A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

BACKGROUND: To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. METHODS: We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). RESULTS: ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. CONCLUSION: The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

Cathepsin L prevents the accumulation of alpha-synuclein fibrils in the cell.

The deposition of α-synuclein (α-Syn) fibrils in neuronal cells has been implicated as a causative factor in Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). α-Syn can be degraded by autophagy, proteasome, and chaperone-mediated autophagy, and previous studies have suggested the potency of certain cathepsins, lysosomal proteases, for α-Syn degradation. However, no studies have comprehensively evaluated all cathepsins. Here, we evaluated the efficacy of all 15 cathepsins using a cell model of α-Syn fibril propagation and found that overexpression of cathepsin L (CTSL) was the most effective in preventing the accumulation of α-Syn aggregates. CTSL-mediated degradation of α-Syn aggregates was dependent on the autophagy machinery, and CTSL itself promoted autophagy flux. Interestingly, CTSL was effective in autophagic degradation of wild-type (WT) α-Syn, but not in the case of A53T and E46K missense mutations, which are causative for familial PD. These results suggest that CTSL is a potential therapeutic strategy for sporadic PD pathology in WT α-Syn.

Roles of Stress Response in Autophagy Processes and Aging-Related Diseases.

The heat shock factor 1 (HSF1)-mediated stress response pathway and autophagy processes play important roles in the maintenance of proteostasis. Autophagy processes are subdivided into three subtypes: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. Recently, molecular chaperones and co-factors were shown to be involved in the selective degradation of substrates by these three autophagy processes. This evidence suggests that autophagy processes are regulated in a coordinated manner by the HSF1-mediated stress response pathway. Recently, various studies have demonstrated that proteostasis pathways including HSF1 and autophagy are implicated in longevity. Furthermore, they serve as therapeutic targets for aging-related diseases such as cancer and neurodegenerative diseases. In the future, these studies will underpin the development of therapies against various diseases.

Odontogenic Cutaneous Sinus Tract in a 10-Year-Old Girl: A Case Report of a Rare Entity.

Odontogenic cutaneous sinus tract (OCST) is defined as pulp necrosis caused by dental caries or trauma that forms a fistula on the body surface as a drainage channel for the infected pulp. OCST can be difficult to diagnose because subjective symptoms, such as pain in the affected tooth, may be minimal. In addition, lesions in the cervical region are extremely rare. In this report, we discuss the case of a 10-year-old girl who presented with inflammation, edema, and purulent exudation on the right neck. Her symptoms resembled those of lateral cervical cysts and fistulas. However, upon evaluation, she was diagnosed with OCST. Although OCST is an important differential diagnosis for head and neck lesions, it is often overlooked. OCST should be considered in the differential diagnosis of neck masses and fistulas.

The Role of Neuropeptide Y in the Nucleus Accumbens.

Neuropeptide Y (NPY), an abundant peptide in the central nervous system, is expressed in neurons of various regions throughout the brain. The physiological and behavioral effects of NPY are mainly mediated through Y1, Y2, and Y5 receptor subtypes, which are expressed in regions regulating food intake, fear and anxiety, learning and memory, depression, and posttraumatic stress. In particular, the nucleus accumbens (NAc) has one of the highest NPY concentrations in the brain. In this review, we summarize the role of NPY in the NAc. NPY is expressed principally in medium-sized aspiny neurons, and numerous NPY immunoreactive fibers are observed in the NAc. Alterations in NPY expression under certain conditions through intra-NAc injections of NPY or receptor agonists/antagonists revealed NPY to be involved in the characteristic functions of the NAc, such as alcohol intake and drug addiction. In addition, control of mesolimbic dopaminergic release via NPY receptors may take part in these functions. NPY in the NAc also participates in fat intake and emotional behavior. Accumbal NPY neurons and fibers may exert physiological and pathophysiological actions partly through neuroendocrine mechanisms and the autonomic nervous system.

Gastrointestinal endoscopic practice during COVID-19 pandemic: a multi-institutional survey.

Introduction. An on-going coronavirus disease 2019 (COVID-19) has become a challenge all over the world. Since an endoscopy unit and its staff are at potentially high risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we conducted a survey for the management of the gastrointestinal endoscopic practice, personal protective equipment (PPE), and risk assessment for COVID-19 during the pandemic at multiple facilities.Methods. The 11-item survey questionnaire was sent to representative respondent of Department of Gastroenterology, Osaka City University Hospital, and its 19 related facilities.Results. A total of 18 facilities submitted valid responses and a total of 373 health care professionals (HCPs) participated. All facilities (18/18: 100%) were screening patients at risk for SARS-CoV-2 infection before endoscopy. During the pandemic, we found that the total volume of endoscopic procedures decreased by 44%. Eleven facilities (11/18: 61%) followed recommendations of the Japan Gastroenterological Endoscopy Society (JGES); consequently, about 35%-50% of esophagogastroduodenoscopy and colonoscopy were canceled. Mask (surgical mask or N95 mask), face shield/goggle, gloves (one or two sets), and gown (with long or short sleeves) were being used by endoscopists, nurses, endoscopy technicians, and endoscope cleaning staff in all the facilities (18/18: 100%). SARS-CoV-2 infection risk assessment of HCPs was conducted daily in all the facilities (18/18: 100%), resulting in no subsequent SARS-CoV-2 infection in HCPs.Conclusion. COVID-19 has had a dramatic impact on the gastrointestinal endoscopic practice. The recommendations of the JGES were appropriate as preventive measures for the SARSCoV-2 infection in the endoscopy unit and its staff.

Identification of characteristic proteins at late-stage erythroid differentiation in vitro.

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.

Ubiquitin, Autophagy and Neurodegenerative Diseases.

Ubiquitin signals play various roles in proteolytic and non-proteolytic functions. Ubiquitin signals are recognized as targets of the ubiquitin-proteasome system and the autophagy-lysosome pathway. In autophagy, ubiquitin signals are required for selective incorporation of cargoes, such as proteins, organelles, and microbial invaders, into autophagosomes. Autophagy receptors possessing an LC3-binding domain and a ubiquitin binding domain are involved in this process. Autophagy activity can decline as a result of genetic variation, aging, or lifestyle, resulting in the onset of various neurodegenerative diseases. This review summarizes the selective autophagy of neurodegenerative disease-associated protein aggregates via autophagy receptors and discusses its therapeutic application for neurodegenerative diseases.

Effect of the Lactococcus Lactis 11/19-B1 Strain on Atopic Dermatitis in a Clinical Test and Mouse Model.

Some lactic acid bacteria (LAB) are known to improve atopic dermatitis (AD) through the regulation and stimulation of the host immune system. In this study, we found that ingestion of yogurt containing Lactococcus lactis 11/19-B1 strain (L. lactis 11/19-B1) daily for 8 weeks significantly improved the severity scoring of atopic dermatitis (SCORAD) system score from 38.8 ± 14.4 to 24.2 ± 12.0 in children suffering from AD. We tried to identify which LAB species among the five species contained in the test yogurt contributed to the improvement in AD pathology using an AD mouse model induced by repeated application of 1-fluoro-2, 4-dinitrobenzene (DNFB). AD-like skin lesions on the dorsal skin and ear were most improved by L. lactis 11/19-B1 intake among the five LAB species. In addition, analysis of CD4+ T cell subsets in Peyer's patches (PPs) and cervical lymph nodes (CLNs) indicated that the intake of L. lactis 11/19-B1 generally suppressed all subsets related to inflammation, i.e., Th1, Th2 and Th17, instead of activating the suppressive system, Treg, in the AD mouse model. Histological observations showed ingestion of L. lactis 11/19-B1 significantly suppressed severe inflammatory findings, such as inflammatory cell filtration, epidermal erosion and eosinophil infiltration. These results suggest that the immunomodulatory effects of L. lactis 11/19-B1 contribute to improvements in AD pathology.

Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that is widely expressed in the central nervous system, including the cerebral cortex, nucleus accumbens (NAc) and hypothalamus. We previously analyzed the behavior of transgenic mice exclusively expressing an unedited RNA isoform of the 5-HT2C receptor. These mice showed decreased NPY gene expression in the NAc and exhibited behavioral despair, suggesting that NAc NPY neurons may be involved in mood disorder; however, their role in this behavior remained unknown. Therefore, in the present study, we investigated the functional role of NAc NPY neurons in anxiety-like behavior by examining the impact of specific ablation or activation of NAc NPY neurons using NPY-Cre mice and Cre-dependent adeno-associated virus. Diphtheria toxin-mediated ablation of NAc NPY neurons significantly increased anxiety-like behavior in the open field and elevated plus maze tests, compared with before toxin treatment. Moreover, chemogenetic activation of NAc NPY neurons reduced anxiety-like behavior in both behavioral tests compared with control mice. These results suggest that NPY neurons in the NAc are involved in the modulation of anxiety in mice.

α-Synuclein Promotes Maturation of Immature Juxtaglomerular Neurons in the Mouse Olfactory Bulb.

α-Synuclein (αSyn), the major constituent of Lewy bodies and Lewy neurites, is generally expressed in presynapses and is involved in synaptic function. However, we previously demonstrated that some neurons, including those in the olfactory bulb, show high αSyn expression levels in the cell body under normal conditions. αSyn is also known to be important for adult neurogenesis. Thus, in present study, we examined the role of αSyn in juxtaglomerular neurons (JGNs) with high αSyn expression in the mouse olfactory bulb. Most αSyn-enriched JGNs expressed sex-determining region Y-box 2 (Sox2), which functions to maintain neural immature identity. Interestingly, in αSyn homozygous (-/-) knockout (KO) mice, Sox2-positive JGNs were significantly increased compared with heterozygous (+/-) KO mice. Following global brain ischemia using wild-type mice, there was also a significant decrease in Sox2-positive JGNs, and in the co-expression ratio of Sox2 in αSyn-enriched JGNs. By contrast, the co-expression ratio of neuronal nuclei (NeuN, mature neuronal marker) was significantly increased in αSyn-enriched JGNs. However, this ischemia-induced decrease of Sox2-positive JGNs was not observed in αSyn homozygous KO mice. Overall, these data suggest that αSyn functions to promote the maturation of immature JGNs in the mouse olfactory bulb.

Suppression of autophagic activity by Rubicon is a signature of aging.

Autophagy, an evolutionarily conserved cytoplasmic degradation system, has been implicated as a convergent mechanism in various longevity pathways. Autophagic activity decreases with age in several organisms, but the underlying mechanism is unclear. Here, we show that the expression of Rubicon, a negative regulator of autophagy, increases in aged worm, fly and mouse tissues at transcript and/or protein levels, suggesting that an age-dependent increase in Rubicon impairs autophagy over time, and thereby curtails animal healthspan. Consistent with this idea, knockdown of Rubicon extends worm and fly lifespan and ameliorates several age-associated phenotypes. Tissue-specific experiments reveal that Rubicon knockdown in neurons has the greatest effect on lifespan. Rubicon knockout mice exhibits reductions in interstitial fibrosis in kidney and reduced α-synuclein accumulation in the brain. Rubicon is suppressed in several long-lived worms and calorie restricted mice. Taken together, our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging.

Expression of α-synuclein is regulated in a neuronal cell type-dependent manner.

α-Synuclein, the major component of Lewy bodies (LBs) and Lewy neurites (LNs), is expressed in presynapses under physiologically normal conditions and is involved in synaptic function. Abnormal intracellular aggregates of misfolded α-synuclein such as LBs and LNs are pathological hallmarks of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). According to previous studies using pathological models overexpressing α-synuclein, high expression of this protein in neurons is a critical risk factor for neurodegeneration. Therefore, it is important to know the endogenous expression levels of α-synuclein in each neuronal cell type. We previously reported differential expression profiles of α-synuclein in vitro and in vivo. In the wild-type mouse brain, particularly in vulnerable regions affected during the progression of idiopathic PD, α-synuclein is highly expressed in neuronal cell bodies of some early PD-affected regions, such as the olfactory bulb, the dorsal motor nucleus of the vagus, and the substantia nigra pars compacta. Synaptic expression of α-synuclein is mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory synapse marker protein. In contrast, α-synuclein expression in inhibitory synapses differs among brain regions. Recently accumulated evidence indicates the close relationship between differential expression profiles of α-synuclein and selective vulnerability of certain neuronal populations. Further studies on the regulation of α-synuclein expression will help to understand the mechanism of LB pathology and provide an innovative therapeutic strategy to prevent PD and DLB onset.

Establishment and characterization of immortalized erythroid progenitor cell lines derived from a common cell source.

Immortalized erythroid progenitor cell lines, which exhibit potential for enucleated red blood cell (RBC) production, are expected to serve as an in vitro source of RBCs. These erythroid progenitor cell lines have previously been established from a variety of sources; however, large numbers of cell lines have not been established, characterized, and compared from a common cell source. In the present study, 37 cell lines were established from human bone marrow cells from a single donor. The time required for the establishment of each cell line varied greatly from 46 to 246 days. Of these lines, five were selected and their characteristics were analyzed. The cell lines established at the earliest time point showed better results in terms of both karyotype and differentiation potential than those established the latest. Moreover, obvious differences were noted even when cell lines were established at the earliest time point from the same source. These results suggest that it is important to select the best cell lines from ones established at the earliest time point for generating cell lines with low genomic abnormality and high differentiation ability. We have successfully generated an adult type of cell line with 50% cells carrying a normal karyotype and with 25% enucleation efficiency. These findings could be valuable in the development of an optimal method for establishing cell lines.

RNA Editing of Serotonin 2C Receptor and Alcohol Intake.

Serotonin 2C receptor (5-HT2 CR) belongs to the superfamily of seven transmembrane domain receptors coupled to G proteins (GPCR). It is broadly distributed in the CNS and its expression is relatively high in the limbic system including the amygdala, nucleus accumbens (NAc), hippocampus, and hypothalamus. Based on its expression patterns and numerous pharmacological studies, 5-HT2 CR is thought to be involved in various brain functions including emotion, appetite, and motor behavior. Here, we review 5-HT2 CR and its relationship with alcohol intake with a particular focus on the involvement of 5-HT2 CR mRNA editing and its association with alcohol preference in mice. RNA editing is a post-transcriptional modification mechanism. In mammals, adenosine is converted to inosine by the deamination enzymes ADAR1 and ADAR2. 5-HT2 CR is the only GPCR subjected to RNA editing within the coding region. It has five editing sites in exon 5 that encode the second intracellular loop. Consequently, three amino acids residues (I156, N158, and I160) of the unedited receptor (INI) may be altered to differently edited isoforms, resulting in a change of receptor activity such as 5-HT potency and G-protein coupling. 5-HT2 CR in the NAc is involved in enhanced alcohol drinking after chronic alcohol exposure and alterations in 5-HT2 CR mRNA editing is important in determining the alcohol preference using different strains of mice and genetically modified mice. RNA editing of this receptor may participate in the development of alcoholism.

An intubated 7-month-old infant with a retropharyngeal abscess and multidrug-resistant Streptococcus mitis.

The profile of antimicrobial resistance (ie, antibiogram) may be disparate between children and adults. An infant developed severe deep neck infection with a multidrug-resistant microbe. The microbe was more drug-resistant in children than in adults, in our hospital. Treatment of a child should be guided by the antibiogram obtained from children.

A Double-Blind Controlled Study to Evaluate the Effects of Yogurt Enriched with Lactococcus lactis 11/19-B1 and Bifidobacterium lactis on Serum Low-Density Lipoprotein Level and Antigen-Specific Interferon-γ Releasing Ability.

In order to clarify the effects of the Lactococcus lactis (L. lactis) 11/19-B1 strain, a double-blind controlled study of yogurt fermented with the strain was carried out. For the study, two kinds of yogurt, the control and test yogurt, were prepared; the control yogurt was fermented with Streptococcus thermophiles, Lactobacillus delbrueckii subspecies bulgaricus, and Lactobacillus acidophilus, and the test yogurt was enriched with L. lactis 11/19-B1 and Bifidobacterium lactis (B. lactis) BB-12 strains. Seventy-six volunteers who had not received treatment with pharmaceuticals were randomly divided into two groups with each group ingesting 80 g of either the test or control yogurt every day for 8 weeks. Before and after yogurt intake, fasting blood was taken and blood sugar, blood lipids, and anti-cytomegalovirus cellular immunity were estimated. In the test yogurt group, low-density lipoprotein (LDL) was significantly decreased (159.1 ± 25.7 to 149.3 ± 24.4; p = 0.02), but this effect was not observed in the control yogurt group. When the test yogurt group was divided into two groups based on LDL levels of over or under 120 mg/dL, this effect was only observed in the high LDL group. No LDL-lowering effect of B. lactis BB-12 strain was previously reported; therefore, the hypocholesterolemic effects observed in this study are thought to be caused by the L. lactis 11/19-B1 strain alone or its combination with the B. lactis BB-12 strain.

Ethanol Preference and Drinking Behavior Are Controlled by RNA Editing in the Nucleus Accumbens.

RNA editing plays critical roles in normal brain function, and alteration of its activity causes various disorders. We previously found that chronic consumption of ethanol was associated with increased levels of RNA editing of serotonin 2C receptor in the nucleus accumbens (NAc). However, it remains unknown whether RNA editing in the NAc modulates alcohol addiction through the brain reward system. To investigate the involvement of NAc RNA editing in alcohol addiction, we generated NAc-specific knockout mice of the double-stranded RNA-specific adenosine deaminase ADAR2 using AAV-GFP/Cre and conducted a battery of behavioral tests including anxiety- and depression-like behaviors. In addition, NAc-specific ADAR2 knockout mice were exposed to ethanol vapor for 20 days, followed by ethanol-drinking and conditioned place preference (CPP) tests. NAc-specific ADAR2 knockout mice showed a significant decrease in locomotor activity in the open field test although they did not develop anxiety- and depression-like behaviors. In addition, the enhancements of ethanol intake and ethanol preference that are usually observed after chronic ethanol vapor exposure were significantly reduced in these mice. These results suggest that ADAR2-mediated RNA editing in the NAc is involved in determination of alcohol preference after chronic alcohol consumption.

DHA Mitigates Autistic Behaviors Accompanied by Dopaminergic Change in a Gene/Prenatal Stress Mouse Model.

Autism Spectrum Disorder (ASD) is characterized by impairments in social interaction, social communication, and repetitive and stereotyped behaviors. Recent work has begun to explore gene × environmental interactions in the etiology of ASD. We previously reported that prenatal stress exposure in stress-susceptible heterozygous serotonin transporter (SERT) KO pregnant dams in a mouse model resulted in autism-like behavior in the offspring (SERT/S mice). The association between prenatal stress and ASD appears to be affected by maternal SERT genotype in clinical populations as well. Using the mouse model, we examined autistic-like behaviors in greater detail, and additionally explored whether diet supplementation with docosahexaenoic acid (DHA) may mitigate the behavioral changes. Only male SERT/S mice showed social impairment and stereotyped behavior, and DHA supplementation ameliorated some of these behaviors. We also measured monoamine levels in the SERT/S mice after three treatment paradigms: DHA-rich diet continuously from breeding (DHA diet), DHA-rich diet only after weaning (CTL/DHA diet) and control diet only (CTL diet). The dopamine (DA) content in the striatum was significantly increased in the SERT/S mice compared with wild-type (WT) mice, whereas no difference was observed with noradrenaline and serotonin content. Moreover, DA content in the striatum was significantly reduced in the SERT/S mice with the DHA-rich diet provided continuously from breeding. The results indicate that autism-associated behaviors and changes in the dopaminergic system in this setting can be mitigated with DHA supplementation.

[Injection of High Concentration Glucose Solution for Pleural Coating Reduces Postoperative Recurrence of Spontaneous Pneumothorax;A Short-term Retrospective Study].

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) is the standard treatment for patients with spontaneous pneumothorax (SP). However, postoperative recurrence is not infrequent even with an absorbable covering sheet used to reinforce the visceral pleura. Recent reports suggest that intraoperative injection of a highly concentrated glucose solution into the thoracic cavity provides effective prophylaxis against postoperative SP recurrence. Since September 2015, we have been injecting 50 ml of 50 % glucose solution intraoperatively for pleural coating (GPC) around an absorbable sheet to prevent postoperative SP recurrence. METHODS: We evaluated 340 patients who underwent VATS between February 2011 and June 2017(88 patients:GPC group, 252:non-GPC group), and we retrospectively analyzed the efficacy of GPC in preventing postoperative SP recurrence. RESULTS: One year postoperative recurrence rates of GPC and non-GPC groups were 9.0 and 17.9%,respectively. The log-rank test revealed GPC as a significant factor in preventing postoperative recurrence (p=0.020). No severe adverse events occurred in either group. Minor postoperative complications, viz., high blood sugar, high volume of chest tube drainage occurred in the GPC group. CONCLUSION: Application of GPC is beneficial in reducing postoperative recurrence of SP.