Perfluoropolyether-Based Gut-Liver-on-a-Chip for the Evaluation of First-Pass Metabolism and Oral Bioavailability of Drugs.

In the evolving field of drug discovery and development, multiorgans-on-a-chip and microphysiological systems are gaining popularity owing to their ability to emulate in vivo biological environments. Among the various gut-liver-on-a-chip systems for studying oral drug absorption, the chip developed in this study stands out with two distinct features: incorporation of perfluoropolyether (PFPE) to effectively mitigate drug sorption and a unique enterohepatic single-passage system, which simplifies the analysis of first-pass metabolism and oral bioavailability. By introducing a bolus drug injection into the liver compartment, hepatic extraction alone could be evaluated, further enhancing our estimation of intestinal availability. In a study on midazolam (MDZ), PFPE-based chips showed more than 20-times the appearance of intact MDZ in the liver compartment effluent compared to PDMS-based counterparts. Notably, saturation of hepatic metabolism at higher concentrations was confirmed by observations when the dose was reduced from 200 µM to 10 µM. This result was further emphasized when the metabolism was significantly inhibited by the coadministration of ketoconazole. Our chip, which is designed to minimize the dead volume between the gut and liver compartments, is adept at sensitively observing the saturation of metabolism and the effect of inhibitors. Using genome-edited CYP3A4/UGT1A1-expressing Caco-2 cells, the estimates for intestinal and hepatic availabilities were 0.96 and 0.82, respectively; these values are higher than the known human in vivo values. Although the metabolic activity in each compartment can be further improved, this gut-liver-on-a-chip can not only be used to evaluate oral bioavailability but also to carry out individual assessment of both intestinal and hepatic availability.

Rehabilitation facilitates functional improvement following intravenous infusion of mesenchymal stem cells in the chronic phase of cerebral ischemia in rats.

The primary objective of this study was to investigate the potential facilitating effects of daily rehabilitation for chronic cerebral ischemia following the intravenous infusion of mesenchymal stem cells (MSC) in rats. The middle cerebral artery (MCA) was occluded by intraluminal occlusion using a microfilament (MCAO). Eight weeks after MCAO induction, the rats were used as a chronic cerebral ischemia model. Four experimental groups were studied: Vehicle group (medium only, no cells); Rehab group (vehicle + rehabilitation), MSC group (MSC only); and Combined group (MSC + rehabilitation). Rat MSCs were intravenously infused eight weeks after MCAO induction, and the rats received daily rehabilitation through treadmill exercise for 20 min. Behavioral testing, lesion volume assessment using magnetic resonance imaging (MRI), and histological analysis were performed during the observation period until 16 weeks after MCAO induction. All treated animals showed functional improvement compared with the Vehicle group; however, the therapeutic efficacy was greatest in the Combined group. The combination therapy is associated with enhanced neural plasticity shown with histological analysis and MRI diffusion tensor imaging. These findings provide behavioral evidence for enhanced recovery by combined therapy with rehabilitation and intravenous infusion of MSCs, and may form the basis for the development of clinical protocols in the future.

Actin Alpha 2, Smooth Muscle (ACTA2) Is Involved in the Migratory Potential of Malignant Gliomas, and Its Increased Expression at Recurrence Is a Significant Adverse Prognostic Factor.

Malignant glioma is a highly invasive tumor, and elucidating the glioma invasion mechanism is essential for developing novel therapies. We aimed to highlight actin alpha 2, smooth muscle (ACTA2) as potential biomarkers of brain invasion and distant recurrence in malignant gliomas. Using the human malignant glioma cell line, U251MG, we generated ACTA2 knockdown (KD) cells treated with small interfering RNA, and the cell motility and proliferation of the ACTA2 KD group were analyzed. Furthermore, tumor samples from 12 glioma patients who underwent reoperation at the time of tumor recurrence were utilized to measure ACTA2 expression in the tumors before and after recurrence. Thereafter, we examined how ACTA2 expression correlates with the time to tumor recurrence and the mode of recurrence. The results showed that the ACTA2 KD group demonstrated a decline in the mean motion distance and proliferative capacity compared to the control group. In the clinical glioma samples, ACTA2 expression was remarkably increased in recurrent samples compared to the primary samples from the same patients, and the higher the change in ACTCA2 expression from the start to relapse, the shorter the progression-free survival. In conclusion, ACTA2 may be involved in distant recurrence in clinical gliomas.

Therapeutic efficacy of intravenous infusion of mesenchymal stem cells in rat perinatal brain injury.

BACKGROUND: Perinatal brain injury is multifactorial and primarily associated with brain prematurity, inflammation, and hypoxia-ischemia. Although recent advances in perinatal medicine　have improved the survival rates of preterm infants, neurodevelopmental disorders remain a significant complication. We tested whether the intravenous infusion of mesenchymal stem cells (MSCs) had therapeutic efficacy against perinatal brain injury in rats. METHODS: Pregnant rats at embryonic day (E) 18 received lipopolysaccharide and the pups were born at E21. On postnatal day (PND) 7, the left common carotid artery of each pup was ligated, and they were exposed to 8% oxygen for 2 h. They were randomized on PND10, and MSCs or vehicle were intravenously infused. We performed behavioral assessments, measured brain volume using MRI, and performed histological analyses on PND49. RESULTS: Infused MSCs showed functional improvements in our model. In vivo MRI revealed that MSC infusion increased non-ischemic brain volume compared to the vehicle group. Histological analyses showed that cortical thickness, the number of NeuN+ and GAD67+ cells, and synaptophysin density in the non-ischemic hemisphere in the MSC group were greater than the vehicle group, but less than the control group. CONCLUSIONS: Infused MSCs improve sensorimotor and cognitive functions in perinatal brain injury and enhance neuronal growth. IMPACT: Intravenous infusion of MSCs improved neurological function in rats with perinatal brain injury, including motor, sensorimotor, cognitive, spatial, and learning memory. Infused MSCs increased residual (non-ischemic) tissue volume, number of neuronal cells, GABAergic cells, and cortical synapses in the contralesional (right) hemisphere. Intravenous administration of MSC might be suitable for the treatment of perinatal brain injury.

A conditional gene expression system in Porphyromonas gingivalis for study of the secretion mechanisms of lipoproteins and T9SS cargo proteins.

The Gram-negative anaerobe, Porphyromonas gingivalis, is known to be a pathogen associated with chronic periodontitis. P. gingivalis possesses virulence factors such as fimbriae and gingipain proteinases. Fimbrial proteins are secreted to the cell surface as lipoproteins. In contrast, gingipain proteinases are secreted into the bacterial cell surface via the type IX secretion system (T9SS). The transport mechanisms of lipoproteins and T9SS cargo proteins are entirely different and remain unknown. Therefore, using the Tet-on system developed for the genus Bacteroides, we newly created a conditional gene expression system in P. gingivalis. We succeeded in establishing conditional expression of nanoluciferase and its derivatives for lipoprotein export, of FimA for a representative of lipoprotein export, and of T9SS cargo proteins such as Hbp35 and PorA for representatives of type 9 protein export. Using this system, we showed that the lipoprotein export signal, which has recently been found in other species in the phylum Bacteroidota, is also functional in FimA, and that a proton motive force inhibitor can affect type 9 protein export. Collectively, our conditional protein expression method is useful for screening inhibitors of virulence factors, and may be used to investigate the role of proteins essential to bacterial survival in vivo.

A practical protocol for high-spatial-resolution magnetic resonance angiography for cerebral arteries in rats.

BACKGROUND: Magnetic resonance angiography (MRA) is an important tool in rat models of cerebrovascular disease. Although MRA has long been used in rodents, the image quality is typically not as high as that observed in clinical practice. Moreover, studies on MRA image quality in rats are limited. This study aimed to develop a practical high-spatial-resolution MRA protocol for imaging cerebral arteries in rats. NEW METHOD: We used the "half position method" regarding coil placement and modified the imaging parameters and image reconstruction method. We applied this new imaging method to measure maturation-related signal changes on rat MRAs. RESULTS: The new practical high-spatial-resolution MRA imaging protocol obtained a signal intensity up to 3.5 times that obtained using a basic coil system, simply by modifying the coil placement method. This method allowed the detection of a gradual decrease in the signal in cerebral vessels with maturation. COMPARISON WITH EXISTING METHODS: A high-spatial-resolution MRA for rats was obtained with an imaging time of approximately 100 min. Comparable resolution and image quality were obtained using the new protocol with an imaging time of 30 min CONCLUSIONS: The new practical high-spatial-resolution MRA protocol can be implemented simply and successfully to achieve high image quality with an imaging time of approximately 30 min. This protocol will benefit researchers performing MRA imaging in cerebral artery studies in rats.

Intestinal Permeability of Drugs in Caco-2 Cells Cultured in Microfluidic Devices.

Microfluidic devices are attracting attention for their ability to provide a biomimetic microenvironment wherein cells are arranged in a particular pattern and provided fluidic and mechanical forces. In this study, we evaluated drug transport across Caco-2 cell layers in microfluidic devices and investigated the effects of fluid flow on drug transport and metabolism. We designed a microfluidic device that comprises two blocks of polydimethylsiloxane and a sandwiched polyethylene terephthalate membrane with pores 3.0 µm in diameter. When cultured in a dynamic fluid environment, Caco-2 cells were multilayered and developed microvilli on the surface as compared with a static environment. Drugs with higher lipophilicity exhibited higher permeability across the Caco-2 layers, as well as in the conventional method using Transwells, and the fluidic conditions had little effect on permeability. In the Caco-2 cell layers cultured in Transwells and microfluidic devices, the basal-to-apical transport of rhodamine 123, a substrate of P-glycoprotein, was greater than the apical-to-basal transport, and the presence of tariquidar, an inhibitor of P-glycoprotein, completely diminished asymmetric transport. Furthermore, fluidic conditions promoted the metabolism of temocapril by carboxylesterases. On the other hand, we showed that fluidic conditions have little effect on gene expression of several transporters and metabolic enzymes. These results provide useful information regarding the application of microfluidic devices in drug transport and metabolism studies.

Intravenous Infusion of Autoserum-Expanded Autologous Mesenchymal Stem Cells in Patients With Chronic Brain Injury: Protocol for a Phase 2 Trial.

BACKGROUND: Brain injuries resulting from motor vehicle accidents and falls, as well as hypoxic insults and other conditions, are one of the leading causes of disability and death in the world. Current treatments are limited but include continuous rehabilitation, especially for chronic brain injury. Recent studies have demonstrated that the intravenous infusion of mesenchymal stem cells (MSCs) has therapeutic efficacy for several neurological diseases, including stroke and spinal cord injury. OBJECTIVE: The objective of our investigator-initiated clinical trial is to assess the safety and potential efficacy of the intravenous infusion of autoserum-expanded autologous MSCs for patients with chronic brain injury. METHODS: The (phase 2) trial will be a single-arm, open-label trial with the primary objective of confirming the safety and efficacy of autoserum-expanded autologous MSCs (STR-01; produced under good manufacturing practices) when administered to patients with chronic brain injury. The estimated number of enrolled participants is 6 to 20 patients with a modified Rankin Scale grade of 3 to 5. The assessment of safety and the proportion of cases in which the modified Rankin Scale grade improves by 1 point or more at 180 days after the injection of STR-01 will be performed after MSC infusion. RESULTS: We received approval for our clinical trial from the Japanese Pharmaceuticals and Medical Devices Agency on December 12, 2017. The trial will be completed on June 11, 2023. The registration term is 5 years. The recruitment of the patients for this trial started on April 20, 2018, at Sapporo Medical University Hospital in Japan. CONCLUSIONS: Our phase 2 study will aim to address the safety and efficacy of the intravenous infusion of MSCs for patients with chronic brain injury. The use of STR-01 has been performed for patients with cerebral infarction and spinal cord injury, providing encouraging results. The potential therapeutic efficacy of the systemic administration of autoserum-expanded autologous MSCs for chronic brain injury should be evaluated, given its safety and promising results for stroke and spinal cord injury. TRIAL REGISTRATION: Japan Medical Association Center for Clinical Trials JMA-IIA00333; https://tinyurl.com/nzkdfnbc. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/37898.

The Impact of Purple-Flesh Potato (Solanum tuberosum L.) cv. "Shadow Queen" on Minor Health Complaints in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Study.

The purple-flesh potato (Solanum tuberosum L.) cultivar "Shadow Queen" (SQ) naturally contains anthocyanins. This randomized, double-blind, placebo-controlled study determines whether ingesting purple potatoes increases the number of mesenchymal stem cells (MSC) and improves stress response, a minor health complaint in healthy adults (registration number: UMIN000038876). A total of 15 healthy subjects (ages: 50-70 years) with minor health complaints were randomly assigned to one of two groups. For 8 weeks, the placebo group received placebo potatoes cv. "Haruka" and the test group received test potato cv. SQ containing 45 mg anthocyanin. The MSC count and several stress responses were analyzed at weeks 0 and 8 of the intake periods. The ingestion of a SQ potato did not affect the MSC count but markedly improved psychological stress response, irritability, and depression as minor health complaints compared with "Haruka". No adverse effects were noted. Hence, an 8-week intake of SQ could improve stress responses.

Enhanced Network in Corticospinal Tracts after Infused Mesenchymal Stem Cells in Spinal Cord Injury.

Although limited spontaneous recovery occurs after spinal cord injury (SCI), current knowledge reveals that multiple forms of axon growth in spared axons can lead to circuit reorganization and a detour or relay pathways. This hypothesis has been derived mainly from studies of the corticospinal tract (CST), which is the primary descending motor pathway in mammals. The major CST is the dorsal CST (dCST), being the major projection from cortex to spinal cord. Two other components often called "minor" pathways are the ventral and the dorsal lateral CSTs, which may play an important role in spontaneous recovery. Intravenous infusion of mesenchymal stem cells (MSCs) provides functional improvement after SCI with an enhancement of axonal sprouting of CSTs. Detailed morphological changes of CST pathways, however, have not been fully elucidated. The primary objective was to evaluate detailed changes in descending CST projections in SCI after MSC infusion. The MSCs were infused intravenously one day after SCI. A combination of adeno-associated viral vector (AAV), which is an anterograde and non-transsynaptic axonal tracer, was injected 14 days after SCI induction. The AAV with advanced tissue clearing techniques were used to visualize the distribution pattern and high-resolution features of the individual axons coursing from above to below the lesion. The results demonstrated increased observable axonal connections between the dCST and axons in the lateral funiculus, both rostral and caudal to the lesion core, and an increase in observable axons in the dCST below the lesion. This increased axonal network could contribute to functional recovery by providing greater input to the spinal cord below the lesion.

Pharmacological Difference Between Platelet Aggregations in Cardioembolic Stroke Patients with Direct Oral Anticoagulants: A Pilot Study.

Background Selecting the appropriate direct oral anticoagulants (DOACs) for embolic ischemic stroke patients, especially on concurrent antiplatelet therapy, is important. However, a limited number of studies have reported on the pharmacological differences in platelet aggregation of each DOAC. We aimed to evaluate the antiplatelet effects of selected DOACs, by comparing dabigatran (a direct oral thrombin inhibitor) and factor Xa (FXa) inhibitors (apixaban and rivaroxaban) in patients who had suffered a cardioembolic stroke. Methods We retrospectively evaluated 12 patients diagnosed with a cardioembolic stroke who took any DOAC without an antiplatelet drug and underwent platelet aggregation tests within 60 days from the onset of symptoms. The platelet aggregation tests were analyzed by both light transmission aggregometry and VerifyNow®. Results Six patients (50%) took dabigatran, while the other six (50%) took an FXa inhibitor (n = 4 for apixaban and n = 2 for rivaroxaban). From the light transmission aggregometry analysis, it was found that the maximal extent of aggregation for adenosine diphosphate (ADP) was significantly higher with dabigatran than with FXa inhibitors, and the ED50 value of ADP on platelet aggregation was significantly lower with dabigatran than with FXa inhibitors. Moreover, the VerifyNow® analyses revealed that P2Y12 reaction units were significantly higher with dabigatran than with FXa inhibitors. Conclusions Dabigatran had little impact on platelet aggregation compared to FXa inhibitors in patients who had suffered a cardioembolic stroke with atrial fibrillation, and who took DOACs for secondary prevention within 60 days from the onset.

Repeated intravenous infusion of mesenchymal stem cells for enhanced functional recovery in a rat model of chronic cerebral ischemia.

OBJECTIVE: Stroke is a major cause of long-term disability, and there are few effective treatments that improve function in patients during the chronic phase of stroke. Previous research has shown that single systemic infusion of mesenchymal stem cells (MSCs) improves motor function in acute and chronic cerebral ischemia models in rats. A possible mechanism that could explain such an event includes the enhanced neural connections between cerebral hemispheres that contribute to therapeutic effects. In the present study, repeated infusions (3 times at weekly intervals) of MSCs were administered in a rat model of chronic stroke to determine if multiple dosing facilitated plasticity in neural connections. METHODS: The authors induced middle cerebral artery occlusion (MCAO) in rats and, 8 weeks thereafter, used them as a chronic stroke model. The rats with MCAO were randomized and intravenously infused with vehicle only (vehicle group); with MSCs at week 8 (single administration: MSC-1 group); or with MSCs at weeks 8, 9, and 10 (3 times, repeated administration: MSC-3 group) via femoral veins. Ischemic lesion volume and behavioral performance were examined. Fifteen weeks after induction of MCAO, the thickness of the corpus callosum (CC) was determined using Nissl staining. Immunohistochemical analysis of the CC was performed using anti-neurofilament antibody. Interhemispheric connections through the CC were assessed ex vivo by diffusion tensor imaging. RESULTS: Motor recovery was better in the MSC-3 group than in the MSC-1 group. In each group, there was no change in the ischemic volume before and after infusion. However, both thickness and optical density of neurofilament staining in the CC were greater in the MSC-3 group, followed by the MSC-1 group, and then the vehicle group. The increased thickness and optical density of neurofilament in the CC correlated with motor function at 15 weeks following induction of MCAO. Preserved neural tracts that ran through interhemispheric connections via the CC were also more extensive in the MSC-3 group, followed by the MSC-1 group and then the vehicle group, as observed ex vivo using diffusion tensor imaging. CONCLUSIONS: These results indicate that repeated systemic administration of MSCs over 3 weeks resulted in greater functional improvement as compared to single administration and/or vehicle infusion. In addition, administration of MSCs is associated with promotion of interhemispheric connectivity through the CC in the chronic phase of cerebral infarction.

Possible role of intravenous administration of mesenchymal stem cells to alleviate interstitial cystitis/bladder pain syndrome in a Toll-like receptor-7 agonist-induced experimental animal model in rat.

BACKGROUND: Interstitial cystitis/bladder pain syndrome (IC/BPS) categorized with and without Hunner lesions is a condition that displays chronic pelvic pain related to the bladder with no efficacious treatment options. There are strong associations suggested between Hunner-type IC and autoimmune diseases. Recently, we established an animal model of Hunner-type IC using a Toll-like receptor-7 (TLR7) agonist. Intravenous infusion of mesenchymal stem cells (MSCs) can be used to treat injury via multimodal and orchestrated therapeutic mechanisms including anti-inflammatory effects. Here, we investigated whether infused MSCs elicit therapeutic efficacy associated with the TLR7-related anti-inflammatory pathway in our Hunner-type IC model. METHODS: Voiding behaviors were monitored 24 h prior to the Loxoribine (LX), which is a TLR7 agonist instillation in order to establish a Hunner-type IC model (from - 24 to 0 h) in female Sprague-Dawley rats. LX was instilled transurethrally into the bladder. At 0 h, the initial freezing behavior test confirmed that no freezing behavior was observed in any of the animals. The LX-instilled animals were randomized. Randomized LX-instilled rats were intravenously infused with MSCs or with vehicle through the right external jugular vein. Sampling tissue for green fluorescent protein (GFP)-positive MSCs were carried out at 48 h. Second voiding behavior tests were monitored from 72 to 96 h. After the final evaluation of the freezing behavior test at 96 h after LX instillation (72 h after MSC or vehicle infusion), histological evaluation with H&E staining and quantitative real-time polymerase chain reaction (RT-PCR) to analyze the mRNA expression levels of inflammatory cytokines were performed. RESULTS: Freezing behavior was reduced in the MSC group, and voiding behavior in the MSC group did not deteriorate. Hematoxylin-eosin staining showed that mucosal edema, leukocyte infiltration, and hemorrhage were suppressed in the MSC group. The relative expression of interferon-ß mRNA in the bladder of the MSC group was inhibited. Numerous GFP-positive MSCs were distributed mainly in the submucosal and mucosal layers of the inflammatory bladder wall. CONCLUSION: Intravenous infusion of MSCs may have therapeutic efficacy in a LX-instilled Hunner-type IC rat model via a TLR7-related anti-inflammatory pathway.

Intravenous Infusion of Mesenchymal Stem Cells Promotes the Survival of Random Pattern Flaps in Rats.

BACKGROUND: Surgical reconstruction options of soft-tissue defects often include random pattern skin flaps. Flap survival depends on flap size and rotation arc and can be challenging regarding flap perfusion, leading to wound healing complications, insufficient wound coverage, and even flap loss. Therefore, novel approaches that promote skin flap survival are required. Bone marrow-derived mesenchymal stem cells intravenous infusion is therapeutically effective in various experimental disease models by means of multimodal and orchestrated mechanisms including anti-inflammatory and immunomodulatory effects, and by means of microvasculature reestablishment. METHODS: A modified McFarlane-type rodent skin flap model was used. After skin flap surgery, intravenous infusion of mesenchymal stem cells or vehicle was performed. In vivo optical near-infrared imaging using indocyanine green was performed, followed by histologic analysis, including hematoxylin and eosin and Masson trichrome staining, and gene expression analysis. RESULTS: The flap survival area was greater in the mesenchymal stem cell group. In vivo optical near-infrared perfusion imaging analysis suggested that skin blood perfusion was greater in the mesenchymal stem cell group. Ex vivo histologic analysis demonstrated that the skin structure was more clearly observed in the mesenchymal stem cell group. The dermal thickness was greater in the mesenchymal stem cell group, according to the Masson trichrome staining results. The authors observed a higher expression of fibroblast growth factor 2 mRNA in the tissues of the mesenchymal stem cell group using quantitative reverse-transcription polymerase chain reaction. CONCLUSION: These results suggest that intravenous infusion of bone marrow-derived mesenchymal stem cells promotes skin survival of random pattern flaps, which is associated with increased blood perfusion and higher expression of fibroblast growth factor 2.

Repeated infusion of mesenchymal stem cells maintain the condition to inhibit deteriorated motor function, leading to an extended lifespan in the SOD1G93A rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative fatal disorder in which motor neurons within the brain and spinal cord degenerate. A single infusion of mesenchymal stem cells (MSCs) delays disease progression by protecting motor neurons and restoring the blood-spinal cord barrier in the SOD1G93A transgenic ALS rat model. However, the therapeutic effect of a single infusion of MSCs is transient and does not block disease progression. In this study, we demonstrated that repeated administration of MSCs (weekly, four times) increased the survival period, protected motor functions, and reduced deterioration of locomotor activity compared to a single infusion and vehicle infusion, after which rats displayed progressive deterioration of hind limb function. We also compared the days until gait ability was lost in rats and found that the repeated-infused group maintained gait ability compared to the single-infusion and vehicle-infusion groups. These results suggest that repeated administration of MSCs may prevent the deterioration of motor function and extend the lifespan in ALS.

Intravenous infusion of auto serum-expanded autologous mesenchymal stem cells in spinal cord injury patients: 13 case series.

BACKGROUND: Although spinal cord injury (SCI) is a major cause of disability, current therapeutic options remain limited. Recent progress in cellular therapy with mesenchymal stem cells (MSCs) has provided improved function in animal models of SCI. We investigated the safety and feasibility of intravenous infusion of MSCs for SCI patients and assessed functional status after MSC infusion. METHODS: In this phase 2 study of intravenous infusion of autologous MSCs cultured in auto-serum, a single infusion of MSCs under Good Manufacturing Practice (GMP) production was delivered in 13 SCI patients. In addition to assessing feasibility and safety, neurological function was assessed using the American Spinal Injury Association Impairment Scale (ASIA), International Standards for Neurological and Functional Classification of Spinal Cord (ISCSCI-92). Ability of daily living was assessed using Spinal Cord Independence Measure (SCIM-III). The study protocol was based on advice provided by the Pharmaceuticals and Medical Devices Agency in Japan. The trial was registered with the Japan Medical Association (JMA-IIA00154). RESULTS: No serious adverse events were associated with MSC injection. There was neurologic improvement based on ASIA grade in 12 of the 13 patients at six months post-MSC infusion. Five of six patients classified as ASIA A prior to MSC infusion improved to ASIA B (3/6) or ASIA C (2/6), two ASIA B patients improved to ASIA C (1/2) or ASIA D (1/2), five ASIA C patients improved and reached a functional status of ASIA D (5/5). Notably, improvement from ASIA C to ASIA D was observed one day following MSC infusion for all five patients. Assessment of both ISCSCI-92, SCIM-III also demonstrated functional improvements at six months after MSC infusion, compared to the scores prior to MSC infusion in all patients. CONCLUSION: While we emphasize that this study was unblinded, and does not exclude placebo effects or a contribution of endogenous recovery or observer bias, our observations provide evidence supporting the feasibility, safety and functional improvements of infused MSCs into patients with SCI.

Intravenous Infusion of Mesenchymal Stem Cells Enhances Therapeutic Efficacy of Reperfusion Therapy in Cerebral Ischemia.

OBJECTIVE: Reperfusion therapy is a standard therapeutic strategy for acute stroke. Non-favorable outcomes are thought to partially result from impaired microcirculatory flow in ischemic tissue. Intravenous infusion of mesenchymal stem cells (MSCs) reduces stroke volume and improves behavioral function in stroke. One suggested therapeutic mechanism is the restoration of the microvasculature. The goal of this study was to determine whether infused MSCs enhance the therapeutic efficacy of reperfusion therapy following stroke in rats. METHODS: First, to establish a transient middle cerebral artery occlusion (MCAO) model displaying approximately identical neurologic function and lesion volume as seen in permanent MCAO (pMCAO) at day 7 after stroke induction, we transiently occluded the MCA for 90, 110, and 120 minutes. We found that the 110-minute occlusion met these criteria and was used as the transient MCAO (tMCAO) model. Next, 4 MCAO groups were used to compare the therapeutic efficacy of infused MSCs: (1) pMCAO+vehicle, (2) tMCAO+vehicle, (3) pMCAO+MSC, and (4) tMCAO+MSC. Our ischemic model was a unique ischemic model system in which both pMCAO and tMCAO provided similar outcomes during the study period in the groups without MSC infusion groups. Behavioral performance, ischemic volume, and regional cerebral blood flow (rCBF) using arterial spin labeling-magnetic resonance imaging and histologic evaluation of microvasculature was performed. RESULTS: The behavioral function, rCBF, and restoration of microvasculature were greater in group 4 than in group 3. Thus, infused MSCs facilitated the therapeutic efficacy of MCA reperfusion in this rat model system. CONCLUSIONS: Intravenous infusion of MSCs may enhance therapeutic efficacy of reperfusion therapy.

Intravenous infusion of mesenchymal stem cells delays disease progression in the SOD1G93A transgenic amyotrophic lateral sclerosis rat model.

ALS is a devastating neurodegenerative disease with few curative strategies. Both sporadic and familial ALS display common clinical features that show progressive paralysis. The pathogenesis remains unclear, but disruption of the blood-spinal cord barrier (BSCB) may contribute to the degeneration of motor neurons. Thus, restoration of the disrupted BSCB and neuroprotection for degenerating motor neurons could be therapeutic targets. We tested the hypothesis that an intravenous infusion of MSCs would delay disease progression through the preservation of BSCB function and increased expression of a neurotrophic factor, neurturin, in SOD1G93A ALS rats. When the open-field locomotor function was under 16 on the Basso, Beattie, and Bresnahan (BBB) scoring scale, the rats were randomized into two groups; one received an intravenous infusion of MSCs, while the other received vehicle alone. Locomotor function was recorded using BBB scoring and rotarod testing. Histological analyses, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), were performed. The MSC group exhibited reduced deterioration of locomotor activity compared to the vehicle group, which displayed progressive deterioration of hind limb function. We observed the protection of motor neuron loss and preservation of microvasculature using Evans blue leakage and immunohistochemical analyses in the MSC group. Confocal microscopy revealed infused green fluorescent protein+ (GFP+) MSCs in the spinal cord, and the GFP gene was detected by nested PCR. Neurturin expression levels were significantly higher in the MSC group. Thus, restoration of the BSCB and the protection of motor neurons might be contributing mechanisms to delay disease progression in SOD1G93A ALS rats.

Outcome of an outpatient specialty clinic for chronic epipharyngitis.

OBJECTIVE: In Japan, chronic epipharyngitis became a subject of interest in the 1960s and is currently garnering renewed attention. Previous studies have focused only on the similarities between the immunological characteristics of the tonsil and epipharynx and reported the efficacy of epipharyngeal abrasive therapy (EAT) in patients with IgA nephropathy. However, endoscopic findings of chronic epipharyngitis have not yet been fully evaluated, and. this study aimed to elucidate those findings. METHODS: The study period was from November 2016 to October 2017. Two hundred and twelve new patients visited the specialty outpatient clinic for EAT. Age distribution and mean age of patients, sex, chief complaint, diagnosis at other departments and outcomes were retrospectively reviewed based on medical records. Band-limited light endoscopy was performed, and the findings were videotaped in 102 of the 212 new patients, who underwent endoscopic EAT for the first time. RESULTS: The study included 32 men and 70 women with a mean age of 46.0 years (range, 22-83 years). The most common complaint was postnasal drip (42 patients), followed by pharyngeal pain (12 patients), and throat discomfort (11 patients). The outcomes of 74 patients who continued treatment until the last session were; complete cure in 48.6% of cases, marked improvement in 21.6%, improvement in 16.2%, and no change in 13.5%. Band-limited light endoscopic findings included black spots (73%), granular changes (76%), vessel truncations (92%), crust/mucus adhesion (54%), adenoidal hypertrophy (31%) and tonsil cysts (7%). With regard to the appearance of the mucous membranes, 48% patients had an ivory-like-colored mucous membrane, 72% had a green vascular network, and 89% had a dark red to reddish-brown appearance. Six to nine months' EAT remarkably improved their symptoms with resolution of the endoscopic findings in 86% of the patients. CONCLUSION: Nasal endoscopy using band-limited light is useful for diagnosis and management of chronic epipharyngitis. We believe that this study not only provides information to help the diagnosis of chronic epipharyngitis but also contributes to treat sick patients suffering from chronic epipharyngitis.

Complete Genome Sequence of a Japanese Clinical Isolate of Haemophilus influenzae Type a Strain TAMBA230.

Haemophilus influenzae causes severe infections such as pneumonia and meningitis. Here, we report the complete genome of H. influenzae type a strain TAMBA230, which was isolated in 2019 from a patient exhibiting bacteremia. This represents the first case in Japan of an H. influenzae type a strain associated with invasive infection.