A novel intranuclear RNA vector system for long-term stem cell modification.

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells, while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction.

Revision of anterior cruciate ligament reconstruction with allografts in patients younger than 40 years old: a 2 to 4 year results.

PURPOSE: The purpose of this study is first to report the outcomes, at 4 years follow-up, in revision ACL surgery using allografts in patients younger than 40 years old, and then compared soft tissue allografts to bone tendon allografts. METHODS: This retrospective study included 47 patients who underwent ACL revision surgery with fresh-frozen allografts. Patellar tendon allograft or tibialis anterior allograft was used. Twenty-seven patients undergoing ACL revision with patellar tendon allograft were compared retrospectively with twenty-two patients undergoing the same procedure with soft tissue tibialis anterior allograft. Lysholm, IKDC, and KT-1000 values were obtained preoperatively and postoperatively. RESULTS: The average patient follow-up was 4.6 years (±2.5). The mean age at time of the revision was 34 years old (±6.3). Overall, patients reported the overall condition of their knee as excellent or good in 85% of the patients (10 excellent, 33 good). Based on their experience, 85% would have the surgery again if they had the same problem in the other knee. Both subgroups experienced significant improvement in Lysholm, IKDC, and KT-1000 values, with no difference found between groups at final follow-up. CONCLUSION: Revision ACL with allografts has excellent and good results in 85% of patients younger than 40 years old. No statistical difference was seen between soft tissue (tibialis anterior) and patellar tendon allograft. LEVEL OF EVIDENCE: IV.

Intravascular papillary endothelial hyperplasia of the digit: MRI features with histological correlation.

To clarify the magnetic resonance (MR) features of the pure form of intravascular papillary endothelial hyperplasia, MR images (MRIs) from five patients were retrospectively reviewed and compared with histological findings. The images showed a heterogeneous, iso- to slightly high signal intensity mass on T1-weighted images and a mass with a central heterogeneous, iso- to slightly high signal intensity area completely or incompletely surrounded by peripheral high signal intensity areas on T2-weighted images. Heterogeneous enhancement was observed after gadolinium administration. Histological studies indicated that the central heterogeneous area on T2-weighted images corresponded to thrombi (organized and/or hyalinized) and/or papillary endothelial proliferation, and also that the peripheral high signal intensity area corresponded to vascular blood space and/or papillary endothelial proliferation. The pure form of intravascular papillary endothelial hyperplasia showed relatively characteristic features on MRIs.

Treatment of chronic patellar tendinopathy with autologous bone marrow stem cells: a 5-year-followup.

The purpose of this study is to determine if patients with chronic patellar tendinopathy will improve clinically after the inoculation of bone marrow mononuclear cells (BM-MNCs). Eight patients with chronic patellar tendinopathy were included. Patients averaged 24 years old (range 14-35). All patients were refractory to conservative treatment for at least 6 months before the procedure. BM-MNCs were harvested from the iliac bone crest and inoculated under ultrasound guide in the patellar tendon lesion. Improvement was assessed through established clinical scores and ultrasound. At 5-year followup, statistically significant improvement was seen for most clinical scores. Seven of eight patients said they would have the procedure again if they had the same problem in the opposite knee and were completely satisfied with the procedure. Seven of 8 patients thought that the results of the procedure were excellent. According to our results, inoculation of BM-MNCs could be considered as a potential therapy for those patients with chronic patellar tendinopathy refractory to nonoperative treatments.

Biochemical changes associated with in vivo RbcL fragmentation by reactive oxygen species under chilling-light conditions.

During physiological stress, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation is accelerated, which is considered to be one of the key factors responsible for photosynthetic decline. A recent study has shown that the large subunit (RbcL) of Rubisco is directly fragmented by hydroxyl radicals in Cucumis sativus leaves under chilling-light conditions. In the present study, we investigated biochemical aspects associated with this in vivo RbcL fragmentation by reactive oxygen species. RbcL fragmentation was observed in C. sativus and Phaseolus vulgaris, but not in Solanum lycopersicum, Glycine max, Oryza sativa, Triticum aestivum, Spinacia oleracea or Arabidopsis thaliana. In C. sativus and P. vulgaris, RbcL fragmentation followed the fragmentation of PsaB, while in the other species, PsaB fragmentation did not occur. In C. sativus and P. vulgaris, the activities of antioxidant enzymes decreased dramatically under chilling-light conditions, and the proportion of uncarbamylated Rubisco increased. These data suggest that in vivo RbcL fragmentation under chilling-light conditions is associated with a combination of events, namely, inactivation of antioxidant enzymes, destruction of photosystem I and an increase of uncarbamylated Rubisco, which can produce hydroxyl radicals via the Fenton reaction at the catalytic site of RbcL.

Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O(2)(-)) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs. METHODS: MCECs were freshly isolated and lysed for protein measurement, or cultured to determine mitochondrial morphology and O(2)(-) production. For the ex vivo hyperglycaemia experiments, human coronary endothelial cells were used. RESULTS: Elongated mitochondrial tubules were observed in MCECs isolated from control mice, whereas mitochondria in MCECs from diabetic mice exhibited augmented fragmentation. The level of optic atrophy 1 (OPA1) protein, which leads to mitochondrial fusion, was significantly decreased, while dynamin-related protein 1 (DRP1), which leads to mitochondrial fission, was significantly increased in MCECs from diabetic mice. Diabetic MCECs exhibited significantly higher O(2)(-) concentrations in cytosol and mitochondria than control MCECs. Administration of the O(2)(-) scavenger TEMPOL to diabetic mice for 4 weeks led to a significant decrease in mitochondrial fragmentation without altering the levels of OPA1 and DRP1 proteins in MCECs. High-glucose treatment for 24 h significantly induced mitochondrial fragmentation, which was restored by TEMPOL treatment. In addition, excess O(2)(-) production, either in cytosol or in mitochondria, significantly increased mitochondrial fragmentation. CONCLUSIONS/INTERPRETATION: These data suggest that lowering the O(2)(-) concentration can restore the morphological change in mitochondria and may help improve mitochondrial function in diabetic MCECs.

Emerging and reemerging influenza virus infections.

Influenza A virus infection occurs in many species. Wild waterfowl harbor the widest variety of influenza A viruses and serve as a constant reservoir for the emergence of new viruses. Highly pathogenic avian influenza, or "fowl plague," has been a known poultry disease for more than 130 years. It continues to emerge and reemerge, but global changes in trade and poultry production have expanded the impact and geographic range of these outbreaks. One subtype of highly pathogenic avian influenza, H5N1, has infected poultry on several continents as well as many people, leading to a human disease that is markedly different from seasonal influenza and that is associated with high mortality.

Local structure studies of Fe-Nb-B metallic glasses using electron diffraction.

Local atomic structures in Fe(84)Nb(7)B(9) and Fe(70)Nb(10)B(20) amorphous alloys were examined by means of electron diffraction with the help of computer calculation. Electron diffraction patterns were taken by using energy-filtered transmission electron microscopy (TEM) to eliminate inelastic scattering. We constructed structure models with 5000 atoms fitting to experimental interference functions. Voronoi polyhedral analyses were performed for the obtained final structure models. Local atomic structures of the alloys were closely related to those of the crystalline phases that appeared on annealing. A difference of stability of two amorphous phases was discussed on the basis of structure models.

Composite adenocarcinoma and large cell neuroendocrine carcinoma of the rectum.

Composite glandular-endocrine tumors of the gastrointestinal tract are rare neoplasms. Even more uncommon are the so-called amphicrine tumors, lesions in which dual epithelial and endocrine differentiation occurs in the same cell. We describe a patient who complained of rectal pain and bleeding with a mixed or composite adenocarcinoma and neuroendocrine carcinoma of the rectum. Histological examination revealed a distinct adenocarcinoma of conventional type with glandular structures admixed intimately with a neuroendocrine carcinoma. The latter component was deeply infiltrative, while the adenocarcinoma occupied the more superficial aspect of the tumor. What was interesting was the immunophenotype of the lesion: cytokeratin (CK) 20 expression was very focal in the adenocarcinoma component and negative in the neuroendocrine carcinoma, while CK 7 was expressed strongly in the adenocarcinoma and only focally in the neuroendocrine component. This cytokeratin profile suggests a possible origin from the anal transitional zone.

Caracterizacao e analise da satisfacao da clientela atendida pela fisioterapia do servico de atencao a saude do trabalhador/UFMG / Characterization and analysis of the satisfaction of clients seen by the physiotherapist of worker`s health care service/UFMG

Em 1999, a Universidade Federal de Minas Gerais (UFMG) criou o Servico de Atencao a Saude do Trabalhador (SAST Pampulha). O presente estudo objetivou descrevero perfil e avaliar a satisfacao dos servidores que receberam tratamento fisioterapeutico no SAST no periodo de julho de 1999 a junho de 2002. Para caracterizar a clientela atendida na fisioterapia, elaborou-se um protocolo contendo variaveis relacionadas as caracteristicas socio-demograficas, ocupacionais e clinicas dos trabalhadores. Para avaliar a satisfacao, foi criado um questionario com 18 questoes de multipla escolha relacionadas ao ambiente fisico do servico, ao profissional, ao tratamento e as expectativas do paciente, uma questao aberta para o entrevistado expor suas criticas e sugestoes. Desde a criacao do SAST Pampulha, em 1999, ate junho de 2002, 307 trabalhadores procuraram o servico de fisioterapia. Deste total 149 concluiram o tratamento e receberam alta, 76 abandonaram o servico, 39 foram somente avaliados e 43 nao partticiparam do estudo. Dos 264 estudados, 103 (40 por cento) eram trabalhadores de servicos administrativos e 197 apresentaram diagnostico clinico de doenca osteomuscular, sendo que as dorsalgias foram responsaveis por (20 por cento) dos casos (n=54). A analise da satisfacao foi realiazada separadamente, a partir da comparacao de dois grupos de trabalhadores, equivalentes quanto ao sexo, idade e escolaridade, de acordo com a conclusao do tratamento (alta e abandono). A relacao terapeuta-paciente parece ter sido o fator que mais motivou o abandono do tratamento ocasionando a insatisfacao com os resultados do mesmo. Espera-se que os resultados deste estudo possam contribuir para a dequacao da dinamica de funcionamento do servico e servir e base para o planejamento e a estruturacao de outos servicos de fisioterapia voltados para a atencao a saude do trabalhador

Adaptation of conduit artery vascular smooth muscle tone to induced hypertension.

We studied the changes in vascular smooth muscle (VSM) cell tone during the adaptation of rat common carotids to induced hypertension. Hypertension was induced in 8 week old male Wistar rats by total ligation of the aorta between the two kidneys. Mean blood pressure increased abruptly from 92 +/- 2mm Hg (mean +/- SE) to 145 +/- 4 mm Hg and remained constant thereafter. Rats were sacrificed 2, 4, 8, and 56 days after surgery and the left common carotid artery was excised for analysis. Pressure-diameter curves were measured in vitro under normal, maximally contracted, and totally relaxed VSM. The VSM tone was analyzed in terms of its basal tone (active stress at low strains) and its myogenic tone (increase in active stress at high strains). Our results show that the capacity of the VSM to develop maximal active stress is not altered in hypertension. Basal tone, however, increases rapidly in the acute hypertension phase (2-8 days postsurgery) and drops to nearly control values at 56 days postsurgery. Also, the onset of myogenic response decreases to lower strains following the step change in pressure, to be restored back to control levels at 56 days postsurgery. We conclude that VSM adaptation is most significant in the acute hypertension phase and acts as a first, rapid defense mechanism for the arterial wall. The VSM tone returns back to normal levels once the slower geometrical and structural remodeling is developed sufficiently to restore the biomechanical environment and function of the arterial wall to control levels.

[A comparison of propofol and midazolam in sedation of mechanically-ventilated postoperative patients].

To compare the quality of sedation using propofol (n = 8) vs midazolam (n = 7) we evaluated their effectiveness, safety, and recovery time after their continuous infusion in patients who required mechanical ventilation after cervical spine surgery. We also calculated the cost of drugs used during the mechanical ventilation. In addition, processed electroencephalogram (pEEG) was monitored employing spectral edge frequency 90 (SEF 90) as an indicator of sedation. Both drugs produced good sedation without any complication. The patients who had received propofol were extubated significantly earlier than those who had received midazolam (P; 35 +/- 18 mins, M; 97 +/- 55 mins). However, the mean drug cost in the propofol group was five times higher than that in the midazolam group (P; yen 15,881 +/- 7,788, M; yen 3,355 +/- 1,187). There was no correlation between the value of SEF 90 and the depth of sedation during mechanical ventilation. In conclusion, propofol exhibited the shorter recovery time after cessation of the continuous infusion than midazolam, but it costed five times compared with midazolam. SEF 90 failed to indicate the depth of sedation during mechanical ventilation.

Significant accumulation of C(4)-specific pyruvate, orthophosphate dikinase in a C(3) plant, rice.

The C(4)-Pdk gene encoding the C(4) enzyme pyruvate, orthophosphate dikinase (PPDK) of maize (Zea mays cv Golden Cross Bantam) was introduced into the C(3) plant, rice (Oryza sativa cv Kitaake). When the intact maize C(4)-Pdk gene, containing its own promoter and terminator sequences and exon/intron structure, was introduced, the PPDK activity in the leaves of some transgenic lines was greatly increased, in one line reaching 40-fold over that of wild-type plants. In a homozygous line, the PPDK protein accounted for 35% of total leaf-soluble protein or 16% of total leaf nitrogen. In contrast, introduction of a chimeric gene containing the full-length cDNA of the maize PPDK fused to the maize C(4)-Pdk promoter or the rice Cab promoter only increased PPDK activity and protein level slightly. These observations suggest that the intron(s) or the terminator sequence of the maize gene, or a combination of both, is necessary for high-level expression. In maize and transgenic rice plants carrying the intact maize gene, the level of transcript in the leaves per copy of the maize C(4)-Pdk gene was comparable, and the maize gene was expressed in a similar organ-specific manner. These results suggest that the maize C(4)-Pdk gene behaves in a quantitatively and qualitatively similar way in maize and transgenic rice plants. The activity of the maize PPDK protein expressed in rice leaves was light/dark regulated as it is in maize. This is the first reported evidence for the presence of an endogenous PPDK regulatory protein in a C(3) plant.

An efficient method for extraction of RNA from rice leaves at different ages using benzyl chloride.

With a conventional method of RNA extraction using an acid guanidinium thiocyanate-water-saturated phenol-chloroform mixture, extraction efficiency of extractable RNA to total RNA (extractable RNA+ residual RNA) in rice leaves at various ages was 54-69%. With a new method, an improvement of the above, using benzyl chloride instead of water-saturated phenol together with further maceration with a small amount of quartz sand, the efficiency was increased to 81-95%. When RNA fractions obtained with the improved method were subjected to agarose gel electrophoresis, intact bands of 25 S and 17 S rRNAs were detected. With a DNA probe for rice rbcS, only a single band was observed on the blotted membrane. These results indicate that the improved extraction method of RNA with benzyl chloride is useful for quantitative and qualitative analysis of RNA in plant tissues such as stiff leaves of rice.

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.

Magnetic resonance imaging follow-up study of bone bruises associated with anterior cruciate ligament ruptures.

PURPOSE: The purpose of this study was to perform a magnetic resonance imaging (MRI) follow-up study of bone bruises in a group of patients with acute anterior cruciate ligament (ACL) ruptures that were reconstructed and followed-up for a minimum of 2 years. TYPE OF STUDY: Cohort study. METHODS: The study group included 21 patients with a mean age of 31 years whose initial MRI scans showed associated bone bruises. Patients were included if they had an acute isolated ACL tear, no documentation of an episode of repeated injury to the affected knee during the follow-up period, and no evidence of cartilaginous injury at the time of arthroscopy. All patients had preoperative MRI scans and underwent arthroscopic ACL reconstruction using a bone-patellar tendon autograft an average of 2 months after injury. The preoperative MRI scans were analyzed using a 3-level grading system based on the appearance and location of bone bruises. A second MRI of the knee was obtained from 24 to 64 months postoperatively (average 34 months). The presence of resolution of bone bruises was determined and correlation with clinical scoring established. RESULTS: This study showed resolution of all type I lesions and 91% of type II lesions (10 of 11). In all type III lesions, an articular cartilage thinning and depression was observed after 2 years of follow-up. In 15 patients (71%), MRI showed that the bone bruises had resolved without apparent sequelae. In the remaining 6 patients (29%), sequelae of the osteochondral lesion were evident on MRI. CONCLUSIONS: According to our clinical data, there was no correlation between scores obtained from patients with resolved lesions against those with osteochondral sequelae. Although long-term clinical implications of these findings are uncertain, a severe occult osteochondral lesion sustained at the time of ACL rupture seems to be persistent on MRI even after a successful reconstruction.

Mechanisms underlying increased release of endothelin-1 from aorta in diabetic rats.

To clarify the mechanism underlying increased endothelin-1 release in diabetic rats, we examined its release from thoracic aortas obtained from streptozotocin-induced diabetic rats. The methoxamine-induced contraction was significantly inhibited by BQ-123 plus BQ-788 (specific antagonists for ET(A) and ET(B) receptors) in diabetic, but not control rats. Preincubation with phosphoramidon also inhibited the methoxamine-induced contraction in diabetic but not control rats. The expression of prepro endothelin-1 mRNA was significantly enhanced in aortas from streptozotocin-induced diabetic rats. These results suggest that the increases in the basal and alpha-agonist-induced release of endothelin-1 in the diabetic state may be due to an overexpression of the mRNA for prepro endothelin-1.

Short-Term biomechanical adaptation of the rat carotid to acute hypertension: contribution of smooth muscle.

The biomechanical adaptation of the arterial wall to hypertension has been studied extensively in recent years; however, the exact biomechanical contribution of vascular smooth muscle cells (VSMCs) during the adaptation process in conduit vessels is not known. We induced hypertension in 8 wk old Wistar rats by total ligation of the aorta between the two kidneys. Mean blood pressure increased from 92 +/- 2 (mean +/- SE) mm Hg to approximately 150 mmHg. Rats were sacrificed 2, 4, and 8 d after surgery and the left common carotid artery was excised for analysis. Wall thickness increased by 18% in 8 d and the opening angle by 32% in 4 d. The elastic properties were measured under normal VSMC tone (i.e., the amount of VSMC tone under normal conditions also called basal VSMC tone or normal resting VSMC tone), under maximally contracted VSMC (NE, 5 x 10(-7) mol/L) and under totally relaxed VSMC conditions (papaverine, 10(-4) mol/L). The most pronounced modifications were the changes in elastic properties related to normal VSMC tone. The functional contraction ratio at 100 mm Hg, defined as the relative contraction under normal conditions (normal VSMC tone), increased by 439% 4 d after the induction of hypertension. The total contraction capacity of the VSMC increased by 38% within 8 d. The changes in normal VSMC tone led to important changes in the mechanical properties of the arterial wall. Under normal VSMC conditions, compliance at mean pressure (148 mm Hg) increased by 159% within 8 d, whereas in the absence of VSMC tone, compliance did not increase significantly. We conclude that in conduit vessels, the VSMC, which is the sensing and effecting element of the adaptation process, is subjected to large-scale changes during the early phase of arterial adaptation to acute hypertension.

A case of massive subchorionic thrombohematoma diagnosed by ultrasonography and magnetic resonance imaging.

Massive subchorionic thrombohematoma is uncommon but associated with a poor perinatal prognosis. Placental enlargement was detected in a 25-year-old Japanese primipara woman with fetal growth retardation and oligohydramnios at 23 weeks' gestation. Ultrasonography (USG) showed an abnormal sonolucency within the placenta at 28 weeks' gestation, but could not give an unequivocal differentiation from placental abnormalities such as hematomas, cysts and other tumors. Magnetic resonance imaging (MRI) pointed to a large hematoma in the subchorionic region. Simultaneously, the amniotic fluid was brownish colored. From these findings, it was possible to have prenatal diagnosis of massive subchorionic thrombohematoma. At 32 weeks' gestation, the fetus died in utero and was stillborn 3 days later. Pathological findings for the placenta revealed a large hematoma diffused between the villous chorion and the chorionic plate, with wide necrosis of placental tissue, likely due to formation of multiple thrombi. The clinical and pathological findings were compatible with massive subchorionic thrombohematoma. MRI might be useful for the detection of massive subchorionic thrombohematoma and help its clinical management in combination with USG and pulse Doppler imaging.