Pathogen Profiles in Outpatients with Non-COVID-19 during the 7th Prevalent Period of COVID-19 in Gunma, Japan.

The identification of pathogens associated with respiratory symptoms other than the novel coronavirus disease 2019 (COVID-19) can be challenging. However, the diagnosis of pathogens is crucial for assessing the clinical outcome of patients. We comprehensively profiled pathogens causing non-COVID-19 respiratory symptoms during the 7th prevalent period in Gunma, Japan, using deep sequencing combined with a next-generation sequencer (NGS) and advanced bioinformatics technologies. The study included nasopharyngeal swabs from 40 patients who tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) using immuno-chromatography and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Comprehensive pathogen sequencing was conducted through deep sequencing using NGS. Additionally, short reads obtained from NGS were analyzed for comprehensive pathogen estimation using MePIC (Metagenomic Pathogen Identification Pipeline for Clinical Specimens) and/or VirusTap. The results revealed the presence of various pathogens, including respiratory viruses and bacteria, in the present subjects. Notably, human adenovirus (HAdV) was the most frequently detected virus in 16 of the 40 cases (40.0%), followed by coryneforms, which were the most frequently detected bacteria in 21 of the 40 cases (52.5%). Seasonal human coronaviruses (NL63 type, 229E type, HKU1 type, and OC43 type), human bocaviruses, and human herpesviruses (human herpesvirus types 1-7) were not detected. Moreover, multiple pathogens were detected in 50% of the subjects. These results suggest that various respiratory pathogens may be associated with non-COVID-19 patients during the 7th prevalent period in Gunma Prefecture, Japan. Consequently, for an accurate diagnosis of pathogens causing respiratory infections, detailed pathogen analyses may be necessary. Furthermore, it is possible that various pathogens, excluding SARS-CoV-2, may be linked to fever and/or respiratory infections even during the COVID-19 pandemic.

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase (RdRp) Region and VP1 Gene in Human Norovirus Genotypes GII.P6-GII.6 and GII.P7-GII.6.

To understand the evolution of GII.P6-GII.6 and GII.P7-GII.6 strains, the prevalent human norovirus genotypes, we analysed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than for the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

Comparison of angiogenic, cytoprotective, and immunosuppressive properties of human amnion- and chorion-derived mesenchymal stem cells.

Although mesenchymal stem cells (MSCs) can be obtained from the fetal membrane (FM), little information is available regarding biological differences in MSCs derived from different layers of the FM or their therapeutic potential. Isolated MSCs from both amnion and chorion layers of FM showed similar morphological appearance, multipotency, and cell-surface antigen expression. Conditioned media obtained from amnion- and chorion-derived MSCs inhibited cell death caused by serum starvation or hypoxia in endothelial cells and cardiomyocytes. Amnion and chorion MSCs secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Transplantation of human amnion or chorion MSCs significantly increased blood flow and capillary density in a murine hindlimb ischemia model. In addition, compared to human chorion MSCs, human amnion MSCs markedly reduced T-lymphocyte proliferation with the enhanced secretion of PGE2, and improved the pathological situation of a mouse model of acute graft-versus-host disease. Our results highlight that human amnion- and chorion-derived MSCs, which showed differences in their soluble factor secretion and angiogenic/immuno-suppressive function, could be ideal cell sources for regenerative medicine.

Systemic transplantation of allogenic fetal membrane-derived mesenchymal stem cells suppresses Th1 and Th17 T cell responses in experimental autoimmune myocarditis.

We have reported that systemic administration of autologous bone marrow or allogenic fetal membrane (FM)-derived mesenchymal stem cells (MSCs) similarly attenuated myocardial injury in rats with experimental autoimmune myocarditis (EAM). Since rat EAM is a T-helper (Th) cell-mediated autoimmune disease, and recent evidence has indicated that both autologous and allogenic MSCs exert an immunosuppressive effect on Th cell activity, we focused on Th cell differentiation in allogenic FM-MSC administered EAM rats. EAM was induced in Lewis rats by injecting porcine cardiac myosin (day 0). Allogenic FM-MSCs, obtained from major histocompatibility complex mismatched ACI rats, were intravenously injected (5 × 10(5)cells/rat) on days 7, 10, or 14 (MSCd7, MSCd10, or MSCd14 groups, respectively). At day 21, echocardiography confirmed that reduced ejection fraction in the untreated EAM group (63 ± 2%) was significantly improved in the MSCd10 and MSCd14 groups (74 ± 1 and 75 ± 2%, respectively, P<0.01). CD68 immunostaining revealed that prominent macrophage infiltration in the myocardium of the EAM group (1466 ± 93 cells/mm(2)) was significantly decreased in the MSCd10 group (958 ± 139 cells/mm(2), P<0.05). To evaluate Th cell differentiation, we used flow cytometry to determine the percentage of interferon (IFN)-γ positive Th1 and interleukin (IL)-17 positive Th17 cells in peripheral CD4-positive Th cells. The percentage of Th1 cells at day 16 was significantly lower in the MSCd10 (1.3 ± 0.2%) and MSCd14 (1.6 ± 0.3%) groups compared to the EAM group (2.4 ± 0.3%, P<0.05), as was the percentage of Th17 cells in the MSCd10 group (1.9 ± 0.5%) compared to the EAM group (2.2 ± 0.9%, P<0.05). At day 21, infiltrating Th17 cells in myocardium were significantly decreased in the MSCd10 group (501 ± 132 cells/mm(2), P<0.05) compared to EAM (921 ± 109 cells/mm(2)). In addition, human CD4+ Th cells co-cultured with human FM-MSCs exhibited reduced Th1 and Th17 cell-differentiation and proliferation, with increased expression of immunosuppressive molecules including indoleamine 2,3-dioxygenase 2 and IL-6 in co-cultured FM-MSCs. These results suggest that intravenous administration of allogenic FM-MSCs ameliorates EAM via the suppression of Th1/Th17 immunity.

Sustained-release adrenomedullin ointment accelerates wound healing of pressure ulcers.

Pressure ulcers are one of the most common complications in elderly, incontinent or paralyzed patients. For the healing of pressure ulcers, the development of granulation tissue and reepithelialization is required. Adrenomedullin (AM), an endogenous vasodilator peptide, is reported to stimulate the proliferation and migration of various cells including endothelial cells, fibroblasts and keratinocytes. Therefore, we hypothesized that AM might accelerate the healing process of pressure ulcers in which these cells were involved. We developed a sustained-release ointment containing human recombinant AM, and applied it in a mouse model of pressure ulcer twice a day for 14 days. Human AM was efficiently absorbed in wound area, but its blood concentration was negligible. AM ointment significantly reduced the wound area on day 5 to 7 after injury. In addition, AM ointment accelerated the formation of granulation tissue and angiogenesis as well as lymphangiogenesis after 7 days of treatment. Immunological analysis revealed that Ki-67-positive proliferating cells in granulation tissue expressed AM receptors. In summary, sustained-release AM significantly improved wound healing of pressure ulcers through acceleration of granulation and induction of angiogenesis and lymphangiogenesis. Therefore, sustained-release AM ointment may be a novel therapeutic agent for pressure ulcers.

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats.

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10(5) cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.

Therapeutic time window of cannabidiol treatment on delayed ischemic damage via high-mobility group box1-inhibiting mechanism.

Cannabidiol decreases cerebral infarction and high-mobility group box1 (HMGB1) in plasma in ischemic early phase. However, plasma HMGB1 levels in ischemic delayed phase reach higher concentration with the progressing brain injury. In this study, we investigated the therapeutic time window of cannabidiol on functional deficits, glial HMGB1 and plasma HMGB1 levels in a 4 h mouse middle cerebral artery (MCA) occlusion model. Cannabidiol-treated mice were divided into 3 groups as follows: group (a) treated from day 1, group (b) treated from day 3, group (c) treated from day 5 after MCA occlusion. Moreover, minocycline, microglia inhibitor, and fluorocitrate, an inhibitor of astroglial metabolism, were used to compare with cannabidiol-treated group. Repeated treatment with cannabidiol from 1 and 3 d at the latest after cerebral ischemia improved functional deficits and survival rates. However, cannabidiol from 5 d could not improve the ischemic damage as well as fluorocitrate-treated group. Moreover, both group (a), group (b) and minocycline but not group (c) and fluorocitrate-treated group had a decrease in the number of Iba1 expressing HMGB1 positive cells and HMGB1 levels in plasma. Cannabidiol may provide therapeutic possibilities for the progressing brain injury via HMGB1-inhibiting mechanism.

Adrenomedullin induces lymphangiogenesis and ameliorates secondary lymphoedema.

AIMS: Adrenomedullin (AM) is a multifunctional peptide hormone that plays a significant role in vasodilation and angiogenesis. Lymphoedema is a common but refractory disorder that is difficult to be treated with conventional therapy. We therefore investigated whether AM promotes lymphangiogenesis and improves lymphoedema. METHODS AND RESULTS: The effects of AM on lymphatic endothelial cells (LEC) were investigated. AM promoted proliferation, migration, and network formation of cultured human lymphatic microvascular endothelial cells (HLMVEC). AM increased intracellular cyclic adenosine monophosphate (cAMP) level in HLMVEC. The cell proliferation induced by AM was inhibited by a cAMP antagonist and mitogen-activated protein kinase kinase (MEK) inhibitors. Phosphorylated extracellular signal-regulated kinase (ERK) in HLMVEC was increased by AM. Continuous administration of AM (0.05 microg/kg/min) to BALB/c mice with tail lymphoedema resulted in a decrease in lymphoedema thickness. AM treatment increased the number of lymphatic vessels and blood vessels in the injury site. CONCLUSION: AM promoted LEC proliferation at least in part through the cAMP/MEK/ERK pathway, and infusion of AM induced lymphangiogenesis and improved lymphoedema in mice.

Allogeneic injection of fetal membrane-derived mesenchymal stem cells induces therapeutic angiogenesis in a rat model of hind limb ischemia.

Bone marrow-derived mesenchymal stem cells (BM-MSC) have been demonstrated to be an attractive therapeutic cell source for tissue regeneration and repair. However, it remains unknown whether or not allogeneic transplantation of mesenchymal stem cells (MSC) derived from fetal membranes (FM), which are generally discarded as medical waste after delivery, has therapeutic potential. FM-MSC were obtained from Lewis rats and had surface antigen expression and multipotent potential partly similar to those of BM-MSC. Compared with BM-MSC, FM-MSC secreted a comparable amount of hepatocyte growth factor despite a small amount of vascular endothelial growth factor. FM-MSC and BM-MSC both expressed major histocompatibility complex (MHC) class I but not MHC class II antigens and did not elicit allogeneic lymphocyte proliferation in mixed lymphocyte culture. FM-MSC or BM-MSC obtained from Lewis rats were injected into a MHC-mismatched August-Copenhagen-Irish rat model of hind limb ischemia. Three weeks after injection, blood perfusion and capillary density were significantly higher in the FM-MSC and BM-MSC groups than in the phosphate-buffered saline group, and allogeneic FM-MSC and BM-MSC were still observed. In nonischemic hind limb tissues, allogeneic FM-MSC and BM-MSC injection were associated with a comparatively small amount of T lymphocyte infiltration, compared with the injection of allogeneic splenic lymphocytes. In conclusion, allogeneic FM-MSC injection did not elicit a lymphocyte proliferative response and provided significant improvement in a rat model of hind limb ischemia, comparable to the response to BM-MSC. Thus, allogeneic injection of FM-MSC may be a new therapeutic strategy for the treatment of severe peripheral vascular disease. Disclosure of potential conflicts of interest is found at the end of this article.

Delta9-tetrahydrocannabinol (Delta9-THC) prevents cerebral infarction via hypothalamic-independent hypothermia.

Delta(9)-tetrahydrocannabinol (Delta(9)-THC), a primary psychoactive constituent of cannabis, has been reported to act as a neuroprotectant via the cannabinoid CB(1) receptor. In this study, Delta(9)-THC significantly decreased the infarct volume in a 4 h mouse middle cerebral artery occlusion mouse model. The neuroprotective effect of Delta(9)-THC was completely abolished by SR141716, cannabinoid CB(1) receptor antagonist, and by warming the animals to 31 degrees C. Delta(9)-THC significantly decreased the rectal temperature, and the hypothermic effect was also inhibited by SR141716 and by warming to 31 degrees C. At 24 h after cerebral ischemia, Delta(9)-THC significantly increased the expression level of CB(1) receptor in both the striatum and cortex, but not in the hypothalamus. Warming to 31 degrees C during 4 h cerebral ischemia did not increase the expression of CB(1) receptor at the striatum and cortex in MCA-occluded mice. These results show that the neuroprotective effect of Delta(9)-THC is mediated by a temperature-dependent mechanism via the CB(1) receptor. In addition, warming to 31 degrees C might attenuate both the neuroprotective and hypothermic effects of Delta(9)-THC through inhibiting the increase in CB(1) receptor in both the striatum and cortex but not in the hypothalamus, which may suggest a new thermoregulation mechanism of Delta(9)-THC.

High-cholesterol feeding aggravates cerebral infarction via decreasing the CB1 receptor.

We examined how feeding conditions affect the CB1 receptor and cerebral infarction caused by cerebral ischemia. Mice were divided into the following three groups: normal diet (ND), caloric restriction (CR) and high-cholesterol-enriched diet (HCD), and were kept for 6 weeks. After 6 weeks, we measured both serum and brain cholesterol and the expression level of cannabinoid CB1 receptor within the brain in intact mice. In addition, middle cerebral artery (MCA) was occluded for 2 h following reperfusion. Serum cholesterol significantly increased in the HCD group in comparison with both the ND and CR groups. However, brain cholesterol decreased in the HCD group. Then, the expression level of CB1 receptor significantly decreased in the HCD group, while that of the CR group clearly increased in comparison with the ND group in intact mice. In MCA-occluded mice, The HCD group produced the most severe cerebral infarction, while cerebral infarction was significantly decreased in the CR group. These results suggest that CR prevents infarction by increasing CB1 receptor expression, while high-cholesterol feeding aggravates cerebral infarction both by hypercholesterolemia in serum and by decreasing CB1 receptor expression modulated by hypocholesterolemia within the brain.

Rehabilitative training tasks improve spatial learning impairment in the water maze following hypoxic-ischemic insult in neonatal rats.

We recently reported that hypoxic-ischemic (HI) insult to the brain of 7-d-old rats resulted in a slowly progressive learning and memory disability, which started at around 5 wk after HI, a time frame that is representative of human adolescence. The purpose of the present study was to examine whether physical or mental exercises can prevent this late-onset, slowly progressing disability. Wistar rats were subjected to left carotid ligation followed by 2 h of hypoxic stress (8% O2 and 92% N(2) at 33 degrees C). Sham-control rats were subjected to the same procedure without ligation and hypoxic stress. Six weeks after the HI, the animals were divided into four groups: pretraining control, no training control, pretraining HI, and no training HI groups. We used the plus maze, eight-arm radial maze, and choice reaction time task as the rehabilitative training. Sixteen weeks after the HI, the water maze task was performed over 5 d to evaluate spatial learning ability; thereafter, cerebral morphology of the animals was examined. There were no differences in swimming length and latency between the pretraining control and no training control groups. Swimming length and latency in the pretraining HI group were significantly shorter and swifter than those in the no training HI group. The infarct areas on the left cerebral hemisphere were equivalent between pretraining HI and no training HI groups at each sectional slice. Rehabilitative training tasks prevented the neonatal HI-induced late-onset slowly progressive learning and memory disability.

Implantation of encapsulated glial cell line-derived neurotrophic factor-secreting cells prevents long-lasting learning impairment following neonatal hypoxic-ischemic brain insult in rats.

OBJECTIVE: Implantation of encapsulated glial cell line-derived neurotrophic factor-secreting cells into brain parenchyma reduces histological brain damage following hypoxic-ischemic stress in neonatal rats. We examined the effect of glial cell line-derived neurotrophic factors on long-term learning and memory impairment and morphological changes up to 18 weeks after hypoxic-ischemic stress in neonatal rats. STUDY DESIGN: Baby hamster kidney cells were transfected with expression vector either including (glial cell line-derived neurotrophic factor-hypoxic-ischemic group; n = 10) or not including (control-hypoxic-ischemic group; n = 8) human glial cell line-derived neurotrophic factor cDNA, encapsulated in semipermeable hollow fibers, and implanted into the left brain parenchyma of 7-day-old Wistar rats. Two days after implantation the rats received hypoxic-ischemic stress, and their behavior was then examined in several learning tasks: the 8-arm radial maze, choice reaction time, and water maze tasks, which examine short-term working memory, attention process, and long-term reference memory, respectively. The rats were killed 18 weeks after the hypoxic-ischemic insult for evaluation of brain damage. Two additional control groups were used: the control group (n = 15), which underwent no treatment, and the glial cell line-derived neurotrophic factor group (n = 6), which underwent implantation of the glial cell line-derived neurotrophic factor capsule but did not undergo hypoxic-ischemic stress. RESULTS: The decrease in the size of the cerebral hemisphere was significantly less in the glial cell line-derived neurotrophic factor-hypoxic-ischemic group, compared with the control-hypoxic-ischemic group, and improved performance was observed in all three tasks for the glial cell line-derived neurotrophic factor-hypoxic-ischemic group: for the control-hypoxic-ischemic group versus the glial cell line-derived neurotrophic factor-hypoxic-ischemic group, respectively, in the 8-arm radial maze test, average number of correct choices was 6.2 +/- 0.1 versus 6.9 +/- 0.1 ( P < .01); in the choice reaction time test, average reaction time for a correct response was 2.35 +/- 0.1 seconds versus 1.97 +/- 0.09 seconds ( P < .01); in the water maze test, average swimming length was 1120.0 +/- 95.2 cm versus 841.6 +/- 92.1 cm ( P < .01). All results for the glial cell line-derived neurotrophic factor group were similar to those for the control group. CONCLUSION: Glial cell line-derived neurotrophic factor treatment is effective in not only reducing brain damage but also inhibiting learning and memory impairment, following hypoxic-ischemic insult in neonatal rats. No adverse effects in learning and memory tests were observed in the glial cell line-derived neurotrophic factor group.