody ameliorated the clinical symptoms in mSOD1 mice. Histochemical analysis revealed that the anti-RGMa antibody significantly decreased mutant SOD1 protein accumulation in the motor neurons of mSOD1 mice via inhibition of actin depolymerization. In vitro analysis revealed that the anti-RGMa antibody inhibited the cellular uptake of the mutant SOD1 protein, presumably by reinforcing the neuronal actin barrier. Collectively, these data suggest that RGMa leads to the collapse of the neuronal actin barrier and promotes aberrant protein deposition, resulting in exacerbation of the ALS pathology.

Survival benefit of bursectomy in patients with resectable gastric cancer: interim analysis results of a randomized controlled trial.

BACKGROUND: Bursectomy is regarded as a standard surgical procedure during gastrectomy for serosa-positive gastric cancer in Japan. There is little evidence, however, that bursectomy has clinical benefit. We conducted a randomized controlled trial to demonstrate non-inferiority of treatment with the omission of bursectomy. METHODS: Between July 2002 and January 2007, 210 patients with cT2-T3 gastric adenocarcinoma were intraoperatively randomized to radical gastrectomy and D2 lymphadenectomy with or without bursectomy. The primary endpoint was overall survival (OS). Secondary endpoints were recurrence-free survival, operative morbidity, and levels of amylase in drainage fluid on postoperative day 1. Two interim analyses were performed, in September 2008 and August 2010. RESULTS: Overall morbidity (14.3%) and mortality (0.95%) rates were the same in the two groups. The median levels of amylase in drainage fluid on postoperative day 1 were similar in the two groups (P = 0.543). In the second interim analysis, the 3-year OS rates were 85.6% in the bursectomy group and 79.6% in the non-bursectomy group. The hazard ratio for death without bursectomy was 1.44 (95% confidence interval [CI] 0.79-2.61; P = 0.443 for non-inferiority). Among 48 serosa-positive (pT3-T4) patients, the 3-year OS was 69.8% for the bursectomy group and 50.2% for the non-bursectomy group, conferring a hazard ratio for death of 2.16 (95% CI 0.89-5.22; P = 0.791 for non-inferiority). More patients in the non-bursectomy group had peritoneal recurrences than in the bursectomy group (13.2 vs. 8.7%). CONCLUSIONS: The interim analyses suggest that bursectomy may improve survival and should not be abandoned as a futile procedure until more definitive data can be obtained.