Depression prevention in healthcare workers during the COVID-19 pandemic.

BACKGROUND: Few studies have assessed depression in healthcare workers (HCWs) in Japan owing to the coronavirus disease 2019 (COVID-19) pandemic, and no studies have proposed effective interventions to help support their mental health. AIMS: To test the hypothesis that enhancing access to mental healthcare professionals helps to improve HCWs' mental health. METHODS: This cross-sectional study assessed depressive symptoms in HCWs at three hospitals in Osaka prefecture between May and July, 2020. The survey obtained information on HCWs' mental state and related situations/perceptions. Multivariable logistic regression analysis was performed to identify factors associated with depressive symptoms. RESULTS: Of the 3291 eligible HCWs, 1269 (39%) completed the survey. Of all HCWs, 87 (7%) were physicians, and 700 (55%) were nurses. A total of 181 (14%) HCWs had moderate-to-severe symptoms of depression. Being a frontline worker was not significantly associated with depressive symptoms (odds ratio: 0.86 [95% confidence intervals: 0.54-1.37], P = 0.50). The unwillingness to consult with anyone was significantly associated with more severe depressive symptoms (1.70 [1.10-2.63], P < 0.01). HCWs who had no opportunity to confide in family/friends (1.66 [1.10-2.52], P < 0.01) or colleagues/supervisors (3.19 [2.22-4.58], P < 0.001) were significantly more likely to have depressive symptoms. CONCLUSIONS: Being a frontline HCW in a Japanese hospital treating patients with COVID-19 was not significantly associated with having depressive symptoms. The study highlights that encouraging daily communication with close persons (family, friends, colleagues and supervisors), rather than improving access to mental health professionals, might help to prevent depression in HCWs during the COVID-19 pandemic.

Calcineurin inhibitors exacerbate coronary arteritis via the MyD88 signalling pathway in a murine model of Kawasaki disease.

Calcineurin inhibitors (CNIs) have been used off-label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in wild-type, severe combined immunodeficiency (SCID), caspase-associated recruitment domain 9 (CARD9)-/- and myeloid differentiation primary response gene 88 (MyD88)-/- mice. We also performed in-vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1-mediated coronary arteritis in a dose-dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9-/- mice but not in MyD88-/- mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88-dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.

BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

Treatment with YIGSR peptide ameliorates mouse tail lymphedema by 67 kDa laminin receptor (67LR)-dependent cell-cell adhesion.

Impaired microcirculation can cause lymphatic leakage which leads to a chronic swelling in the tissues of the body. However, no successful treatment gives any protection against lymphedema due to the lack of well-revealed pathophysiology of secondary lymphedema. Binary image of laminin immunohistochemical expression revealed that distribution of laminin expression localized during surgically induced lymphedema. 67 kDa laminin receptor (67LR) mRNA expression showed a peak at during lymphedema exacerbation. Since the response of 67LR molecules may affect the prevention of inflammation and edema, here we have hypothesized that 67LR ligand of YIGSR peptide could permit reconstructive environment for amelioration of lymphedema and evaluated the effect of YIGSR in a mouse tail model of lymphedema. Indeed, intra-abdominal injections of YIGSR for the first 3 days after inducing lymphedema in the mouse tail model reduced the tail lymphedema on day 14 by 27% (P = 0.035). Histology showed that YIGSR treatment protected lymphedema impairment in epidermis and dermis, and it also inhibited the expansion of intercellular spaces and enhanced especially cell adhesion in the basement membrane as revealed by transmission electron microscopy. Interestingly, the treatment also reduced the local expression of transforming growth factor (TGF)ß. Further elucidation of the mechanisms of 67LR-facilitated lymphangiogenesis contributes to find potential targets for the treatment of lymphedema.

Stimulating peristalsis improves esophagogastric junction observation during sedated esophagogastroduodenoscopy in children and adolescents.

Background and study aims: Sedation impairs full visualization of the esophagogastric junction (EGJ) and Z line (the squamocolumnar junction) during esophagogastroduodenoscopy (EGD). The aim of this study was to determine whether induction of esophageal peristalsis could improve the ability to evaluate the Z line in children and adolescents. Patients and methods: Study 1: Consecutive patients (10-15 years) undergoing EGD with propofol or midazolam sedation were enrolled. The proportion of Z line observed was compared between the two groups. Study 2: The effect of an air infusion near the EGJ following deflation of the stomach to induce esophageal peristalsis was investigated in the patients (15-18 years), undergoing EGD with propofol sedation. The proportion of Z line observed was compared between the stimulated group and control group. Results: Study 1: 149 patients were evaluated; 87 received propofol (43 boys; average age 13.2 years (range, 10-15)) and 62 received midazolam (30 boys; average age 12.8 years (range, 10-15)). The proportion of the Z line visualized was low but was greater with propofol vs. midazolam sedation (36.8% vs 16.1%, P=0.0059). Study 2: 102 patients were evaluated; 62 had induction of peristalsis (34 boys; average age 16.2 years (range, 15-18)) and 40 controls (20 boys; average age 16.8 years (range, 15-18)). Complete visualization of the Z line achieved in 95% (59 of 62) following induction of peristalsis vs. 37.5% (15 of 40) of controls (P>0.001). Conclusions: Induction of esophageal peristalsis greatly improved visualization of the Z line during sedated EGD in children and adolescents.

Recurrent atrial fibrillation after high-dose methylprednisolone therapy in a girl with lupus-associated hemophagocytic syndrome.

Hemophagocytic syndrome (HPS) is a serious complication of systemic lupus erythematosus (SLE). A 15-year-old female with lupus-nephritis developed HPS. Bone marrow study showed florid thrombophagocytosis. There was no associated infection. High-dose methylprednisolone therapy ameliorated HPS. However, atrial fibrillation (Af) repeated after the infusion and required direct-current cardioversion. No underlying diseases were found in the heart and endocrine system. Chest roentgenogram and echocardiography were normal. Electrocardiogram showed slightly prolonged PR interval in sinus rhythm. Af occurred at high circulating levels of interferon-γ and interleukin (IL)-10, but not IL-6, IL-2, tumor necrosis factor-α, C-reactive protein or catecholamines. This is the first observation that high-dose corticosteroid induced Af in a case of lupus-HPS. Af is unusual in SLE children without cardiac disease, while conduction defect occurs associated with lupus-myocarditis. Lupus-HPS may be an aggressive SLE subset with cardiac involvement. High-dose corticosteroid infusion controls lupus activity, but could disclose the cardiac stress in lupus-HPS patients.

Unique activation status of peripheral blood mononuclear cells at acute phase of Kawasaki disease.

Although Kawasaki disease (KD) is characterized by a marked activation of the immune system with elevations of serum proinflammatory cytokines and chemokines at acute phase, the major sources for these chemical mediators remain controversial. We analysed the activation status of peripheral blood mononuclear cells (PBMCs) by flow cytometry, DNA microarray and quantitative reverse transcription-polymerase chain reaction. The proportions of CD69+ cells in both natural killer cells and gammadeltaT cells at acute-phase KD were significantly higher than those at convalescent-phase KD. Microarray analysis revealed that five genes such as NAIP, IPAF, S100A9, FCGR1A and GCA up-regulated in acute-phase KD and the pathways involved in acute phase KD were related closely to the innate immune system. The relative expression levels of damage-associated molecular pattern molecule (DAMP) (S100A9 and S100A12) genes in PBMCs at acute-phase KD were significantly higher than those at convalescent-phase KD, while those of TNFA, IL1B and IL6 genes were not significantly different between KD patients and healthy controls. Intracellular production of tumour necrosis factor-alpha, interleukin-10 and interferon-gamma in PBMCs was not observed in KD patients. The present data have indicated that PBMCs showed a unique activation status with high expression of DAMP genes but low expression of proinflammatory cytokine genes, and that the innate immune system appears to play a role in the pathogenesis and pathophysiology of KD.

NEMO mutation as a cause of familial occurrence of Behçet's disease in female patients.

Behçet's disease is a chronic, relapsing, multisystem inflammatory disease of unknown etiology. Nuclear factor κB (NF-κB) essential modulator (NEMO) that is required for the activation of NF-κB plays an important role in inflammation. To investigate the role of NEMO in the pathogenesis of Behçet's disease, we analyzed NEMO gene and its expression pattern in tissues in a family with Behçet's disease. We found a heterozygous mutation (1217A> T, D406V) in a 6-year-old girl and her mother. Skewed X-chromosome inactivation was not observed in the peripheral blood mononuclear cells as well as in oral and intestinal mucosa of the patients. Accordingly, there was a significant proportion of peripheral blood monocytes that did not produce sufficient intracellular tumor necrosis factor-α with the stimulation of lipopolysaccharide. Heterozygous NEMO mutation is a cause of familial occurrence of Behçet's disease in female patients.

Gingipains from Porphyromonas gingivalis synergistically induce the production of proinflammatory cytokines through protease-activated receptors with Toll-like receptor and NOD1/2 ligands in human monocytic cells.

Gingipains (HRgpA, RgpB and Kgp) are cysteine proteinases and virulence factors of Porphyromonas gingivalis, the major causative bacterium of periodontal disease. To study synergistic effects of gingipains and signalling via Toll-like receptors (TLRs) and NOD1/2, we investigated effects of a gingipain on the secretion of proinflammatory cytokines from monocytic THP-1 cells in the presence of pathogen-associated molecular patterns (PAMPs). Gingipains stimulated interleukin (IL)-8's secretion from THP-1 cells, which was completely inhibited by proteinase inhibitors of gingipain and increased in the presence of PAMPs. Synergistic effects of gingipains and PAMPs were also seen in the secretion of IL-6 and MCP-1 and reduced to about 50% the secretion of IL-8 from THP-1 cells treated with siRNA targeting either protease-activated receptor (PAR)-1, -2 or -3. PAR agonist peptides mimicked the synergistic effects of gingipains with PAMPs. These results indicate that gingipains stimulate the secretion of cytokines from monocytic cells through the activation of PARs with synergistic effects by PAMPs. This is the first report of synergism of signalling via PARs, and TLRs or NOD1/2. The host defence system against P. gingivalis may be triggered through the activation of PARs by gingipains and augmented by PAMPs from this pathogen via TLRs or NOD1/2.

Synergism between TLRs and NOD1/2 in oral epithelial cells.

Oral epithelium is the first barrier against oral bacteria in periodontal tissue. Oral epithelial cells constitutively express Toll-like receptors (TLRs) and NOD1/2, functional receptors which induce the production of antibacterial factors such as peptidoglycan recognition proteins (PGRPs) and beta-defensin 2, but not pro-inflammatory cytokines such as interleukin (IL)-8. In this study, we hypothesized that innate immune responses in the oral epithelium are enhanced in inflamed tissue. We found that NOD1 and NOD2 agonists, in combination with TLR agonists, synergistically induced production of PGRPs and of beta-defensin 2 in human oral epithelial cells via NF-kappaB. In contrast, co-stimulation with NOD1/2 and TLR ligands had no effect on the production of pro-inflammatory cytokines (IL-6, IL-8, and monocyte chemoattractant protein-1). These findings indicate that, in innate immune responses to invading microbes, a combination of signaling through TLRs and NODs leads to the synergistic activation of antibacterial responses in the oral epithelium.

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation.

BACKGROUND AND OBJECTIVE: While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-kappaB (NF-kappaB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins. MATERIAL AND METHODS: Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-kappaB were measured using enzyme-linked immunosorbent assay. RESULTS: The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-kappaB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-kappaB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts. CONCLUSION: These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.

Intracellular changes of metal elements by fucoidan extracted from brown seaweed (Cladosiphon okamuranus).

This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.

Genomic loss and epigenetic silencing of very-low-density lipoprotein receptor involved in gastric carcinogenesis.

Homozygous loss in the genomic sequence, a mechanism for inactivating tumor-suppressor genes (TSGs) in cancer, has been used as a tag for the identification of novel TSGs, and array-based comparative genomic hybridization (array-CGH) has a great potential for high-throughput identification of this change. We identified a homozygous loss of the very-low-density lipoprotein receptor (VLDLR) gene (9p24.2) from genome-wide screening for copy-number alterations in 32 gastric cancer (GC) cell lines using array-CGH. Although previous reports demonstrated mRNA or protein expression of VLDLR in various cancers including GC, the association between genomic losses or epigenetic silencing of this gene and carcinogenesis has never been reported before. Homozygous deletion of VLDLR was also seen in primary GCs, albeit infrequently, and about half of GC cell lines showed lost or reduced VLDLR expression. The VLDLR expression was restored in gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. According to methylation analyses, hypermethylation of the VLDLR promoter region, which all of GC lines without its expression showed, occurred in some primary GCs. Restoration of VLDLR type I expression in GC cells reduced colony formation. These results suggest that not only the expression of VLDLR but also genetic or epigenetic silencing of this gene may contribute to tumor formation and be involved in gastric carcinogenesis.

Predominant activation and expansion of V gamma 9-bearing gamma delta T cells in vivo as well as in vitro in Salmonella infection.

Gamma delta T cell receptor-positive cells (gamma delta T cells) have recently been implicated to play a role in the protection against infectious pathogens. Serial studies on gamma delta T cells in 14 patients with salmonella infection have revealed that the proportions of gamma delta T cells (mean +/- SD: 17.9 +/- 13.2%) in salmonella infection were significantly increased (P less than 0.01) compared with 35 normal controls (5.0 +/- 2.6%) and 13 patients with other bacterial infections (4.0 +/- 1.4%). Expansion of gamma delta T cells was more prominent in the systemic form (28.9 +/- 10.8%) than in the gastroenteritis form (10.5 +/- 7.9%) of salmonella infection (P less than 0.01). Most in vivo-expanded gamma delta T cells expressed V gamma 9 gene product. Increased activated (HLA-DR+) T cells were observed in all the six patients with the systemic form and four of the seven with gastroenteritis form. Especially in the six with systemic form, gamma delta T cell activation was significantly higher than alpha beta T cell activation at the early stage of illness (P less than 0.01). When peripheral blood lymphocytes from normal individuals were cultured with live salmonella, gamma delta T cells were preferentially activated and expanded and most of them expressed V gamma 9. Purified gamma delta T cells also responded to live salmonella in vitro. The present study suggests that human gamma delta T cells play a role in the protection against salmonella infection in vivo.

Functional TLRs and NODs in human gingival fibroblasts.

Since human gingival fibroblasts are the major cells in periodontal tissues, we hypothesized that gingival fibroblasts are endowed with receptors for bacterial components, which induce innate immune responses against invading bacteria. We found clear mRNA expression of Toll-like receptors (TLR)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MD-2, MyD88, NOD1, and NOD2 in gingival fibroblasts. Gingival fibroblasts constitutively expressed these molecules. Upon stimulation with chemically synthesized ligands mimicking microbial products for these receptors, the production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, was markedly up-regulated. Furthermore, the production of pro-inflammatory cytokines induced by TLR and NOD ligands was significantly inhibited by an RNA interference assay targeted to NF-kappaB. These findings indicate that these innate immunity-related molecules in gingival fibroblasts are functional receptors involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis.

Toll-like receptors, NOD1, and NOD2 in oral epithelial cells.

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of beta-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

An elderly female who survived more than 30 years following a diagnosis of Takayasu's arteritis, complicated by fatal intestinal amyloidosis.

Compared to young patients with Takayasu's arteritis (TA), little information about elderly patients with TA has been reported. Additionally, no reports were found regarding TA cases with complications of intestinal amyloidosis. This is a case report of an elderly female, who developed intestinal amyloidosis, during late-stage TA. After years of outpatient management, she developed sudden severe dyspnea with pulmonary effusion, requiring hospitalization. After this event, betamethasone was replaced by methotrexate (MTX) for the next 34 months, but it seemed ineffective. After 1.5 years, she developed intractable diarrhea, followed by increases in BUN and serum creatinine (Cr), requiring several courses of hemodialysis. Colonoscopy revealed the presence of amyloid in her intestine, although she died of complicated sepsis caused by MRSA infection. This may be the first paper describing intestinal amyloidosis in a TA patient. Additionally, her case is rare in that she lived more than 30 years after the onset and diagnosis of TA.

Left ventricular chamber size predicts the race time of Japanese participants in a 100 km ultramarathon.

OBJECTIVE: As a subanalysis of an echocardiographic study performed on 291 Japanese participants in a 100 km ultramarathon, to estimate predictors of race time. METHODS: A total of 247 male participants in a 100 km ultramarathon (age 20-73 years) were examined by echocardiography. Correlations between age, body surface area, monthly running distance, or echocardiographic variables and the race time were examined. RESULTS: According to simple regression analysis, age (r = 0.299, p < 0.0001), monthly running distance (r = -0.388, p < 0.0001), left ventricular end diastolic diameter (r = -0.300, p < 0.0001), and left ventricular end systolic diameter (r = -0.325, p < 0.0001) correlated significantly with the race time. When multiple regression analysis was performed, age (f = 2.364), monthly running distance (f = -0.113), and left ventricular end systolic diameter (f = -2.361) remained significant predictors of the race time. CONCLUSION: Left ventricular diameter predicts the race time for a 100 km ultramarathon, in addition to age and amount of training.