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Acupuncture is a non-pharmacological therapy used clinically for mood disorders. Relief of physical symptoms with acupuncture treatment may lead to relief of depressive symptoms and improvement of quality of life (QoL). Few studies have examined the effect of acupuncture on the physical symptoms and QoL of patients with mood disorders.To examine the effect of acupuncture on physical symptoms and QoL of patients with treatment-resistant major depressive disorder (MDD) and bipolar disorder (BD).This prospective, single-arm, longitudinal study included patients with MDD and BD from an outpatient psychiatric clinic. Acupuncture was performed weekly for 12 weeks in combination with regular treatment, with fixed acupoints and individualized treatment for each patient. Psychiatric symptoms were evaluated using the Himorogi Self-Rating Depression Scale (HSDS) and Himorogi Self-Rating Anxiety Scale (HSAS). Physical symptoms such as physical pain, gastrointestinal symptoms, and sleep disorders were evaluated using the Japanese version of the Somatic Symptom Scale-8 (SSS-8) and Visual Analog Scale (VAS). QoL was evaluated using the 8-item Short-Form (SF-8) Health Survey.A total of 36 patients (15 MDD and 21 BD patients) were analyzed. After 12 weeks of acupuncture, HSDS and HSAS scores significantly decreased (Acupuncture may improve not only psychiatric symptoms but also physical symptoms and QoL in patients with treatment-resistant mood disorders. Further studies are required for confirmation of the preliminary data collected thus far.

ObjectiveIn patients with bipolar disorder (BD), rapid cycling (RC) presents a risk for a more severe illness, while euthymia (EUT) has a better prognosis. This study focused on the progression of RC and EUT, which are contrasting phenomenology, and aimed to clarify the influence of patient backgrounds and prescription patterns on these different progressions, using a large sample from the first and second iterations of a multicenter treatment survey for BD in psychiatric clinics (MUSUBI).MethodsIn the cross-sectional study (MUSUBI), a questionnaire based on a retrospective medical record survey of consecutive BD cases (N = 2,650) was distributed. The first survey was conducted in 2016, and the second one in 2017. The questionnaire collected information on patient backgrounds, current episodes, and clinical and prescribing characteristics.ResultsIn the first survey, 10.6% of the participants had RC and 3.6% had RC for two consecutive years, which correlated with BP I (Bipolar disorder type I), suicidal ideation, duration of illness, and the use of lithium carbonate and antipsychotic medications. Possible risk factors for switching to RC were comorbid developmental disorders and the prescription of anxiolytics and sleep medication. Moreover, 16.4% of the participants presented EUT in the first survey, and 11.0% presented EUT for two consecutive years. Possible factors for achieving EUT included older age; employment; fewer psychotic symptoms and comorbid personality disorders; fewer antidepressants, antipsychotics, and anxiolytics, and more lithium prescriptions.ConclusionRC and EUT generally exhibit conflicting characteristics, and the conflicting social backgrounds and factors contributing to their outcomes were distinctive. Understanding these clinical characteristics may be helpful in clinical practice for management of patients with BD.

BackgroundBipolar disorder is a psychiatric disorder that causes recurrent manic and depressive episodes, leading to decreased levels of social functioning and suicide. Patients who require hospitalization due to exacerbation of bipolar disorder have been reported to subsequently have poor psychosocial functioning, and so there is a need to prevent hospitalization. On the other hand, there is a lack of evidence regarding predictors of hospitalization in real-world clinical practice.MethodsThe multicenter treatment survey on bipolar disorder (MUSUBI) in Japanese psychiatric clinics was an observational study conducted to provide evidence regarding bipolar disorder in real-world clinical practice. Psychiatrists were asked, as part of a retrospective medical record survey, to fill out a questionnaire about patients with bipolar disorder who visited 176 member clinics of the Japanese Association of Neuro-Psychiatric Clinics. Our study extracted baseline patient characteristics from records dated between September and October 2016, including comorbidities, mental status, duration of treatment, Global Assessment of Functioning (GAF) score, and pharmacological treatment details. The incidence and predictors of hospitalization among patients with bipolar disorder over a 1-year period extending from that baseline to September–October 2017 were examined.ResultsIn total, 2,389 participants were included in our study, 3.06% of whom experienced psychiatric hospitalization over the course of 1 year from baseline. Binomial logistic regression analysis revealed that the presence of psychiatric hospitalization was correlated with bipolar I disorder, lower baseline GAF scores, unemployment, substance abuse and manic state.ConclusionsOur study revealed that 3.06% of outpatients with bipolar disorder were subjected to psychiatric hospitalization during a 1-year period that extended to September–October 2017. Our study suggested that bipolar I disorder, lower baseline GAF scores, unemployment, substance abuse and baseline mood state could be predictors of psychiatric hospitalization. These results may be useful for clinicians seeking to prevent psychiatric hospitalization for bipolar disorder.

Background Childbearing-aged female patients and elderly patients with bipolar disorder need special attention for pharmacological treatments, but current guidelines provide little information on their pharmacological treatment. In particular, the risk/benefit balance of pharmacological treatment for childbearing-aged females with bipolar disorder is a growing concern. Therefore, we aimed to address the effect of age and sex on psychotropic drug prescription for outpatients with bipolar disorder. Methods The MUlticenter treatment SUrvey for BIpolar disorder in Japanese psychiatric clinics (MUSUBI) study was conducted, and data on age, sex, and details of pharmacological treatment were collected. Results A total of 3106 outpatients were included in this study. Among young females (age ≤ 39), 25% were prescribed valproate. There was no significant difference in the frequency and daily dose of valproate prescription for young females among all groups. Valproate prescriptions were significantly less frequent among young males and more frequent among middle-aged males. Lithium prescriptions were significantly less frequent among young females and more frequent among older males (age ≥ 65) and older females. Lamotrigine prescriptions were significantly more frequent among young males and young females and less frequent among older males and older females. Carbamazepine prescriptions were significantly less frequent among young males and more frequent among older males. Conclusions Biased information about the risk and safety of valproate and lithium for young females was suggested, and further study to correct this bias is needed. Older patients were prescribed lithium more commonly than lamotrigine. Further studies are needed to determine the actual pharmacotherapy for elderly individuals.

Objective Rapid cycling (RC) presents a risk of greater severity in bipolar disorder (BD), whereas patients with one-year euthymia (OYE) have better prognosis. The purpose of this study was to identify the clinical background and prescription characteristics of patients in the two opposing states of current RC and OYE from a large sample (N = 2609) in a multicenter treatment survey on BD in psychiatric clinics (MUSUBI). Methods MUSIBI was a cross-sectional study wherein questionnaires, based on a retrospective medical record survey of consecutive cases of BD, were distributed to 176 outpatient clinics. The questionnaire collected information on patient background, current episode, and clinical and prescription characteristics. OYE was defined as the presence of a euthymic state for at least 12 months. Results In this study, current RC (9.7% frequency) was significantly higher in females, had a younger age of onset, functional impairments, and a higher rate of neurodevelopmental disorder and physical comorbidity compared to non-RC patients. OYE (19.4% frequency) was associated with a lower proportion of females, older age, higher occupational status, and lower rate of suicide ideation, psychotic symptoms, personality disorder, and alcohol or substance abuse. Mood stabilizers were prescribed in ≥80% of cases, while antipsychotics were prescribed in half of the cases (more in RC and less in OYE). Antidepressant prescription rates were lower in OYE than in RC. Conclusions RC and OYE generally show opposing characteristics, but the details of the opposite parameters are distinctive. Clinicians can help predict the progression of BD by understanding the clinical background and characteristics of these opposing clinical features.

Background To provide updates on clinical practice of acute pancreatitis (AP) in Japan, we conducted a nationwide epidemiological survey. Methods This study consisted of a two-staged survey; the number of AP patients was estimated by the first-stage survey and their clinical features were examined by the second-stage survey. We surveyed AP patients who had visited hospitals in 2016. Results The estimated number of AP patients in 2016 was 78,450, with an overall incidence of 61.8 per 100,000 persons. We obtained detailed clinical information of 2994 AP patients, including 706 (23.6%) severe cases classified according to the Japanese severity criteria. The male-to-female sex ratio was 2.0, and the mean age at onset was 59.9 years in males and 66.5 years in females. Alcohol was the most common etiology (42.8%) in males and gallstones in females (37.7%). The AP-associated mortality was 6.1% in severe AP cases, which was decreased by 40% compared to the 2011 survey. Antibiotics were administered to most cases, with carbapenem being frequently used. Enteral nutrition was given in 31.8% of severe cases, but majority cases received after 48 h. Among the 107 patients who received intervention for walled-off necrosis, five patients received surgery-first approach, 66 received endoscopic ultrasound-guided transluminal drainage, and 19 underwent step-up approach. Conclusions We clarified the current status of AP in Japan including the significant reduction of mortality in severe cases, shift to endoscopic approaches for walled-off necrosis, and poor compliance of the recommendations in the guidelines including management of enteral nutrition and antibiotic administration.

Takashi Tsuboi,1,2 Takefumi Suzuki,2,3 Takaharu Azekawa,4 Naoto Adachi,4 Hitoshi Ueda,4 Kouji Edagawa,4 Eiichi Katsumoto,4 Yukihisa Kubota,4 Eiichiro Goto,4 Seiji Hongo,4 Yoichiro Watanabe,4 Masaki Kato,2,5 Norio Yasui-Furukori,2,6 Reiji Yoshimura,2,7 Atsuo Nakagawa,2,8 Toshiaki Kikuchi,2,8 Koichiro Watanabe1,2 1Department of Neuropsychiatry, Kyorin University School of Medicine, Tokyo, Japan; 2The Japanese Society of Clinical Neuropsychopharmacology, Tokyo, Japan; 3Department of Neuropsychiatry, University of Yamanashi Faculty of Medicine, Yamanashi, Japan; 4The Japanese Association of Neuro-Psychiatric Clinics, Tokyo, Japan; 5Department of Neuropsychiatry, Kansai Medical University, Osaka, Japan; 6Department of Psychiatry, Dokkyo Medical University, Tochigi, Japan; 7Department of Psychiatry, University of Occupational and Environmental Health, Fukuoka, Japan; 8Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, JapanCorrespondence: Takashi TsuboiDepartment of Neuropsychiatry, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka-Shi, Tokyo 181-8611, JapanTel +81 422 47 5511Fax +81 422 45 4697Email takashi.tsuboi.0821@gmail.comPurpose: The aim of this study was to identify factors associated with non-remission in bipolar disorder.Patients and Methods: The multicenter treatment survey for bipolar disorder in psychiatric outpatient clinics (MUSUBI) study used a questionnaire administered at 176 clinics throughout Japan from September to October 2016. Clinic psychiatrists performed a retrospective medical record survey of consecutive cases with bipolar disorder. Patients were considered to be in remission if they met all of the following criteria: they were not in a mixed state, their manic or depressive symptoms were either borderline or nonexistent (corresponding to 2 or 1 points on the Clinical Global Impressions Scale, Bipolar Version), and their psychiatrists clinically considered them to be in remission. Enrolled patients were classified into remitters group and non-remitters group and demographic and clinical characteristics were contrasted between the groups. Non-remitters were compared with remitters, using a series of logistic regression analyses.Results: A total of 3130 patients (1420 men; mean age: 50.3 years) were included in this study; 1307 patients (41.8%) were in remission. Of the remaining 1823 patients, 1260 (40.3%) had mild to severe depression, 261 (8.3%) suffered from manic or hypomanic episodes, and 302 (9.6%) were in a mixed state. Logistic regression analyses found the following eight factors to be significantly correlated with non-remission in patients with bipolar disorder: female gender, younger age, unemployed status, rapid cycling pattern, comorbid alcohol/substance abuse, poorer social function, lithium non-use, and antidepressant use.Conclusion: The MUSUBI study, the largest nationwide investigation on bipolar disorder, identified eight clinically relevant factors associated with non-remission in bipolar patients. They have important clinical implications; further prospective studies are necessary to replicate these findings and to guide better managements for those in serious needs.Keywords: bipolar disorder, non-remission, nationwide study, mood stabilizer, antipsychotics

Pancreatic ductal adenocarcinoma (PDAC), which accounts for majority of pancreatic cancers, is one of the most lethal human malignancies. Most patients are diagnosed at an advanced stage after symptom development. Early diagnosis of PDAC in asymptomatic subjects is important to improve prognosis. Diabetes mellitus (DM) is a risk factor for PDAC, and DM, especially new-onset DM, has attracted attentions as a diagnostic clue to PDAC. However, the impact of DM as a diagnostic opportunity on the prognosis of PDAC is unclear. We here retrospectively reviewed 489 PDAC patients and compared the clinical characteristics and prognosis according to the opportunities for PDAC diagnosis. PDAC was diagnosed upon presentation of symptoms, such as pain and jaundice, in 318 cases including 151 DM patients, upon new-onset or exacerbation of long-standing DM in 53 asymptomatic patients, and upon incidental detection by medical check-up or follow-up/work-up of other diseases in 118 asymptomatic patients. Asymptomatic patients including those with DM had smaller tumors, earlier disease stage, and higher resectability rates than symptomatic patients. Asymptomatic patients diagnosed in association with DM had better prognosis (median survival time, 771 days) than those diagnosed due to symptoms (343 days, P < 0.001), and similar to those diagnosed by incidental detection (869 days). The survival advantage was not evident in symptomatic patients with DM-associated signs. In conclusion, patients diagnosed in association with DM at asymptomatic stages had better prognosis than those diagnosed with symptoms. DM-associated signs might provide a clue to the early diagnosis of PDAC among asymptomatic subjects.

There is limited evidence regarding predictors of long-term clinical outcomes in patients with bipolar disorder (BD). The objective of this study was to describe 3-year clinical outcomes and identify their predictors from participants in the multicenter treatment survey for BD in psychiatric outpatient clinics (MUSUBI).The MUSUBI was a naturalistic study investigating patients with BD in real-world clinical practice. Our study extracted data regarding 1647 outpatients with BD from 2016, 2017, and 2019 as baseline, 1-year, and 3-year assessments. As clinical outcomes, we assessed the presence of time ill (depressive and manic) during the 1 year prior to the 3-year assessment and durable remission (53 weeks or more) prior to the 3-year assessment.Participants with durable remission prior to the 3-year assessment had significant associations with diagnosis of a personality disorder and duration of continuous remission at baseline. Regarding the presence of depressive symptoms during the 1 year prior to the 3-year assessment, work status, Global Assessment of Functioning (GAF) scores, suicidal ideation, and duration of continuous remission at baseline had significant associations with this outcome.At the 3-year assessment, 19.3% of participants (318/1647) achieved durable remission, while 47.5% of them (782/1647) were not remitted. Our findings can help clinicians predict the illness course of BD by understanding demographic and clinical characteristics.

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV and IBV, respectively). It has a monobasic site, which is cleaved by some host enzymes. The cleavage is essential to activating the virus, but the enzyme or enzymes in the respiratory tract have not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK cells (MDCK-TMPRSS2 and MDCK-HAT). ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multistep growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs for ICV infection. IMPORTANCE Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, which possesses receptor-binding, receptor-destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme or enzymes in the respiratory tract have not been identified. This study revealed that transmembrane protease serine S1 member 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of ICV infection.

Purpose. To clarify the spread of Mycoplasma pneumoniae infections in semi-closed settings such as schools and family homes using molecular typing methods. Methodology. We retrospectively searched for school- and family-based clusters of M. pneumoniae infections based on information regarding patients from whom M. pneumoniae strains had been isolated between 2011 and 2013 in Yamagata, Japan. The molecular typing profile, including the P1 type and the four-locus (Mpn13, 14, 15 and 16) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) type, was obtained from our previous study. Results. We identified 11 school-based clusters involving 71 patients and 16 family-based clusters involving 38 patients, including 14 duplications between these types of clusters. A total of 95 M. pneumoniae strains isolated from those patients were divided into 4 genotypes: 33 strains of type 4-5-7-2, 1; 31 of type 4-5-7-3, 1; 24 of type 3-5-6-2, 2c; and 7 of type 3-5-6-2, 2a. Of the 11 school-based clusters, 6 clusters (54.5%) consisted of multiple genotypes, and the remaining 5 clusters consisted of a single genotype. Moreover, the presence of multiple genotypes was identified in three classrooms of a school. On the other hand, in 14 (87.5%) of the 16 family-based clusters, the genotypes of the M. pneumoniae strains isolated from each family member were identical. Conclusion. The spread of M. pneumoniae infection in schools is likely polyclonal, since M. pneumoniae strains are brought into schools from various sites, such as family homes, which are important sites of disease transmission.

A 79-year-old woman was referred for pancreatic tail cancer with multiple liver metastases. The pancreatic tail tumor was diagnosed as acinar cell carcinoma (ACC) histologically by endoscopic ultrasound-guided fine-needle aspiration. Because of multiple liver metastases, S-1 chemotherapy was administered, resulting in a partial response to chemotherapy one year later. After approximately three years, liver atrophy and esophageal varices developed. We suspected S-1 as the cause of the liver cirrhosis. S-1 cessation minimized ascites and improved the esophageal varices. Although S-1 can potentially treat ACC, we should be watchful for liver cirrhosis caused by its long-term administration.

Yusuke Konno,1,2 Yoshihisa Fujino,2 Atsuko Ikenouchi,1,3 Naoto Adachi,4 Yukihisa Kubota,4 Takaharu Azekawa,4 Hitoshi Ueda,4 Koji Edagawa,4 Eiichi Katsumoto,4 Eiichiro Goto,4 Seiji Hongo,4 Masaki Kato,5,6 Takashi Tsuboi,5,7 Norio Yasui-Furukori,5,8 Atsuo Nakagawa,5,9 Toshiaki Kikuchi,5,9 Koichiro Watanabe,5,7 Reiji Yoshimura1,5 1Department of Psychiatry, University of Occupational and Environmental Health, Kitakyushu, Japan; 2Department of Environmental Epidemiology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan; 3Medical Center for Dementia, University Hospital of Occupational and Environmental Health, Kitakyushu, Japan; 4The Japanese Association of Neuro-Psychiatric Clinics, Tokyo, Japan; 5The Japanese Society of Clinical Neuropsychopharmacology, Tokyo, Japan; 6Department of Neuropsychiatry, Kansai Medical University, Osaka, Japan; 7Department of Neuropsychiatry, Kyorin University School of Medicine, Tokyo, Japan; 8Department of Psychiatry, Dokkyo Medical University, Tochigi, Japan; 9Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, JapanCorrespondence: Yusuke KonnoDepartment of Psychiatry, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishiku, Kitakyushu, 807-8555, JapanTel +81-93-691-7401Fax +81-93-692-4894Email yu-konno@med.uoeh-u.ac.jpObjective: The objective of this study was to clarify the relationship between mood episode and employment in patients with bipolar disorder to help improve their employment status.Methods: All medical records of patients with bipolar disorder who visited 176 member clinics of the Japanese Association of Neuro-Psychiatric Clinics in September–October 2016 were investigated in September–October 2017. Details of the medical care received were investigated using a survey sheet, which included employment status. Odds ratios of mood episodes for employment status were analyzed using a logistic regression model.Results: Among patients aged 60 years or less, 2292 described their occupation. On univariate analysis, odds ratios were statistically significant for depressive episode (OR = 2.68 [1.50– 4.78] p = 0.001) and manic episode (OR = 2.64 [1.07– 6.47] p = 0.034), whereas no significant difference was noted for mixed episode (OR = 1.72 [0.69– 4.33] p = 0.246). On multivariate analysis, odds ratios were statistically significant for depressive episode (OR = 2.16 [1.13– 4.13], p = 0.020) and manic episode (OR = 3.55 [1.36– 9.25], p = 0.010), whereas no significant difference was noted for mixed episode (OR = 1.83 [0.65– 5.14] p = 0.254).Conclusion: Employment status among these patients with bipolar disorder receiving outpatient treatment was 43.5%. Compared to remission episodes, manic and depressive episodes were associated with a higher risk of unemployment.Keywords: mood disorder, continuous employment, occupational health, Japan

A 71-year-old man underwent surgery for a pancreatic neuroendocrine tumor. Follow-up imaging showed swelling of the remnant pancreas, and he was histologically diagnosed with autoimmune pancreatitis based on endoscopic ultrasonography-guided fine-needle aspiration specimens. After two years, a tumor appeared on the liver surface. Although we planned to perform laparoscopic partial hepatectomy, the intraoperative findings showed that the tumor was located in the diaphragm. Partial resection of the diaphragm was performed, and the final diagnosis was an immunoglobulin G4-related inflammatory pseudotumor in the diaphragm. To our knowledge, this is the first reported case of an immunoglobulin G4-related diaphragmatic inflammatory pseudotumor.

A 71-year-old man underwent surgery for a pancreatic neuroendocrine tumor. Follow-up imaging showed swelling of the remnant pancreas, and he was histologically diagnosed with autoimmune pancreatitis based on endoscopic ultrasonography-guided fine-needle aspiration specimens. After two years, a tumor appeared on the liver surface. Although we planned to perform laparoscopic partial hepatectomy, the intraoperative findings showed that the tumor was located in the diaphragm. Partial resection of the diaphragm was performed, and the final diagnosis was an immunoglobulin G4-related inflammatory pseudotumor in the diaphragm. To our knowledge, this is the first reported case of an immunoglobulin G4-related diaphragmatic inflammatory pseudotumor.

Background Several evidence-based practice guidelines have been developed to better treat bipolar disorder. However, the articles cited in these guidelines were not sufficiently based on real-world clinical practice. Methods The MUlticenter treatment SUrvey on BIpolar disorder in Japanese psychiatric clinics (MUSUBI) is a study conducted to accumulate evidence on the real-world practical treatment of bipolar disorder. Psychiatrists were asked to complete a questionnaire about patients with bipolar disorder by performing a retrospective medical record survey. The questionnaire included patient characteristics (age, gender, height, weight, academic background, and occupational status), comorbidities, mental status, treatment period, Global Assessment of Functioning (GAF) score, and details of pharmacological treatment. Results Data on 2705 patients were included in this study. The proportion of patients receiving antidepressant prescriptions was 40.9%. The most commonly used antidepressant was duloxetine, and the most frequently used antidepressant class was selective serotonin reuptake inhibitors (SSRIs). Binomial logistic regression analysis and bivariate analysis revealed that the usage of antidepressants was correlated with low prescription rates for mood stabilizers, high prescription rates for anxiolytics and hypnotics, and low GAF scores. In addition, patients in a depressive state had a significantly higher rate of antidepressant prescriptions than patients with other mental states. Conclusions Approximately 40% of patients in Japan with a diagnosis of bipolar disorder have received antidepressants. Antidepressants were most often prescribed in combination with mood stabilizers, antipsychotics or both. Patients who were prescribed antidepressants received fewer mood stabilizers, more anxiolytics, and more hypnotics than those who did not receive antidepressant prescriptions.

Objective This study aims to clarify the relevant factors influencing practitioners' methods of prescribing medications for bipolar disorder, in a nation-wide survey in Japan. Methods The clinical records of 3130 outpatients with bipolar disorder were consecutively reviewed from 176 psychiatric outpatient clinics. Fifteen parameters, that is, five patients' including five general characteristics (sex, age, education, occupation, and social adjustment), five patients' aspects of mental functioning (onset age, comorbid mental illness, rapid-cycling, psychopathologic severity, and followed-up years), and five practitioners' characteristics (sex, age, specialist experience, clinic standing years, and location), were evaluated. The number of psychotropic drugs (mood stabilizers, antidepressants, antipsychotic drugs, anxiolytics, and hypnotics) was used as an index of pharmacotherapy. Converted data from each practitioner-unit were analyzed. Results Seven factors (patient's social adjustment, patient's psychopathology, patient's comorbid mental disorders, patient's followed-up years, doctor's age, clinic running years, and patient's education years) were correlated to the number of psychotropic drugs. Multiple regression analysis showed that the severity of illness (poor social adjustment, and comorbid mental illness) and an intractable disease course (long followed-up years), were significantly associated with the number of psychotropic drugs. Conclusion Our findings indicated that patient-related conditions affected psychotropic polypharmacy more strongly than did practitioner-related conditions.

Pancreatic cancer is one of the most dangerous solid tumors, but its early diagnosis is difficult. The abnormality of the main pancreatic duct (MPD), such as a single localized stricture and upstream dilatation, might be useful in the early detection of pancreatic cancer. However, these findings are often observed in benign inflammatory cases. This study aimed to clarify whether early pancreatic cancer presenting MPD abnormalities has characteristic features different from those of benign cases. This is a single-center, retrospective study. We analyzed 20 patients who underwent pancreatectomy presenting with a single, localized MPD stricture without identifiable masses on imaging: 10 patients with pancreatic ductal adenocarcinoma (cancer group; 6 with stage 0 and 4 with stage I) and 10 patients with benign strictures (benign group; 8 with inflammation and 2 with low-grade pancreatic intraepithelial neoplasms). Pancreatectomy was performed in these benign cases because high-grade intraepithelial neoplasm was suspected. Although the proportion of patients with diabetes mellitus tended to be higher in the cancer group (6/10) than that in the benign group (1/10) (P = 0.058), other clinical characteristics were not different between the groups. Preoperative cytological malignancies were detected in four patients in the cancer group (4/10) but not in the benign group (P = 0.09). Focal parenchymal atrophy and fat replacement were more frequently detected on computed tomography in the cancer group (7/10) than in the benign group (1/10) (P = 0.02). In conclusion, focal parenchymal atrophy and fat replacement may provide clues for the early diagnosis of pancreatic cancer.

OBJECTIVE There is limited information available on the prescription of psychotropic agents to patients with bipolar I (BD-I) and bipolar II disorder (BD-II). The purpose of this study was to investigate the characteristics of drug therapy in BD-I and BD-II outpatients, particularly with regard to antidepressants. METHODS In 2017, the MUlticenter treatment SUrvey for BIpolar disorder in Japanese psychiatric clinics (MUSUBI) study collected data on current mental status, medications, and other factors from 2774 outpatients with BD-I or BD-II. RESULTS There were significant differences in the rates of prescriptions for mood stabilizers, antipsychotics and antidepressants. Mood stabilizers and antipsychotics were prescribed at higher rates to patients with BD-I (mood stabilizers; BD-I 86.0%, BD-II 80.8%, p

Influenza C virus is a pathogen that causes acute respiratory illness in children and results in the hospitalization of infants. The antigenicity of the hemagglutinin esterase (HE) glycoprotein is highly stable, and it is not yet known whether antigenic changes contribute to the worldwide transmission and the occurrence of outbreaks of influenza C virus. Here, we performed antigenic analysis of 84 influenza C viruses isolated in Yamagata, Japan, during a 4-year period from 2015 to 2018 and analyzed sequence data for strains of the virus from Japan and many other parts of the world. Antigenic and phylogenetic analyses revealed that 83 strains belonged to the C/Sao Paulo lineage, and two sublineage strains, the Aichi99 sublineage and Victoria2012 sublineage, cocirculated between 2016 and 2018. Aichi99 sublineage strains exhibiting decreased reactivity with the monoclonal antibody YA3 became predominant after 2016, and these strains possessed the K190N mutation. Residue 190 is located in the 190-loop on the top side of the HE protein within a region that is known to show variation that does not impair the biological activity of the protein. The Aichi99 sublineage strains possessing the K190N mutation were detected after 2012 in Europe, Australia, the USA, and Asia as well as Japan. These observations suggest that antigenic variants with K190N mutations have circulated extensively around the world and caused outbreaks in Japan between 2016 and 2018. Our study indicated that the 190-loop is an important antigenic region, and the results suggested that changes in the 190-loop have contributed to the extensive transmission of the virus.

Purpose. To determine the timing of the emergence of macrolide-resistant mutations after macrolide treatment in individuals with Mycoplasma pneumoniae infections. Methodology. Between October 2011 and December 2013, serial pharyngeal swab specimens were collected before and after macrolide treatment from 21 otherwise healthy children infected with M. pneumoniae without macrolide-resistant mutations. The copy numbers of a M. pneumoniae gene and the proportion of clones showing macrolide-resistance mutations were determined for each specimen. Results. After macrolide treatment (10–15 mg kg−1 day−1 clarithromycin for 5–10 days or 10 mg kg−1 day−1 azithromycin for 3 days), fever resolved in 19 (90 %) of 21 children within 1 to 2 days, and the M. pneumoniae gene copy number decreased in all but one specimen in the second set of specimens relative to the number in the corresponding initial specimens. None of the second specimens, which were collected 2–4 days after initiation of macrolide treatment, showed mutations in the 23S rRNA gene. However, the proportion of mutant clones with A2063G and A2064G mutations in the specimens collected 7–24 days after initiation of treatment increased to 100 %. We identified a family in which three members had M. pneumoniae infections. The analysis of transmission in this household indicated that the M. pneumoniae harbouring a macrolide-resistant mutation that developed in the index patient after macrolide treatment was not transmitted to the family members. Conclusion. A macrolide-resistant population might develop in individual patients up to 24 days after initiation of macrolide treatment. However, the decrease in M. pneumoniae load after macrolide administration effectively reduces interpersonal transmission.

Aim: S16, a monoclonal antibody against the hemagglutinin-esterase fusion (HEF) glycoprotein of influenza C virus, reacts with SV40 large T antigen (LT) and a host cellular component(s). The aim is to determine the location of S16 linear epitope on LT and the amino acid sequence of S16 epitope. Materials & methods: BHK-21 cells expressing wild-type and mutant LTs, HEFs or GFPs, each of which was tagged with a FLAG epitope, were analyzed by immunoblotting using S16. Results & conclusions: An amino acid sequence 98-FNEENL-103 on LT forms a linear epitope recognized by S16. The sequence of S16 epitope was defined as F[NAT]EE[NYA]L, excluding FAEEAL and FTEEAL. This finding will be of help in identifying a host cellular component(s) crossreactive with S16.

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable, however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability.

A 32-year-old woman was referred due to abdominal pain and elevated liver enzymes. Computed tomography and magnetic resonance imaging showed ectopic opening of the common bile duct (CBD) into the duodenal bulb. Esophagogastroduodenoscopy showed a hemispheric bulge in the duodenal bulb. Endoscopic retrograde cholangiopancreatography (ERCP) revealed the bulge to be cystic dilatation of the CBD. ERCP also showed no communication between the ventral and dorsal pancreatic ducts. We diagnosed the patient with ectopic opening of the CBD accompanied by choledochocele and pancreas divisum. Endoscopic incision was performed for the treatment of the choledochocele. The patient's symptoms and elevated liver enzymes improved after treatment.

From November 2009 to December 2013 in the Philippines, 15 influenza C viruses were isolated, using MDCK cells, from specimens obtained from children with severe pneumonia and influenza-like illness (ILI). This is the first report of influenza C virus isolation in the Philippines. In addition, from January 2008 to December 2013, 7 influenza C viruses were isolated from specimens that were obtained from children with acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein showed that 19 strains (12 from the Philippines and 7 from Japan) were similar to the influenza C virus reference strain C/Sao Paulo/378/82 (SP82). Phylogenetic analysis of the HE gene showed that the strains from the Philippines and Japan formed distinct clusters within an SP82-related lineage. The clusters that included the Philippine and Japanese strains were shown to have diverged from a common ancestor around 1993. In addition, phylogenetic analysis of the internal genes showed that all strains isolated in the Philippines and Japan had emerged through reassortment events. The composition of the internal genes of the Philippine strains was different from that of the Japanese strains, although all strains were classified into an SP82-related lineage by HE gene sequence analysis. These observations suggest that the influenza C viruses analyzed here had emerged through different reassortment events; however, the time and place at which the reassortment events occurred were not determined.

Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for Mycoplasma pneumoniae strains isolated between 2004 and 2014 in Yamagata, Japan. The results were examined by considering the combination of the P1 type and prevalence of macrolide resistance-associated mutations. Four-locus (Mpn13-16) MLVA classified 347 strains into 9 MLVA types, including 3 major types: 3-5-6-2, 4-5-7-2, and 4-5-7-3. All type 3-5-6-2 strains (77 strains) were P1 type 2 variants (2a or 2c), while types 4-5-7-2 (181 strains) and 4-5-7-3 (75 strains) were P1 type 1. MLVA type 4-5-7-2 strains circulated and were dominant until 2010, accounting for 88.4% of the 121 strains isolated between 2004 and 2010. The prevalence of types 4-5-7-3 and 3-5-6-2 strains increased rapidly in 2011 and 2012, respectively, resulting in cocirculation of 3 MLVA types, including type 4-5-7-2, between 2011 and 2013. The prevalence of macrolide resistance-associated mutations in MLVA types 4-5-7-2, 4-5-7-3, and 3-5-6-2 strains was 59.7% (108/181), 25.3% (19/75), and 0% (0/77), respectively. Because the prevalence of macrolide resistance-associated mutations differed by current MLVA types in Yamagata, continued surveillance combined with molecular typing and identification of macrolide resistance-associated mutations is necessary.

CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site. IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of the influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo . We previously demonstrated that the influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike that of the influenza A virus M2 protein, plays a role in virus replication.

Objective The study evaluated relationships between frontal lobe function in patients with schizophrenia and both their social adjustment and medication, using 22-channel near-infrared spectroscopy (NIRS). Methods One hundred ninety-nine stable patients with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision schizophrenia, whose medication had not been changed within the preceding 3 months and who were able to visit our clinics for NIRS, were the study subjects. As a comparator, 144 healthy volunteers who underwent a physical examination and the Mini-International Neuropsychiatric Interview also received NIRS. Results The main outcomes evaluated were frontal lobe oxyhemoglobin concentration (OxHb) measured by NIRS, current medication, social adjustment, and scores on the Brief Psychiatric Rating Scale. The OxHb in schizophrenic patients (0.878 ± 1.1801 mMmm; n = 199) was significantly lower than that in the healthy volunteers (2.085 ± 1.7480 mM mm: n = 100) (p

We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417–427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain. IMPORTANCE The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.

Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.

To clarify the longitudinal molecular epidemiology of coxsackievirus A16, phylogenetic analysis based on the VP1 region of 220 isolates in Yamagata, Japan was performed. The resultant phylogenetic tree indicates that the Yamagata isolates and reference strains can be readily genotyped into three genogroups, and 0, 12 and 208 isolates belonged to the first, second, and third genogroups, respectively. The first genogroup includes only the prototype strain, the second strains that had disappeared by the end of the 20th century and the third comprises those that have been circulating since then in local communities, such as Yamagata.

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.

SummaryMeasles among healthcare workers (HCWs) is associated with a significant risk of nosocomial transmission to susceptible patients. When a measles case occurs in the healthcare setting, most guidelines recommend exhaustive measures. To evaluate the effects of measures against measles transmission in the healthcare setting precisely, it is essential to determine whether secondary transmission generally occurs. This study describes, for the first time, the actual secondary transmission rate for a measles-infected HCW in a ward with no special air ventilation capacity. The routine treatment of a number of immunocompromised patients occurs in this ward, and thus patients as well as HCWs have a thorough understanding and practice of standard and extended precautions. Our paired serum sample study revealed that none of the people in the ward exposed to the HCW at the catarrhal stage over a period of 4 days exhibited elevated levels of antibodies against measles. We suggest that strict adherence to standard and expanded precautions among patients and HCWs may be effective for preventing the transmission of a highly airborne disease, such as measles.

The purpose of this study was to evaluate the frequency of human metapneumovirus (hMPV) infection in adults and to determine the association between the levels of serum antibody titers and the susceptibility to reinfection. Serum samples collected at the periodic occupational medical checkup for employees of a hospital were subjected to an ELISA test. Of the 289 subjects, 288 (99.7%) had hMPV antibody titers that were more than 1:100 in May 2006. The percentage of subjects with a titer of ≥ 3,200 was significantly higher in adults aged 40-65 years old (30.2-31.5%) compared to young adults 20-39 years old (13.6%) (P < 0.05). To investigate the longitudinal course of the hMPV antibody titer, a total of 649 serum samples collected from 59 subjects who had participated in all biannual medical checkups between 2001 and 2006 were tested. We found that ten serum pairs showed a greater than fourfold increase in hMPV antibody titers. Additionally, the 5-year reinfection rate was estimated at 16.9% (10 of 59 subjects). The baseline titer before the fourfold increase ranged from 1:100 to 1:3,200, and the titer returned to baseline levels 2 or 3 years after the fourfold increase. The antibody titer of the person with the baseline titer of 1:100 showed a greater than fourfold increase twice within a year. Sixty to 80% of adults had an ELISA titer of 1:800 to 1:1,600, suggesting that such an antibody titer is not enough to protect from hMPV infection and that reinfection could occur among adults.

Seven hexon hypervariable regions (HVRs) of adenoviruses (Ads) were identified by comparing the regions among different serotypes; however, no one has compared HVR sequences among the identical serotypes, except for adenovirus type 3 (Ad3). To examine a variability between the HVRs for each serotype, we compared the sequences of Ad1-6 isolates, respectively, isolated between 1988 and 2007 in Yamagata, Japan. We selected 23-43 isolates randomly and sequenced 894-987 bp regions. Except for strains with insertions and deletions, the sequence identities among Ad1-6 were 99-100%, excluding that between the two Ad5 groups (approx. 94%). Even the insertions and deletions were likely to be established, as these changes were repeatedly observed. The obtained phylogenetic tree indicated that Ad isolates and reference strains branched depending on serotype. The Yamagata isolates had similar sequences or amino acid arrangements to the reference strains as well as to other strains isolated in different areas. HVRs have been stably conserved as serotype-specific regions for a long period with only minor genomic variations. Therefore, we herein recommend that these regions be hereafter referred to as "serotype-specific regions", which might be a more appropriate title with which to characterize the epidemiological nature of these sites than the current "HVRs".

RNA segment 7 of influenza C virus encodes two non-structural (NS) proteins, NS1 and NS2. The influenza C virus NS2 protein has been proposed to possess nuclear export activity like that of influenza A and B virus NS2 proteins (NEP). In the present study, we investigated the kinetics and localization of the NS2 protein in influenza C virus-infected cells, and analysed whether NS2 is present in virions. Immunofluorescent staining analysis of the infected cells indicated that NS2 was localized in the nucleus immediately after synthesis and predominantly in the cytoplasm in the later stages of infection. Confocal microscopy revealed that a part of the NS2 protein was colocalized with nucleoprotein NP/vRNP in the cytoplasm and on the cell membrane in the late stages of infection. The NS2 protein was detected in influenza C virions purified by gradient centrifugations and/or affinity chromatography. Trypsin treatment demonstrated that the NS2 protein was present inside the viral envelope. Furthermore, glycerol gradient analysis of detergent-solubilized virions revealed that the NS2 protein cosedimented with vRNPs. These data suggest that the influenza C virus NS2 protein is incorporated into virions, where it associates with vRNP.

Although well over 200 viral agents have been implicated in acute respiratory infections (ARIs) among children, no system able to detect such a wide range of viruses has been established. Between January 2004 and December 2005, a modified microplate method, including HEF, HEp-2, Vero E6, MDCK, RD-18S, and GMK cell lines (HHVe6MRG plate), was adopted to isolate viruses. A total of 1,551 viruses were isolated, representing both outbreaks and sporadic cases, from 4,107 nasopharyngeal specimens, at monthly isolation rates of 22.3 to 52.6%. Influenza, parainfluenza, respiratory syncytial (RS), and mumps viruses, and human metapneumovirus, enterovirus, parechovirus, rhinovirus, adenovirus, herpesvirus, and cytomegalovirus were all isolated. The use of multiple cell lines increased the isolation rates of most of these viruses. The findings showed that ARIs due to a number of respiratory viruses occurred across all seasons in succession and/or concurrently in children in the community. These data will help clinicians determine in which seasons and for which age groups they should use the rapid diagnostic test kits available for influenza virus, RS virus, and adenovirus. In conclusion, we verified that the modified microplate method was able to clarify the etiology and epidemiology of numerous viruses isolated from children with ARI.

Reverse genetics has been documented for influenza A, B, and Thogoto viruses belonging to the family Orthomyxoviridae . We report here the reverse genetics of influenza C virus, another member of this family. The seven viral RNA (vRNA) segments of C/Ann Arbor/1/50 were expressed in 293T cells from cloned cDNAs, together with nine influenza C virus proteins. At 48 h posttransfection, the infectious titer of the culture supernatant was determined to be 2.51 × 10 3 50% egg infectious doses/ml, which is lower than the number of influenza C virus-like particles (VLPs) (10 6 /ml) generated using the same system. By generating influenza C VLPs containing a given vRNA segment, we showed that each of the vRNA segments was similarly synthesized in the plasmid-transfected cells but that some segments were less efficiently incorporated into the VLPs. This finding leads us to speculate that the differences in incorporation efficiency into VLPs between segments might be a reason for the inefficient production of infectious viruses. Second, we generated a mutant recombinant virus, rMG96A, which possesses an Ala→Thr mutation at residue 24 of the M1 protein, a substitution demonstrated to be involved in the morphology (filamentous or spherical) of the influenza C VLPs. As expected, rMG96A exhibited a spherical morphology, whereas recombinant wild-type of C/Ann Arbor/1/50, rWT, exhibited a mainly filamentous morphology. Membrane flotation analysis of the cells infected with rWT or rMG96A revealed a difference in the ratio of membrane-associated M1 proteins, suggesting that the affinity of M1 protein to the cell membrane is a determinant for virion morphology.

A number of epidemiological studies have shown human metapneumovirus (hMPV) to be one of the most important viral agents associated with acute respiratory infections in humans. However, due to the difficulty in growing the virus, all epidemiological studies of hMPV infection have been performed on the basis of the molecular method. Thus, the development of a cell line suitable for the isolation of hMPV from clinical specimens is a crucial step for further research. Using the Vero E6 cell line, which could be stably maintained for 1 month without passage or medium change, we succeeded in isolating 79 strains from 4,112 specimens obtained in Yamagata, Japan, in 2004 and 2005. The total isolation rate was 1.9% (79/4,112). The monthly distribution revealed that hMPV infections occurred between February and April in 2004 and throughout most of the year in 2005. Phylogenetic analysis indicated that subgenogroup B2 was predominant in 2004, whereas three subgenogroups, A2, B1, and B2, had cocirculated in 2005. Although multiple subgenogroups cocirculated in 2005, each individual subgenogroup strain was found to predominate at specific sites. An infectivity assay of hMPV strains also indicated that the infection efficiency in Vero E6 cells was better than that in LLC-MK2 cells. Finally, we found that Vero E6 cells are useful for the isolation of hMPVs and that this utility might aid further research into hMPVs beyond the epidemiological data shown in this study.

During the period from January to July 2004, a total of 131 influenza C viruses were detected by cell culture or reverse transcription-PCR (RT-PCR) from specimens that were obtained from children with acute respiratory symptoms in 10 prefectures across Japan. Influenza C virus was identified most frequently in the Miyagi (1.4%, 45 of 3,226 specimens) and Yamagata (2.5%, 31 of 1,263 specimens) prefectures, and the frequency in this year was the highest since 1990. Phylogenetic analysis of the hemagglutinin esterase gene of the 13 strains isolated in nine prefectures revealed that genetically similar strains belonging to the Kanagawa/1/76-related lineage dominantly spread throughout Japan. During the 2004 influenza season, influenza C virus coexisted with epidemics of influenza A virus (H3 strain), and 12 cases were identified from patients who had been diagnosed with influenza-like illness (7 were detected by RT-PCR, and 5 were detected by culture). A comparison of specimens that were found positive by culture with those found positive only by RT-PCR shows that the amount of virus in PCR-positive specimens tended to be lower than in isolation-positive specimens. Although the mean peak temperature in patients in the PCR-positive group was slightly lower, there were no significant differences in characteristics between specimens (i.e., kind of specimen, period from onset to specimen collection, age distribution of patients, and severity of illness). These results suggest that an epidemic of influenza C virus occurred on a national scale during this period and that RT-PCR can be an effective supplemental tool for the evaluation of clinical and epidemiological information.

A 50-year-old male was referred to our hospital for the evaluation of hyperproteinemia. Fluorodeoxyglucose positron emission tomography revealed high fluorodeoxyglucose uptake in the pancreas, bilateral lacrimal glands, submandibular glands, parotid glands, bilateral pulmonary hilar lymph nodes, and kidneys. Laboratory data showed an elevation of hepatobiliary enzymes, renal dysfunction, and remarkably high immunoglobulin (Ig) G levels, without elevated serum IgG4. Abdominal computed tomography revealed swelling of the pancreatic head and bilateral kidneys. Endoscopic retrograde cholangiopancreatography showed an irregular narrowing of the main pancreatic duct in the pancreatic head and stricture of the lower common bile duct. Histological examination by endoscopic ultrasonography-guided fine-needle aspiration revealed findings of lymphoplasmacytic sclerosing pancreatitis without IgG4-positive plasma cells. Abnormal laboratory values and the swelling of several organs were improved by the treatment with steroids. The patient was diagnosed as having type 1 autoimmune pancreatitis (AIP) based on the International Consensus Diagnostic Criteria. Therefore, we encountered a case of compatible type 1 AIP without elevated levels of serum IgG4 or IgG4-positive plasma cells. This case suggests that AIP phenotypes are not always associated with IgG4.

Influenza A/H3N2 viruses have increased the number of glycosylation sites on the head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of the addition of oligosaccharide chains to the HA of the H3N2 virus on its biological activities was investigated. We constructed wild-type HA and six mutant HAs of A/Aichi/2/68 virus with two or one to six glycosylation sites on the head, respectively. The glycosylation sites of mutated HAs correspond to representative H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86 and A/Sydney/5/97). We analyzed their intracellular transport, receptor-binding and cell fusion activities. The results showed that all the mutated HAs were transported to the cell surface, as well as wild-type HA, but some of them decreased in the receptor-binding activity. By contrast, the cell-fusion activity was not affected. Reactivity of mutated HAs, having four or five oligosaccharides with human serum collected in 1976, was much lower than that of the wild-type HA. These observations raised the possibility that the addition of new oligosaccharides to the head of the HA of the H3N2 virus may have provided the virus with an ability to evade antibody pressures.

The hemagglutinin-esterase (HE) glycoprotein of the influenza C virus causes cell fusion under acidic conditions. Because the influenza C virus grows more efficiently at 34 °C than at 37 °C, we examined whether the cell fusion activity of HE synthesized at 34 °C was higher than that at 37 °C. COS-1 cells, transfected with HE cDNA of C/Ann Arbor/1/50 virus, were incubated at 34 or 37 °C, and the cell fusion activity was analyzed after incubation for 24 h at each temperature. The results showed that cell fusion was observed with HE synthesized at 34 °C but not at 37 °C, although both HEs were transported to the cell surface. The fusion pore formation process did not proceed sufficiently when HE was synthesized at 37 °C. Comparison of the acetylesterase activities and antigenic structures between the HE synthesized at 34 °C and that at 37 °C showed that there were little or no differences in their properties. In contrast, the hemadsorption test with human erythrocytes suggested that the cell surface expression of HE synthesized at 37 °C was less efficient than that at 34 °C.

We have succeeded in generating virus-like particles (VLPs) of influenza C virus from cloned cDNAs. A cDNA of green fluorescent protein (GFP) gene flanked by the 5' and 3' noncoding regions of the segment 5 of influenza C virus was inserted between an RNA polymerase I promoter and terminator of the Pol I vector in antisense orientation. This plasmid DNA was transfected into 293T cells together with plasmids encoding the viral proteins of C/Ann Arbor/1/50 (AA/50) or C/Yamagata/1/88 (YA/88). Transfer of the supernatants of the 293T cells to HMV-II cells with helper virus resulted in the GFP expression in the HMV-II cells, and the quantification of the GFP-positive HMV-II cells indicated the presence of 106 VLPs/ml supernatant. Cords with 50−300 μm in length were observed on the transfected 293T cells, though they were not observed when the plasmid for M1 protein of AA/50 was replaced with that of C/Taylor/1233/47 (TAY/47). This finding provided direct evidence for the previous hypothesis by Nishimura et al. (1994) that M1 protein is involved in the formation of cord-like structures protruded from the YA/88-infected cells.

To characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94-, Tokyo/6/98-, and Shiga/T30/98-related lineage) of the Yamagata/16/88-like lineage. The NS genes of the viruses belonging to the Harbin/7/94-related lineage have additional three nucleotides at positions 439–447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88- and Victoria/2/87-like lineages, but were closely related to that of the Yamagata/16/88-like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87-like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98-like virus, and two strains of Harbin/7/94-related lineage had acquired the various gene segments from Shiga/T30/98-like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses. J. Med. Virol. 74:132–140, 2004. © 2004 Wiley-Liss, Inc.

Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized disease entity characterized by high-serum IgG4 concentration and IgG4-producing plasma cell production with fibrotic or sclerotic changes in affected organs. We aimed to clarify the roles of Epstein-Barr virus (EBV) in patients with IgG4-RDs.A retrospective clinical study at the Yamagata University School of Medicine, Yamagata, Japan.The patient group consisted of four males and four females with an average age of 62 years (range: 48-73). Expression of IgG4, latent member protein 1, EBV nuclear antigens-2, and EBV-encoded RNA in affected salivary glands from patients with IgG4-RD was examined by using immunohistochemistry and in situ hybridization. The copy number of EBV DNA in the salivary glands was also investigated by real-time polymerase chain reaction.All patients had hard masses in the salivary or lacrimal glands, or both, bilaterally. Serum concentrations of IgG4 were elevated in all cases (mean 589.1, range 129-1750), and IgG4-positive plasmacytes were observed in the involved salivary glands. Four patients developed potentially life-threatening systemic involvement after initial salivary gland swelling. EBV-associated molecules (EBNA and EBER) were overexpressed in the affected salivary glands. The copy number of EBV DNA was significantly higher in patients with potentially life-threatening systemic involvement than in patients without systemic involvement (P 0.05).These results suggest that the copy number of EBV DNA could be useful as diagnostic findings in IgG4-RD to predict potentially life-threatening systemic involvement.4.

Single preoperative biliary drainage for malignant perihilar biliary stricture occasionally fails to control jaundice and cholangitis. Multiple biliary drainage is required in such cases, but their clinical background is unclear. We determined the clinical characteristics associated with the requirement for multiple biliary drainage. The consecutive 122 patients with malignant perihilar biliary stricture were enrolled in a single-center retrospective study. Preoperative biliary drainage was initially performed on the future remnant hepatic lobe. Additional drainage was performed if jaundice failed to improve or cholangitis developed in undrained hepatic lobes. Detailed clinical characteristics and the number of preoperative biliary drainage procedures required before operation were analyzed. Thirty-one patients (25.4 %) initially underwent multiple biliary drainage. However, 69 (56.7 %) required multiple biliary drainage by the time of the operation. In the univariate analysis, the initial serum bilirubin level, cholangitis, percutaneous portal vein embolization, history of inserted endoscopic biliary stenting, length of preoperative period, operative procedure, and Bismuth classification were significant factors. In the multivariate analysis using these factors, Bismuth classification was independently associated with the requirement for multiple biliary drainage. The number of patients who required multiple biliary drainage was higher in those with Bismuth-II (91.9 %), Bismuth-IIIa (65.7 %), and Bismuth-IV (92.9 %) than in those with Bismuth-I (22.2 %) and Bismuth-IIIb (18.2 %). Patients with Bismuth-II, Bismuth-IIIa, and Bismuth-IV are at higher risk for multiple biliary drainage. A strategy based on the Bismuth classification for performing preoperative biliary drainage is important for patients with malignant perihilar biliary stricture.