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Abstract Arthropathy is a common complication in haemophilia and decreases quality of life. It has been known that concentrations of β-hydroxybutyrate (BHB) in blood are increased by a ketogenic diet, and elevated levels of circulating BHB restricts the progression of inflammation-mediated joint pathological changes. We hypothesized that elevation of blood BHB concentrations could be effective for reducing the progression of bleeding-induced arthropathy by moderating the inflammatory responses of macrophages. In this study, we investigated whether BHB alleviates the arthropathy caused by repeated intra-articular blood injection in rats. To increase blood BHB levels, rats were fed with ketogenic diet. Repeated intra-articular blood injection induced significant joint swelling, whereas ketogenic diet intake significantly increased blood BHB concentrations and ameliorated the joint swelling. The periarticular tissue-fibrosis observed in the control diet intake group appeared to be significantly alleviated in the ketogenic diet intake group. In addition, the IL-1β, which is involved in the progression of arthropathy, levels in the supernatants of blood-exposed macrophages derived from THP-1 cell line were significantly suppressed by BHB supplementation. In summary, BHB moderated the pathological joint changes caused by intra-articular blood exposure.

Introduction: CD34-positive endothelial progenitor cells (EPCs) promote angiogenesis and are a promising tool for regenerative cell therapy of ischemic diseases. However, the number and quality of CD34-positive cells decrease owing to various external and internal factors; thus, an efficient method is needed to establish CD34-positive EPCs. The generation of functional cells by reprogramming, that is, manipulating cell fate via gene transfer and/or treatment with chemical compounds, has recently been reported. Therefore, we aimed to generate CD34-positive cells by the reprogramming of endothelial cells (ECs). Methods: Based on previous reports, seven candidate chemical compounds were selected to reprogram human umbilical vein ECs (HUVECs) to CD34-positive cells. Following stimulation with the chemical compounds, the expression of CD34 was evaluated using quantitative PCR, flow cytometry, and immunocytochemistry. Results: HUVECs treated with the compounds exhibited increased CD34 expression. We cultured cells in alternate media lacking one of the seven compounds and found no CD34 expression in cells treated with PD0325901-free media, suggesting that PD0325901—a MEK inhibitor—mainly contributed to the increase in CD34 expression. We found that 98% of cells were CD34-positive after PD0325901 treatment alone for 7 d. Western blotting revealed that the phosphorylation of ERK was suppressed in PD0325901-treated cells. No upregulation of CD34 was observed in fibroblast cell lines, even after PD0325901 treatment. These results suggested that PD0325901 induces CD34-positive cells by inhibiting ERK phosphorylation in ECs. Conclusions: CD34 expression was strongly induced in ECs by treatment with the MEK inhibitor PD0325901 in vitro. Our study provides a useful reference for the establishment of CD34-positive EPCs and will contribute to the development of regenerative therapies, especially for ischemic diseases.

Background: Japanese people sleep less compared to other countries around the world. Using a large nationally representative survey in 2019 and 2022, we investigated whether sleep duration and nonrestorative sleep (NRS) among Japanese people have improved or worsened due to the COVID-19 pandemic. Methods: Data were drawn from the Comprehensive Survey of Living Conditions, a nationwide cross-sectional sample based on self-administered questionnaires. We analyzed 426,510 people in 2019 and 375,578 people in 2022 aged ≥20 living in the community. The generalized estimating equations of the multivariable Poisson regression models were used to estimate adjusted prevalence of NRS by survey year. Potential confounders included gender, age, marital status, family size, housing tenure, equivalent household expenditures, education, employment status, illness under treatment, lifestyle behaviors (i.e., smoking, drinking, dietary, and fitness habits), mental health, and sleep duration. Results: Among the study participants, 35.7% slept less than 6 hours and 20.9% had NRS. Regarding sleep duration, the prevalence of sleep duration of less than 6 hours was significantly lower in 2022 than in 2019 for both men and women. By gender and age, the prevalence of short sleep duration (

Introduction: Liver sinusoidal endothelial cells (LSECs) are specialized vascular endothelial cells that play an important role in the maintenance of biological homeostasis. However, the lack of versatile human LSECs has hindered research on LSECs and development of medical technologies for liver diseases including hemophilia A. In this study, we developed a technique to induce LSEC differentiation from human bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: To induce LSECs from human BM-MSCs, cytokines and chemical compounds associated with signaling implicated in LSEC differentiation and liver development were screened. Then LSEC-related genes and proteins expression in the differentiated cells were analyzed by qPCR and flow cytometry analysis, respectively. LSEC-related functions of the differentiated cells were also examined. Results: We found that the gene expression of LSEC markers, such as LYVE1, was considerably increased by culturing human BM-MSCs with bone morphogenetic protein 4, fibroblast growth factor 8b, transforming growth factor-β signal inhibitor, and cyclic AMP. Furthermore, the differentiated cells expressed LSEC marker proteins and clearly demonstrated LSEC-specific functions, such as the uptake of hyaluronic acid. Conclusions: Our result indicate that the functional LSEC-like cells were successfully generated from human BM-MSCs using our established protocol.

BackgroundSmall-member households are increasing worldwide. However, most previous studies have focused on older people and living alone. Using the latest national survey data, we investigated a dose–response relationship between household size and serious psychological distress (SPD).MethodsWe analyzed data from the 2019 Comprehensive Survey of Living Conditions in Japan. The study participants were 405,560 community-dwelling adults aged 20 or older. Household size was classified into 5 or more, 3 or 4, two, and one (i.e., living alone). SPD was defined as ≥13 points based on the Kessler 6-item Psychological Distress Scale. We used multivariable logistic regressions and included age, education, equivalent household expenditures, housing tenure, employment contract, smoking, and illness under treatment as covariates.ResultsAfter stratified analyses by age and gender, a dose–response relationship between smaller household size and more common SPD was significant for younger, but not for older people (p-trend was

Abstract Background In patients with hemophilia (PwH), bleeding often occurs in joints and muscles, and early detection of hemorrhage is important to prevent the onset and progression of mobility impairment. Complex-Image analysis such as ultrasonography, computed tomography, and magnetic resonance imaging are used to detect bleeding. On the other hand, no simple and rapid method to detect the active bleeding has been reported. Local inflammatory responses occur when blood leaks from damaged vessels, and the temperature at the site of active bleeding could be expected to increase in these circumstances, leading to an increase in surrounding skin temperature. Therefore, the purpose of this study was to investigate whether the measurement of skin temperature using infrared thermography (IRT) can be used as a diagnostic aid to detect active bleeding. Methods Fifteen PwH (from 6 to 82 years old) complaining of discomfort such as pain were examined. Thermal images were obtained simultaneously at the affected sides and comparable unaffected sides. The average skin temperature of the affected side and of the unaffected side were measured. The temperature differences were calculated by subtracting the average skin temperature at the unaffected side from the affected side. Results In eleven cases with active bleeding, the skin temperature at the affected side was more than 0.3 °C higher (0.3 °C to 1.4 °C) compared to the unaffected side. In two cases without active bleeding, there were no significant differences in skin temperature between the affected and unaffected sides. In two cases with previous rib or thumb bone fracture, the skin temperature at the affected side was 0.3 °C or 0.4 °C lower than that of the unaffected side, respectively. In two cases with active bleeding in which longitudinal evaluation was conducted, the difference in skin temperature decreased after hemostatic treatment. Conclusion The analysis of skin temperature deference using IRT was a useful supportive tool to readily assess musculoskeletal abnormalities and bleeding in PwH as well as to determine the success of the hemostatic treatment.

Introduction Haemophilic arthropathy, a serious complication of haemophilia, results from recurrent joint bleeding, causing progressive joint damage and severely impacting patient quality of life. Rehabilitation therapy (RT) effectively addresses declining physical function due to joint degradation, but pain during RT can hinder its success. Therefore, an effective pain-alleviating treatment method is required. The single-joint hybrid assistive limb (HAL-SJ), a powered exoskeleton, measures bioelectric potential during muscle contraction and provides motorised support, potentially alleviating pain.Objective This study outlines our protocol for a randomised, prospective, single-blind (evaluator) trial aimed to investigate the effects of HAL-SJ on pain reduction during RT, kinesiophobia and other physical functions in patients with haemophilia.Methods and analysis This two-group comparison intervention study will include 24 male patients aged 12–85 years diagnosed with a bleeding disorder necessitating RT for pain and physical function improvement. The primary outcome measures pain changes during the first and second RT session in patients receiving HAL-SJ-assisted RT compared with traditional RT without HAL-SJ. The secondary outcomes include kinesiophobia (Japanese version of the Tampa Scale for Kinesiophobia), standing position gait (zebris FDM-T treadmill), range of motion (manual goniometer) and body surface temperature (infrared thermography camera) during the study period of up to 3 months or until the end of 10 RTs. RT intensity remains below that required to move the affected joint against gravity, given HAL-SJ’s muscular support. The follow-up period extends to 1 month after the last RT. Intergroup study variables are compared by an unpaired t-test or Mann-Whitney test. Intragroup comparisons of secondary outcomes are analysed by a paired t-test or Wilcoxon signed-rank test.Ethics and dissemination This study was approved by the accreditation committee of Nara Medical University Hospital. The study results will disseminate through publication in a peer-reviewed journal.Trial registration number jRCTs052220076.

Background: There is limited evidence of a protective effect of Internet use for incident disability (ID) during the COVID-19 pandemic. We investigated the association between frequency of Internet use (FIU) and ID among community-dwelling older people. Methods: We used longitudinal data from the 2019 and 2022 surveys, including 7,913 residents aged ≥65 without disability at baseline. ID was defined as a new public long-term care insurance certification. FIU at baseline was categorized into daily, weekly, monthly, yearly, and non-users. Changes in FIU before and during the COVID-19 pandemic were categorized into continuing frequent (i.e., daily or weekly), continuing moderate (i.e., monthly or yearly), increase in frequency, from non-users to users, decrease in frequency, from users to non-users, and continuing non-users. Covariates included age, gender, education, perceived economic situation, family structure, body mass index, chronic medical conditions, dietary variety, working status, walking time, and cognitive functioning. Multivariable Poisson regression models were used to estimate adjusted cumulative incidence ratio (aCIR) and 95% confidence interval (CI) for ID. Results: During the 3-year follow-up, 132 of 4,453 people aged 65–74, 595 of 3,460 people aged ≥75, 287 of 3,660 men, and 440 of 4,253 women developed ID. For FIU at baseline, among people aged ≥75 or men, there was a dose-response relationship between more frequent Internet use at baseline and a lower risk of ID (P-trend was 0.005 in people aged ≥75, and

Introduction Emicizumab prophylaxis substantially reduces bleeding episodes in patients with haemophilia A (HA). The haemostatic efficacy of emicizumab in patients with HA is estimated as approximately 15% based on mimic activity of factor (F) VIII. Although it has been proven effective in preventing bleeding, its haemostatic effect during breakthrough bleeding or surgery is considered insufficient. Therefore, haemostatic management of emicizumab-treated patients with HA without inhibitors frequently requires FVIII replacement therapy. In haemostatic management of emicizumab-treated patients with HA, conventional FVIII dosage calculations are used in clinical practice without considering the coagulant effects of emicizumab.Methods and analysis In the CAGUYAMA study, 100 patients with HA without inhibitors will be enrolled for a maximum duration of 1 year, and samples of 30 events following the concomitant use of FVIII concentrates (30±5 U/kg) with emicizumab will be collected. An ‘event’ is defined as obtaining blood samples at preadministration and postadministration of FVIII concentrates during a breakthrough bleeding or a surgical procedure. Global coagulation assays will be used to measure the coagulation potential of the obtained samples. Clot waveform analysis (CWA) is used to identify the primary end-point, that is, the degree of improvement in the maximum coagulation rate at preadministration and post-administration of fixed-dose FVIII concentrations. The parameter obtained from CWA, which is triggered by an optimally diluted mixture of prothrombin time reagent and activated partial thromboplastin time reagent, is reported to be an excellent marker for assessing the degree of improvement of the coagulation potential in emicizumab-treated plasmas.Ethics and dissemination The CAGUYAMA study was approved by the Japan-Certified Review Board of Nara Medical University (Approval ID; nara0031). The study results will be communicated through publication in international scientific journals and presentations at (inter)national conferences.Trial registration number jRCTs051210137.

Introduction: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. Methods: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. Results: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. Conclusions: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Abstract Background Higher smoking prevalence in people with serious psychological distress (SPD) is well-recognized. However, gender and age differences in the association between heaviness of cigarette smoking and SPD have not been fully investigated. Methods We used anonymized data from a nationally representative survey in Japan (33,925 men and 37,257 women). SPD was measured using the Kessler 6-item Psychological Distress Scale and defined as ≥ 13 points. Multiple logistic regression analyses stratified by gender and age-groups (20–44 years, 45–64 years, and ≥ 65 years) were used to estimate adjusted odds ratio (aOR) and 95% confidence interval (CI) for SPD. Results After adjusting for sociodemographic confounders including education, equivalent household expenditures, and employment contract, women had a significant association between heavier smoking and more frequent SPD: compared to never-smokers, aORs (95% CIs) of ex-smokers, current light smokers who smoked 1–10 cigarettes per day (CPD), current moderate smokers 11–20 CPD, and current heavy smokers ≥ 21 CPD were 1.22 (0.92–1.63), 1.52 (1.25–1.84), 1.75 (1.46–2.09), and 2.22 (1.59–3.10), respectively (P-trend

Abstract Background Bleeding in people with hemophilia A can be life threatening, and intra‐articular bleeds can result in joint damage. Most clinical studies focus on treated bleeds, while bleeds not treated with coagulation factor(s) (untreated bleeds) are underreported. Objectives We assessed the incidence of untreated bleeds during a noninterventional study (NIS) wherein people with hemophilia A, with or without factor VIII (FVIII) inhibitors, were managed according to standard practice. Patients/Methods Using the Bleed and Medication Questionnaire, we prospectively collected data from three cohorts: Cohort A, adults/adolescents (age ≥12 years) with FVIII inhibitors; Cohort B, children (aged

Introduction Persons with haemophilia A (PwHA) commonly experience regular bleeding into joints, which may result in joint damage and complications such as degenerative arthritis. Emicizumab has previously demonstrated efficacy in reducing the occurrence of joint bleeds and target joints, along with having a favourable safety profile; however, data on the long-term effects on joint health are lacking. The AOZORA study will evaluate the long-term safety and joint health of paediatric PwHA without factor (F)VIII inhibitors taking emicizumab; here, we report the details of the study protocol and baseline data.Methods and analysis AOZORA is a multicentre, open-label, phase IV clinical study in Japan that aims to enrol approximately 30 PwHA aged

Abstract Regular prophylaxis has markedly improved the treatment for patients with hemophilia A, especially after the introduction of highly purified factor VIII (FVIII) concentrates. However, frequent intravenous infusions and the development of FVIII inhibitors remain as unsolved difficulties. To overcome these unmet needs, a bispecific antibody mimicking activated FVIII has been developed in Japan. This bispecific antibody, emicizumab, recognizes activated factor IX (FIXa) and activated factor X (FXa), and promotes FIXa‐catalyzed activation of FX in the absence of FVIII. Emicizumab initially reacts with FIXa generated by the action of factor VIIa/tissue factor complexes. Subsequently, thrombin generation is enhanced in the presence of higher amounts of FIXa derived from FXIa‐dependent mechanisms. Hence, emicizumab‐driven FXa and thrombin generation is maintained by a FXI activation loop in the intrinsic coagulation pathway. Reactions downstream of emicizumab are regulated by natural anticoagulants including activated protein C, antithrombin, and tissue factor pathway inhibitor. Phase 3 studies (HAVEN 1‐4 and HOHOEMI studies) demonstrated a remarkable reduction in bleeding rates together with a high percentage of patients with zero treated bleeds irrespective of the presence of inhibitors. In general, emicizumab proved to be well tolerated, although isolated thromboembolic and thrombotic microangiopathic complications were observed in the HAVEN 1 studies, and 3 out of a total of 400 patients developed neutralizing antidrug antibodies. In addition, several questions remain to be discussed with respect to open‐use clinical practice, including when to start treatment, how to monitor therapy, and optimum dosage for surgical procedures and immune tolerance induction.

Background: Civil servants and physicians play an important role in combating COVID-19. However, it is unclear whether the number of civil servants and physicians is associated with rapid COVID-19 vaccine uptake among older people (i.e., smoother rollout of priority vaccination for older people). Methods: Using Poisson regression models of the generalized estimating equations, we examined the ecological association of the number of civil servants and physicians with prefectural-level rapid COVID-19 vaccination in older people. Prefectural-level data were based on publicly available government surveys. The outcome variable was the proportion of fully vaccinated people aged 65 and older on the day with the largest standard deviation across 47 prefectures (i.e., July 6, 2021). The explanatory variable was the number of civil servants and physicians per population by prefecture. Results: After adjusting for population density, influenza vaccination coverage, socioeconomic factors, natural environmental factors, health indicators, and the number of civil servants and physicians, in all 3 models, prefectures with the highest number of civil servants and physicians had faster COVID-19 vaccine uptake than prefectures with the lowest number. A significant trend between higher staffing levels and more rapid vaccination was observed for the number of physicians in all 3 models, but for the number of civil servants only in one model. Conclusion: We found that COVID-19 vaccine uptake among older people was more rapid in prefectures with more civil servants and physicians per population, with the number of physicians having a stronger association. This study may point the way to future areas of research on vaccine policies that include other age groups and infectious diseases.

Background: Infantile pertussis is a major concern and causes a significant health burden worldwide. Maternal adult tetanus, diphtheria, and acellular pertussis (Tdap) vaccination is very effective way of preventing infantile pertussis. However, Tdap has not been approved by the Japanese government or been included in Japan’s national immunization program (NIP). We carried out a questionnaire survey to investigate whether Japanese women would accept the Tdap vaccination if they were provided with appropriate information. Methods: The questionnaire survey was administered to pregnant women who visited the Obstetrics and Gynecology Department and mothers who visited the Pediatrics Department of Nara Medical University, Kashihara City, Japan, between October 2018 and May 2019. The questionnaire included information about pertussis infection and maternal vaccination, followed by seven questions. Results: A total of 943 participants answered the questionnaire (481 pregnant women and 462 mothers). Before reading the information, just 4.6% of participants knew that infantile pertussis can be prevented by maternal vaccination. After reading the information, 93.0% and 92.6% of participants thought that the maternal Tdap vaccine should be approved by the Japanese government and be included in the NIP, respectively. Although only 67.6% of participants wished to have the maternal Tdap vaccine without government financial support after reading the information, 92.5% said they would have the vaccine with government support (P

Acquired hemophilia A (AHA), a rare bleeding disorder caused by neutralizing autoantibodies against coagulation factor VIII (FVIII), occurs in both men and women without a previous history of bleeding. Patients typically present with an isolated prolonged activated partial thromboplastin time due to FVIII deficiency. Neutralizing antibodies (inhibitors) are detected using the Nijmegen-modified Bethesda assay. Approximately 10% of patients do not present with bleeding and, therefore, a prolonged activated partial thromboplastin time should never be ignored prior to invasive procedures. Control of acute bleeding and prevention of injuries that may provoke bleeding are top priorities in patients with AHA. We recommend treatment with bypassing agents, including recombinant activated factor VII, activated prothrombin complex concentrate, or recombinant porcine FVIII in bleeding patients. Autoantibody eradication can be achieved with immunosuppressive therapy, including corticosteroids, cyclophosphamide and rituximab, or combinations thereof. The median time to remission is 5 weeks, with considerable interindividual variation. FVIII activity at presentation, inhibitor titer and autoantibody isotype are prognostic markers for remission and survival. Comparative clinical studies to support treatment recommendations for AHA do not exist; therefore, we provide practical consensus guidance based on recent registry findings and the authors’ clinical experience in treating patients with AHA.

Background: Community health activities by public health nurses (PHNs) are known to improve lifestyle habits of local residents, and may encourage the practice of infectious disease prevention behaviors during the COVID-19 pandemic. We investigated the association between prefecture-level COVID-19 incidence rate and the number of PHNs per population in Japan, by the COVID-19 variant type. Methods: Our data were based on government surveys where prefectural-level data are accessible to the public. The outcome variable was the COVID-19 incidence rate (i.e., the cumulative number of COVID-19 cases per 100,000 population for each variant type in 47 prefectures). The explanatory variable was the number of PHNs per 100,000 population by prefecture. Covariates included socioeconomic factors, regional characteristics, healthcare resources, and health behaviors. The generalized estimating equations of the multivariable Poisson regression models were used to estimate adjusted incidence rate ratio (IRR) and 95% confidence interval (CI) for the COVID-19 cases. We performed stratified analyses by variant type (i.e., wild type, alpha variant, and delta variant). Results: A total of 1,705,224 confirmed COVID-19 cases (1351.6 per 100,000 population) in Japan were reported as of September 30, 2021. The number of PHNs per 100,000 population in Japan was 41.9. Multivariable Poisson regression models showed that a lower number of PHNs per population was associated with higher IRR of COVID-19. Among all COVID-19 cases, compared to the highest quintile group of the number of PHNs per population, the adjusted IRR of the lowest quintile group was consistently significant in the models adjusting for socioeconomic factors (IRR: 3.76, 95% CI: 2.55–5.54), regional characteristics (1.73, 1.28–2.34), healthcare resources (3.88, 2.45–6.16), and health behaviors (2.17, 1.39–3.37). These significant associations were unaffected by the variant type of COVID-19. Conclusion: We found that the COVID-19 incidence rate was higher in prefectures with fewer PHNs per population, regardless of the COVID-19 variant type. By increasing the number of PHNs, it may be possible to contain the spread of COVID-19 in Japan and provide an effective human resource to combat emerging infectious diseases in the future.

Background: The relationship between leisure activities (LA) in old age and prevention of disability has not been fully investigated, and age and gender differences of these relationships are unknown. This study aimed to investigate whether physical and cognitive LA predicted incident disability among community-dwelling older adults by age and gender. Methods: We prospectively observed 8,275 residents aged 65 or above without disability at baseline for 3 years. Incident disability was defined as a new certification of the public long-term care insurance system. LA were classified into two types: physical LA and cognitive LA. The frequency of LA was categorized into frequent (i.e., once a week or more), moderate (i.e., monthly or yearly), and non-engagement. Covariates included age, gender, family number, education, perceived economic situation, body mass index, chronic medical conditions, alcohol consumption, smoking status, regular dental visits, depression, cognitive functioning, and social participation. Multivariable Poisson regression models were used to estimate adjusted cumulative incidence ratio (CIR) and 95% confidence interval (CI) for incident disability. We performed stratified analyses by age groups (i.e., the young-old aged 65–74 and the old-old aged 75–97) and gender (i.e., men and women). Results: The 3-year cumulative incidence of disability was 7.5%. After adjustment for covariates and mutual adjustment for both types of LA, a significant dose-response relationship between more frequent LA and lower risk of incident disability was found in young-old physical LA (P-trend < 0.001), in old-old cognitive LA (P-trend = 0.012), in male cognitive LA (P-trend = 0.006), and in female physical LA (P-trend = 0.030). Compared with people without LA, adjusted CIR (95% CI) of frequent LA was 0.47 (0.30–0.74) in young-old physical, 0.75 (0.58–0.96) in old-old cognitive, 0.65 (0.46–0.89) in male cognitive, and 0.70 (0.52–0.95) in female physical. Regarding the effect modification according to age and gender, only interaction between age and physical LA significantly prevented incident disability (P for interaction = 0.019). Conclusion: We found age differences in the association of physical LA with incident disability among community-dwelling older adults. An effective measure to prevent long-term care in the community would be to recommend frequent physical LA for the young-old.

Abstract: Emicizumab (ACE910), a recombinant humanized bispecific monoclonal antibody, provides factor VIII (FVIII) cofactor bridging function to restore hemostasis in people with hemophilia A. In a phase 1 trial involving 18 Japanese patients with severe hemophilia A, once-weekly subcutaneous administration of emicizumab 0.3, 1, or 3 mg/kg (cohorts 1, 2, and 3, respectively) was well tolerated and substantially reduced annualized bleeding rates (ABRs) in the presence or absence of FVIII inhibitors. The current study represents an open-label, long-term extension of the previously reported 12-week phase 1 study, in which 16 of 18 patients continued to receive emicizumab for up to 33.3 months. Long-term emicizumab treatment was well tolerated, with no thromboembolic events reported and no neutralizing antiemicizumab antibodies developing during the course of the study. Plasma concentrations of emicizumab increased in a dose-proportional manner, with activated partial thromboplastin times remaining short. In cohorts 1, 2, and 3, respectively, median ABRs remained low at 1.4, 0.2, and 0 compared with 4.4, 0, and 0 in the 12-week study. Overall, 8 patients experienced no bleeding events (6 patients with and 2 patients without FVIII inhibitors); dose up-titration resulted in further reduction in ABRs in patients with suboptimal bleeding control; and the episodic use of clotting factors to control bleeding was reduced. In conclusion, long-term emicizumab treatment demonstrated a favorable safety profile with encouraging efficacy, irrespective of the presence of FVIII inhibitors, in patients with hemophilia A. This study was registered at www.clinicaltrials.jp as #JapicCTI-132195.

Emicizumab is a humanized bispecific antibody that binds simultaneously to factor (F) IXa and FX replacing the cofactor function of FVIIIa. Because emicizumab recognizes FIX/FIXa and FX/FXa, a question may arise whether emicizumab competes with antithrombin (AT) and/or tissue factor pathway inhibitor (TFPI), thereby enhancing overall hemostatic potential by blocking their antihemostatic effects. To address this question, we performed enzymatic assays using purified coagulation factors to confirm whether emicizumab interferes with the action of AT on FIXa or FXa, or with the action of TFPI on FXa. In those assays, we found no interference of emicizumab on the actions of AT and TFPI. We next assessed emicizumab's influences on the anticoagulation actions of AT or TFPI in thrombin generation assays triggered with FXIa or tissue factor (TF) in AT-depleted or TFPI-depleted plasma supplemented with AT or TFPI in vitro. In those assays, we employed anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab instead of emicizumab itself so as to prevent emicizumab's FVIIIa cofactor activity from boosting thrombin generation. Consequently, we found that neither anti-FIXa, anti-FX monospecific antibody, nor the mixture of the two interfered with the anticoagulation actions of AT or TFPI in plasma. Although emicizumab can bind to FIXa and FXa, our results showed no interference of emicizumab with the action of AT or TFPI on FIXa or FXa. This indicates that the presence of emicizumab is irrelevant to the action of AT and TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI.

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

Development of factor VIII (fVIII)-neutralizing antibodies, called inhibitors, is a challenging problem in the management of hemophilia A patients. We explored the possibility of pretreatment with apoptotic fVIII-expressing embryonic stem (ES) cells to prevent the development of fVIII inhibitors. Murine ES cells integrated with the human F8 gene were differentiated into embryoid bodies, dissociated to a single cell suspension, subjected to hypo-osmotic shock to induce apoptosis, and intraperitoneally injected into hemophilia A mice. Inhibitors were induced by periodic intraperitoneal injections of recombinant human fVIII (rhfVIII). In the groups in which intraperitoneal injections of rhfVIII began at 1-3 weeks after pretreatment, the titers of inhibitors were significantly lower after the third administration of rhfVIII compared with that in the control group in which apoptotic Ainv18 ES cells (without the human F8 gene) were used for pretreatment, and continued to show lower levels until the sixth administration of rhfVIII. These results suggest that pretreatment with apoptotic hfVIII-expressing ES cells might be promising for the prevention of fVIII inhibitor development in hemophilia A patients.

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3-5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

We previously reported that cell-based therapies using isolated hepatocytes including hepatocyte transplantation and liver tissue engineering approaches provide therapeutic benefits to hemophilia. For clinical application of these approaches, it is important to establish an active hepatocyte proliferation system that enables providing a sufficient number of hepatocytes. We also reported that human hepatocytes, which were transplanted into the liver of urokinase-type plasminogen activator transgenic severe combined immunodeficiency (uPA/SCID) mice, were able to proliferate while retaining their ability to produce coagulation factor IX. The objective of this study was to explore the functionalities of other coagulation and anticoagulation factors of the propagated human hepatocytes in uPA/SCID mice. Human hepatocytes were transplanted into the liver of uPA/SCID mice, and the propagation status of human hepatocytes in the mice was monitored by the increase in serum human albumin levels and immunohistochemical evaluation on the liver sections. Using uPA/SCID livers with various stages of human hepatocyte propagation, we analyzed the gene expression levels of coagulation factors (prothrombin, factor VII, factor X, and factor VIII) and anticoagulation factors (protein C and protein S) by real-time polymerase chain reaction (PCR) using human-specific primers. As a result, the total amount of raw messenger RNA expression levels increased in all genes analyzed according to the progress of hepatocyte propagation and proliferation. Except for factor VIII, the gene expression levels of the highly repopulated uPA/SCID mouse livers with human hepatocyte showed higher levels than those of normal human livers, indicating that propagated human hepatocytes in the uPA/SCID system possess full functions to produce most of the coagulation-related factors. The current work demonstrated that human hepatocytes can be propagated in experimental animals while maintaining normal gene expression levels of coagulation-related factors. It could be speculated that the propagated cells serve as a cell source for the treatment of various types of coagulation factor deficiencies.

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5–2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.

Acquired hemophilia A (AHA) is a rare bleeding disorder characterized by autoantibodies directed against circulating coagulation factor (F) VIII. Typically, patients with no prior history of a bleeding disorder present with spontaneous bleeding and an isolated prolonged aPTT. AHA may, however, present without any bleeding symptoms, therefore an isolated prolonged aPTT should always be investigated further irrespective of the clinical findings. Control of acute bleeding is the first priority, and we recommend first-line therapy with bypassing agents such as recombinant activated FVII or activated prothrombin complex concentrate. Once the diagnosis has been achieved, immediate autoantibody eradication to reduce subsequent bleeding risk should be performed. We recommend initial treatment with corticosteroids or combination therapy with corticosteroids and cyclophosphamide and suggest second-line therapy with rituximab if first-line therapy fails or is contraindicated. In contrast to congenital hemophilia, no comparative studies exist to support treatment recommendations for patients with AHA, therefore treatment guidance must rely on the expertise and clinical experience of specialists in the field. The aim of this document is to provide a set of international practice guidelines based on our collective clinical experience in treating patients with AHA and contribute to improved care for this patient group.