The added value of tomosynthesis in endoscopic retrograde cholangiography with radiography for the detection of choledocholithiasis.

OBJECTIVE: The diagnostic performance of endoscopic retrograde cholangiography (ERC) with radiography is imperfect. We assessed the value of adding tomosynthesis to ERC with radiography for the detection of choledocholithiasis. METHODS: This study included 102 consecutive patients (choledocholithiasis/non-choledocholithiasis, n = 57/45), who underwent both radiography and tomosynthesis for ERC in the same examination and were not diagnosed with malignancy. The reference standard for the existence of choledocholithiasis was confirmed by endoscopic stone extraction during ERC, intraoperative cholangiography, or follow up with magnetic resonance cholangiopancreatography (n = 78, 11, and 13, respectively). A gastroenterologist and a radiologist independently evaluated the radiographs and the combination of tomosynthesis and radiographic images in a blinded and randomised manner. Receiver operating characteristic analysis was used for statistical analysis. RESULTS: The areas under the receiver operating characteristic curve for combined tomosynthesis and radiography were significantly higher than those for radiography alone for both readers: Reader 1/Reader 2, 0.929/0.956 [95% confidence interval (CI), 0.861-0.965/0.890-0.983) vs 0.803/0.769 (95% confidence interval, 0.707-0.873/0.668-0.846), respectively (p = 0.0047/< 0.0001). CONCLUSION: Adding tomosynthesis to radiography improved the diagnostic performance of ERC for detection of choledocholithiasis. Advances in knowledge: Adding tomosynthesis to radiography improves detection of choledocholithiasis and tomosynthesis images can be obtained easily after radiographs and repeated immediately.

Temporary Internal Fixation Using C1 Lateral Mass Screw and C2 Pedicle Screw (Goel-Harms Technique) without Bone Grafting for Chronic Atlantoaxial Rotatory Fixation.

BACKGROUND: The primary treatment strategy for chronic atlantoaxial rotatory fixation (chro-AARF) is traction followed by bracing or application of a halo device. However, to complete these conservative therapies, patient cooperation is mandatory. If conservative therapy fails, surgery is required for reduction and prevention of recurrence. It has been considered that surgery for atlantoaxial rotatory fixation necessitates solid bony fusion. However, once bony fusion is achieved, loss of range of motion is problematic. Here, we report a patient with chro-AARF who was successfully treated with temporary internal fixation using a C1 lateral mass screw and C2 pedicle screw (Goel-Harms technique) without any grafting of bone or use of bone substitute materials. CASE DESCRIPTION: A 9-year-old boy with chro-AARF was referred to our institution. He had a history of pervasive developmental disorders. He did not cooperate for the completion of conservative therapy and could not tolerate this therapy. Therefore, the orthopedic staff and his parents considered surgery. Under general anesthesia, reduction was easily performed. The Goel-Harms screw-rod construct was completed as a temporary internal fixator without any grafting of bone or use of bone substitute materials. After 6 months, the screw-rod construct was removed. Removal of the screw-rod construct was performed easily without complication. There was no ankylosis of the C1-2 joint, and cervical range of motion was maintained 2.8 years after removal of the construct. CONCLUSIONS: When conservative therapy cannot be continued, Goel-Harms surgery as a temporary internal fixator without bone grafting might be a suitable alternative for selected patients with chro-AARF.

Surgical Management of the Pregnant Patient With Lumbar Disc Herniation in the Latter Stage of the Second Trimester.

STUDY DESIGN: Case report. OBJECTIVE: To report on a pregnant woman successfully treated with microendoscopic discectomy in the left lateral position under general anesthesia at 24-week gestation. SUMMARY OF BACKGROUND DATA: Treatment for lumbar disc herniation in pregnant women poses a particular challenge due to the complexity of the clinical situation. Review of the literature emphasizes timely diagnosis with adequate management specific for each gestational period. A surgical approach mandates consideration of the physiologic parameters of pregnancy and the effects of these stressors on the fetus. METHODS: A 38-year-old primigravid woman presented with persistent and incapacitating low back and left leg pain. Magnetic resonance imaging demonstrated a herniated disc at L4-5 with a severely compressed left L5 nerve root. Symptoms were resistant to conservative treatment (acetaminophen; 1200 mg/day) and nerve root block with corticosteroids (1 mg/0.5 mL of betamethasone plus 0.5 mL of 1% lidocaine) provided only transient pain relief. Operative management with surgical discectomy was discussed. Anesthesiologists, obstetricians, and neonatologists were consulted for preoperative planning, focusing on appropriate anesthesia, ideal positioning for surgical access, and provision for emergent fetal care. Surgery was ultimately performed in the left lateral position, in contrast to the oft-used prone position. Microendoscopic discectomy was performed under general anesthesia at 24-week gestation. RESULTS: The patient experienced complete relief from pain after surgical intervention and delivered a healthy baby at 39-week gestation after normal labor. Our methods, used in accordance with our preoperative simulation, resulted in a satisfactory outcome for both mother and child. CONCLUSION: Although previously published cases noted the safety of operating in the prone position under epidural anesthesia, we performed minimally invasive microendoscopic discectomy in the left lateral position in combination with general anesthesia and found that this is a safe and preferable alternative for pregnant patients in the latter stage of the second trimester. LEVEL OF EVIDENCE: N/A.

Encapsulated mesenchymal stromal cells for in vivo transplantation.

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish.

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.

Delayed intrathecal delivery of RhoA siRNA to the contused spinal cord inhibits allodynia, preserves white matter, and increases serotonergic fiber growth.

RhoA is a key regulator of the actin cytoskeleton that is upregulated after spinal cord injury (SCI). We analyzed different methods for siRNA delivery and developed siRNAs targeting RhoA (siRhoA) for SCI treatment. Cy 3.5-labeled siRNA delivered at the time of SCI yielded fluorescence in several cell types in the injury site. Intraspinal injections of chemically stabilized siRhoA into the spinal cord of injured rats reduced RhoA protein levels after 1 week and improved hindlimb walking over 6 weeks. To explore a less invasive route, we tested intrathecal injection of Cy 3.5-labeled siRNA via lumbar puncture 1 day after SCI, which resulted in robust uptake in the T9-T10 injury site. Lumbar injection of siRhoA 1 day after SCI reduced RhoA mRNA and protein levels 3 days after injection. Although siRhoA treatment did not yield significant improvement in locomotion, it decreased tactile hypersensitivity significantly compared to controls. Histological analysis at 8 weeks showed significant improvement in white matter sparing with siRhoA compared to control siRNA. siRhoA treatment also resulted in less accumulation of ED1+macrophages, increased PKC-γ immunoreactivity in the corticospinal tract rostral to the injury site, and increased serotonergic fiber growth 12 mm caudal to the contusion site. The ability of siRhoA to preserve white matter and promote serotonergic axonal regrowth caudal to the injury site is likely to suppress allodynia. This provides justification for considering clinical development of RhoA inhibitors to treat SCI sub-acutely to reduce allodynia, which occurs frequently in SCI patients.

Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells.

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.

A novel method to isolate mesenchymal stem cells from bone marrow in a closed system using a device made by nonwoven fabric.

Bone marrow stromal cells (BMSCs) include cells with multidirectional differentiation potential described as mesenchymal stem cells. For clinical use, it is important to develop a way to isolate BMSCs from bone marrow in a closed system without centrifugation. After screening 200 biomaterials, we developed a device containing a nonwoven fabric filter composed of rayon and polyethylene. The filter selectively traps BMSCs among mononuclear cells in bone marrow based on affinity, not cell size. The cells are then recovered by the retrograde flow. Using canine and human bone marrow cells, the biological properties of BMSCs isolated by the device were compared with those obtained by conventional methods using centrifugation. The total number isolated by the device was larger, as was the number of CD106(+)/STRO-1(+) double-positive cells. The cells showed osteogenic, chondrogenic, and adipogenic differentiation potential in vitro. Finally, the direct transplantation of cells isolated by the device without in vitro cultivation accelerated bone regeneration in a canine model of osteonecrosis in vivo. The proposed method is rapid and efficient, does not require a biological clean area, and will be useful for the clinical application of mesenchymal stem cells in bone marrow.

Cell-specific epigenetic regulation of ChM-I gene expression: crosstalk between DNA methylation and histone acetylation.

The expression of the chondromodulin-I (ChM-I) gene, a cartilage-specific gene, is regulated by the binding of Sp3 to the core promoter region, which is inhibited by the methylation of CpG in the target genome in the osteogenic lineage, osteosarcoma (OS) cells. The histone tails associated with the hypermethylated promoter region of the ChM-I gene were deacetylated by histone deacetylase 2 (HDAC2) in three ChM-I-negative OS cell lines. Treatment with an HDAC inhibitor induced the binding of Sp3 in one cell line, which became ChM-I-positive. This process was associated with acetylation instead of the dimethylation of histone H3 at lysine 9 (H3-K9) and, surprisingly, the demethylation of the core promoter region. The demethylation was transient, and gradually replaced by methylation after a rapid recovery of histone deacetylaion. These results represent an example of the plasticity of differentiation being regulated by the cell-specific plasticity of epigenetic regulation.

Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.

Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is potentially silenced by DNA methylation during in vitro expansion.

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A-positive cells were Ki67-negative and senescence associated beta-galactosidase-positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty-five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.

In vitro transformation of mesenchymal stem cells by oncogenic H-rasVal12.

Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.

Expression of claudin7 is tightly associated with epithelial structures in synovial sarcomas and regulated by an Ets family transcription factor, ELF3.

Synovial sarcoma, a soft tissue sarcoma that develops in adults, is pathologically subclassified into monophasic spindle synovial sarcoma and biphasic synovial sarcoma with epithelial components. The molecular mechanism building the epithelial components in biphasic synovial sarcoma is totally unknown. Here we investigated claudins, critical molecules in the tight junction, in biphasic synovial sarcoma. Expression profiles of 21 claudins in 17 synovial sarcoma tumor samples, including 9 biphasic tumors, identified claudin4, claudin7, and claudin10 as biphasic tumor-related claudins, and immunohistochemical analyses demonstrated the localization of these claudins in the epithelial component in biphasic tumors, with claudin7 the most closely associated with the epithelial component. The mRNA expression and protein localization of claudin7 coincided with those of the ELF3, an epithelia-specific member of the Ets family of transcription factors. Luciferase reporter assays demonstrated that the presence of the Ets-binding site at -150 in the promoter region of the claudin7 gene was critical for the transcriptional activity, and gel shift and chromatin immunoprecipitation assays confirmed the binding of ELF3 to the Ets site at -150. Inhibition of ELF3 expression by small interfering RNA simultaneously down-regulated the mRNA expression of the claudin7 gene, and the introduction of ELF3 expression in claudin7-negative cell lines induced mRNA expression of the claudin7 gene. Therefore, the induction of claudin7 expression by ELF3 appears critical to the formation of the epithelial structures in biphasic synovial sarcoma.

Regeneration of osteonecrosis of canine scapho-lunate using bone marrow stromal cells: possible therapeutic approach for Kienböck disease.

We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze-thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of beta-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.

A variant of the SYT-SSX2 fusion gene in a case of synovial sarcoma.

Synovial sarcoma is a malignant soft tissue tumor harboring a tumor-specific fusion gene, SYT-SSX, of which exon 10 of the SYT gene is fused to exon 6 of the SSX gene is the common form. Here we report a case of synovial sarcoma with a novel form of the SYT-SSX2 fusion transcript, in which 75 bases were inserted at the common fusion junction. Computer analyses revealed that 15 bases were from intron 10 of the SYT gene, and 10 from the end of intron 4, and 50 from exon 5 of the SSX2 gene. Precise analyses of genomic breakpoints in SYT and SSX2 loci revealed that the reciprocal translocation creating the fusion gene was associated with a large deletion in both loci. The structure of SYT-SSX2 suggests that the fusion transcript in this case was created using a cryptic splicing acceptor site 15 bases upstream of the genomic fusion point, incorporating intronic sequences in mature mRNA. Reexamination of two variant SYT-SSX2 genes reported previously revealed that unknown sequences inserted at the common junction points were derived from intron sequences, as in the present case.