Calcineurin inhibitors exacerbate coronary arteritis via the MyD88 signalling pathway in a murine model of Kawasaki disease.

Calcineurin inhibitors (CNIs) have been used off-label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in wild-type, severe combined immunodeficiency (SCID), caspase-associated recruitment domain 9 (CARD9)-/- and myeloid differentiation primary response gene 88 (MyD88)-/- mice. We also performed in-vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1-mediated coronary arteritis in a dose-dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9-/- mice but not in MyD88-/- mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88-dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.

Effects of transforming growth factor beta 1 on the plasminogen activation system, collagen and integrin synthesis, and proliferation of rabbit mandibular condylar chondrocytes.

The objective of this study was to identify the mechanism by which mandibular condyle chondrocytes regulate the extracellular matrix. Primary rabbit condylar chondrocytes were isolated, cultured, and treated with transforming growth factor beta 1 (TGF-ß1). Cells were then assayed for the following: urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1), collagen types I and II, ß1 integrin expression, and proliferative activity. TGF-ß1 induced synthesis of collagen type II, αVß1 integrin, and PAI-1. TGF-ß1 induced the growth of chondrocytes and suppressed the synthesis of uPA. Chondrocyte regulation of the extracellular matrix is mediated by TGF-ß1. Synthesis of collagen type II, αVß1 integrin, and PAI-1 is induced, while uPA is suppressed. Also, TGF-ß1 induces cellular growth.

Clinical experience of esophageal perforation occurring with endoscopic submucosal dissection.

Esophageal perforation occurring during or after endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) is a rare, but serious complication. However, reports of its characteristics, including endoscopic imaging and management, have not been fully detailed. To analyze and report the clinical presentation and management of esophageal perforations occurred during or after EMR/ESD. Four hundred seventy-two esophageal neoplasms in 368 patients were treated (171 EMR; ESD 306) at Northern Yokohama Hospital from 2003 to 2012. Esophageal perforation occurred in a total of seven (1.9%) patients, all of whom were male and had undergone ESD. The etiology of perforation was: three (42.9%) intraoperative; three (42.9%) balloon dilatation for stricture prevention; one (14.2%) due to food bolus impaction. All cases were managed non-operatively based on the comprehensive assessment of clinical severity, extent of the injury, and the time interval from perforation to treatment onset. Conservative management included (i) bed rest and continuous monitoring to determine the need for operative intervention; (ii) fasting and intravenous fluid infusion/ tube feeding; and (iii) intravenous antibiotics. All defects closed spontaneously, save one case where closure was achieved by endoscopic clipping. Surgery was not required. Conservative management for esophageal perforation during advanced endoscopic resection is may be possible when there is no delay in diagnosis or treatment. Decision-making should be governed purely by multidisciplinary discussion.

Microvascular caliber changes in intramucosal and submucosally invasive esophageal cancer.

BACKGROUND AND STUDY AIM: Intrapapillary capillary loops (IPCLs) show distinct pattern changes corresponding to tumor progression and depth of invasion, important for in vivo characterization of superficial squamous cell carcinoma (SCC). We examined the relation between invasion depth and histopathologic IPCL diameter. PATIENTS AND METHODS: Prospectively, before lesion resection, magnification endoscopy and narrow band imaging were used to identify IPCL patterns of type V1 (corresponding to tumors limited to the mucosa; 10 patients) and type Vn (submucosally invading tumors; 10 patients). Post-resection, IPCL samples (type I [normal mucosa], n = 103; V1, n = 113; Vn, n = 100) were stained with hematoxylin & eosin, CD34, and desmin, and vessel diameter measured using light microscopy. RESULTS: Mean (standard deviation [SD]) histopathologic calibers of IPCLs of types I, V1, and Vn were significantly different, being 7.7 (2.8) µm, 21.9 (7.4) µm, and 65.2 (22.9) µm; type 1 vs. V1, P < 0.001; V1 vs. Vn, P < 0.001. CONCLUSIONS: Magnification endoscopy observation of IPCLs allows in vivo discrimination between intramucosal and submucosally invasive cancer.

Submucosal endoscopic tumor resection for subepithelial tumors in the esophagus and cardia.

BACKGROUND AND STUDY AIMS: Resection of submucosal tumors by means of endoscopy has been reported using a variety of techniques, but cannot be performed safely in tumors originating from the muscularis propria. Using the submucosal tunnel created by the technique of peroral endoscopic myotomy (POEM), we report the first series describing the new technique of submucosal endoscopic tumor resection (SET) for tumors of the esophagus and cardia. PATIENTS AND METHODS: SET was attempted in nine consecutive patients with tumors (size >2cm) of either the esophagus or cardia with clinical indications for lesion removal. Following creation of a submucosal tunnel from 5 cm above the tumor, as described previously, the tumor was dissected from the overlying mucosa/submucosa and then carefully removed from the muscular layer using triangle-tip and insulated-tip knives. Following specimen retrieval through the tunnel, the orifice was closed by clips. RESULTS: Of the nine patients, two had tumors that were too large (60 mm and 75 mm, respectively) to allow safe removal due to loss of endoscopic overview. All remaining tumors (maximal tumor extension 12-30 mm) could be resected safely using this method. No complications occurred and follow-up was unremarkable. On histology, all tumors were resected completely (one gastrointestinal stromal tumor, five leiomyomas). The technique had to be modified in one patient with an aberrant pancreas. CONCLUSIONS: SET is a promising new technique for selected submucosal tumors in the esophagus and cardia up to a size of 4 cm and should be studied further.

Gemcitabine synergistically enhances the effect of adenovirus gene therapy through activation of the CMV promoter in pancreatic cancer cells.

Adenovirus-mediated gene therapy shows remarkable promise as a new strategy for advanced pancreatic cancer, but satisfactory clinical results have not yet been obtained. To improve this gene therapy, we investigated the effects of gemcitabine (GEM) on transgene expression by adenoviral vectors and their biological effects. We used Ad-lacZ and adenoviral vector-expressing NK4 (Ad-NK4) as representative adenoviral vectors. These vectors express beta-galactosidase (beta-gal) and NK4 (which inhibits the invasion of cancer cells), respectively, under the control of the CMV promoter. Cells were infected with the individual adenoviruses and then treated with GEM. GEM increased beta-gal mRNA expression and beta-gal activity, and increased NK4 expression in both culture media and within infected cells, in dose-dependent manners. The increased expression of NK4 delivered by Ad-NK4 had biological effects by inhibiting the invasion of cancer cells. GEM also enhanced NK4 expression in SUIT-2 cells transfected with an NK4-expressing plasmid, suggesting that GEM enhanced CMV promoter activity. In in vivo experiments, NK4 expression within subcutaneously implanted tumors was increased in GEM-treated mice compared with control mice. These results suggest that adenovirus-mediated gene therapy with GEM may be a promising approach for treating pancreatic cancer, and that this combination therapy may decrease the risks of side effects.

Recombinant Sendai virus vectors for activated T lymphocytes.

T-lymphocyte-directed gene therapy has potential as a treatment of subjects with immunological disorders. One current limitation of this therapeutic strategy is low gene transfer efficiency, even when complex procedures are used. We report herein that a recombinant Sendai virus vector (SeV) was able to overcome this issue. Using jellyfish enhanced green fluorescent protein gene (EGFP), we found that SeV was able to transduce and express a foreign gene specifically and efficiently in activated murine and human T cells, but not in naive T cells, without centrifugation or reagents including polybrene and protamine sulfate; the present findings were in clear contrast to those demonstrated with the use of retroviruses. The transduction was selective in antigen-activated T cells, while antigen-irrelevant T cells were not transduced, even under bystander activation from specific T-cell responses by antigens ex vivo. Receptor saturation studies suggested a possible mechanism of activated T-cell-specific gene transfer, ie, SeV might attach to naive T cells but might be unable to enter their cytoplasm. We therefore propose that the SeV vector system may prove to be a potentially important alternative in the area of T-cell-directed gene therapy used in the clinical setting.

Random multi-recombinant PCR for the construction of combinatorial protein libraries.

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries. For the construction of a 'random shuffling library', RM-PCR was used to shuffle six DNA fragments each encoding 25 amino acids; this affords many different fragment sequences whose every position has an equal probability to encode any of the six blocks. For the construction of the 'alternative splicing library', RM-PCR was used to perform different alternative splicings at the DNA level, which also yields different block sequences. DNA sequencing of the RM-PCR products in both libraries revealed that most of the sequences were quite different, and had a long open reading frame without a frame shift or stop codon. Furthermore, no distinct bias among blocks was observed. Here we describe how to use RM-PCR for the construction of combinatorial DNA libraries, which encode protein libraries that would be suitable for selection experiments in the global protein space.

Hypoxia-induced angiogenesis of cultured human salivary gland carcinoma cells enhances vascular endothelial growth factor production and basic fibroblast growth factor release.

The angiogenic activity of two human salivary gland tumor cell lines, ACCS from adenoid cystic carcinoma and IT-2 from mucoepidermoid carcinoma, was examined by stimulating tube formation by bovine capillary endothelial cells (BCE). ACCS and IT-2 were cultured in 20 or 3% oxygen, representing normoxic and hypoxic conditions, respectively, and conditioned medium (CM) was obtained from each culture. The BCE tubes stimulated by hypoxic CM were 1.59 (ACCS) and 1.42 (IT-2) times longer than those stimulated by normoxic CM. The tube-forming activity of CM was inhibited by preincubation with either anti-vascular endothelial growth factor (VEGF) IgG or anti-basic fibroblast growth factor (bFGF) IgG, suggesting that both VEGF and bFGF with angiogenic activity were present in the CM. This was confirmed by ELISA, which also demonstrated increased concentrations of both proteins in the hypoxic CM. Northern blot analysis showed an increased VEGF mRNA level in both carcinoma cells with hypoxia, while hypoxia did not affect the bFGF mRNA level in either cell line. The results suggest that both VEGF and bFGF are major angiogenesis factors in salivary gland tumors, and hypoxia-induced angiogenesis results from upregulation of VEGF and increased release of bFGF.

Sp1 decoy transfected to carcinoma cells suppresses the expression of vascular endothelial growth factor, transforming growth factor beta1, and tissue factor and also cell growth and invasion activities.

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the angiogenic factors produced by tumor cells, vascular endothelial growth factor is considered to be the most potent and pathologically important. The synthesis of this growth factor has been shown to be modulated through Sp1 function following stimulation by tumor necrosis factor alpha (TNF-alpha). Oligodeoxynucleotides (ODNs) were synthesized with either the consensus sequence for Sp1 binding (Sp1 decoy ODNs) or a mutated form of this sequence (mt-Sp1 decoy ODNs). Using the hemagglutinating virus of Japan (HVJ)-liposome method, we transferred these ODNs into cultured cancer cells (A549 and U251 cells). The TNF-alpha-mediated expression of both VEGF and transforming growth factor beta1 and tissue factor (TF) by the cancer cells could be simultaneously suppressed to less than 30% by transfection of Sp1 decoy ODNs but not by mt-Sp1 decoy ODNs. In addition, in vitro invasiveness, synthesis of mRNA for urokinase-type plasminogen activator, and cell proliferation of both cell lines were also inhibited to 40% by the transfection of only Sp1 decoy ODNs. These results suggested that the Sp1 decoy strategy would be effective for regulating tumor growth by simultaneously reducing cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness.

Characterization and distribution of a new enterotoxin-related superantigen produced by Staphylococcus aureus.

Staphylococcal enterotoxins (SEs) are a family of structurally related pyrogenic exotoxins consisting of the five prototypic SEs (types A to E) and three newly characterized SEs (types G to I) produced by Staphylococcus aureus (S. aureus). They also work as superantigens and cause food poisoning and shock symptoms in humans. In this study, we cloned a new variant gene of the seg and characterized its superantigenic properties and distribution among the clinical isolates of S. aureus. The gene encodes a 233 amino acid protein which is highly homologous to SEG (97.7%). The variant SEG (SEGv) expressed by the cloned gene exerted mitogenic activity on human peripheral blood mononuclear cells at the concentration of 100 pg/ml. T cells bearing Vbeta3, 12, 13.1, 13.2, 14 and 15 were preferentially expanded after stimulation with the recombinant protein. The mRNA of the variant seg gene was detected in the total RNA of the organisms bearing this gene. By PCR, 27 out of 48 clinical isolates of S. aureus (56%) possessed either the seg or variant seg gene. These findings suggest that SEG, or SEGv, is one of the most frequently produced superantigen exotoxins by S. aureus and may participate in the inflammatory process of the host by activating a distinct set of Vbeta families of T cells.

Clinical role for a superantigen in Yersinia pseudotuberculosis infection.

Yersinia pseudotuberculosis is an enteric pathogen that causes a variety of clinical symptoms in the human. Recently, we reported the production of a superantigen (Y. pseudotuberculosis-derived mitogen, YPM) by this organism and characterized the gene structure of ypm. To further study the potential pathogenic role of YPM in Y. pseudotuberculosis infection, we assayed IgG anti-YPM antibodies and T cell antigen receptor-Vbeta expression of the T cells in peripheral blood and in mesenteric lymph node in patients acutely infected with Y. pseudotuberculosis. 20 out of 33 patients (61%) had an elevated antibody titer compared with healthy controls (P = 0.0001). Patients with systemic symptoms such as lymphadenopathy, transient renal dysfunction, and arthritis had significantly higher titers of anti-YPM than patients with gastrointestinal tract symptoms alone. T cells bearing the Vbeta3 gene segment were significantly increased (P = 0.009) among acute phase patients compared with healthy children. During the convalescence phase of the illness, there was a reduction in the abnormal level of Vbeta3 T cells. Moreover, in the mesenteric lymph node, an elevated level of Vbeta3 T cells compared with peripheral blood and a sequence diversity in the junctional region of the T cell antigen receptor beta-chain containing Vbeta3 element was observed in one patient. Together, these findings suggest that YPM was produced in vivo and played an important role in the pathogenesis of Y. pseudotuberculosis infection.

Isovolumetric relaxation flow in patients with ischemic heart disease.

OBJECTIVES: The purpose of this study was to clarify the characteristics of flow during the isovolumetric relaxation period and to analyze the relation between these flow patterns and standard hemodynamic indexes. BACKGROUND: Outward motion of the left ventricle during the isovolumetric relaxation period has been observed by cineangiography. However, there is little information about blood flow during this period. METHODS: Seventy-nine patients with ischemic heart disease were examined by pulsed Doppler echocardiography and cardiac catheterization. All patients were classified into three groups according to the observed patterns of isovolumetric relaxation flow: group I (n = 41), flow directed toward the apex; group II (n = 21), flow directed toward the base, and group III (n = 17), low velocity flow (< 12 cm/s) without a dominant direction. Patients in group I were further classified into group Ia (n = 19) with normal ventriculograms and group Ib (n = 22) with asynergy. RESULTS: Left ventricular ejection fraction and negative first derivative of left ventricular pressure were significantly lower in group II (49 +/- 9% and 1,274 +/- 212 mm Hg/s, respectively) and group III (38 +/- 8% and 1,147 +/- 280 mm Hg/s, respectively) than in group Ia (68 +/- 7% and 1,727 +/- 358 mm Hg/s), each p < 0.01. Time constant was significantly prolonged in group II (49 +/- 6 ms) and group III (48 +/- 6 ms) compared with that in group Ia (41 +/- 6 ms), p < 0.05. On left ventriculography, patterns of outward wall motion during the isovolumetric relaxation period were associated with the patterns of isovolumetric relaxation flow. CONCLUSIONS: Changes in left ventricular relaxation are accompanied by alterations in isovolumetric relaxation flow. It is therefore useful to evaluate isovolumetric relaxation flow when investigating early diastolic ventricular function.

[Left ventricular blood flow dynamics caused by ventricular premature contraction: pulsed Doppler echocardiographic study].

Cardiac function at the time of ventricular premature contractions (VPC) is influenced by the coupling interval or the site of those origin. Clinical and experimental studies of the effects of VPC on intracardiac pressure dynamics have been performed; however, little is known about left ventricular blood flow dynamics. This study was attempted to determine the characteristics of blood flow dynamics in respect to the site of origin of VPC using pulsed Doppler echocardiography. The subjects consisted of 18 cases with VPC but without apparent organic heart disease. Seven cases had VPCs with a left bundle branch block pattern suggesting possible origin in the right ventricle. The other 11 cases had VPCs with a right bundle branch block pattern indicating the left ventricular origin. With the probe in the apical position, the blood flow patterns of the left ventricular outflow, central and inflow tracts were examined. The results were as follows; 1. Except for one case with shortened coupling interval, all six cases with VPCs originated from the right ventricle showed preservation of left ventricular ejection flow. 2. In two of the three cases with VPC which originated from the left ventricle and with left axis deviation, systolic flow in the left ventricular central area showed "back flow" to the apex. Ejection flow at the outflow tract was markedly diminished or disappeared in all three cases. 3. In all eight cases with VPC which originated from the left ventricle and with right axis deviation, ejection flow was slightly disturbed both in the left ventricular outflow and in the central area. 4. Ejection flow volume assessed by velocity integral indicated similar dynamics as did the ejection flow velocity. 5. In left ventriculography, asynchrony due to dyskinetic motion of the anteroapical wall was observed at the times of VPCs with left axis deviation. In conclusion, the patterns of left ventricular ejection flow dynamics depend on the site of origin of VPCs. This disturbed flow is more apparent in VPCs originating from the left ventricle compared to the right ventricle. This is especially true in cases with left axis deviation, in which VPCs arise from the posterior site of the left ventricle.