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Abstract To evaluate the antibody response following the initial four doses of mRNA vaccines (BNT162b2 or mRNA-1273) in SARS-CoV-2-naïve healthy adults and investigate factors influencing antibody titer increases, this prospective cohort study was conducted in Japan from March 2021. The study included participants who received either the 1st and 2nd doses (n = 467), 3rd dose (n = 157), or 4th dose (n = 89). Blood samples were collected before and up to 6 months after each dose, and anti-receptor-binding domain antibody levels were measured. Multivariate analysis (usin multiple linear regression or linear mixed models) revealed several factors significantly associated with higher post-vaccination antibody levels, including mRNA-1273 vaccine (after the 1st and 2nd dose), male gender (after the 3rd and 4th doses), younger age (after the 1st and 2nd dose), non-smoking status (after the 2nd dose), non-use of immunosuppressive agents (after the 1st dose), higher pre-vaccination antibody titers (after the 2nd, 3rd, and 4th doses), and higher post-vaccination fever (after the 2nd and 4th doses). Furthermore, longer intervals since the last dose were significantly associated with higher antibody levels after the 3rd and 4th doses. These findings provide valuable insights for optimizing vaccination strategies.

Abstract Background Adults infected with Plasmodium spp. in endemic areas need to be re-evaluated in light of global malaria elimination goals. They potentially undermine malaria interventions but remain an overlooked aspect of public health strategies. Methods This study aimed to estimate the prevalence of Plasmodium spp. infections, to identify underlying parasite species, and to assess predicting factors among adults residing in an endemic area from the Democratic Republic of Congo (DRC). A community-based cross-sectional survey in subjects aged 18 years and above was therefore carried out. Study participants were interviewed using a standard questionnaire and tested for Plasmodium spp. using a rapid diagnostic test and a nested polymerase chain reaction assay. Logistic regression models were fitted to assess the effect of potential predictive factors for infections with different Plasmodium spp. Results Overall, 420 adults with an estimated prevalence of Plasmodium spp. infections of 60.2% [95% CI 55.5; 64.8] were included. Non-falciparum species infected 26.2% [95% CI 22.2; 30.5] of the study population. Among infected participants, three parasite species were identified, including Plasmodium falciparum (88.5%), Plasmodium malariae (39.9%), and Plasmodium ovale (7.5%) but no Plasmodium vivax. Mixed species accounted for 42.3% of infections while single-species infections predominated with P. falciparum (56.5%) among infected participants. All infected participants were asymptomatic at the time of the survey. Adults belonging to the “most economically disadvantaged” households had increased risks of infections with any Plasmodium spp. (adjusted odds ratio, aOR = 2.87 [95% CI 1.66, 20.07]; p

IntroductionDuchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding gene that leads to muscle necrosis and degeneration with chronic inflammation during growth, resulting in progressive generalized weakness of the skeletal and cardiac muscles. We previously demonstrated the therapeutic effects of systemic administration of dental pulp mesenchymal stromal cells (DPSCs) in a DMD animal model. We showed preservation of long-term muscle function and slowing of disease progression. However, little is known regarding the effects of cell therapy on the metabolic abnormalities in DMD. Therefore, here, we aimed to investigate the mechanisms underlying the immunosuppressive effects of DPSCs and their influence on DMD metabolism.MethodsA comprehensive metabolomics-based approach was employed, and an ingenuity pathway analysis was performed to identify dystrophy-specific metabolomic impairments in the mdx mice to assess the therapeutic response to our established systemic DPSC-mediated cell therapy approach.Results and DiscussionWe identified DMD-specific impairments in metabolites and their responses to systemic DPSC treatment. Our results demonstrate the feasibility of the metabolomics-based approach and provide insights into the therapeutic effects of DPSCs in DMD. Our findings could help to identify molecular marker targets for therapeutic intervention and predict long-term therapeutic efficacy.

Abstract Background Chagas disease can lead to life-threatening cardiac manifestations. Regional factors, including genetic characteristics of circulating Trypanosoma cruzi (T. cruzi), have attracted attention as likely determinants of Chagas disease phenotypic expression and Chagas cardiomyopathy (CCM) progression. Our objective was to elucidate the differential transcriptomic signatures of cardiomyocytes resulting from infection with genetically discrete T. cruzi strains and explore their relationships with CCM pathogenesis and progression. Methods HL-1 rodent cardiomyocytes were infected with T. cruzi trypomastigotes of the Colombian, Y, or Tulahuen strain. RNA was serially isolated post-infection for microarray analysis. Enrichment analyses of differentially expressed genes (fold-change ≥ 2 or ≤ 0.5) highlighted over-represented biological pathways. Intracellular levels of reactive oxygen species (ROS) were compared between T. cruzi-infected and non-infected HL-1 cardiomyocytes. Results We found that oxidative stress-related gene ontology terms (GO terms), ‘Hypertrophy model’, ‘Apoptosis’, and ‘MAPK signaling’ pathways (all with P

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.

Abstract Background Duchenne muscular dystrophy (DMD) is an incurable genetic disease characterized by degeneration and necrosis of myofibers, chronic inflammation, and progressive muscle weakness resulting in premature mortality. Immunosuppressive multipotent mesenchymal stromal cell (MSC) therapy could be an option for DMD patients. We focused on amnion-derived mesenchymal stromal cells (AMSCs), a clinically viable cell source owing to their unique characteristics, such as non-invasive isolation, mitotic stability, ethical acceptability, and minimal risk of immune reaction and cancer. We aimed to identify novel immunomodulatory effects of AMSCs on macrophage polarization and their transplantation strategies for the functional recovery of skeletal and cardiac muscles. Methods We used flow cytometry to analyze the expression of anti-inflammatory M2 macrophage markers on peripheral blood mononuclear cells (PBMCs) co-cultured with human AMSCs (hAMSCs). hAMSCs were intravenously injected into DMD model mice (mdx mice) to assess the safety and efficacy of therapeutic interventions. hAMSC-treated and untreated mdx mice were monitored using blood tests, histological examinations, spontaneous wheel-running activities, grip strength, and echocardiography. Results hAMSCs induced M2 macrophage polarization in PBMCs via prostaglandin E2 production. After repeated systemic hAMSC injections, mdx mice exhibited a transient downregulation of serum creatin kinase. Limited mononuclear cell infiltration and a decreased number of centrally nucleated fibers were indicative of regenerated myofibers following degeneration, suggesting an improved histological appearance of the skeletal muscle of hAMSC-treated mdx mice. Upregulated M2 macrophages and altered cytokine/chemokine expressions were observed in the muscles of hAMSC-treated mdx mice. During long-term experiments, a significant decrease in the grip strength in control mdx mice significantly improved in the hAMSC-treated mdx mice. hAMSC-treated mdx mice maintained running activity and enhanced daily running distance. Notably, the treated mice could run longer distances per minute, indicating high running endurance. Left ventricular function in DMD mice improved in hAMSC-treated mdx mice. Conclusions Early systemic hAMSC administration in mdx mice ameliorated progressive phenotypes, including pathological inflammation and motor dysfunction, resulting in the long-term improvement of skeletal and cardiac muscle function. The therapeutic effects might be associated with the immunosuppressive properties of hAMSCs via M2 macrophage polarization. This treatment strategy could provide therapeutic benefits to DMD patients.

Abstract Background Since the late twentieth century, Chagas disease gained global attention to suppress the vector burden as a main control strategy in endemic countries. In Central America, multi-national initiative successfully achieved significant reduction in the estimated disease prevalence as well as elimination of the region’s principal vector species at the time in 2012. While the last decade has witnessed significant changes in ecosystem—such as urbanization and replacement of the main vector species—that can possibly affect the vector’s habitation and residual transmission, the up-to-date vector burden in the region has not been evaluated thoroughly due to the cessation of active vector surveillance. The aim of this study was to update the risk of vector-borne Trypanosoma cruzi infection in El Salvador, the top Chagas disease-endemic country in Central America. Methods A nationwide vector survey was conducted in the domestic environment of El Salvador from September 2018 to November 2020. The selection of the houses for inspection was based on expert purposeful sampling. Infection for T. cruzi was examined by microscopic observation of the insects’ feces, followed by a species confirmation using PCR. The data were analyzed using R software version 4.1.3. Proportion estimates with 95% confidence intervals were inferred using the Jeffrey’s method provided under the epiR package. Results A total of 1529 Triatoma dimidiata was captured from 107 houses (infestation rate, 34.4%; 107/311) in all the fourteen departments of the country visited within the period; prevalence of T. cruzi infection was as high as 10% (153/1529). In the country, domestic T. dimidiata infestation was distributed ubiquitously, while T. cruzi infection rates varied across the departments. Five out of fourteen departments showed higher infection rates than the average, suggesting sporadic high-risk areas in the country. Conclusions Our comprehensive study revealed substantial T. cruzi infection of T. dimidiata across the country, indicating potential active transmission of the disease. Therefore, strengthened surveillance for both vector and human infection is required to truly eliminate the risk of T. cruzi transmission in Central America.

Abstract We report on a 52‐year‐old Brazilian immigrant woman with past histories of chronic kidney disease and uveitis, presenting with symptomatic atrioventricular block. Her country of origin being endemic for Trypanosoma cruzi infection, we suspected Chagas disease as the aetiology, diagnosis of which was confirmed by serological tests. Further systemic workup identified an emerging nodular lesion in the lung, which turned out to be a sarcoid epithelioid granuloma on biopsy. Involvement of the kidneys and eyes was suggestive of systemic extension of the lung sarcoidosis. Although imaging modalities did not detect inflammatory foci in the myocardium, the rare coexistence of histologically proven sarcoidosis raised the intriguing concept of cardiac manifestation having arisen from two possibly overlapping aetiologies: Chagas disease and cardiac sarcoidosis. The case highlights a treatment dilemma increasingly likely to be encountered in this globalized world, and also raises the potential, but intriguing, association of these two diseases.

More people with a history of prior infection are receiving SARS-CoV-2 vaccines. Understanding the level of protection granted by ‘hybrid immunity’, the combined response of infection- and vaccine-induced immunity, may impact vaccination strategies through tailored dosing. A total of 36 infected (‘prior infection’) and 33 SARS-CoV-2 ‘naïve’ individuals participated. Participants provided sera six months after completing a round of BNT162b2 vaccination, to be processed for anti-spike antibody measurements and the receptor binding domain-ACE2 binding inhibition assays. The relationships between antibody titer, groups and age were explored. Anti-spike antibody titers at 6 months post-vaccination were significantly higher, reaching 13- to 17-fold, in the ‘prior infection’ group. Semi-log regression models showed that participants with ‘prior infection’ demonstrated higher antibody titer compared with the ‘naïve’ even after adjusting for age. The enhancement in antibody titer attributable to positive infection history increased from 8.9- to 9.4- fold at age 30 to 19- to 32-fold at age 60. Sera from the ‘prior infection’ group showed higher inhibition capacity against all six analyzed strains, including the Omicron variant. Prior COVID-19 led to establishing enhanced humoral immunity at 6 months after vaccination. Antibody fold-difference attributed to positive COVID-19 history increased with age, possibly because older individuals are prone to symptomatic infection accompanied by potentiated immune responses. While still pending any modifications of dosing recommendations (i.e. reduced doses for individuals with prior infection), our observation adds to the series of real-world data demonstrating the enhanced and more durable immune response evoked by booster vaccinations following prior infection.

This study assessed the immunogenicity and safety of the BNT162b2 mRNA vaccine in lung cancer patients receiving anticancer treatment. We enrolled lung cancer patients receiving anticancer treatment and non-cancer patients; all participants were fully vaccinated with the BNT162b2 vaccine. Blood samples were collected before the first and second vaccinations and 4 ± 1 weeks after the second vaccination. Anti-severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein S1 subunit receptor-binding domain antibody titers were measured using the Architect SARS-CoV-2 IgG II Quant and Elecsys Anti-SARS-CoV-2 S assays. Fifty-five lung cancer patients and 38 non-cancer patients were included in the immunogenicity analysis. Lung cancer patients showed significant increase in the geometric mean antibody concentration, which was significantly lower than that in the non-cancer patients after the first (30 vs. 121 AU/mL, p

ABSTRACT Past severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an important determinant of protection from reinfection and of postvaccine immune responses. Herein, we conducted a follow-up analysis of health care workers previously infected with coronavirus disease 2019 (COVID-19) with the aim of evaluating different immunoassays for their capability in detecting the waning anti-SARS-CoV-2 immune responses and accuracy in documenting past SARS-CoV-2 infections. We evaluated serum antinucleocapsid antibody levels in convalescent individuals following a 1.5-year interval from SARS-CoV-2 infection. Three different commercial immunoassays that qualitatively measure serum antibodies targeting the SARS-CoV-2 nucleocapsid protein, namely, the Abbott Architect SARS-CoV-2 IgG, the Euroimmun anti-SARS-CoV-2 NCP enzyme-linked immunosorbent assay (ELISA) IgG, and the Roche Elecsys anti-SARS-CoV-2, were tested for comparison of detectability. A total of 38 individuals consented to participating in this follow-up analysis. From assay to assay, seropositivity rate at 18 months from infection varied from lowest at 42% to highest at 92%. The Roche Elecsys immunoassay, dependent on the dual-antigen antibody detection method and tuned for the detection of high avidity antibodies, was most capable of accurately documenting past SARS-CoV-2 infections. Different immunoassays showed variable capability of determining previous infection status under waning antibody concentrations. Immunoassays with lower detection limits are to be selected, and adjusted thresholds are to be considered in order to maximize the tests’ performance. IMPORTANCE Past SARS-CoV-2 infection is an important determinant of protection from reinfection and of postvaccine immune responses. Our results show that different immunoassays, by design, harbor variable capability of tracking SARS-CoV-2 infection under waning antibody concentrations. With each recovered patient standing at a unique time point along the decline curve of antibodies, precise estimation of COVID-19 cumulative incidence remains a challenge. Since future surveillance studies will be targeting more than ever heterogenous cohorts, selecting the appropriate immunoassay is crucial in order to assure reliable decisions about an individual’s previous infection status.

Duchenne muscular dystrophy (DMD) is a severe congenital disease associated with mutation of the dystrophin gene. Supplementation of dystrophin using recombinant adeno-associated virus (rAAV) has promise as a treatment for DMD, although vector-related general toxicities, such as liver injury, neurotoxicity, and germline transmission, have been suggested in association with the systemic delivery of high doses of rAAV. Here, we treated normal or dystrophic dogs with rAAV9 transduction in conjunction with multipotent mesenchymal stromal cell (MSC) injection to investigate the therapeutic effects of an rAAV expressing microdystrophin (μDys) under conditions of immune modulation. Bone-marrow-derived MSCs, rAAV-CMV-μDys, and a rAAV-CAG-luciferase (Luc) were injected into the jugular vein of a young dystrophic dog to induce systemic expression of μDys. One week after the first injection, the dog received a second intravenous injection of MSCs, and on the following day, rAAV was intravenously injected into the same dog. Systemic injection of rAAV9 with MSCs pretreatment improves gene transfer into normal and dystrophic dogs. Dystrophic phenotypes significantly improved in the rAAV-μDys-injected dystrophic dog, suggesting that an improved rAAV-μDys treatment including immune modulation induces successful long-term transgene expression to improve dystrophic phenotypes.

Abstract Background Multipotent mesenchymal stromal cells (MSCs) are potentially therapeutic for muscle disease because they can accumulate at the sites of injury and act as immunosuppressants. MSCs are attractive candidates for cell-based strategies that target diseases with chronic inflammation, such as Duchenne muscular disease (DMD). We focused on the anti-inflammatory properties of IL-10 and hypothesized that IL-10 could increase the typically low survival of MSCs by exerting a paracrine effect after transplantation. Methods We developed a continuous IL-10 expression system of MSCs using an adeno-associated virus (AAV) vector. To investigate the potential benefits of IL-10 expressing AAV vector-transduced MSCs (IL-10-MSCs), we examined the cell survival rates in the skeletal muscles after intramuscular injection into mice and dogs. Systemic treatment with IL-10-MSCs derived from dental pulp (DPSCs) was comprehensively analyzed using the canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients. Results In vivo bioluminescence imaging analysis revealed higher retention of IL-10-MSCs injected into the hindlimb muscle of mice. In the muscles of dogs, myofiber-like tissue was formed after the stable engraftment of IL-10-MSCs. Repeated systemic administration of IL-10-DPSCs into the CXMDJ model resulted in long-term engraftment of cells and slightly increased the serum levels of IL-10. IL-10-hDPSCs showed significantly reduced expression of pro-inflammatory MCP-1 and upregulation of stromal-derived factor-1 (SDF-1). MRI and histopathology of the hDPSC-treated CXMDJ indicated the regulation of inflammation in the muscles, but not myogenic differentiation from treated cells. hDPSC-treated CXMDJ showed improved running capability and recovery in tetanic force with concomitant increase in physical activity. Serum creatine kinase levels, which increased immediately after exercise, were suppressed in IL-10-hDPSC-treated CXMDJ. Conclusions In case of local injection, IL-10-MSCs could maintain the long-term engraftment status and facilitate associated tissue repair. In case of repeated systemic administration, IL-10-MSCs facilitated the long-term retention of the cells in the skeletal muscle and also protected muscles from physical damage-induced injury, which improved muscle dysfunction in DMD. We can conclude that the local and systemic administration of IL-10-producing MSCs offers potential benefits for DMD therapy through the beneficial paracrine effects of IL-10 involving SDF-1.

Abstract Background Duchenne muscular dystrophy (DMD) is an inherited progressive disorder that causes skeletal and cardiac muscle deterioration with chronic inflammation. Dental pulp stem cells (DPSCs) are attractive candidates for cell-based strategies for DMD because of their immunosuppressive properties. Therefore, we hypothesized that systemic treatment with DPSCs might show therapeutic benefits as an anti-inflammatory therapy. Methods To investigate the potential benefits of DPSC transplantation for DMD, we examined disease progression in a DMD animal model, mdx mice, by comparing them with different systemic treatment conditions. The DPSC-treated model, a canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients, also underwent comprehensive analysis, including histopathological findings, muscle function, and locomotor activity. Results We demonstrated a therapeutic strategy for long-term functional recovery in DMD using repeated DPSC administration. DPSC-treated mdx mice and CXMDJ showed no serious adverse events. MRI findings and muscle histology suggested that DPSC treatment downregulated severe inflammation in DMD muscles and demonstrated a milder phenotype after DPSC treatment. DPSC-treated models showed increased recovery in grip-hand strength and improved tetanic force and home cage activity. Interestingly, maintenance of long-term running capability and stabilized cardiac function was also observed in 1-year-old DPSC-treated CXMDJ. Conclusions We developed a novel strategy for the safe and effective transplantation of DPSCs for DMD recovery, which included repeated systemic injection to regulate inflammation at a young age. This is the first report on the efficacy of a systemic DPSC treatment, from which we can propose that DPSCs may play an important role in delaying the DMD disease phenotype.

Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies.

ABSTRACT The prompt rollout of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine is facilitating population immunity, which is becoming more dominant than natural infection-mediated immunity. In the midst of coronavirus disease 2019 (COVID-19) vaccine deployment, understanding the epitope profiles of vaccine-elicited antibodies will be the first step in assessing the functionality of vaccine-induced immunity. In this study, the high-resolution linear epitope profiles of Pfizer-BioNTech COVID-19 mRNA vaccine recipients and COVID-19 patients were delineated by using microarrays mapped with overlapping peptides of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The vaccine-induced antibodies targeting the RBD had a broader distribution across the RBD than that induced by the natural infection. Half-maximal neutralization titers were measured in vitro by live virus neutralization assays. As a result, relatively lower neutralizability was observed in vaccine recipient sera, when normalized to a total anti-RBD IgG titer. However, mutation panel assays targeting the SARS-CoV-2 variants of concern have shown that the vaccine-induced epitope variety, rich in breadth, may grant resistance against future viral evolutionary escapes, serving as an advantage of vaccine-induced immunity. IMPORTANCE Establishing vaccine-based population immunity has been the key factor in attaining herd protection. Thanks to expedited worldwide research efforts, the potency of mRNA vaccines against the coronavirus disease 2019 (COVID-19) is now incontestable. The next debate is regarding the coverage of SARS-CoV-2 variants. In the midst of vaccine deployment, it is of importance to describe the similarities and differences between the immune responses of COVID-19 vaccine recipients and naturally infected individuals. In this study, we demonstrated that the antibody profiles of vaccine recipients are richer in variety, targeting a key protein of the invading virus, than those of naturally infected individuals. Vaccine-elicited antibodies included more nonneutralizing antibodies than infection-elicited antibodies, and their breadth in antibody variations suggested possible resilience against future SARS-CoV-2 variants. The antibody profile achieved by vaccinations in naive individuals provides important insight into the first step toward vaccine-based population immunity.

Carbohydrate sulfotransferase 14 (CHST14) encodes dermatan 4-O-sulfotransferase 1, a critical enzyme for dermatan sulfate (DS) biosynthesis. Musculocontractural Ehlers-Danlos syndrome (mcEDS) is associated with biallelic pathogenic variants of CHST14 and is characterized by malformations and manifestations related to progressive connective tissue fragility. We identified myopathy phenotypes in Chst14-deficient mice using an mcEDS model. Decorin is a proteoglycan harboring a single glycosaminoglycan chain containing mainly DS, which are replaced with chondroitin sulfate (CS) in mcEDS patients with CHST14 deficiency. We studied the function of decorin in the skeletal muscle of Chst14-deficient mice because decorin is important for collagen-fibril assembly and has a myokine role in promoting muscle growth. Although decorin was present in the muscle perimysium of wild-type (Chst14+/+) mice, decorin was distributed in the muscle perimysium as well as in the endomysium of Chst14–/– mice. Chst14–/– mice had small muscle fibers within the spread interstitium; however, histopathological findings indicated milder myopathy in Chst14–/– mice. Myostatin, a negative regulator of protein synthesis in the muscle, was upregulated in Chst14–/– mice. In the muscle of Chst14–/– mice, decorin was downregulated compared to that in Chst14+/+ mice. Chst14–/– mice showed altered cytokine/chemokine balance and increased fibrosis, suggesting low myogenic activity in DS-deficient muscle. Therefore, DS deficiency in mcEDS causes pathological localization and functional abnormalities of decorin, which causes disturbances in skeletal muscle myogenesis.

ABSTRACT We describe the results of testing health care workers, from a tertiary care hospital in Japan that had experienced a coronavirus disease 2019 (COVID-19) outbreak during the first peak of the pandemic, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody seroconversion. Using two chemiluminescent immunoassays and a confirmatory surrogate virus neutralization test, serological testing revealed that a surprising 42% of overlooked COVID-19 diagnoses (27/64 cases) occurred when case detection relied solely on SARS-CoV-2 nucleic acid amplification testing (NAAT). Our results suggest that the NAAT-positive population is only the tip of the iceberg and the portion left undetected might potentially have led to silent transmissions and triggered the spread. A questionnaire-based risk assessment was further indicative of exposures to specific aerosol-generating procedures (i.e., noninvasive ventilation and airway suctioning) having mediated transmission and served as the origins of the outbreak. Our observations are supportive of a multitiered testing approach, including the use of serological diagnostics, in order to accomplish exhaustive case detection along the whole COVID-19 spectrum. IMPORTANCE We describe the results of testing frontline health care workers, from a hospital in Japan that had experienced a COVID-19 outbreak, for SARS-CoV-2-specific antibodies. Antibody testing revealed that a surprising 42% of overlooked COVID-19 diagnoses occurred when case detection relied solely on PCR-based viral detection. COVID-19 clusters have been continuously striking the health care system around the globe. Our findings illustrate that such clusters are lined with hidden infections eluding detection with diagnostic PCR and that the cluster burden in total is more immense than actually recognized. The mainstays of diagnosing infectious diseases, including COVID-19, generally consist of two approaches, one aiming to detect molecular fragments of the invading pathogen and the other to measure immune responses of the host. Considering antibody testing as one trustworthy option to test our way through the pandemic can aid in the exhaustive case detection of COVID-19 patients with variable presentations.

Regenerative medicine aims to restore human functions by regenerating organs and tissues using stem cells or living tissues for the treatment of organ and tissue defects or dysfunction. Clinical trials investigating the treatment of cerebral infarction using mesenchymal stem cells, a type of somatic stem cell therapy, are underway. The development and production of regenerative medicines using somatic stem cells is expected to contribute to the treatment of cerebral infarction, a central nervous system disease for which there is no effective treatment. Numerous experimental studies have shown that cellular therapy, including the use of human dental pulp stem cells, is an attractive strategy for patients with ischemic brain injury. This review describes the basic research, therapeutic mechanism, clinical trials, and future prospects for dental pulp stem cell therapy, which is being investigated in Japan in first-in-human clinical trials for the treatment of patients with acute cerebral ischemia.

Hepatocyte growth factor (HGF) has neuroprotective effects against ischemia-induced injuries. Dental pulp stem cell (DPSC) transplantation attenuates tissue injury in the brain of rats with post-transient middle cerebral artery occlusion. We sought to determine whether DPSCs that overexpress HGF can enhance their therapeutic effects on brain damage post-ischemia/reperfusion injury. Treatment with DPSCs overexpressing HGF reduced infarct volumes compared to unmodified DPSC treatment at 3 and 7 days post-transient middle cerebral artery occlusion. The use of unmodified DPSCs and DPSCs overexpressing HGF was associated with improved motor function compared to that with administration of vehicle at 7 days post-transient middle cerebral artery occlusion. DPSCs overexpressing HGF significantly inhibited microglial activation and pro-inflammatory cytokine production along with suppression of neuronal degeneration. Post-reperfusion, DPSCs overexpressing HGF attenuated the decreases in tight junction proteins, maintained blood-brain barrier integrity, and increased microvessel density in peri-infarct areas. The administration of DPSCs overexpressing HGF during the acute phase of stroke increased their neuroprotective effects by modulating inflammation and blood-brain barrier permeability, thereby promoting improvements in post-ischemia/reperfusion brain injury. Keywords: focal cerebral ischemia, hepatocyte growth factor, dental pulp stem cells, gene transfer, ex vivo therapy, intravenous transplantation, neuroprotection

Hypophosphatasia is an inherited disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP), the major symptom of which is hypomineralization of the bones and teeth. We had recently demonstrated that TNALP-deficient (Akp2−/−) mice, which mimic the phenotype of the severe infantile form of hypophosphatasia, can be treated by intramuscular injection of a self-complementary (sc) type 8 recombinant adeno-associated virus (rAAV8) vector expressing bone-targeted TNALP with deca-aspartates at the C terminus (TNALP-D10) via the muscle creatine kinase (MCK) promoter. In this study, we focused on the efficacy of this scAAV8-MCK-TNALP-D10 treatment on the mandibular bone and teeth in neonatal Akp2−/− mice. Upon scAAV8-MCK-TNALP-D10 injection, an improvement of mandibular growth was observed by X-ray analysis. Micro-computed tomography analysis revealed progressive mineralization of the molar root in the treated Akp2−/− mice, and morphometric parameters of the alveolar bone were improved. These results suggest that the mandibular bones and teeth of hypophosphatasia were effectively treated by muscle directed rAAV-mediated TNALP-D10 transduction. Our strategy would be promising for future hypophosphatasia gene therapy because it induces dentoalveolar mineralization and reduces the risk of tooth exfoliation. Keywords: hypophosphatasia, AAV vector, alkaline phosphatase, gene therapy, mandibular

Interleukin (IL)-10 is a contributing factor to neuroprotection of mesenchymal stem cell (MSC) transplantation after ischemic stroke. Our aim was to increase therapeutic effects by combining MSCs and ex vivo IL-10 gene transfer with an adeno-associated virus (AAV) vector using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague-Dawley rats underwent 90 min MCAO followed by intravenous administration of MSCs alone or IL-10 gene-transferred MSCs (MSC/IL-10) at 0 or 3 hr after ischemia reperfusion. Infarct lesions, neurological deficits, and immunological analyses were performed within 7 days after MCAO. 0-hr transplantation of MSCs alone and MSC/IL-10 significantly reduced infarct volumes and improved motor function. Conversely, 3-hr transplantation of MSC/IL-10, but not MSCs alone, significantly reduced infarct volumes (p < 0.01) and improved motor function (p < 0.01) compared with vehicle groups at 72 hr and 7 days after MCAO. Immunological analysis showed that MSC/IL-10 transplantation significantly inhibits microglial activation and pro-inflammatory cytokine expression compared with MSCs alone. Moreover, overexpressing IL-10 suppressed neuronal degeneration and improved survival of engrafted MSCs in the ischemic hemisphere. These results suggest that overexpressing IL-10 enhances the neuroprotective effects of MSC transplantation by anti-inflammatory modulation and thereby supports neuronal survival during the acute ischemic phase.

Duchenne muscular dystrophy (DMD) is an X-linked muscle disorder characterized by primary muscle degeneration. Patients with DMD reveal progressive muscle weakness leading to ambulatory dysfunction. Novel outcome measures are needed for more sensitive evaluation of therapeutic effects in clinical trials. Multiple parameters of acceleration and angular velocity are used as efficient indicators to quantify the motion of subjects, and these parameters have been recently applied for evaluation of motor function in DMD. In the present study, we evaluated gait in a dystrophic dog model, CXMDJ, by measuring three-axial acceleration and angular velocity over the course of months. Hybrid sensors were placed on the dorsal thoracic and lumbar regions of dogs to detect a wide range of acceleration (±8 G) and angular velocity (±1000 degrees per second). Multiple parameters showed lower values in dystrophic dogs compared to wild-type (WT) dogs, and declined over the course of months. Acceleration magnitude (AM) at the thoracic region in dystrophic dogs was prominently lower compared with WT dogs, even at the age of 2 months, the onset of muscle weakness, whereas AM at the lumbar region drastically declined throughout the disease course. The angular velocity index in the vertical direction in the lumbar region increased in dystrophic dogs, suggesting waddling at the girdle. These parameters also accordingly decreased with exacerbation of clinical manifestations and a decrease in spontaneous locomotor activity. The AM of dystrophic dogs was analyzed with magnetic resonance imaging to look for a correlation with crus muscle involvement. Results showed that acceleration and angular velocity are multifaceted kinematic indices that can be applied to assess outcomes in clinical trials for hereditary neuromuscular disorders including DMD.

Hypophosphatasia (HPP) is an inherited disease caused by genetic mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). This results in defects in bone and tooth mineralization. We recently demonstrated that TNALP-deficient (Akp2−/−) mice, which mimic the phenotype of the severe infantile form of HPP, can be treated by intravenous injection of a recombinant adeno-associated virus (rAAV) expressing bone-targeted TNALP with deca-aspartates at the C-terminus (TNALP-D10) driven by the tissue-nonspecific CAG promoter. To develop a safer and more clinically applicable transduction strategy for HPP gene therapy, we constructed a self-complementary type 8 AAV (scAAV8) vector that expresses TNALP-D10 via the muscle creatine kinase (MCK) promoter (scAAV8-MCK-TNALP-D10) and examined the efficacy of muscle-directed gene therapy. When scAAV8-MCK-TNALP-D10 was injected into the bilateral quadriceps of neonatal Akp2−/− mice, the treated mice grew well and survived for more than 3 months, with a healthy appearance and normal locomotion. Improved bone architecture, but limited elongation of the long bone, was demonstrated on X-ray images. Micro-CT analysis showed hypomineralization and abnormal architecture of the trabecular bone in the epiphysis. These results suggest that rAAV-mediated, muscle-specific expression of TNALP-D10 represents a safe and practical option to treat the severe infantile form of HPP.

Profiles of recombinant adeno-associated virus (rAAV)-mediated transduction show interspecies differences for each AAV serotype. Robust long-term transgene expression is generally observed in rodents, whereas insufficient transduction is seen in animals with more advanced immune systems. Non-human primates, including the common marmoset, could provide appropriate models for neuromuscular diseases because of their higher brain functions and physiological resemblance to humans. Strategies to induce pathologies in the neuromuscular tissues of non-human primates by rAAV-mediated transduction are promising; however, transgene expression patterns with rAAV transduction have not been elucidated in marmosets. In this study, transduction of adult marmoset skeletal muscle with rAAV9 led to robust and persistent enhanced green fluorescent protein (EGFP) expression that was independent of the muscle fiber type, although lymphocyte infiltration was recognized. Systemic rAAV injection into pregnant marmosets led to transplacental fetal transduction. Surprisingly, the intraperitoneal injection of rAAV1 and rAAV9 into the neonatal marmoset resulted in systemic transduction and persistent transgene expression without lymphocyte infiltration. Skeletal and cardiac muscle were effectively transduced with rAAV1 and rAAV9, respectively. Interestingly, rAAV9 transduction led to intense EGFP signaling in the axons of the corpus callosum. These transduction protocols with rAAV will be useful for investigating gene functions in the neuromuscular tissues and developing gene therapy strategies.