Yusuke Sawada

High Transgene Expression by Lentiviral Vectors Causes Maldevelopment of Purkinje Cells In Vivo

Lentiviral vectors are promising as gene-transfer vehicles for gene therapy targeted to intractable brain diseases. Although lentiviral vectors are thought to exert little toxicity on infected cells, the adverse influence of viral infection on vulnerable developing neurons has not been well studied. Here, we examined whether lentiviral vector infection and subsequent transgene expression affected the morphological and functional maturation of vigorously developing cerebellar Purkinje cells in vivo. Lentiviral vectors expressing GFP under the control of the murine stem cell virus (MSCV) promoter were injected into the cerebellar cortex of neonatal rat pups. Three weeks after treatment, GFP-expressing Purkinje cells were compared with control Purkinje cells from phosphate-buffered saline-injected rats. Analysis of the dendritic tree showed that total dendrite length in GFP-expressing Purkinje cells was almost 80% that in control Purkinje cells. Electrophysiological examination showed that short-term synaptic plasticity at parallel fiber–Purkinje cell synapses and climbing fiber–Purkinje cell synapses was significantly altered in GFP-expressing Purkinje cells. In contrast, maldevelopment of infected Purkinje cells was substantially attenuated when lentiviral vectors with much weaker promoter activity were used. These results suggest that the maldevelopment of Purkinje cells was mainly caused by subsequent expression of a high amount of GFP driven by the strong MSCV promoter. Thus, the use of lentiviral vectors carrying a strong promoter may require particular precautions when applying them to neurological disorders of infants.

Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje neurons to Bergmann glia

Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.

Prediction of extravasation in pelvic fracture using coagulation biomarkers

PURPOSE To evaluate the usefulness of coagulation biomarkers, which are easy and quick to analyze in emergency settings, for prediction of arterial extravasation due to pelvic fracture. PATIENTS AND METHODS The medical records of pelvic fracture patients transferred to the emergency department of Gunma University Hospital between December 2009 and May 2015 were reviewed. Patients were divided into two groups, those with (Extra(+)) and without (Extra(-)) arterial extravasation on enhanced CT or angiography. Levels of fibrin degradation products (FDP), D-dimer, fibrinogen, the ratio of FDP to fibrinogen, the ratio of D-dimer to fibrinogen, systolic blood pressure, heart rate, the Glasgow Coma Scale, pH, base excess, hemoglobin and lactate levels, the pattern of pelvic injury, and injury severity score were measured at hospital admission, and compared between the two groups. Parameters with a significant difference between the two groups were used to construct receiver operating characteristic (ROC) curves. RESULTS The study included 29 patients with pelvic fracture. FDP, D-dimer, the ratio of FDP to fibrinogen and the ratio of D-dimer to fibrinogen were the most useful parameters for predicting arterial extravasation due to pelvic fracture. FDP, D-dimer, the ratio of FDP to fibrinogen, the ratio of D-dimer to fibrinogen, and hemoglobin and lactate levels were significantly higher in the Extra(+) group than in the Extra(-) group (FDP, 354.8μg/mL [median] versus 96.6μg/mL; D-dimer, 122.3μg/mL versus 42.1μg/mL; the ratio of FDP to fibrinogen, 3.39 versus 0.42; the ratio of D-dimer to fibrinogen, 1.14 versus 0.18; hemoglobin, 10.5g/dL versus 13.5g/dL; lactate, 3.5mmol/L versus 1.7mmol/L). The area under the ROC curves for FDP, D-dimer, the ratio of FDP to fibrinogen, the ratio of D-dimer to fibrinogen, hemoglobin and lactate levels were 0.900, 0.882, 0.918, 0.900, 0.815 and 0.765, respectively. CONCLUSION Coagulation biomarkers, and hemoglobin and lactate levels could be useful to predict the existence of arterial extravasation due to pelvic fracture. The ratio of FDP to fibrinogen and the ratio of D-dimer to fibrinogen were the most accurate markers. Coagulation biomarkers may enable more rapid and specific treatment for pelvic fracture.

FDP/fibrinogen ratio reflects the requirement of packed red blood cell transfusion in patients with blunt trauma

Purpose To find factors that predict the requirement of packed red blood cells (pRBC) transfusion in patients with blunt trauma on arrival at the hospital. Methods We conducted blood tests in trauma patients whose trauma severity was suspected as being 3 and over in the Abbreviated Injury Scale. Patients were divided into the blood transfusion (BT) and control groups according to the requirement of pRBC transfusion within 24 h after arrival. Results We analyzed 347 patients (BT group, n = 14; control group, n = 333). On univariate analysis, there were significant differences in Glasgow Coma Scale (GCS), rate of positive FAST (focused assessment with sonography for trauma) finding, hematocrit, international normalized ratio of prothrombin time, activated partial thromboplastin time, fibrinogen (Fib), and level of fibrin degradation products (FDP). On multivariable analysis, positive FAST finding, GCS, Fib, and FDP influenced the requirement of pRBC transfusion. In the area under the receiver operating characteristic curve analysis, Fib and FDP were markers that predicted the requirement of pRBC transfusion. The FDP/Fib ratio had a better correlation with the requirement of pRBC transfusion than FDP or Fib. Conclusions The FDP/Fib ratio can be easily measured and may be a predictor of the need for pRBC transfusion.

Reversible Hypopituitarism Associated with Intravascular Large B-Cell Lymphoma: Case Report of Successful Immunochemotherapy

Intravascular large B-cell lymphoma (IVLBCL) is a rare subtype of diffuse large B-cell lymphoma. There have been only a limited number of reports regarding pituitary dysfunction associated with IVLBCL. We present a 71-year-old woman with hypopituitarism without any hypothalamic/pituitary abnormalities as assessed by magnetic resonance imaging. She presented with edema, abducens palsy, and elevated levels of lactate dehydrogenase and soluble interleukin-2 receptor. Provocative testing showed that the peaks of luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone and adrenocorticotropic hormone were evoked to normal levels by simultaneous administration of luteinizing hormone-releasing hormone, thyrotropin-releasing hormone and corticotropin-releasing hormone, but the responses of these four pituitary hormones showed a delayed pattern. She was diagnosed with IVLBCL with cerebrospinal invasion by pathological findings of the bone marrow, skin, and cerebrospinal fluid. She achieved hematological remission after immunochemotherapy. Pituitary function was also restored without hormonal replacement, and the improvement of the pituitary function was confirmed by dynamic testing. We reviewed the literature with respect to hypopituitarism associated with IVLBCL. There were less than 20 case reports and most of the patients died. Endocrinological course was described in only two cases, and both of them required hormonal supplementation. To our knowledge, this is the first case of hypopituitarism induced by IVLBCL that was successfully managed by immunochemotherapy alone. This case suggests that early diagnosis and treatment of IVLBCL might improve anterior pituitary function and enable patients to avoid hormone replacement therapy.

Can the Wish to Receive Intensive Treatment in Elderly Patients with Respiratory Tract Infection Be Predicted

Objective Almost no Japanese elderly patients have an advance directive (AD). Our aim was to determine whether or not the wish to receive intensive care in elderly patients with respiratory tract infection could be predicted from the prehospital data. Methods In this retrospective study, we reviewed patients ≥65 years of age with respiratory tract infection who had been transferred to our hospital by ambulance between September 2014 and August 2016. The patients were divided into two groups according to whether or not they wished to receive intensive treatment. We placed patients without a wish to receive intensive treatments (WITs) in Group A and patients with a WITs in Group B. We then analyzed parameters that could be determined in the prehospital phase and compared the findings between the groups. Results Thirty-seven patients were in Group A, and 67 patients were in Group B. None of the patients in this study had an AD. There were significant differences in the age, rate of residence in an extended care facility, frequency of inability to care for oneself fully, frequency of dementia, number of prescribed drugs, and Glasgow coma scale (GCS) on a univariate analysis. A logistic regression analysis showed that the inability to care for oneself fully [odds ratio (OR): 4.521, 95% confidence interval (CI): 2.024-10.096, p<0.001] and a low GCS (OR 0.885, 95%CI 0.838-0.935, p<0.001) were related to a WITs. Conclusion Elderly patients who are unable to care for themselves and who have a low GCS in the prehospital stage are likely not to want intensive treatment.

Report of Four Cases with Equestrian Injury: Therapeutic Approach and Outcome

Equestrianism is associated with a risk of severe trauma due to falls and/or direct injury from the horse, depending on the mechanism of injury. This article presents four cases of equestrian injury treated in Gunma University Hospital: Case 1: hepatic injury (fall and kick by the horse); Case 2: left hemopneumothorax and pulmonary contusion with multiple rib fractures (fall and trampling by the horse); Case 3: lumbar compression fracture (fall); and Case 4: scrotum injury (horse bite). Equestrian injuries may be high-energy traumas. Therefore, adhering to relevant primary care guidelines may prevent mortality by trauma.

Post-traumatic hypopituitarism: report of a child case

We report a case of post‐traumatic hypopituitarism in a 9‐year‐old boy who was injured in a car accident.

Study of cerebellar development and regeneration, using the lentiviral vector

KIAA0319 gene is associated with the onset of dyslexia. KIAA0319 encodes a transmembrane protein that is reported to be involved in the regulation of neuronal migration in the developing brain. Because KIAA0319 mRNA is also expressed in the adult brain, it may have other functions than the regulation of neuronal migration. In this study we examined the function of KIAA0319 on neuronal morphology. We found that primary cultured neurons expressing full-length KIAA0319 protein formed complex neurites and fine branches. On the other hand, expression of a mutant KIAA0319 protein that lacked the intracellular region had less effect. It was thus likely that KIAA0319 was involved in the regulation of neuronal morphology, presumably by interacting with a certain intracellular molecule(s). We next performed yeast two-hybrid screening and identified radixin, a member of the ERM (ezrin, radixin, moesin) protein family, as a potential binding partner of KIAA0319. ERM proteins are adapter molecules that link membrane proteins to actin cytoskeletone and involved in the regulation of cellular morphology and movement. Pull-down experiments indicated that radixin specifically bound to the intracellular region of KIAA0319. We are now trying to clarify the physiological significance of the interaction in primary cultured neurons, which may lead to better understanding of the molecular mechanisms involved in dyslexia.