Ubiquitin‐negative,eosinophilic neuronal cytoplasmic inclusions associated with stress granules and autophagy: An immunohistochemical investigation of two cases

Identification of the proteinaceous components of the pathological inclusions is an important step in understanding the associated disease mechanisms. We immunohistochemically examined two previously reported cases with eosinophilic neuronal cytoplasmic inclusions (NCIs) (case 1, Mori et al. Neuropathology 2010; 30: 648–53; case 2, Kojima et al. Acta Pathol Jpn 1990; 40: 785–91) using 67 antibodies against proteins related to cytoskeletal constituents, ubiquitin‐proteasome system, autophagy‐lysosome pathway and stress granule formation. Regional distribution pattern of eosinophilic NCIs in case 1 was substantially different from that in case 2. However, NCIs in both cases were immunonegative for ubiquitin and p62 and were immunopositive for stress granule markers as well as autophagy‐related proteins, including valosin‐containing protein. Considering that eukaryotic stress granules are cleared by autophagy and valosin‐containing protein function, our findings suggest that eosinophilic NCIs in the present two cases may represent the process of autophagic clearance of stress granules.     

Clinical improvements in higher brain function and rapid functional recovery in a case of cerebellar hemorrhage treated by neurocognitive rehabilitation

Cerebellar stroke rarely causes disorders of higher brain function such as cognitive deficits and emotional dysfunction; hence, the prognosis of these patients is uncertain. We report the case of a 34-year-old patient with cerebellar hemorrhage due to arteriovenous malformation causing higher brain dysfunction who was able to return to full-time employment after neurocognitive rehabilitation. Cerebellar stroke caused by nonatherosclerotic diseases or diaschisis may lead to cognitive deficits but these symptoms can be reversed by appropriate therapy. It is important to assess higher brain function in cerebellar stroke patients and to tailor neurocognitive rehabilitation programs appropriately to promote functional recovery.     

High prevalence of equine-like G3P[8] rotavirus in children and adults with acute gastroenteritis in Thailand

Group A rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. This study aims to clarify the distribution of G/P types and genetic characteristics of RVAs circulating in Thailand. Between January 2014 and September 2016, 1867 stool specimens were collected from children and adults with acute gastroenteritis in six provinces in Thailand. RVAs were detected in 514/1867 (27.5%) stool specimens. G1P[8] (44.7%) was the most predominant genotype, followed by G3P[8] (33.7%), G2P[4] (11.5%), G8P[8] (7.0%), and G9P[8] (1.3%). Unusual G3P[9] (0.8%), G3P[10] (0.4%), G4P[6] (0.4%), and G10P[14] (0.2%) were also detected at low frequencies. The predominant genotype, G1P[8] (64.4%), in 2014 decreased to 6.1% in 2016. In contrast, the frequency of G3P[8] markedly increased from 5.5% in 2014 to 65.3% in 2015 and 89.8% in 2016. On polyacrylamide gel electrophoresis, most (135/140; 96.4%) of the G3P[8] strains exhibited a short RNA profile. Successful determination of the nucleotide sequences of the VP7 genes of 98 G3P[8] strains with a short RNA profile showed that they are all equine-like G3P[8] strains. On phylogenetic analysis of genome segments of two representative Thai equine-like G3P[8] strains, it was noteworthy that they possessed distinct NSP4 genes, one bovine-like and the other human-like. Thus, we found that characteristic equine-like G3P[8] strains with a short RNA electropherotype are becoming highly prevalent in children and adults in Thailand.     

Carbon monoxide-bound hemoglobin-vesicles for the treatment of bleomycin-induced pulmonary fibrosis

Carbon monoxide (CO) has potent anti-inflammatory and anti-oxidant effects. We report herein on the preparation of a nanotechnology-based CO donor, CO-bound hemoglobin-vesicles (CO-HbV). We hypothesized that CO-HbV could have a therapeutic effect on idiopathic pulmonary fibrosis (IPF), an incurable lung fibrosis, that is thought to involve inflammation and the production of reactive oxygen species (ROS). Pulmonary fibril formation and respiratory function were quantitatively evaluated by measuring hydroxyproline levels and forced vital capacity, respectively, using a bleomycin-induced pulmonary fibrosis mice model. CO-HbV suppressed the progression of pulmonary fibril formation and improved respiratory function compared to saline and HbV. The suppressive effect of CO-HbV on pulmonary fibrosis can be attributed to a decrease in ROS generation by inflammatory cells, NADPH oxidase 4 and the production of inflammatory cells, cytokines and transforming growth factor-β in the lung. This is the first demonstration of the inhibitory effect of CO-HbV on the progression of pulmonary fibrosis via the anti-oxidative and anti-inflammatory effects of CO in the bleomycin-induced pulmonary fibrosis mice model. CO-HbV has the potential for use in the treatment of, not only IPF, but also a variety of other ROS and inflammation-related disorders.     

A case of Krukenberg carcinoma metastasized from colon cancer resembling mucinous cystadenocarcinoma of the ovary

We report a case of a 44-year-old woman with bilateral ovarian carcinoma that had metastasized from the colon and mimicked primary mucinous cystadenocarcinoma. Macroscopically, both ovarian tumors were large, multiloculated cystic masses with abundant mucinous content. Histologically, they were lined with mucinous epithelium with mild to moderate nuclear atypia and showed stromal invasion and surface involvement. At first, the tumors were diagnosed as bilateral primary ovarian mucinous cystadenocarcinomas. However, three months after surgery, a large villous tumor was discovered in the ascending colon by colonoscopic examination and was surgically resected. Histologically, the colonic tumor was a villous adenomatous tumor with invasive components of mucinous adenocarcinoma composed of well-differentiated adenocarcinoma and exhibited abundant extracellular mucin production. As a villous adenomatous component was present in the mucosal area, the colonic tumor was considered a primary tumor. Therefore, the original diagnosis of bilateral ovarian tumors was revised for consistent with metastasis from the colon carcinoma, in line with the findings of immunohistochemistry and loss of heterozygosity analysis. This case highlights the importance of considering the possibility of metastatic tumors from the gastrointestinal tract in the diagnosis of mucinous ovarian tumors.     

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate

Hepatitis C virus (HCV) is a global disease burden, and a preventive vaccine is needed to control or eradicate the virus. Despite the advent of effective antiviral therapy, this treatment is not accessible to many patients and does not prevent reinfection, making chronic hepatitis C an ongoing global health problem. Thus, development of a prophylactic vaccine will represent a significant step toward global eradication of HCV. HCV exhibits high genetic variability, which leads frequently to immune escape. However, a considerable challenge faced in HCV vaccine development is designing an antigen that elicits broadly neutralizing antibodies. Here, we characterized the immunogenicity of a vaccine based on a soluble, secreted form of the E1E2 envelope heterodimer (sE1E2.LZ). Sera from mice immunized with sE1E2.LZ exhibited an anti-E1E2–specific response comparable to mice immunized with membrane-bound E1E2 (mbE1E2) or a soluble E2 ectodomain (sE2). In competition-inhibition ELISA using antigenic domain-specific neutralizing and nonneutralizing antibodies, sera from sE1E2.LZ-immunized mice showed nearly identical or stronger competition toward neutralizing antibodies when compared with mbE1E2. In contrast, sera from mice immunized with sE2, and to a lesser extent mbE1E2, competed more effectively with nonneutralizing antibodies. An assessment of neutralization activity using both HCV pseudoparticles and cell culture–derived infectious HCV showed that immunization with sE1E2.LZ elicited the broadest neutralization activity of the three antigens, and sE1E2.LZ induced neutralization activity against all genotypes. These results indicate that our native-like soluble glycoprotein design, sE1E2.LZ, induces broadly neutralizing antibodies and serves as a promising vaccine candidate for further development.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5), and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct acting antivirals (DAAs), but several issues have blunted their effectiveness in eradicating HCV. In particular, multiple public health and financial barriers (7, 8) restrict access to DAAs in areas with high incidence of infection and DAAs do not prevent reinfection. Moreover, HCV infection is largely asymptomatic and often does not generate sterilizing immunity, thereby contributing to reinfection or continued disease progression (7, 9, 10). Collectively, these issues have resulted in a continued rise in HCV infections.Acute HCV infections can be cleared by host immunity in ∼25% of cases. Among individuals who clear their first infection, the rate of clearance rises to 80% for subsequent infections, indicating an effective immune memory response (11–14). This type of natural protective immunity to HCV requires the induction of broadly neutralizing antibodies to E1E2 ectodomains and T cell responses to the structural and nonstructural proteins (15–17). The above clinical observations suggest that, if a vaccine candidate could induce broadly neutralizing antibody and cell-mediated immune responses equivalent to that seen in spontaneous clearance, such a vaccine would be highly effective at preventing HCV infection. An HCV vaccine therefore remains an essential proactive measure to protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (17, 18).A number of challenges exist that have thus far limited progress toward developing a prophylactic vaccine against HCV. One major challenge in developing a successful vaccine for HCV has been the remarkable genetic diversity of the virus which has six major genotypes (genotypes 1 to 6), in addition to two less-common genotypes (19) (genotypes 7 and 8), and intragenotypic diversity resulting in 90 total subtypes (20). Moreover, shielding of important neutralizing epitopes with glycans (21, 22), and the presence of immunodominant nonneutralizing epitopes (23–26) deflect the immune response from conserved regions that mediate virus neutralization. Multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, but their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited. In particular, immunological assessment in chimpanzees of an E1E2 vaccine produced superior immune responses as compared with E2 administered alone and resulted in sterilizing immunity against homologous virus challenge (27, 28), but with less cross-neutralization capacity against heterologous isolates (29). In addition, an E1E2 formulation tested in humans is well-tolerated (30). However, due to the limited neutralization breadth observed in the human clinical trial (31, 32), using native E1E2 as a vaccine is not likely to provide sufficient protection from HCV infection. Rather, optimization of E1E2 to improve its immunogenicity and capacity to elicit bnAbs through rational design appears to be the preferred path for developing an effective B cell-based vaccine (33).An additional bottleneck contributing to the difficulty in generating protective B cell immune responses required for an effective HCV vaccine is preparation of a homogeneous E1E2 antigen. HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (34–36). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (37) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (36, 38, 39) and interactions between E1 and E2 ectodomains (40–42). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and scavenger receptor type B class I (SR-B1) coreceptors as part of a multistep entry process on the surface of hepatocytes (43–46). Neutralizing antibody (nAb) responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (25, 47–52). A significant impediment to the uniform production of an immunogenic E1E2 heterodimer that could be utilized for vaccine development is the association of the antigen with the membrane via the TMDs (36, 53). Progress has been made in the production and purification of the membrane-bound E1E2 complex via immunoaffinity purification (54, 55) or the use of tags that allow protein A (56) or anti-Flag (57) chromatography. While these methods produce high-quality samples, they all involve harsh elution conditions. How such conditions might influence sample quality at a scale required for vaccine trials is unclear. Furthermore, intracellular expression and membrane extraction limits the ability to produce large quantities of sufficient homogeneity required for both basic research and vaccine production.In contrast, viral glycoproteins of influenza hemagglutinin (58), respiratory syncytial virus (RSV) (59), SARS-CoV-2 (60), and others (61, 62) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. In addition, HIV gp120-gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (63, 64). Recent efforts have made strides toward liberating the E1E2 complex from the membrane in its native form (65, 66). In particular, our previous work (66) showed that a soluble E1E2 (sE1E2) using the Fos/Jun leucine zipper (LZ) coiled-coil as a scaffold (sE1E2.LZ) is antigenically intact, as the protein is recognized by E1E2-specific mAbs AR4A and AR5A (67). Moreover, sE1E2.LZ elicited nAbs in mice immunized with the antigen, making this scaffold a promising potential platform for engineering of additional HCV vaccine candidates.Here, we describe the immunogenicity of our native-like secreted E1E2 construct sE1E2.LZ and compare it with the membrane-bound E1E2 complex (mbE1E2) and a secreted form of the E2 ectodomain (sE2). Immunization of mice with sE1E2.LZ produced sera possessing anti-E1E2 antibodies at levels comparable to mice immunized with mbE1E2 or sE2. Moreover, the antibody response in sE1E2.LZ-immunized mice is skewed more toward nAbs relative to non-nAbs than the other two antigens. Remarkably, sera from sE1E2.LZ-immunized mice exhibited broader neutralization activity than either mbE1E2 or sE2 when assessed using both pseudotyped HCV particles (HCVpp) and cell culture-derived HCV (HCVcc), suggesting that this sE1E2 platform represents a favorable starting point for developing scaffolded E1E2 vaccine candidates.     

Prevalence of Burnout among Internal Medicine and Primary Care Physicians before and during the COVID-19 Pandemic in Japan

Objective To evaluate the change in the prevalence of burnout during the COVID-19 pandemic among internists and primary care physicians in Japan, and to identify factors associated with the exacerbation of burnout among these populations during this period. Methods This was a cross-sectional study based on two web-based surveys conducted in January 2020 (before the declaration of the COVID-19 pandemic) and June 2020 (during the pandemic). The participants were internists and primary care physicians of the Japanese Chapter of the American College of Physicians. The main outcome was the change in the prevalence of burnout between before and during the “first wave” of the pandemic. We also examined factors associated with the exacerbation of burnout during this period. Results Among the 283 respondents in the first survey and 322 in the second survey, 98 (34.6%) and 111 (34.5%) reported symptoms of burnout, respectively. In June 2020, 82 respondents (25.5%) reported that their level of burnout exacerbated compared to January 2020. Only the experience of self-quarantine was associated with the exacerbation of burnout [odds ratio (OR) 3.12; 95% confidence interval (CI) 1.49-6.50; p=0.002], while being a woman, being a resident physician, and an experience of having worked in a prefecture under a state of emergency were not. Conclusions No marked change in the prevalence of burnout among internists and primary care physicians in Japan was observed during the COVID-19 pandemic as a whole. However, self-quarantine was associated with the exacerbation of the burnout level.     

Work and family characteristics as socioeconomic determinants in long sickness absence: the Japanese civil servants study

Long sickness absence is more common among low socioeconomic status (SES) groups than high SES groups. This study aimed to evaluate whether work and family characteristics contribute to SES and sex differences in long sickness absence (7 days or more). The participants were 3080 civil servants working for a local Japanese government. In both sexes, low-grade employees were likely to take long sickness absence, with a statistically significant association for men (age-adjusted OR of lowest-grade employees for long sickness absence: 2.30 (95% Confidence Interval (CI): 1.32–4.02)). After adjusting for all variables, SES differences in long sickness absence in men decreased to OR 1.98 (CI 1.10–3.55) but remained significant; in men, being without a spouse was significantly associated with long sickness absence. Employees working long hours had lower OR for long sickness absence after adjusting for all variables in both sexes. Conversely, poor sleep quality and longstanding illness significantly increased OR for long sickness absence. In conclusion, SES differences in sickness absence were explained partly by work and family characteristics, longstanding illness, and poor sleep quality; however, other factors that were not evaluated in this study may also be associated with SES differences.