"Evaluation of neprilysin activity in Adipose-Derived stem cells from Alzheimer's disease patients".

INTRODUCTION: The antibody drugs targeting ß-amyloid in Alzheimer's disease pose risks of inflammation and vascular damage. It is known that neprilysin, an endogenous enzyme responsible for ß-amyloid degradation, is reduced in areas with ß-amyloid deposition. Supplementation of neprilysin could potentially contribute to Alzheimer's disease treatment. When considering the use of adipose tissue-derived stem cells (ADSCs) for Alzheimer's disease therapy, it is crucial to ensure that Alzheimer's disease patient-derived ADSCs maintain neprilysin activity. If so, the use of autologous ADSCs may lead to a treatment with minimal risks of rejection or infection. Therefore, we investigated the neprilysin activity in Alzheimer's disease patient-derived adipose tissue-derived stem cells to assess their potential in Alzheimer's disease treatment. METHODS: Five Alzheimer's disease patients (MSC1-5) and two Chronic Obstructive Pulmonary Disease (COPD) patients (MSC6-7) were enrolled. ADSCs were cultured for 6 days with varying seeding densities. On the 3rd day, the medium was replaced, and on the 6th day, ADSCs were harvested. Cells were stained for PE-Cy7 Mouse IgG1 κ Isotype control and PE-Cy Mouse Anti-Human CD10, and CD10 expression was assessed by flow cytometry. Ethical approval and informed consent were obtained. RESULTS: Neprilysin activity, crucial for ß-amyloid degradation, was assessed in ADSCs. Positivity rates for CD10 expression in ADSCs from Alzheimer's patients were consistently high: 99.6%, 99.5%, 99.9%, 99.3%, 99.8%, and 100.0%. Control ADSCs from COPD patients (MSC6-7) exhibited comparable positivity rates. Flow cytometry plots for all seven cases are presented in Figures 1-7. DISCUSSION: This study confirms the presence and maintenance of neprilysin activity in ADSCs from Alzheimer's disease patients. The high positivity rates for CD10 expression in these cells suggest that neprilysin, a key enzyme in ß-amyloid degradation, remains active. The implications are significant, as ADSCs offer immune-compatible and low infection risk advantages. The study underscores the potential of autologous ADSCs as a therapeutic approach in Alzheimer's disease. Their ability to naturally harbor neprilysin activity, coupled with their safety profile, makes them a promising candidate for further exploration. While acknowledging the need for larger, more diverse cohorts and long-term studies, these findings contribute to the growing body of evidence supporting the development of stem cell-based interventions in Alzheimer's disease treatment.

Perspectives on Stem Cell-Based Regenerative Medicine with a Particular Emphasis on Mesenchymal Stem Cell Therapy.

Regenerative medicine is a medical treatment that aims to restore lost human body functions by regenerating missing or dysfunctional organs and tissues using stem cells, etc. There are three major types of stem cells used in regenerative medicine: induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and mesenchymal stem cells (MSCs). MSCs are expected to be widely applied to regenerative medicine because of their ability to differentiate into various types of cells, repair cells and tissues; anti-inflammatory effects; secretion of various growth factors; and resolution of abnormally accumulated protein amyloid. MSCs can be derived from bone marrow, dental pulp, and other sources, but adipose tissue-derived stem cells (ADSCs) may be superior in that they can be harvested with the least amount of invasion, and therefore, a sufficient amount of stem cells can be cultured relatively easily. When MSCs are administered systemically by intravenous infusion, they tend to accumulate at the site of disease, a property known as "homing," which is extremely advantageous for clinical applications. In Japan, stem cell therapy can be performed only after the research or treatment plan has been reviewed and approved by the "Committee for Specific Approval of Regenerative Medicine" and submitted to the Ministry of Health, Labor and Welfare for approval in accordance with the "Act on Securing the Safety of Regenerative Medicine" and after approval by the ethics committee of the facility where the therapy is performed. In this review, the characteristics of MSCs, the actual status of their clinical application, and their future prospects are presented.

Repeated infusion of autologous adipose tissue-derived stem cells for Parkinson's disease.

BACKGROUND: Mesenchymal stem cells are expected to have a therapeutic effect on progressive neurodegenerative diseases for which there is currently no fundamental treatment. AIMS OF THE STUDY: The aim is to confirm that repeated infusion of autologous adipose tissue-derived stem cells (ADSCs) can be safely administered to patients with Parkinson's disease, and to investigate the effects of this as a pilot study. METHODS: Three patients with Parkinson's disease received five or six repeated infusions of ADSCs at intervals of approximately one month. Observations were based on medical examinations by a neurologist and interviews with the patient and caregivers. The severity of Parkinson's disease was assessed using the Hoehn & Yahr staging scale and Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS). RESULTS: No adverse events were observed during the observation period from the start of treatment to six months after the end of the last dose. MDS-UPDRS improved in all three patients. CONCLUSIONS: Repeated administration of Autologous ADSCs for Parkinson's disease was safe and feasible. The results of this pilot study provide insight into the value of further research.

Hypothesis: Intravenous administration of mesenchymal stem cells is effective in the treatment of Alzheimer's disease.

We propose the intravenous administration of autologous adipose-derived stem cells as a new treatment for Alzheimer's disease. We hypothesize that the stem cells will secrete neprilysin in the brain to break down and remove amyloid deposits in the Alzheimer's brain. We have shown a case of skin amyloid deposition that disappeared after stem cell administration and confirmed that the stem cells administered had neprilysin activity. In addition to neprilysin secretion, other mechanisms of action of stem cells include nerve regeneration, nerve repair, growth factor secretion, anti-inflammatory effects, and angiogenesis. The harvesting of adipose-derived stem cells is minimally invasive, and intravenous administration can be safely repeated. We hope that the efficacy of this new treatment will be verified and that it will bring a ray of hope to patients suffering from this incurable disease.

Neutrophil extracellular traps in neuropathy with anti-neutrophil cytoplasmic autoantibody-associated microscopic polyangiitis.

To clarify the roles of neutrophils in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitic neuropathy, we studied neutrophil extracellular traps (NETs) in peripheral nerve vasculitis. Stored nerve samples from 17 patients with microscopic polyangiitis (MPA) were immunohistochemically analyzed using antibodies for citrullinated histone H3 (citH3) and various neutrophil enzymes. We defined merged citH3 and extracellularly released myeloperoxidase (MPO) as NET formation. We also compared NET formation between MPO-ANCA-positive/negative MPA and rheumatoid arthritis (RA)-associated vasculitic neuropathy. NETs were identified mostly in vasculitic small arterioles of 6 of 12 MPO-ANCA-positive MPA patients, and their frequency was higher (p < 0.05) than in ANCA-negative patients. NETs were not found in vasculitic neuropathy with RA or patients with chronic inflammatory demyelinating polyradiculoneuropathy. NETs were also observed in the peripheral nervous system of MPA patients as well as in the lung and kidney. These results suggest that NETs may be involved in the pathogenesis of MPA neuropathy.

Influences of hyperlipidemia history on stroke outcome; a retrospective cohort study based on the Kyoto Stroke Registry.

BACKGROUND: Although hyperlipidemia is known as a risk factor of stroke, its effects on the outcome are unknown. The aim of the study is to clarify the influences of hyperlipidemia on the stroke early outcome by estimating odds ratio (OR) for sequelae requiring care and hazard ratio (HR) for death. METHODS: A total of 12617 stroke patients registered in the Kyoto Stroke Registry with information on a hyperlipidemia history. We compared patients who had hyperlipidemia history and patients who hadn't. The OR for remaining sequelae requiring certain care on 30 day after stroke was calculated using a logistic regression in stroke as a whole and in each stroke subtype; cerebral infarction (CI), cerebral hemorrhage (CH) and subarachnoid hemorrhage (SAH). The HR for death within 30 day after stroke was estimated by the Cox regression. RESULTS: The OR (95% confidence interval) for remaining sequelae 30 days after stroke was 0.66 (0.60-0.73, p < 0.001) in patients with hyperlipidemia history compared with patients without hyperlipidemia history. After stratified by stroke subtypes, it was 0.75 (0.67-0.85, p < 0.001) in CI, 0.59 (0.45-0.77, p < 0.001) in CH and 0.77 (0.43-1.38, p = 0.767) in SAH. The HR (95% confidence interval) for death was 0.39 (0.31-0.48, p < 0.001) in patients with hyperlipidemia history comparing patients without hyperlipidemia history. After stratified by stroke subtypes, it was 0.45 (0.32-0.63, p < 0.001) in CI, 0.64 (0.44-0.93, p = 0.018) in CH and 0.76 (0.47-1.23, p = 0.264) in SAH. Each value was adjusted for age and sex. CONCLUSIONS: This study suggests that the outcome is favorable for patients with hyperlipidemia history in terms of both remaining sequelae and HR for death. A factor which increases the incidence of the disease could influence on the severity of the disease in a favorable way.

Weekly variations of stroke occurrence: an observational cohort study based on the Kyoto Stroke Registry, Japan.

OBJECTIVES: Understanding the temporal pattern of stroke onset and exploring the possible triggers are important strategies to reducing the incidence of stroke. If stroke occurs frequently on a specific day of the week, it is assumed that other factors, that is, 'triggering factors', induce stroke. The aim of the study is to investigate differences in the incidences of stroke among days of the week. DESIGN: Hospital-based registry stroke over an 11-year period. SETTING: Kyoto Prefecture, Japan. PARTICIPANTS: A total of 13,788 patients with stroke identified from January 1999 to December 2009 inclusive in the entire Kyoto Prefecture and registered in the Kyoto Stroke Registry (KSR). MAIN OUTCOME MEASURES: Patients with stroke were classified into seven groups based on the day of the week on which stroke developed. We confirmed the differences in the incidence among days using the χ(2) test and then performed multinomial logistic analysis referring to the stroke incidence on Sunday to calculate the OR and 95% CI of the stroke occurrence on each day of the week. RESULTS: The OR (95% CI) for stroke occurring on Monday, Tuesday, Wednesday, Thursday, Friday and Saturday was 1.157 (1.030 to 1.293), 1.101 (0.981 to 1.236), 1.059 (0.943 to 1.188), 1.091 (0.972 to 1.225), 1.053 (0.938 to 1.205) and 1.074 (0.956 to 1.205), respectively. After stratification by stroke subtypes, cerebral infarction occurred more frequently on Monday than on Sunday (OR and 95% CI were 1.189 and 1.034 to 1.366, p=0.014) independent of age and gender. There was no significant day of the week variation in cerebral haemorrhage or subarachnoid haemorrhage. CONCLUSIONS: Some factors that arise periodically appear to affect the incidence of cerebral infarction, which gradually develops over years, and this suggests an aetiological mechanism different from the conventional cumulative effect of risk factors due to long-term exposure. We propose a hypothesis that there is a 'triggering factor' for the development of cerebral infarction.

Speech disturbance at stroke onset is correlated with stroke early mortality.

BACKGROUND: Speech disturbance is a common symptom of stroke and is important as a prompt identifier of the event. The frequency of the symptom among each stroke subtype, differences between patients with and without speech disturbance and its correlation to early mortality remain unclear. METHODS: The Kyoto prefecture of Japan has established a registry to enroll new stroke patients in cooperation with the Kyoto Medical Association and its affiliated hospitals. It is named the Kyoto Stroke Registry (KSR). We confirmed the existence or absence of speech disturbance in 1693 stroke patients registered to the KSR and investigated associations between speech disturbance and other characteristics. RESULTS: Speech disturbance was observed in 52.6% of cerebral infarction (CI), 47.5% of cerebral hemorrhage (CH), and 8.0% of subarachnoid hemorrhage (SAH) cases. Characteristics showing statistically significant differences between patients with and without speech disturbance and patients were age, blood pressure, history of hypertension, arrhythmia and diabetes mellitus, habit of tobacco and alcohol, and paresis. Mortality rates of patients with/without speech disturbance were 5.2%/1.2% for CI, 12.5% /4.1% for CH, and 62.5%/ 9.0% for SAH. Adjusted hazard ratios were 2.63 (1.14-6.13, p = 0.024) in CI, 4.15 (1.41-12.23, p = 0.010) in CH, and 20.46 (4.40-95.07, p < 0.001) in SAH). CONCLUSION: Speech disturbance was frequently observed in stroke patients at the onset and therefore could be useful to identify the problem at the earliest stage. Hazard ratio for death was higher in stroke patients with speech disturbance than patients without. Speech disturbance is a prompt predictor of stroke early mortality.Hiromi Nakano, Yoshiyuki Watanabe, Tatsuyuki Sekimoto, Kouichiro Shimizu, Akihiko Nishizawa, Atsushi Okumura and Masahiro Makino contributed equally to this work.

The eye response test alone is sufficient to predict stroke outcome--reintroduction of Japan Coma Scale: a cohort study.

OBJECTIVES: Prompt assessment of consciousness levels is vitally important during the emergency care of stroke patients. The Japan Coma Scale (JCS) is a one-axis coma scale published in 1974 with outstanding simplicity. The hypothesis is that JCS is sufficient to predict stroke outcome. The aim of the study was to verify the predictability of JCS, which should help JCS attain international recognition. DESIGN: A cohort study. SETTING: A prefectural stroke registry. PARTICIPANTS: We analysed 13 788 stroke patients identified from January 1999 to December 2009 inclusive in the entire Kyoto prefecture and registered in the Kyoto Stroke Registry (KSR). MAIN OUTCOME MEASURES: We investigated the relationship between consciousness levels, based on JCS at stroke onset and activities of daily living (ADL) at 30 days or deaths within 30 days in a large population-based stroke registry. We calculated Spearman's coefficient for the correlation between JCS and the ADL scale, generated estimated survival curves by the Kaplan-Meier method and finally compared HRs for death within 30 days after onset, comparing patients with different conscious levels based on JCS. RESULTS: A total of 13 406 (97.2%) patients were graded based on JCS. JCS correlated to the ADL scale with Spearman's correlation coefficient of 0.61. HRs for death within 30 days were 1 (reference) (95% CIs), 5.55 (4.19 to 7.37), 9.54 (7.16 to 12.71) and 35.21 (26.10 to 44.83) in those scored as JCS0, JCS1, JCS2 and JCS3, respectively. CONCLUSIONS: Using a single test of eye response, JCS has outstanding merits as a coma scale, that is, simplicity and applicability. The present study adds predictability for early outcome in stroke patients. JCS is valuable, especially in an emergency setting, when a prompt assessment of consciousness levels is needed.

Different profiles of onion bulb in CIDP and CMT1A in relation to extracellular matrix.

Hypertrophic neuropathy is usually intractable, and chronic inflammatory demyelinating polyneuropathy (CIDP) and Charcot-Marie-Tooth disease Type 1A (CMT1A) are the representative disorders. The two disorders are sometimes confused both clinically and pathologically. The aim of this study was to clarify the differences in the pathology of large onion bulbs, focusing on the extracellular matrix (ECM) proteins. Nine patients with CIDP and 14 with CMT1A were included. The opened interspaces in OB were frequently shown in CMT1A patients. In CIDP, interspaces of OB packed with collagen fibers were prominent. The mean ratio of opened OB was significantly increased in CMT1A (37.9%) compared to CIDP patients (10.6%) (p = 0.003). Among the ECM examined, tenascin-C (TNC) showed a distinct difference in the pattern of immunoreactivity of OB. The mean ratio of OB showing TNC immunoreactivity was significantly larger in CIDP (29.7%, p = 0.005) than in CMT1A (5.0%). TNC immunoreactivity was confined to the area around myelin sheaths in CMT1A. The increased deposition of collagen fibers in CIDP suggests the activity of nerve regeneration. TNC expression in Schwann cell lamellae comprising OB may also suggest the activity of regeneration. Schwann cell phenotypes in CIDP may be different from CMT1A regarding the production of ECM proteins.

Characteristics, risk factors and mortality of stroke patients in Kyoto, Japan.

OBJECTIVES: The aim of the study was to evaluate the characteristics, risk factors and outcome of recent stroke patients in Kyoto, Japan. DESIGN: We analysed stroke patients in the registry with regard to their characteristics, risk factors and mortality. Cox proportional hazards regressions were used to calculate adjusted HRs for death. SETTINGS: The Kyoto prefecture of Japan has established a registry to enrol new stroke patients in cooperation with the Kyoto Medical Association and its affiliated hospitals PARTICIPANTS: The registry now has data on 14 268 patients enrolled from 1 January 1999 to 31 December 2009. Of these, 12 774(89.5%) underwent CT, 9232 (64.7%) MRI, 2504 (17.5%) angiography and 342 (2.4%) scintigraphy. Excluding 480 (3.3%) unclassified patients, 13 788 (96.6%) patients formed the basis of further analyses which were divided into three subtypes: cerebral infarction (CI), cerebral haemorrhage (CH) and subarachnoid haemorrhage (SAH). RESULTS: A total of 13 788 confirmed stroke patients in the study cohort comprised 9011 (86.3%) CI, 3549 (25.7%) CH and 1197 (8.7%) SAH cases. The mean age ±SD was 73.3±11.8, 69.1±13.6 and 62.7±13.5 in the CI, CH and SAH cases, respectively. Men were predominant in the CI and CH cases, whereas women were predominant in the SAH cases. The frequencies of risk factors were different among the subtypes. Mortality was worst in SAH, followed by CH, and least in CI. HRs for death adjusted for age, sex, histories of hypertension, arrhythmia, diabetes mellitus and hyperlipaemia and use of tobacco and/or alcohol showed a significant (p<0.001) difference among CI (as reference), CH (3.71; 3.11 to 4.43) and SAH (8.94; 7.21 to 11.11). CONCLUSIONS: The characteristics, risk factors and mortality were evaluated in a quantitative manner in a large Japanese study cohort to shed light on the present status of stroke medicine.

Vomiting should be a prompt predictor of stroke outcome.

BACKGROUND: To predict the outcome of stroke at an acute stage is important but still difficult. Vomiting is one of the commonest symptoms in stroke patients. The aim of this study is threefold: first, to examine the percentage of vomiting in each of the three major categories of strokes; second, to investigate the association between vomiting and other characteristics and third, to determine the correlation between vomiting and mortality. METHODS: We investigated the existence or absence of vomiting in stroke patients in the Kyoto prefecture cohort. We compared the characteristics of patients with and without vomiting. We calculated the HR for death in both types of patients, adjusted for age, sex, blood pressure, arrhythmia, tobacco and alcohol use and paresis. RESULTS: Of the 1968 confirmed stroke patients, 1349 (68.5%) had cerebral infarction (CI), 459 (23.3%) had cerebral haemorrhage (CH) and 152 (7.7%) had subarachnoid haemorrhage (SAH). Vomiting was seen in 14.5% of all stroke patients. When subdivided according to stroke type, vomiting was observed in 8.7% of CI, 23.7% of CH and 36.8% of SAH cases. HR for death and 95% CI were 5.06 and 3.26 to 7.84 (p<0.001) when all stroke patients were considered, 5.27 and 2.56 to 10.83 (p<0.001) in CI, 2.82 and 1.33 to 5.99 (p=0.007) in CH and 5.07 and 1.87 to 13.76 (p=0.001) in SAH. CONCLUSIONS: Compared with patients without vomiting, the risk of death was significantly higher in patients with vomiting at the onset of stroke. Vomiting should be an early predictor of the outcome.

Two subtypes of Churg-Strauss syndrome with neuropathy: the roles of eosinophils and ANCA.

The aim of this study was to clarify the differences in the pathogenesis of neuropathy between myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-positive and -negative patients with Churg-Strauss syndrome (CSS). Eight MPO-ANCA-positive and 14 MPO-ANCA-negative patients were included. In addition to the standard histology, nerve biopsies were examined, employing immunohistochemistry for eosinophil major basic protein and electron microscopy. The groups did not differ significantly in clinical profiles, including the peak disability score and number of blood eosinophils. In nerve biopsies, necrotizing vasculitis was found in 63% (5/8) of the ANCA-positive and 21% (3/14) of the ANCA-negative patients. Fibrinoid necrosis of vessel walls was noted in 4 ANCA-positive patients (50%), and in one ANCA-negative patient (p = 0039). In contrast, a large number of eosinophilic infiltrations in the epineurium was shown in 36% (5/14) of the ANCA-negative patients, with no eosinophilic infiltrations shown in ANCA-positive patients. In 3 ANCA-negative patients, endoneurial eosinophils were seen where focal axonal loss and capillary dilatation were occasionally noted. There may be 2 pathogenetic mechanisms of neuropathy with CSS: ANCA-related vascular fibrinoid necrosis, and a toxic eosinophilic effect on nerve fibers which is independent of ANCA. Therapy targeting activated eosinophils may be a possible treatment for intractable neuropathy of CSS.