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Competing Interests: The author has no conflict of interest to declare.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis caused by variants in COL7A1-encoded type VII collagen, a major component of anchoring fibrils. In this study, we developed an ex vivo gene therapy for RDEB using autologous mesenchymal stromal cells (MSCs). On the basis of our previous studies, we first attempted to isolate MSCs from the blister fluid of patients with RDEB and succeeded in obtaining cells with a set of MSC characteristics from all 10 patients. We termed these cells blister fluid-derived MSCs. Blister fluid-derived MSCs were genetically modified and injected into skins of type VII collagen-deficient neonatal mice transplanted onto immunodeficient mice, resulting in continuous and widespread expression of type VII collagen at the dermal-epidermal junction, particularly when administered into blisters. When injected intradermally, the efforts were not successful. The gene-modified blister fluid-derived MSCs could be cultured as cell sheets and applied to the dermis with an efficacy equivalent to that of intrablister administration. In conclusion, we successfully developed a minimally invasive and highly efficient ex vivo gene therapy for RDEB. This study shows the successful application of gene therapy in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions., (Copyright © 2023. Published by Elsevier Inc.)

Competing Interests: None.

Competing Interests: None.

Competing Interests: Dr Ozaki reported personal fees from Medical Network Systems Inc. and 10.13039/501100004095Kyowa Kirin co. ltd. outside the submitted work. Dr Tanimoto reported personal fees from Medical Network Systems Inc. and Bionics co. ltd., outside the submitted work. No other disclosures were reported.

Competing Interests: The author declare that there is no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Competing Interests: Dr Ozaki reported personal fees from Medical Network Systems Inc. and Kyowa Kirin co. ltd. outside the submitted work. Dr Tanimoto reported personal fees from Medical Network Systems Inc. and Bionics co. ltd., outside the submitted work. No other disclosures were reported.

The relationship between age-related brain atrophy and long-chain polyunsaturated fatty acid (LCPUFA) intake is not fully understood. This study investigated the association of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) intake and brain atrophy in non-demented older Japanese people (n = 810, aged 60-89 years) using data sets of a 2-year longitudinal study. Brain volumes were measured using 3D-MRI in the baseline and follow-up periods. The associations of multivariate-adjusted changes in brain volumes with baseline LCPUFA intake were assessed using a general linear model. Higher ARA intake was associated with a smaller decrease in frontal cortex volumes, which was accompanied by a lower risk of cognitive decline among the participants. In the subgroup analysis for low DHA and EPA intake, accounting for one-third of Japanese intake, DHA and EPA intake was positively correlated with preservation of the temporal cortex volume. These findings suggest that appropriate intake of LCPUFA may decelerate age-related brain atrophy and lead to the maintenance of brain health in older people., (Copyright © 2022. Published by Elsevier Inc.)

Immunity-related GTPases (IRGs), also known as p47 GTPases, are a family of interferon-inducible proteins that play roles in immunity defense against intracellular pathogens. Although the molecular functions of IRGs have been well studied, the function of the family member, IRGC1, remains unclear. IRGC1 is unique among IRGs because its expression is not induced by interferon and it is expressed predominantly in the testis. Further, IRGC1 is well conserved in mammals unlike other IRGs. Here, we knocked out (KO) Irgc1 in mice using the CRISPR/Cas9 system and found that the fertility of Irgc1 KO males was severely impaired because of abnormal sperm motility. Further analyses with a transmission electron microscope revealed that the fibrous sheath (FS), an accessory structure of the sperm tail, was disorganized in Irgc1 KO mice. In addition, IRGC1 was detected in the sperm tail and fractionated with FS proteins. These results suggest that IRGC1 is a component of the FS and is involved in the correct formation of the FS., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development., (Copyright © 2019. Published by Elsevier Inc.)

Background: Subcellular localization of a photosensitizer is known to determine the therapeutic efficacy of photodynamic therapy (PDT). Cell membrane is an optimal target that promises an effective treatment outcome., Objectives: We previously developed a novel photosensitizer named porphyrus envelope (PE) by combining hemagglutinating virus of Japan envelope (HVJ-E) with lipidated protoporphyrin IX (PpIX lipid). In the current study, the cellular localization of PE and its ability to induce multiple anti-tumor effect were characterized., Materials and Methods: The localization and uptake of PpIX lipid in cells were evaluated with confocal laser scanning microscopy and a cell-based fluorescent assay, respectively. The ability of PE to suppress the migration and proliferation of cancer cells was assessed using a scratch-wound assay. The synergistic effect of PDT and HVJ-E treatment was evaluated using an in vitro experiment with PC-3 cells., Results: PE localized along the cell membrane and PpIX lipid accumulated selectively in the prostate cancer cells within 10min. Also, PE maintained the ability to undergo fusion and induce cancer cell death even after light irradiation at the dose for PDT. Incubation with PE resulted in delayed migratory and proliferative activity of PC-3 cells. PE-mediated PDT was twice as effective when cells were further incubated with PE following PDT., Conclusions: PE allows rapid drug delivery targeting the cell membrane. Because the cytotoxicity of HVJ-E was maintained, synergistic effect of HVJ-E and the photochemical reactions resulted in highly effective killing of prostate cancer cells in vitro and thus represents a promising treatment for prostate cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study aimed to assess the suitability of luteal blood flow analyses measured by color Doppler ultrasonography (CDUS), to predict pregnancy at pre- and post-embryo transfer (ET) in dairy cows, and to compare with the established criterion like luteal size and plasma progesterone (P4) concentrations. Lactating Holstein cows (n = 65) with spontaneous (n = 34) or synchronized estrus (n = 31) were examined. Cows with a CL greater than or equal to 20 mm in diameter (n = 58) received embryo transfer on Day 7 (Day 0 = estrus). Brightness mode images were captured for calculation of the CL area, luteal cavity area, and dominant follicle area on Days 3, 5, 7, and 14. Color Doppler ultrasonography examinations were conducted to determine the blood flow area (BFA) within the CL at the maximum diameter and the time-averaged maximum velocity (TAMV) of the base of the spiral artery on the same days. Plasma P4 concentrations were determined from blood samples collected at each ultrasound examination. Pregnancy was diagnosed by an ultrasound on Day 30. There was no significant difference in the proportion of cows received embryo (91.2% vs. 87.1%, P = 0.70) and pregnancy rate (58.1% vs. 59.3%, P = 1.00) between the spontaneous estrus and synchronized groups. The BFA values of the pregnant group (n = 34) were approximately 1.42 and 1.54 times higher than those of the nonpregnant group (n = 24) on Days 7 (0.54 ± 0.04 cm(2) vs. 0.38 ± 0.02 cm(2); P < 0.01) and 14 (0.80 ± 0.23 cm(2) vs. 0.52 ± 0.22 cm(2); P < 0.01), respectively. The TAMV of the pregnant group was approximately 1.45 times higher than that of the nonpregnant group on Day 14 (57.8 ± 3.5 cm/s vs. 40.0 ± 3.3 cm/s; P < 0.01). However, no differences were found in the CL area, CL tissue area, dominant follicle area, and plasma P4 concentrations among these groups. In addition, the best logistic regression model to predict pregnancy included scores for BFA on Day 7, BFA and TAMV on Day 14. Setting the cutoff value of BFA at 0.43 cm(2) yielded the highest sensitivity (79.4%) and specificity (75.0%) on Day 7, indicating the effectiveness of using BFA data for predicting pregnancy on Day 7. Furthermore, setting the cutoff value at one obtained from a sample with BFA 0.63 cm(2) and TAMV 50.60 cm/s yielded the highest sensitivity (85.3%) and specificity (91.7%) on Day 14. In conclusion, the evaluations of BFA on Day 7, and paired BFA and TAMV on Day 14 represent reliable predictors of pregnancy in the cow., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: In the clinic, it is often very difficult to treat drug-resistant advanced prostate cancer by conventional treatments. Photodynamic therapy (PDT) is a minimally invasive treatment that takes advantage of photochemical reactions between a photosensitizer and light. On the basis of several of its key characteristics, PDT is considered to be a promising novel method for treating drug-resistant prostate cancer., Objectives: For effective treatment of drug-resistant prostate cancer, we developed a novel agent termed porphyrus envelope, which was produced from PpIX lipid and hemagglutinating virus of Japan envelope (HVJ-E)., Materials and Methods: We inserted PpIX lipid into HVJ-E by centrifugation, and used the resultant porphyrus envelope in PDT of two drug-resistant prostate cancer cell lines, PC-3 and PC-3-DR., Results: Porphyrus envelope enhanced uptake of PpIX, and cytotoxicity of PDT, relative to free PpIX lipid or PpIX induced by 5-ALA., Conclusion: PDT using porphyrus envelope has potential as a method for treating drug-resistant prostate cancer., (Copyright © 2013. Published by Elsevier B.V.)

We report a case involving a ruptured acute type B aortic dissection originating from an aberrant right subclavian artery (ARSA). A thoracic stent-graft was deployed in the distal arch close to the origin of the ARSA; the entry site at the origin of the ARSA was embolized with metallic coils. Perfusion of the left subclavian artery was preserved without a surgical bypass by using a chimney graft. This procedure is a feasible and less invasive treatment for high-risk sternotomy patients and is an effective strategy for acute aortic dissections involving an ARSA., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

We report 2 cases of distal arch aneurysm treated by thoracic endovascular aneurysm repair (TEVAR) with the "double-chimney technique." This technique permitted the implantation of a thoracic stent graft in the ascending aorta over the arch branches while preserving perfusion of innominate and left common carotid arteries without debranching bypasses. The procedure is a feasible and less invasive treatment for distal arch aneurysm with a short proximal neck (<2 cm to the origin of the innominate artery) in patients at high risk when undergoing sternotomy and in emergent cases., (Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: The Lichtenstein inguinal hernia repair is commonly performed and suitable for teaching basic surgical skills. The objective of this study is to evaluate the feasibility of this procedure for surgical training, particularly in regard to patient outcomes., Design: Retrospective case review after introduction of an integrated teaching program., Setting: University teaching hospital., Participants: The Lichtenstein inguinal hernia repair is the standard procedure for adult primary unilateral inguinal hernia since 2003 at Jichi Medical University. We introduced an integrated teaching system of lectures, skill training. and videos to teach the skills for Lichtenstein inguinal hernia repair to residents and junior faculty in 2003. Cases were retrospectively divided into 4 groups based on the experience of the operating surgeon; junior residents (PGY 1-2, group A), senior residents (PGY 3-5, group B), junior faculty (PGY 6-10, group C), and senior faculty (PGY 11 or more, group D). Background, perioperative factors, and outcomes were evaluated among the groups., Results: A total of 246 elective inguinal hernia repairs (group A: 136, group B: 49, group C: 42, group D: 19) were performed. There was a significant difference in the frequency of concomitant diseases (p = 0.012) and anticoagulant therapy (p = 0.031). Average operating time was 80.7 ± 24.9, 72.6 ± 20.8, 63.5 ± 22.0, and 54.7 ± 27.9 (min ± SD) in groups A, B, C, and D, respectively, with a significant difference between groups A and D (p < 0.001). No significant differences were observed in estimated blood loss (p = 0.216) or morbidity (p = 0.294)., Conclusions: The Lichtenstein inguinal hernia repair can be safely performed by residents and junior faculty with the appropriate supervision of senior faculty without any disadvantage to patients. This integrated teaching program for Lichtenstein inguinal hernia repair is effective and feasible for training residents and junior faculty., (Copyright © 2012 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse model compared to the BCG. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality. The BCG prime and HSP65 + IL-12/HVJ vaccine (boost) by the prime-boost method showed a synergistic prophylactic effect in the monkey. Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys.HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the ESR, and augmented the immuneresponses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials., (Copyright © 2011 Published by Elsevier B.V.)

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). Furthermore, the BCG priming and HSP65 + IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials., (Copyright © 2010 Elsevier B.V.)

Recent studies have shown that skin injury recruits bone marrow-derived fibroblasts (BMDFs) to the site of injury to accelerate tissue repair. However, whether uninjured skin can recruit BMDFs to maintain skin homeostasis remains uncertain. Here, we investigated the appearance of BMDFs in normal mouse skin after embryonic bone marrow cell transplantation (E-BMT) with green fluorescent protein-transgenic bone marrow cells (GFP-BMCs) via the vitelline vein, which traverses the uterine wall and is connected to the fetal circulation. At 12 weeks of age, mice treated with E-BMT were observed to have successful engraftment of GFP-BMCs in hematopoietic tissues accompanied by induction of immune tolerance against GFP. We then investigated BMDFs in the skin of the same mice without prior injury and found that a significant number of BMDFs, which generate matrix proteins both in vitro and in vivo, were recruited and maintained after birth. Next, we performed E-BMT in a dystrophic epidermolysis bullosa mouse model (col7a1(-/-)) lacking type VII collagen in the cutaneous basement membrane zone. E-BMT significantly ameliorated the severity of the dystrophic epidermolysis bullosa phenotype in neonatal mice. Type VII collagen was deposited primarily in the follicular basement membrane zone in the vicinity of the BMDFs. Thus, gene therapy using E-BMT into the fetal circulation may offer a potential treatment option to ameliorate genetic skin diseases that are characterized by fibroblast dysfunction through the introduction of immune-tolerated BMDFs.

Over the past decade, many approaches to transferring genes into the skin have been investigated. However, most such approaches have been specifically aimed against genodermatosis, and have not produced sufficient results. The goal of such research is to develop a method in which genes are transferred easily, efficiently and stably into keratinocytes, especially into keratinocyte stem cells, and in which the transgene expression persists without a reaction from the host immune response. Although accidental development of cancer has occurred in trials of gene therapy for X-linked severe combined immunodeficiency (X-SCID), resulting in slowing of the progress of this research, the lessons of these setbacks have been applied to further research. Moreover, combined with the techniques acquired from tissue engineering, recent developments in our knowledge about stem cells will lead to new treatments for genodermatoses. The present review summarizes the methods by which therapeutic genes can be transferred into keratinocytes, with discussion of how gene transfer efficiency can be improved, with particular emphasis on disruption of the skin barrier function. It concludes with discussion of the challenges and prospects of keratinocyte gene therapy, in terms of achieving efficient and long-lasting therapeutic effects.

Data from several studies suggest that the ubiquitin-proteasome system may play a role in the progression of atherosclerosis. Here, we examined the potential role of the deubiquitinating enzyme CYLD (cylindromatosis), mutation of which has been reported to cause familial cylindromatosis. Northern blot analysis revealed expression of CYLD mRNA in the aorta, as well as in cultured human aortic endothelial cells (ECs) and vascular smooth muscle cells. Treatment with recombinant tumor necrosis factor (TNF)-alpha significantly increased CYLD expression in ECs and vascular smooth muscle cells. Immunostaining showed CYLD expression in atherosclerotic lesions from human carotid arteries and up-regulation of CYLD expression in the neointima of rat carotid arteries after balloon injury. Overexpression of CYLD in ECs resulted in inhibition of TNF-alpha-induced nuclear factor-kappaB activity through deubiquitination of TNFR-associated factor 2 (TRAF2), whereas overexpression of catalytically inactive CYLD had no effect. CYLD overexpression also inhibited expression of cyclin D1 and activation of the E2F pathway through deubiquitination of the upstream molecule Bcl-3 and inhibition of its translocation into the nucleus. Overexpressed CYLD also significantly inhibited cell viability. Furthermore, overexpression of CYLD in rat balloon-injured carotid artery attenuated neointimal formation through inactivation of nuclear factor-kappaB and E2F. In conclusion, these data demonstrate that the deubiquitinating enzyme CYLD may inhibit inflammation and proliferation in vascular cells and may represent a novel target for the treatment or prevention of atherosclerosis.

The development of more effective cancer treatments is anticipated. Tumor-targeted drug delivery is an important strategy in cancer therapy. We have developed an HVJ (hemagglutinating virus of Japan; Sendai virus) envelope (HVJ-E) vector using inactivated Sendai virus. The HVJ-E vector has been observed to target a number of cell lines since its hemagglutinin-neuraminidase (HN) protein recognizes the sialic acids of host cells. Thus, to reduce non-specific binding of the HVJ-E vector, we eliminated HN protein using HN-specific short interfering RNA (siRNA). Then, to further increase its tumor-targeting ability, we constructed HN-depleted HVJ containing the F-transferrin chimeric protein. The modified vectors containing Q-dots demonstrated 32-fold greater tumor-targeting efficiency than wild-type HVJ-E.

gamma-Secretase cleaves the transmembrane domains of several integral membrane proteins involved in vasculogenesis. Here, we investigated the role of gamma-secretase in the regulation of postnatal angiogenesis using gamma-secretase inhibitors (GSI). In endothelial cell (EC), gamma-secretase activity was up-regulated under hypoxia or the treatment of vascular endothelial growth factor (VEGF). The treatment of GSI significantly attenuated growth factor-induced EC proliferation and migration as well as c-fos promoter activity in a dose-dependent manner. In vascular smooth muscle cell (VSMC), treatment of GSI significantly attenuated growth factor-induced VEGF and fibroblast growth factor-2 (FGF-2) expression. Indeed, GSI attenuated VEGF-induced tube formation and inhibited FGF-2-induced angiogenesis on matrigel in mice as quantified by FITC-lectin staining of EC. Overall, we demonstrated that gamma-secretase may be key molecule in postnatal angiogenesis which may be downstream molecule of growth factor-induced growth and migration in EC, and regulate the expression of angiogenic growth factors in VSMC.

Despite a number of studies on signal transduction in epidermal keratinocytes, very little is known about how signals move from the cytosol to the nucleus during the course of keratinocyte proliferation and differentiation. In this study, we first compared the expression patterns of the karyopherin alpha (KPNA) subtypes, and found that KPNA2, KPNA3, and KPNA4 were the major subtypes in both normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs). Stimulation with either transforming growth factor (TGF)-beta1 or IFN-gamma for 24 hours resulted in the downregulation of KPNA2 expression specifically in NHEK at both the mRNA and protein levels. Interestingly, IFN-gamma, but not TGF-beta1, specifically downregulated KPNA2 expression at the promoter level, suggesting differential regulation of KPNA2 expression by IFN-gamma and TGF-beta1. We then demonstrated that KPNA2 physically bound to IFN regulatory factor-1 (IRF-1), a transcription factor induced by IFN-gamma, and induced nuclear translocation of IRF-1 in NHEKs. We finally performed microarray and quantitative real-time PCR analysis for the mRNA expression pattern of NHEK with either overexpression or knockdown of KPNA2, and indicated KPNA2 involvement for various epidermal gene regulations such as involucrin. Our data suggest that KPNA2 may play an important role in the signal-transduction pathways that regulate epidermal proliferation and differentiation.

Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood.

Vascular endothelial growth factor (VEGF) is known to play an essential role in vascular development. We have overexpressed VEGF122 or VEGF170, which are equivalent to mouse VEGF120 and VEGF164, in developing Xenopus embryos. Overexpression of VEGF170 but not VEGF122 demonstrated an absence of expression of hematopoietic markers alpha-globin and GATA-1 but only in the posterior portion of the blood island. Interestingly, strong signals of endothelial markers, msr, fli-1, and tie-2, were detectable in those regions, instead of hematopoietic markers. These results suggested both that injection of VEGF170 resulted in disturbance of vasculogenesis in the posterior portion of the blood island, with excessive production of endothelial cells at the expense of blood cells, and that the anterior and posterior portions of the VBI may have distinct characteristics.

We report a rare but notable case of cerebral air embolism complicating transthoracic intrapulmonary injection of an imaging agent used to locate sentinel lymph nodes. After a bolus injection of 2 mL of iopamidol into the peritumoral area with a 23-gauge needle, the patient complained of complete paralysis on his left side. Intraaortic gas was detected by computed tomography immediately after the injection. The patient recovered spontaneously without any additional complication. Surgeons should be aware of this rare but possible complication during sentinel lymph node assessment.

Background: Emphysema is a well-known risk factor for developing air leak or persistent air leak after pulmonary resection. Although quantitative computed tomography (CT) and spirometry are used to diagnose emphysema, it remains controversial whether these tests are predictive of the duration of postoperative air leak., Methods: Sixty-two consecutive patients who were scheduled to undergo major lung resection for cancer were enrolled in this prospective study to define the best predictor of postoperative air leak duration. Preoperative factors analyzed included spirometric variables and area of emphysema (proportion of the low-attenuation area) that was quantified in a three-dimensional CT lung model. Chest tubes were removed the day after disappearance of the air leak, regardless of pleural drainage. Univariate and multivariate proportional hazards analyses were used to determine the influence of preoperative factors on chest tube time (air leak duration)., Results: By univariate analysis, site of resection (upper, lower), forced expiratory volume in 1 second, predicted postoperative forced expiratory volume in 1 second, and area of emphysema (< 1%, 1% to 10%, > 10%) were significant predictors of air leak duration. By multivariate analysis, site of resection and area of emphysema were the best independent determinants of air leak duration. The results were similar for patients with a smoking history (n = 40), but neither forced expiratory volume in 1 second nor predicted postoperative forced expiratory volume in 1 second were predictive of air leak duration., Conclusions: Quantitative CT is superior to spirometry in predicting air leak duration after major lung resection for cancer. Quantitative CT may aid in the identification of patients, particularly among those with a smoking history, requiring additional preventive procedures against air leak.

Background: Gene transfer to skin is an attractive therapeutic approach because of the accessibility of the skin and the high rate of cure for many cutaneous diseases. However, safety concerns over viral vectors and the low efficiency of most non-viral gene transfer techniques have encumbered their clinical application for gene transfer. By contrast, efficient gene transfers into various cell types using microbubble-enhanced ultrasound has been reported., Objectives: The purpose of this study was to investigate whether ultrasound with microbubble enhancement allowed effective transfer of foreign genes into living skin equivalents (LSEs)., Methods: Microbubbles and plasmid DNA encoding green fluorescent protein (GFP) were added to the dermal-epidermal junctions of LSEs, which were then exposed to ultrasound. The LSEs were harvested at different time points to investigate transgene expression using confocal laser microscopy. Transfected LSEs were also transplanted onto nude mice, and the in vivo transgene expression was observed., Results: From days 2 to 7 after transfection, most GFP-positive cells continued to migrate upward from the basal layer, while other GFP-positive cells lagged behind or remained in the basal layer on days 5 and 7. Transfection resulted in 20-30% GFP-positive cells. Multiple transfections further increased the percentage of transfected cells and resulted in multi-layer transgene expression. Grafts from the transfected LSEs survived on nude mice and continued to express GFP up to 2 weeks post-transplantation., Conclusion: Gene transfer into LSE using ultrasound with microbubble enhancement is an effective alternative to viral and non-viral methods.

Study Objectives: To determine the ability of quantitative CT, with special reference to area of emphysema, to predict early postoperative oxygenation capacity and outcome after lung lobectomy for cancer., Methods: Sixty-two consecutive patients scheduled to undergo lung lobectomy for cancer were enrolled in this study. The area of emphysema (< - 910 Hounsfield units) was measured on a three-dimensional CT lung model. Arterial oxygen saturation (Sao(2)) was calculated from Pao(2) measured 1 day before and 1 day after surgery with patients at rest breathing room air. A patient was considered to have recovered at the completion of a standardized management regimen., Results: Postoperative Sao(2) (postSao(2)) was predicted by the baseline value and the area of emphysema with the use of a regression equation. Ten of the 62 patients (16%) had postoperative cardiopulmonary complications (CPCs). The median time to postoperative recovery was 3 days (range, 1 to 17 days). Predicted postSao(2) and predicted postoperative FEV(1) were shown to be significant independent predictors of postoperative CPCs as well as postoperative recovery time., Conclusion: Determining the area of emphysema by quantitative CT is useful in predicting early postoperative oxygenation capacity. Predicted oxygenation capacity and predicted ventilatory capacity independently affect perioperative outcomes. Therefore, using quantitative CT in combination with spirometry may improve risk prediction in patients undergoing lung lobectomy for cancer. However, the role of quantitative CT in grading nonemphysematous lung diseases, such as interstitial lung diseases, must be investigated.

To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector "HVJ-E (hemagglutinating virus of Japan-envelope)" that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.

Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve.

Nuclear factor kappaB (NF-kappaB) is activated in the murine endometrium during implantation period [Am. J. Reprod. Immunol. 51 (2004) 16]. Transient transfection of IkappaBalpha mutant (IkappaBalphaM) cDNA into the mouse uterine cavity using hemagglutinating virus of Japan envelope vector suppressed uterine NF-kappaB activity less than half of that observed in control on days 3.5 and 4.5 p.c. IkappaBalphaM cDNA transfection led to significant delay of implantation. After IkappaBalphaM cDNA transfection, LIF mRNA expression in the uterus was significantly suppressed on days 3.5 and 4.5 p.c. Co-transfection of LIF cDNA with IkappaBalphaM cDNA in the uterus partially rescued the delay of implantation induced by suppression of NF-kappaB activity. Taken together, these findings indicate that NF-kappaB activation determines the timing of the implantation, at least in part, via control of LIF expression.

Background: Colonization of Staphylococcus aureus (S. aureus) on skin is one factor which can worsen atopic dermatitis (AD). The reduction of bacterial colonization in these lesions was reported to be effective for the treatment of subjects with AD. NC/Nga mice are recognized to be a model of AD., Objective: We examined the susceptibility of S. aureus colonization on the skin in NC/Nga mice, as compared with findings in BALB/c mice., Methods: The number of S. aureus on the skin was counted and serum corticosterone, serum interferon-gamma (IFN-gamma) and interleukin (IL)-12 levels were measured. The effects of dexamethasone on number of S. aureus on the skin and serum IFN-gamma and interleukin IL-12 levels were also examined., Results: The number of S. aureus increased in parallel with the severity of the dermatitis in these mice, and the remaining number of S. aureus on the skin after topical treatment of S. aureus suspension was higher than that in BALB/c mice. Serum IFN-gamma and IL-12 concentrations in NC/Nga mice were lower than in BALB/c mice, and the circadian variations of serum corticosterone concentrations in NC/Nga mice tended to reveal higher levels compared with the circadian variations in BALB/c mice. Continuous administration of dexamethasone inhibited the elimination of S. aureus from skin surfaces of BALB/c mice. Serum IFN-gamma and IL-12 concentrations in dexamethasone-treated BALB/c mice were lower than those in vehicle-treated BALB/c mice., Conclusion: Our data support the notion that high levels of circadian variations of endogenous glucocorticoid lead to a lack of protection against bacteria and a persistence of S. aureus colonization on the skin in NC/Nga mice.

When a plasmid encoding the Sleeping Beauty (SB) transposase (pCMV-SB) was cointroduced with luciferase expression plasmid DNA into mouse skeletal muscle at a molar ratio of 4:1, luciferase gene expression was 5 times higher than the expression without pCMV-SB on day 28. This enhancement was not dependent on the presence of transposon (Tn) sequence in luciferase expression plasmid. Southern blot analysis failed to detect luciferase gene insertion into the host genome. Then, expression, a luciferase expression plasmid without Tn, was cointroduced into HeLa cells with or without pCMV-SB. With pCMV-SB, the mRNA amount and the luciferase activity were 1.5 times and 2 times higher, respectively, than without pCMV-SB, even though the cells with pCMV-SB had a smaller copy number of luciferase plasmids than the cells without pCMV-SB. These results suggest that SB transposase enhances the transcription of an exogenous gene regardless of the presence of the Tn sequence.

In this study, we investigated the role of hepatocyte growth factor (HGF) in blood formation during Xenopus development. First, we examined the gene expression of HGF and its receptor, c-met, by whole-mount in situ hybridization during development. Strong signals of HGF as well as c-met were detected early in the developing ventral mesoderm, which later gives rise to the ventral blood island. Furthermore, to study the role of HGF, we blocked the HGF signaling pathway in Xenopus embryos by using truncated c-met lacking the tyrosine kinase domain. Injection of truncated c-met mRNA resulted in a marked decrease in the number of circulating blood cells. Similar results were obtained using morpholino antisense HGF oligonucleotides. Moreover, we also analyzed the expression of several early primitive blood markers in the blood island of these embryos. RNA in situ analysis revealed a significant reduction (or absence) of stem cell leukemia (SCL), alpha-globin, and GATA-1 expression, but not GATA-2 expression. In contrast, no significant difference was observed in the levels of expression of early definitive blood markers, SCL, GATA-2, and GATA-3 in the dorsolateral plate, as analyzed by in situ hybridization. Overall, the present study demonstrated that HGF is necessary for primitive hematopoiesis by regulating the expression of SCL.

Background: Preoperative localization of the sentinel node basin would guide selective lymph node dissection. We tried to identify these nodal stations with indirect computed tomographic lymphography using a conventional extracellular contrast agent, iopamidol., Methods: Eleven consecutive patients scheduled to undergo anatomic resection of suspected lung cancer, without lymphadenopathy, were given a peritumoral injection of undiluted iopamidol under computed tomography guidance, and lymphatic migration was assessed by multidetector-row helical computed tomography., Results: There were no complications such as bleeding, pneumothorax, or allergic reactions. Enhanced nodes were detected in all but 1 patient who had diffuse lymph nodal calcification. Enhanced nodes were identified at 32 ipsilateral intrathoracic nodal stations (20 hilar stations and 12 mediastinal stations). The average length of the longer axis of the enhanced nodes was 4.8 mm (range, 3 to 8 mm), and the average attenuation of the enhanced nodes was 132 (range, 46 to 261) Hounsfield units. In 9 patients with confirmed lung cancer, enhanced nodes appeared at 26 nodal stations, and all apparent enhanced nodes were identified as actual lymph nodes at appropriate position during lymphadenectomy. None of the resected lymph nodes had metastatic involvement., Conclusions: Indirect computed tomographic lymphography with the peritumoral injection of iopamidol effectively depicts the drainage nodes unless they are diffusely calcified. Although further study is required, this method could guide selective lymph node dissection.

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.

Background: Previous studies on intrathoracic lymph node mapping have focused on the validity of a sentinel node concept, but not on the usefulness for sentinel node biopsy., Methods: The subjects were 15 patients clinically diagnosed with N0 nonsmall cell lung cancer. Technetium-99m tin colloid was injected into the peritumoral area 1 day preoperatively and a time course of tracer migration was monitored by scintigraphy. A hand-held gamma probe counter was used to count the intrathoracic lymph node stations. Resected nodes were also counted to assess the accuracy of the intrathoracic counting., Results: Serial scintigraphies showed that the tracer migrated through airways and the appearance resembled hot nodes. On intrathoracic counting, 50% of the nodal stations appeared positive; however, only 23% of these apparently positive nodal stations were ultimately shown to be truly radioactive. The true positive and true negative rates of detecting intrathoracic hot nodes were 100% and 56%, respectively. Because the counts of the nodal stations could include the counts from the hot primary tumor ("shine-through") or airway radioactivity, legitimate hot nodes were identified after dissecting all the apparently positive nodal stations. Two of the 9 patients in whom hot nodes were identified had nodal metastatic disease and actually had tumor cells within the hot nodes. The only complication related to the preoperative injection of technetium-99m was a minor pneumothorax., Conclusions: Although radioisotope intrathoracic lymph node mapping is safe, it appears to be unsuitable for sentinel node biopsy because shine-through and the airway-migrated radioactive tracer complicated the intrathoracic counting. Only serial scintigraphy could distinguish hot nodes from airway migration.

Atherosclerosis has a close relationship to inflammation, particularly T helper type 1 lymphocyte (Th1) response. Interleukin-10 (IL-10), is thought to suppress Th1 response. To target therapeutic strategy for atherosclerosis, we tested whether IL-10 gene transfer suppresses atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice. Four-week-old apoE-KO mice were divided into two groups and either murine IL-10 cDNA plasmid or empty control vector was transferred to the femoral muscle with the use of Hemagglutinating virus of Japan (HVJ)-liposome. At 1 week after transfection, high cholesterol diet was started and continued for 8 weeks. After euthanasia, histological studies of atherosclerotic lesions and quantitative RT-PCR for Th1 cytokines (IL-12 and IFN-gamma) in spleens were performed. IL-10 cDNA gene transfer to the muscle increased plasma IL-10 levels and depressed expression of Th1 cytokines without changing plasma cholesterol levels. IL-10 gene transfer significantly reduced the atherosclerotic plaque area and the macrophage infiltrated area. IL-12 and IFN-gamma mRNA expressions in spleens and plasma IFN-gamma levels were decreased by IL-10 gene transfer. Therefore, IL-10 gene transfer changed the Th1 response and suppressed atherosclerotic lesion formation in apoE-KO mice. IL-10 could be a new target as a therapeutic tool for the treatment of atherosclerosis.

Background: Video-assisted thoracic surgery for lung cancer facilitates early postoperative recovery when patients are treated by critical pathway management. Thus, we developed an original programmed regimen for postoperative management, evaluated the validity of this regimen, and analyzed clinical factors influencing postoperative recovery., Methods: Forty consecutive patients with suspicious lung cancer undergoing anatomic pulmonary resection with video-assisted thoracic surgery were enrolled in this prospective study. After surgery, all patients who underwent anatomic resection were managed using our programmed regimen; a patient was considered recovered when the regimen had been completed., Results: On final pathologic examination, 37 cases were determined to have lung cancer and underwent anatomic resection. The mean number of resected segments was 3.6. There were no complications caused by postoperative management. The mean day of postoperative recovery was 3.7 days and median, 3 days. Significant preoperative factors related to recovery were age, breathlessness, performance status, radiologic emphysema, partial pressure of arterial oxygen, and predictive postoperative forced expiratory volume in 1 second. The overall number of these risk factors was specifically related to postoperative recovery (p < 0.01): the rate of recovery on postoperative day 3 was 100% in patients with no risk, 68% in those with one to three risks, and 22% in those with four to six risks., Conclusions: Our original regimen is useful as a critical pathway for the management of lung cancer patients undergoing video-assisted thoracic surgery. Furthermore, we created specific criteria to identify risk factors related to postoperative recovery that may be useful in planning hospitalization for patients undergoing video-assisted thoracic surgery.

Stability of ribosomal RNA (rRNA) is not only essential for ribosome biogenesis but also crucial to the maintenance of proper translational level for cell viability. rRNA processing (maturation) is one of the key steps to derive functional rRNA, and to date, a large number of factors involved in this process have been identified. We investigated Rps0 binding proteins in fission yeast, Schizosaccharomyces pombe, and revealed that Rps0p is associated with Rps21 protein, similar to that of our previous observation in human cells. We demonstrated that both rps0(+)s and rps21(+) are essential genes for S. pombe analyzed by tetrad dissection assay. To study the functions of both genes, we established disruption strains transformed with inducible rescue plasmids. Using the strains our studies revealed that the loss of rps0(+)s or rps21(+) led to a deficiency of 40S ribosomal subunit formation. Additional functional studies indicate that this phenomenon is likely to be caused by insufficient 18S rRNA stability. The possible role of Rps0p and Rps21 that contribute to 18S rRNA maturation is further discussed.

Study Objectives: A new marking technique was developed to localize small or indistinct pulmonary lesions, involving preoperative radioisotope injection and intraoperative detection with a handheld gamma probe., Setting: National hospital for respiratory disease., Methods: and patients: A technetium suspension (either (99m)Tc tin colloid [2 to 4 mCi, 0.5 to 2.5 mL] or (99m)Tc phytate [2 mCi, 2.0 mL]) was injected preoperatively in the vicinity of the lesion under CT guidance in 25 patients with small or indistinct pulmonary lesions. A handheld gamma probe (Navigator GPS; Tyco Healthcare Japan; Tokyo, Japan) was used to detect the hot spot where radioactivity was localized during surgery. The lesion marked by the radioisotope then was thoracoscopically resected using endostaplers., Results: The preoperative marking procedure took approximately 30 min. A small pneumothorax and mild intrapulmonary bleeding were observed in two patients and one patient, respectively, but no additional treatment was needed for these complications. The lesion was resected soon after the marking procedure in 21 patients, and on the next day in 4 patients. All lesions were easily identified during surgery as radioactive hot spots detected by the handheld gamma probe. Thoracoscopic wedge resection was successfully performed in all patients without complications., Conclusion: This new marking technique for small or indistinct pulmonary lesions (radioisotope injection under CT guidance and intraoperative detection with a handheld gamma probe) seems to be safe, reliable, and convenient.

Neurotransmitter release during and after ischemic event is thought to be involved in excitotoxicity as a pathogenesis for the ischemic brain damage, which is mediated by excessive activation of glutamate receptors and attendant calcium overload. To ascertain the role of transmitter release from nerve terminals in promoting the ischemic neurodegeneration, we delivered antisense oligodeoxynucleotides (ODNs) to synaptotagmin I or synapsin I into the rat brain by using HVJ-liposome gene transfer technique. The antisense ODNs were injected into the lateralventricle in rats 4 days prior to transient forebrain ischemia of 20 min. With a single antisense treatment, long-lasting downregulation of the transmitter release relating protein levels at overall synaptic terminals was achieved. The antisense in vivo knockdown of synaptotagmin I prevented almost completely the ischemic damage of hippocampal CA1 neurons, while the in vivo knockdown of synapsin I markedly promoted the ischemic damage of CA1 pyramidal neurons and extended the injury to relatively resistant CA2/CA3 region. The modulation of ischemic hippocampal damage by the in vivo knockdown of synaptotagmin I or synapsin I suggests that transmitter release from terminals plays an important role in the evolution of ischemic brain damage and therefore the transmitter release strategy by the use of antisense ODNs-HVJ-liposome complex is reliable for neuroprotective therapies.

To overcome some problems of virus vectors, we developed a novel non-viral vector system, the HVJ-envelope vector (HVJ-E). In this study, we investigated the feasibility of gene transfer into the CNS using the HVJ-E both in vitro and in vivo. Using the Venus reporter gene, fluorescence could be detected in cultured rat cerebral cortex neurons and glial cells. In vivo, the reporter gene (Venus) was successfully transfected into the rat brain by direct injection into the thalamus, intraventricular injection, or intrathecal injection, without inducing immunological change. When the vector was injected after transient occlusion of the middle cerebral artery, fluorescence due to EGFP gene or luciferase activity could be detected only in the injured hemisphere. Finally, luciferase activity was markedly enhanced by the addition of 50 U/ml heparin (P<0.01). Development of efficient HVJ-E for gene transfer into the CNS will be useful for research and clinical gene therapy.

Background: We tested the hypothesis that the best time for genetic modification is while the cell viability of the graft is reduced for long-term preservation. The hemagglutinating virus of Japan (HVJ)-liposome method, a nonviral gene transfer technique, was used with a luciferase gene to test the efficacy of protein induction under the critical preservation time. Furthermore, we tested this genetic modification with heat shock protein (HSP) 70 or bcl-2 genes to prevent primary nonfunction (PNF) after long-term preservation., Methods: Orthotopic rat renal transplantation (RT) was performed using the cuff technique in the syngeneic combination of Lew (major histocompatible complex, haplotype: RT1(l)). Rat kidney grafts were preserved for 24 or 48 h in University of Wisconsin (UW) or Ringer's lactate solution using HVJ method with the luciferase gene. Rats with gene-transfected kidneys were re-laparotomized 48 h after transplantation to estimate the lack of arterial flow in the graft and killed for histological evaluation of the degree of PNF luciferase intensity assay. Then, two functional genes (HSP70 or bcl-2) were tested for the occurrence of PNF and histological and immunohistochemical analysis of the grafted kidneys preserved for 48 h in the UW solution., Results: In the kidneys preserved for 24 h, 50% of the Ringer's lactate group had PNF; but all of the UW group had sufficient blood flow. The graft viability was well corrected by the degree of luciferase intensity. The PNF rate was significantly suppressed in the bcl-2 gene-transfer group, and tended to be reduced in the HSP70 group., Conclusions: The HVJ-liposome method effectively induced the foreign gene for kidney grafts even in the cold-preservation solution. Induction of bcl-2 or the HSP70 gene reduced the occurrence of PNF in the rat renal graft. The results suggest that gene transfer not only maintains graft viability, but also graft activation.