Retrieval strategy for ruptured balloon with circumferential tear during angioplasty for arteriovenous fistula in hemodialysis patients.

INTRODUCTION: Balloon angioplasty is a common endovascular procedure. The balloon for angioplasty sometimes ruptures (incidence, 3.6%-10%), and it is constructed such that it ruptures in a longitudinal direction and complications related to rupture are rare. However, on rare occasions, retrieval is challenging, especially in the case of ruptures with a circumferential tear. There is no established method for retrieval and careful retrieval is required due to the risk of embolization by the residual balloon fragment. TECHNIQUE: We describe two cases of balloon rupture in the transverse direction during percutaneous transluminal angioplasty for arteriovenous fistula in hemodialysis patients. In these cases, the balloon ruptured with a circumferential tear and dissected into two parts, and the tip edge remained in the vessel. We inserted an additional introducer at the side of the tip edge, caught the guidewire by a gooseneck snare, and hooked the residual balloon fragment. This also stabilized and increased the stiffness of the guidewire through the "pull-through technique." Then, we reintroduced the gooseneck snare to catch the residual balloon. We then inserted a cobra-head catheter from the first introducer and pushed the residual balloon. We finally retrieved the ruptured balloon by pulling back the gooseneck snare and pushing using the cobra-head catheter simultaneously. RESULTS: We could retrieve the ruptured balloons successfully using this technique and percutaneous transluminal angioplasty was continued in both cases. CONCLUSION: Our technique of retrieval may be suitable for cases of balloon rupture with a circumferential tear during percutaneous transluminal angioplasty. The technique enables less invasive retrieval and continuation of the percutaneous transluminal angioplasty thereafter.

Recurrent gastric volvulus associated with a gastrointestinal stromal tumor: A case report.

INTRODUCTION: Gastric volvulus (GV) is defined as a rotation of the stomach along its short or long axis leading to variable degrees of gastric outlet obstruction. Rotation of the stomach >180° may cause closed loop obstruction and possible strangulation, which often causes acute abdominal pain. Strangulation and gangrene of the twisted stomach sometimes occurs, which demands immediate surgical intervention. We report a case of acute gastric volvulus due to a gastrointestinal stromal tumor (GIST), with multiple recurrences, that eventually required emergency gastrectomy. PRESENTATION OF THE CASE: A 71-year-old woman with a history of recurrent epigastric pain, nausea, and anorexia was diagnosed to have a 70-mm sized submucosal tumor (SMT) in the lesser curvature of the stomach. An elective gastrectomy was planned; however, before the procedure, she visited the emergency room with acute recurrent epigastric pain associated with postural variations. Computed tomography (CT) revealed a GV and the tumor had shifted to the greater curvature. An emergency gastrectomy was performed. The postoperative course was uneventful and pathological examination revealed features consistent with that of GIST. DISCUSSION: GV with GIST has rarely been reported and risk factors such as size or localization are unknown. In this case, GV was probably caused by GIST of the stomach, which was large and heavy enough to rotate the gastric body around the mesenteroaxis. CONCLUSION: Surgical intervention without delay should be planned in similar scenarios accounting for the risk of GV in GIST.

Anterograde bile duct drainage for intractable bile leakage after hepatectomy in a patient with previous pancreatoduodenectomy: A case report.

INTRODUCTION: Endoscopic retrograde drainage is effective for managing bile leakage, which is relatively common after hepatectomy without bile duct reconstruction. However, the procedure is difficult to perform after pancreatoduodenectomy with choledochojejunostomy. We present a case of anterograde bile duct drainage for intractable bile leakage after hepatectomy in a patient with previous pancreatoduodenectomy. PRESENTATION OF CASE: An 80-year-old woman with a history of pancreatoduodenectomy for distal biliary cancer and adjuvant chemotherapy presented with bile leakage. Six years after the pancreatoduodenectomy, she underwent partial hepatectomy for suspected metastasis or intrahepatic cholangiocarcinoma. On the 9th postoperative day, bile leaked from her drainage tube forming an abscess cavity; this continued until the 28th postoperative day. We attempted selective anterograde drainage from the cut surface of the liver under fluoroscopic guidance using a guidewire and Cobra-type catheter. We selectively cannulated the entrance hole of the bile duct. Twenty days after the drainage, the abscess cavity disappeared. After 41 days, the tube was removed, and the patient was discharged. We suggest this procedure as a possible treatment option for difficult bile leakage cases. DISCUSSION: A case of intractable bile leakage after hepatectomy in a patient with a previous history of pancreatoduodenectomy is difficult to manage, and usually needs surgical intervention. The effect of selective cannulation of the entrance hole of the bile duct has not been studied. CONCLUSION: Selective anterograde bile duct drainage for intractable bile leakage after hepatectomy in a patient with a previous history of pancreatoduodenectomy successfully resolved bile duct leakage in our patient.

Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway.

BACKGROUND: We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, ß-actin expression was measured after siRNA ß-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. RESULTS: mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of ß-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and ß-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. CONCLUSIONS: These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

Spontaneous rupture of the urinary bladder due to bacterial cystitis.

We report a case of spontaneous rupture of the urinary bladder (SRUB) due to bacterial cystitis in a 76-year-old woman with chief complaint of abdominal pain a day before presentation. She had fever (38.0°C), and her systolic blood pressure dropped to 70 mmHg; she was referred to our hospital, where she was admitted with a diagnosis of ileus. However, her abdominal pain worsened the following day, and abdominal CT showed free air. Emergency laparotomy was performed for suspicion of digestive tract perforation, which revealed a small hole at the dome of the urinary bladder and another at the peritoneum. Suture repair was performed. We reviewed the abdominal CT on admission and noted that the perforation of the urinary bladder was present during admission, whereas that of the peritoneum occurred the following day. SRUB is rare, and bacterial cystitis rarely causes it; thus, accurate diagnosis and proper treatment are essential.

Released fibroblast growth factor18 from a collagen membrane induces osteoblastic activity involved with downregulation of miR-133a and miR-135a.

We have developed a unique delivery system of growth factors using collagen membranes (CMs) to induce bone regeneration. We hypothesized that fibroblast growth factor18 (FGF-18), a pleiotropic protein that stimulates proliferation in several tissues, can be a good candidate to use our delivery system for bone regeneration. Cell viability, cell proliferation, alkaline phosphatase activity, mineralization, and marker gene expression of osteoblastic differentiation were evaluated after mouse preosteoblasts were cultured with a CM containing FGF-18, a CM containing platelet-derived growth factor, or a CM alone. Furthermore, expression of microRNA, especially miR-133a and miR-135a involving inhibition of osteogenic factors, was measured in preosteoblasts with CM/FGF-18 or CM alone. A sustained release of FGF-18 from the CM was observed over 21 days. CM/FGF-18 significantly promoted cell proliferation, alkaline phosphatase activity, and mineralization compared to CM alone. Gene expression of type I collagen, runt-related transcription factor 2, osteocalcin, Smad5, and osteopontin was significantly upregulated in CM/FGF-18 compared to CM alone, and similar to CM/platelet-derived growth factor. Additionally, CM/FGF-18 downregulated expression of miR-133a and miR-135a. These results suggested that released FGF-18 from a CM promotes osteoblastic activity involved with downregulation of miR-133a and miR-135a.

The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration.

Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (µCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative µCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.

Ex vivo nonviral gene delivery of µ-opioid receptor to attenuate cancer-induced pain.

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 (∼45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the µ-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified ß-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more ß-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

Midline extraperitoneal approach for bilateral widespread retroperitoneal abscess originating from anorectal infection.

INTRODUCTION: Anorectal abscess is one of the most common anorectal conditions encountered in practice. However, such abscesses may rarely extend upward and cause life-threatening medical conditions. PRESENTATION OF CASE: A 53-year-old woman presented with symptoms of anorectal abscess and evidence of severe inflammatory response and acute kidney injury. Computed tomography revealed a widespread abscess extending to the bilateral retroperitoneal spaces. Surgical drainage was performed via a totally extraperitoneal approach through a lower midline abdominal incision, and the patient had a rapid and uncomplicated recovery. DISCUSSION: Although retroperitoneal abscesses originating from the anorectal region are rare, they are life-threating events that require immediate treatment. Percutaneous abscess drainage has been recently evolved; however, surgical drainage is required sometimes that may be challenging, particularly in the case of widespread abscesses, as in our case. CONCLUSION: The midline extraperitoneal approach reported here might be an effective surgical option for patients with bilateral widespread retroperitoneal abscesses.

Donor pretreatment with DHMEQ improves islet transplantation.

BACKGROUND: Currently, pancreatic islet transplantation to achieve normoglycemia in insulin-dependent diabetes mellitus (IDDM) requires two or more donors. This may be due to the inability to transplant functionally preserved and viable islets after isolation. Islets have already been subjected to various harmful stresses during the isolation process leading to apoptosis. One of the intracellular signaling pathways, the transcription factor nuclear factor-kappaB (NF-kappaB)-related pathway, is relevant to the mechanism of beta-cell apoptosis in isolated islets. We attempted to prevent islet apoptosis during isolation by a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). MATERIALS AND METHODS: DHMEQ was injected intraperitoneally into donor mice 2 h prior to isolation. NF-kappaB activation, the functioning of isolated islets, apoptosis after isolation, and cytokine- and apoptosis-related genes were analyzed. After 160 equivalents of islets were transplanted into diabetic mice, graft survival and function were evaluated. RESULTS: Intra-islet NF-kappaB was activated immediately after isolation, and DHMEQ inhibited NF-kappaB activation without deterioration of islet function. DHMEQ significantly prevented apoptosis by inhibiting caspase 3/7 activities and down-regulated Bax, a pro-apoptotic gene. Donor pretreatment with DHMEQ significantly improved engraftment in syngeneic islet transplantation in mice, thus preserving insulin contents in the graft liver, as assessed by functional and histologic analyses. CONCLUSIONS: DHMEQ is a promising agent in islet transplantation because it protects islets from apoptosis during isolation stress. Donor pretreatment with DHMEQ can significantly affect the success of islet engraftment.

Noble gene transduction into pancreatic beta-cells by singularizing islet cells with low doses of recombinant adenoviral vector.

Adenovirus-mediated gene transduction into the intact islets has thus far been limited to the surface cells of islets. We evaluated the efficiency of gene delivery by singularization of islets, followed by self-reorganization into islet-like masses. Adenovirus-mediated gene transduction was performed on dispersed islet cells, obtained by two-step digestion of collagenase and ethylene glycol tetraacetic acid/dispase. Good self-reorganization of islet cells in culture was observed until 120 h in islet cells of a control group, a group with a multiplicity of infection (MOI) of 1, and a group with an MOI of 5, with their sizes of 66.7 +/-14.17, 64.0 +/- 15.14, and 60.8 +/- 23.71 microm, respectively. No significant difference in spontaneous reaggregation capability among the islet cell masses was noticed. However, fragmentation of the reaggregated islet mass was observed in the groups with an MOI of 10 and 50 at 72 and 48 h, respectively. The gene transduction rates at an MOI of 0.5, 1, and 5 into islet cells were 56.1 +/- 1.43, 97.6 +/- 0.92, and 100 +/- 0.00%, respectively. The insulin stimulation indices of the reaggregated islet mass at an MOI of 0.5 and 1 were preserved to the level of a nontransduced islet mass; those at an MOI greater than 5 were significantly low. Efficient adenovirus-mediated gene transduction into islet/beta-cells was achieved by adding a process of dispersion of islets into single cells prior to gene transduction without losing the characteristic ability of islet cells to form a functional islet mass in culture.

Establishment of Immortalized Human Hepatocytes by Introduction of HPV16 E6/E7 and hTERT as Cell Sources for Liver Cell-Based Therapy.

For future cell-based therapies for liver diseases, the shortage of cell sources must be resolved. Immortalized human hepatocytes are expected to be among the new sources. In addition to telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT), inactivation of the p16/RB pathway and/or p53 by E6/E7 of human papillomavirus type 16 (HPV16) has been shown to be useful for efficient immortalization of several human cell types. Here we report the immortalization of human hepatocytes by the introduction of HPV16 E6/E7 and hTERT. Human adult hepatocytes were lentivirally transduced with HPV16 E6/E7 and hTERT. Two human immortalized hepatocyte cell lines were established and were named HHE6E7T-1 and HHE6E7T-2. Those cells proliferated in culture beyond 200 population doublings (PDs). Albumin synthesis and expression of liver-enriched genes were confirmed, but gradually decreased as passages progressed. Karyotype analysis showed that HHE6E7T-1 cells remained near diploid but that HHE6E7T-2 cells showed severe aneuploidy at 150 PDs. Subcutaneous injection of these cells into severe combined immunodeficiency (SCID) mice did not induce tumor development. Intrasplenic transplantation of dedifferentiated HHE6E7T-1 cells over 200 PDs significantly improved the survival of acetaminophen-induced acute liver failure SCID mice. In conclusion, we successfully established immortalized human hepatocytes that retain the characteristics of differentiated hepatocytes. We also showed the reduction of hepatocyte-specific functions in long-term culture. However, the results of intrasplenic transplantation to SCID mice with acetaminophen-induced acute liver failure showed the possibility of HHE6E7T-1 serving as a cell source for hepatocyte transplantation.

Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.

For future cell-based therapies for liver diseases, the shortage of cell sources must be resolved. Immortalized human hepatocytes are expected to be among the new sources. In addition to telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT), inactivation of the p16/RB pathway and/ or p53 by E6/E7 of human papillomavirus type 16 (HPV16) has been shown to be useful for efficient immortalization of several human cell types. Here we report the immortalization of human hepatocytes by the introduction of HPV16 E6/E7 and hTERT. Human adult hepatocytes were lentivirally transduced with HPV16 E6/E7 and hTERT. Two human immortalized hepatocyte cell lines were established and were named HHE6E7T-1 and HHE6E7T-2. Those cells proliferated in culture beyond 200 population doublings (PDs). Albumin synthesis and expression of liver-enriched genes were confirmed, but gradually decreased as passages progressed. Karyotype analysis showed that HHE6E7T-1 cells remained near diploid but that HHE6E7T-2 cells showed severe aneuploidy at 150 PDs. Subcutaneous injection of these cells into severe combined immunodeficiency (SCID) mice did not induce tumor development. Intrasplenic transplantation of dedifferentiated HHE6E7T-1 cells over 200 PDs significantly improved the survival of acetaminophen-induced acute liver failure SCID mice. In conclusion, we successfully established immortalized human hepatocytes that retain the characteristics of differentiated hepatocytes. We also showed the reduction of hepatocyte-specific functions in long-term culture. However, the results of intrasplenic transplantation to SCID mice with acetaminophen-induced acute liver failure showed the possibility of HHE6E7T-1 serving as a cell source for hepatocyte transplantation.

Role of caspases in cleavage of lamin A/C and PARP during apoptosis in macrophages infected with a periodontopathic bacterium.

The periodontopathic bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of periodontal diseases. It has been reported previously that infection with the organism induced apoptosis in the mouse macrophage cell line J774.1. In the present study, the role of caspases during apoptosis in A. actinomycetemcomitans-infected J774.1 cells was examined. A large number of apoptotic cells was detected by flow cytometric analysis in infected J774.1 cells; however, inhibitors of caspase-9, -6 and -3/7 completely blocked the induction of apoptosis. Expression of the cleaved forms of caspase-6 and -7 was detected during apoptosis in infected J774.1 cells. Immunoblot analysis revealed that the caspase-9 inhibitor blocked expression of the cleaved forms of caspase-6 and -7, whilst the caspase-3 inhibitor blocked expression of the cleaved form of caspase-7, but not caspase-6. It is known that lamin A/C and poly(ADP-ribose) polymerase (PARP) are essential nuclear components for maintaining normal cell function and viability, and both were found to be cleaved in the infected J774.1 cells. Immunoblot analysis also revealed that the caspase-6 inhibitor blocked the cleavage of lamin A/C, whilst the caspase-3/7 inhibitor blocked the cleavage of PARP. Taken together, these results suggest that activation of caspases and the subsequent cleavage of lamin A/C and PARP are involved in the morphological changes of apoptotic macrophages infected with A. actinomycetemcomitans.

Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures.

OBJECTIVES: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. RESULTS: EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.

Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans.

We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53.