A variety of gene transfer strategies have been developed to treat inherited, degenerative, and acquired diseases. Among the different vector systems developed so far, recombinant adeno-associated viral (AAV) vectors have shown notable benefits, including prolonged gene expression, transduction of both dividing and nondividing cells, and a lack of pathogenicity caused by wild-type infections. Thanks to these features, the use of AAV vectors as a gene transfer tool has increased dramatically during the past several years, and several recent clinical trials have used AAV vectors. However, AAV vectors are more complicated to produce than are other viral vectors. With steady advances toward clinical application, much effort has been made to isolate novel AAV serotypes and to develop methods for their efficient, scalable, and versatile production and purification. Here we review state of the art methods for AAV vector production and purification, which we have refined in our laboratory.

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The Department of Respiratory Medicine of Nippon Medical School Hospital and the Working Committee of Clinical Simulation Laboratory have held training sessions for chest tube drainage since 2007. The training program consists of the preparation of a training manual, a small-group session, and a review of the process of chest tube drainage using a checklist of steps after the session. A total of 21 medical interns of Nippon Medical School Hospital participated in training sessions from April 2010 through February 2011. A questionnaire survey at the end of the session revealed that most participants rated highly both the explanations given by the instructors and the descriptions in the manual for comprehensibility. Only 3 interns felt that they had successfully acquired the clinical skill, and the other 18 interns felt that they had somewhat acquired the skill. Research after the interns had completed the program of the department showed that 80% of interns had performed chest tube drainage for patients during the rotation. The interns assessed the training program as useful, and some interns felt they could perform the skill with confidence or without anxiety. Other systematic programs of skill training for medical interns are recommended to ensure definite acquisition of basic skills.

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Background: Sleep-disordered breathing (SDB) induces nighttime disturbance of arterial gases, such as carbon dioxide. However, it is still unclear whether nighttime SDB-related gas abnormality is related to respiratory dysregulation in daytime. Therefore, we examined the relationship between the arterial partial pressure of carbon dioxide (PaCO₂) at nighttime and the respiratory response to exercise in daytime.
Methods: Eighteen men (age, mean ± SD; 55 ± 11 years) with heart disease underwent multichannel respiratory monitoring through the night with transdermal measurement of PaCO₂ (PtcCO₂) reflecting PaCO₂ and a cardiopulmonary exercise test in daytime. The ventilatory equivalent (VE)/carbon dioxide production (VCO₂) slope as an index of ventilatory response to exercise and peak oxygen consumption (VO₂) were obtained with a cardiopulmonary exercise test.
Results: Of the 18 patients, 10 patients had obstructive SDB, 5 had central SDB, and 3 patients did not have SDB. The mean apnea-hypopnea index was 21 ± 17. Minimum nighttime saturation of O₂ was positively correlated with peak VO₂, but not with VE/VCO₂. Nighttime PtcCO₂ was not correlated with peak VO₂ but was negatively correlated with the VE/VCO₂ slope of the daytime cardiopulmonary exercise test (r=-0.53).
Conclusion: Nighttime lowering of PaCO₂ in SDB is related to an abnormal ventilatory response to exercise testing in the daytime. This finding suggests that nighttime hyperventilation in SDB alters both nighttime and daytime pathophysiological conditions in patients with heart disease.

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Background: Radical prostatectomy (RP) for localized prostate cancer after transurethral resection of the prostate (TUR-P) is technically difficult because of periprostatic adhesion and changes in the form of the prostate. The advantages of laparoscopic RP (LRP) over retropubic RP (RRP) include a less invasive operation through a small wound, a clearer field of vision, and reduced blood loss, and, therefore, LRP may represent the optimal method for RP after TUR-P. The present study compared clinical, oncological, and pathological outcomes between LRP and RRP after TUR-P at our institution.
Methods: Twenty patients underwent TUR-P for benign prostatic hyperplasia, followed by LRP (12 patients) or RRP (8 patients) after localized prostate cancer was diagnosed at our institution from November 1998 through December 2006. Median patient age was 67.5 years (range, 52-73 years). The median duration of follow-up was 96 months (range, 60-144 months). Operative time, volume of blood loss, duration of indwelling urethral catheter use, degree of urinary incontinence, pathological findings, oncological outcomes, and complications were compared between LRP and RRP.
Results: No significant difference in operative time or amount blood loss was recognized between LRP and RRP. Urinary incontinence in the early and late postoperative periods was significantly more severe after LRP than after RRP. Oncological outcomes and results of pathological examinations were comparable between LRP and RRP.
Conclusion: We found no significant differences in clinical, pathological, and oncological outcomes, except for urinary incontinence, between LRP and RRP.

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Background: The augmentation index (AIx) indicates arterial wave reflection. Clinical studies have shown a relationship between an elevated AIx and cardiovascular disease. This cross-sectional study attempted to clarify the clinical significance of AIx in patients with preserved kidney function.
Patients and Methods: The subjects were 321 patients with preserved kidney function (an estimated glomerular filtration rate ≥60 mL/min/1.73 m2 and normoalbuminuria) but with no history of cardiovascular events. The AIx was determined in the radial artery by means of tonometry, and the relationships of the AIx to kidney function and markers of atherosclerosis were examined.
Results: A significant positive correlation (r=0.30; p<0.001) was found between the AIx and the urinary albumin concentration. The AIx showed a significant positive correlation (r=0.28; p<0.001) with the serum high-sensitivity C-reactive protein concentration, as a marker of inflammation; with the urinary 8-iso-prostaglandin F2α concentration (r=0.31; p<0.001), as a marker of oxidative stress; and with the cardio-ankle vascular index (r=0.17; p<0.01), as a marker of systemic arterial stiffness. Multiple regression analysis indicated that the urinary 8-iso-prostaglandin F2α concentration (t=5.1; p<0.001), serum high-sensitivity C-reactive protein concentration (t=4.9; p<0.001), and urinary albumin concentration (t=3.6; p<0.01) were independent variables for AIx after adjustment.
Conclusion: These findings indicate that the AIx reflects inflammation, oxidative stress, and the urinary albumin concentration in patients with preserved kidney function.

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Lumbar puncture is a medical technique that physicians must learn and is, therefore, considered a basic medical procedure. The lumbar puncture simulators Lumbar-Kun (Lumbar Puncture Simulator) and Lumbar-Kun II (Lumbar Puncture Simulator II) (Kyoto Kagaku, Kyoto, Japan) are teaching aids designed for practicing spinal insertions. We describe and results of a lumbar puncture clerkship course, provided to 5th-year medical students during clinical clerkship activity. The aim of this study was to evaluate the effectiveness of the lumbar puncture clerkship course in the medical education program. Comprehension, technical achievement, and satisfaction were scored by students and instructors using a 6-point Likert scale. Scores for both comprehension and technical achievement were high, but technical achievement scores tended to be higher than comprehension scores. In addition, the scores students gave themselves were higher than the scores they were given by instructors. Student satisfaction was high. The lumbar puncture simulators, Lumbar-Kun and Lumbar-Kun II, achieved excellent overall impressions and represent useful tools for training in lumbar puncture procedures. In addition to the simulators, an appropriate preparatory text and a short lecture before training seemed to increase the educational effect of this lumbar puncture clerkship course for medical students.

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Background: As society ages, early detection of dementia is becoming increasingly important. Many hospitals have opened memory-loss clinics, and various new approaches for early examination and appropriate diagnosis are being tried. However, these memory-loss clinics are ultimately part of the hospital establishment, implying that, in addition to the burdens of time and effort to undergo examinations and consultations, patients might have a certain psychological resistance. With a grant from the Ministry of Education, Culture, Sports, Science and Technology, the Institute of Development and Aging Science at Nippon Medical School has opened a center called the Community Consultation Center for Citizens with Mild Cognitive Impairment and Dementia, which is a dementia-related center outside the regular hospital. This center has been developing a system that makes consultations easier. We performed a retrospective follow-up study that aimed to determine how much this approach contributes to the early detection of dementia compared with outpatient visits to university hospitals.
Methods: Persons who were found to have organic brain syndrome (defined as organic diseases related to dementia, including mild cognitive impairment) after visiting the Consultation Center during the survey period were referred to as the Consultation Center group, and persons who were found to have organic brain syndrome after an initial visit to the Department of Neurology at Nippon Medical School Musashi Kosugi Hospital were referred to as the Hospital group. We compared the groups in terms of sex, age, Mini-Mental State Examination (MMSE) score, and subclassification by means of the t-test and χ² test.
Results: Both the mean MMSE score (p<0.001) and percentage of subjects with an MMSE score of 24 points of higher (p=0.007) were significantly higher in the Consultation Center group than in the Hospital group.
Conclusions: Consultations can be made more casually at the Consultation Center than at hospitals. Our results suggest that more casual consultations contribute to the early detection of dementia.

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Adipose-derived stem cells (ASCs) are a promising cell source and are being investigated for a variety of therapeutic applications. However, standard expansion protocols use fetal bovine serum (FBS) as a growth factor supplement, which is a potential source of undesirable xenogeneic pathogens. For clinical safety, autologous human serum (HS) would be more appropriate. This study compared FBS-supplemented and HS-supplemented media for their enhancement of the proliferation and differentiation potential of human ASCs (hASCs). HS was obtained from the blood of 8 healthy volunteers using collection devices specially designed to derive growth factors from platelets. Growth factors in HS or FBS were measured with enzyme-linked immunosorbent assays. The hASCs were isolated with an established protocol from discarded human fat tissues obtained during a medical procedure and cultured in a medium supplemented with either 10% HS or 10% FBS. The hASCs were collected at several time points for the proliferation assays. The capacity for differentiation into the osteogenic, chondrogenic and adipogenic lineages was assessed qualitatively with the histochemical stains von Kossa, Alcian blue, and Oil red O, respectively, and quantitatively with the qualitative reverse transcriptase polymerase chain reaction. Differences in cell surface marker expression between the HS-supplemented and FBS-supplemented cultures were examined with flow cytometric analysis. Proliferation assays showed that the growth of hASCs was more rapid in HS-supplemented medium than in FBS-supplemented medium. All cells grown in each medium expressed similar patterns of cell surface markers. The ASCs cultured in the HS-supplemented medium proliferated more rapidly than those cultured in the FBS-supplemented medium and retained their differentiation capacity and immunophenotype. These results support the establishment of a safe and rapid expansion protocol with autologous serum for cell-based therapies, such as tissue engineering and regenerative medicine, using hASCs.

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Background: It is important to identify and immunize susceptible healthcare workers to prevent and control hospital infections. Our aim was to evaluate the specific antibodies against the measles, mumps, and rubella viruses and the varicella zoster virus among healthcare workers in a tertiary-care hospital.
Patients and Methods: A total of 284 healthcare workers (89 men and 195 women; mean age, 33.5 ± 11 years), including 111 nurses, 87 physicians, 34 laboratory technicians, and 52 members of the housekeeping staff, of Van Training and Research Hospital were enrolled in this study. Antibodies were detected with an enzyme-linked immunosorbent assay.
Results: The numbers of workers with serological susceptibility to mumps, measles, rubella, or chicken pox were 26 (9.2%), 18 (6.3%), 7 (2.5%), and 5 (1.8%), respectively. Although the difference was not statistical significant, the rate of seroprevalence of antibodies was lowest for measles (90.8%; p>0.05). Susceptibility to measles, mumps, and rubella, and chicken pox was more prevalent among young healthcare workers (p<0.001). Not all healthcare workers born before 1957 were immune to these vaccine-preventable diseases.
Conclusion: These data confirm that screening and vaccination of susceptible healthcare workers is essential regardless of age.

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The annual incidence of pancreatic carcinoma has been increasing worldwide, and the overall 5-year survival rate has remained at approximately 5%. We re-evaluated 30 autopsy cases histologically diagnosed as pancreatic carcinoma from 1994 through 2010 at Nippon Medical School Hospital. The mean patient age was 69.5 years, with no significant differences between male and female patients. The location of the primary tumor was most often the head of the pancreas (46.7%), followed by the body (36.7%) and tail (16.7%). All patients had advanced-stage pancreatic carcinoma at diagnosis, which limited the therapeutic options. Surgical resection, radiation, and surgical resection with chemotherapy were each performed for a single patient, and chemotherapy was performed for 5 patients. The other patients received only symptomatic therapy. The mean survival time from the first medical examination to death was short (5.5 months; range, 1-40 months). The cases were classified into 28 ductal adenocarcinomas, 1 acinar cell carcinoma, and 1 intraductal papillary mucinous neoplasm (IPMN) with an associated invasive carcinoma. Death in most cases was directly related to the pancreatic carcinoma, including cachexia, carcinomatous peritonitis and pleuritis, hepatic failure and ileus due to metastasis, and malignancy-related disorders, such as coagulation disorders and immunodeficiency. The most frequent site of metastasis was the lymph nodes, followed by the liver, peritoneum, spleen, lung and/or pleura, small intestine, adrenal gland, kidney, omentum, diaphragm, and bone. We classified the autopsy cases as showing distant metastasis or local infiltration. All cases with local infiltration were located in the pancreatic head, but no difference was seen in other clinicopathological features between cases with local infiltration and cases with distant metastasis. Thus, the autopsies revealed an extremely poor prognosis for pancreatic carcinoma due to the tumor itself and malignancy-related disorders. The progression pattern (i.e., local infiltration or distant metastasis) may correlate with the location of the primary tumor.

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Splenic cysts are rare, and their treatment remains challenging. A 66-year-old man scheduled to undergo surgical treatment for a pulmonary neoplasm was found with abdominal computed tomography and ultrasonography to have multiple cysts in the body of the spleen. He underwent pulmonary wedge resection, and histological examination showed that the lesion of the left lung was an adenocarcinoma. The patient recovered without complications after the operation. Because the splenic cysts were small and caused no abdominal symptoms, the patient was advised to undergo careful follow-up. Large splenic cysts warrant surgical treatment, whereas careful follow-up is recommended for small asymptomatic splenic cysts.

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Congenital cytomegalovirus (CMV) infection can cause severe permanent disabilities. A mother who is seronegative before conception but acquires infection during pregnancy is a risk factor for congenital infection. We describe a neonate in whom congenital CMV infection was diagnosed at birth and confirmed with DNA quantitation by means of the polymerase chain reaction, was accompanied by cerebral ventriculomegaly and severe hearing loss, and was treated with ganciclovir/valganciclovir for 6 weeks. Initially, cerebral ventriculomegaly and calcification were also found with computed tomography, and severe hearing loss was detected with auditory brainstem response testing. After treatment, CMV DNA decreased in copy number and became undetectable. No marked side effects occurred after treatment. Surprisingly, 1 year after treatment, neurological and motor development was equivalent to that in a healthy infant. Audiometry indicated that auditory ability would improve with rehabilitation, speech and language therapy, and cochlear implantation. Single-photon emission computed tomography showed marked improvement 6 months after treatment. This case provides compelling evidence that a reliable diagnosis of congenital CMV infections coupled with a prompt and appropriate treatment program can prevent permanent disability. It is, therefore, important to establish a more effective strategy for the management of congenital CMV infection.

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We report on a patient with an unusual, slowly enlarging hematoma of the left buttock. A 62-year-old man presented with a 6-year history of an enlarging mass of the left buttock. He had first noted the mass 6 years earlier and had had sciatica of the left lower limb for the last 2 months. He denied any history of antecedent trauma. The lesion extended over 3 gluteal muscles (the gluteus medius, gluteus minimus, and piriformis). On microscopic examination, the lesion showed typical signs of chronic expanding hematoma. The sciatica was relieved after surgical removal of the lesion. The lesion had not recurred at the last follow-up 4 years after the operation. The present case suggested that chronic expanding hematoma can extend into multiple muscles due, perhaps, to long-term growth and the anatomical and functional conditions of the affected muscles. Our case also suggests that chronic expanding hematoma can be a cause of piriformis syndrome.

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A 44-year-old woman had sustained facial lacerations, loss of several teeth, alveolar bone fracture, and severe vertical bone resorption in conjunction with a shattered alveolar bone and marked loss of intraoral function and facial disfigurement in a car accident. She underwent 9 surgical procedures by plastic surgeons to treat the facial lesions, including hold facial scar formation reduction surgery, reduction of fractures of the nasal and cheek bones, and bone transplants. To restore intraoral function and improve facial appearance, plastic surgeons transplanted a rib into the maxillary sinus while dental surgeons simultaneously inserted 5 dental implants. Eleven months after the dental implant surgery, a complete upper denture and a mandibular gingival ceramometal-casting crown were fitted. After insertion of the final prosthodontics, regular follow-up examinations were performed to check dental occlusion, oral hygiene, and the condition of the gingival tissue. In the years since the upper dental implants were fitted, there have been no bone resorption and no functional problems. Transplantation of rib bones is an effective method for maxillary reconstruction and remains effective even after the insertion of dental implants. The patient is extremely satisfied with the results. A means of maintaining oral health over the long term, and of motivating the patient to maintain oral health, should be established.

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L-asparaginase (L-Asp) is an important reagent for acute lymphoblastic leukemia because asparagine is required for the malignant growth of tumor cells, especially lymphoblastic leukemia cells. An allergic response to L-Asp is not unusual because L-Asp is derived from Escherichia coli and is often recognized as a foreign protein. The hypersensitivity induced by L-Asp is of the immediate type in most cases. We report on a 5-year-old girl who was hospitalized for precursor T-cell lymphoblastic leukemia. She was treated according to a Tokyo Children's Cancer Study Group protocol (TCCSG ALL L09-1603 HEX/BFM). During the intensification phase, blisters with erythema developed on the arm proximal to the catheter insertion site owing to a delayed-type hypersensitivity reaction caused by intravenous L-Asp administration. She was treated with additional methylprednisolone, tapered dexamethasone, and an antihistamine for the allergic reaction. No asparaginases other than E. coli L-Asp have been approved for use in Japan. Other asparaginases, such as polyethylene glycol L-Asp and Erwinia L-Asp should be quickly approved for use as alternative chemotherapy reagents in Japan.

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