Venous superdrained gastric tube pull-up procedure for hypopharyngeal and cervical esophageal reconstruction reduces postoperative anastomotic leakage and stricture.

Gastric pull-up is a common procedure to reconstruct the continuity of the upper digestive tract after esophageal resection. However, this technique sometimes causes postoperative anastomotic leakage or stricture, resulting from insufficient blood flow at the distal end. To overcome this problem, additional microvascular venous anastomoses were performed. The purpose of this study was to compare the outcomes of post-surgical anastomotic leakage and stricture in patients with and without additional microvascular venous superdrainage after cervical esophageal and hypopharyngeal resection and gastric tube reconstruction. A total of 29 consecutive patients with esophageal or hypopharyngeal cancer who underwent total esophagectomy and hypopharyngectomy with gastric tube reconstruction in the National Organization Nagasaki Medical Center between April 2014 and May 2016 were analyzed in this study. Of these patients, 20 underwent additional venous anastomoses (superdrainage group), and 9 did not undergo additional procedures (standard group). We compared the frequency of post-surgical stricture and leakage in the two groups retrospectively. Three of nine patients (33.3%) developed postoperative leakage in the standard group, and 1 of 20 (5.0%) did so in the superdrainage group. Six of nine patients (66.7%) showed postoperative anastomotic stricture in the standard group, but none did so in the superdrainage group. Patients who did not undergo additional venous superdrainage were significantly more likely to develop postsurgical leakage (P < 0.05, Chi-square test) and anastomotic stricture (P < 0.001, Chi-square test). Our study revealed that only additional venous anastomoses could reduce the incidence of postoperative anastomotic leakage and stricture. This procedure is of merit to perform after total esophagectomy and hypopharyngectomy with gastric tube reconstruction.

Restricted usage of T cell receptor V alpha-V beta genes in infiltrating cells in the hearts of patients with acute myocarditis and dilated cardiomyopathy.

Prolonged myocardial cell damage initiated by acute myocarditis is thought to be one of the most important etiology of dilated cardiomyopathy. To investigate the immunological mechanisms involved in the pathogenesis of dilated cardiomyopathy, we analyzed the phenotypes of infiltrating cells and examined the expression of perforin in infiltrating cells in the hearts of patients with dilated cardiomyopathy as well as acute myocarditis. We also examined the expression of HLA and intercellular adhesion molecule-1 (ICAM-1) in myocardial tissue of these patients. Furthermore, to evaluate the antigen specificity of infiltrating T cells and persistence of viral genomes in the myocardial tissue, we analyzed the expression of T cell receptor (TCR) V alpha and V beta genes as well as enterovirus genomes by PCR. We found infiltration of perforin-expressing killer cells and enhanced expression of HLA class I and ICAM-1 in the myocardial tissue. We also found that the repertoires of TCR V alpha as well as V beta gene transcripts were restricted, indicating that a specific antigen in the hearts was targeted. Because no enterovirus genomes were detected in all patients, it is strongly suggested that a cell-mediated autoimmune mechanism triggered by virus infection may play a critical role in the pathogenesis of dilated cardiomyopathy. However, we could not exclude the possibility that viruses other than enteroviruses could be pathogenic in these patients.

IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis.

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

Neural correlates of ticklishness in the rat somatosensory cortex.

Rats emit ultrasonic vocalizations in response to tickling by humans. Tickling is rewarding through dopaminergic mechanisms, but the function and neural correlates of ticklishness are unknown. We confirmed that tickling of rats evoked vocalizations, approach, and unsolicited jumps (Freudensprünge). Recordings in the trunk region of the rat somatosensory cortex showed intense tickling-evoked activity in most neurons, whereas a minority of cells were suppressed by tickling. Tickling responses predicted nontactile neural responses to play behaviors, which suggests a neuronal link between tickling and play. Anxiogenic conditions suppressed tickling-evoked vocalizations and trunk cortex activity. Deep-layer trunk cortex neurons discharged during vocalizations, and deep-layer microstimulation evoked vocalizations. Our findings provide evidence for deep-layer trunk cortex activity as a neural correlate of ticklishness.

Molecular cloning of a novel serine/threonine kinase, MRK, possibly involved in cardiac development.

We have isolated a novel member of putative serine/threonine kinase from a rat heart cDNA library using polymerase chain reaction methods. The novel kinase is transcribed as 2.6 kb mRNA encoding for a protein of 629 amino acids with the C-terminal non-catalytic portion. Amino acids analysis revealed that the N-terminal catalytic domain is 87% identical to the male-germ cell associated kinase (MAK), a cdc2-related serine/threonine kinase found to promote meiosis during spermatogenesis. Therefore, we designated this novel kinase as the MAK-related kinase (MRK). MRK protein, with a molecular weight of 66 kD, was shown to phosphorylate itself and the exogenous substrates, histone H1 and myelin basic protein. In addition, phosphoamino acid analysis confirmed the serine/threonine-specific protein kinase activity of MRK. Although MRK was ubiquitous in adult rat tissues, the expression of MRK protein in embryos was restricted primarily to embryonic myocardium during early organogenesis. This finding suggests that MRK may be a participant in cardiac development.

Clinical characteristics of Japanese men with glucokinase gene beta-cell promoter variant.

OBJECTIVE: To study the association between a variant at the position of -30 of beta-cell-specific promoter of the glucokinase gene and glucose tolerance in the Japanese general population and to assess the clinical characteristics of subjects with the variant. RESEARCH DESIGN AND METHODS: The genotype of 657 Japanese men aged 51.0 +/- 8.8 years (mean +/- SD) was analyzed by an allele-specific assay using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The variant allele frequency was 0.188 in subjects with normal glucose tolerance, 0.211 in subjects with impaired glucose tolerance, and 0.176 in diabetic subjects. In subjects with fasting plasma glucose levels <140 mg/dl, homozygous subjects for the promoter variant had significantly higher plasma glucose levels 60 min after oral glucose administration when compared with subjects without the variant allele. A cross-sectional analysis showed age-related elevation of basal glucose levels only in subjects without the promoter variant. Individuals heterozygous for the variant had significantly lower levels of HDL cholesterol than normal subjects. HDL cholesterol values were lower in homozygous people than in normal and heterozygous subjects, although the differences were not statistically significant. CONCLUSIONS: The beta-cell promoter variant in homozygous state was associated with impaired glucose tolerance, but not with diabetes, and low HDL cholesterol levels in Japanese men. It is unlikely that the glucose intolerance associated with the promoter variant is progressive with age.

Codon 972 polymorphism of the insulin receptor substrate-1 gene in impaired glucose tolerance and late-onset NIDDM.

OBJECTIVE: To assess the relevance of a Gly-->Arg substitution in codon 972 of the insulin receptor substrate-1 gene in impaired glucose tolerance (IGT) and NIDDM. RESEARCH DESIGN AND METHODS: The genotype of 1,106 Japanese subjects consisting of 310 subjects with NIDDM, 305 subjects with IGT, and 491 normal control subjects was analyzed by an allele-specific assay using polymerase chain reaction and restriction fragment length polymorphism. RESULTS: The frequency of the variant allele was not different between subjects with NIDDM (0.021) and normal control subjects (0.020). However, subjects with IGT showed a significantly higher prevalence of the variant allele (0.041, P = 0.027). We found two homozygous individuals for the variant; both had IGT with mild insulin resistance. The allelic frequency tended to be lower in normal control subjects aged > 50 years than in younger control subjects. Conversely, in the subjects with IGT or NIDDM, the Gly972Arg substitution was more frequently found in subjects aged > 50 years. Furthermore, NIDDM patients with the variant allele had older ages of diagnosis than patients without the variant. CONCLUSIONS: The codon 972 variant may be associated with IGT and a subset of late-onset NIDDM in the elderly Japanese population.

Inhibitory effects of vesnarinone in the progression of myocardial damage in experimental autoimmune myocarditis in rats.

OBJECTIVES: Vesnarinone, a positive inotropic and immunomodulatory agent, diminishes nitric oxide (NO) levels by suppressing the induction of inducible NO synthase (iNOS) expressed in cytokine-stimulated macrophages and cardiomyocytes. We examined whether vesnarinone exerts inhibitory effects on the progression of myocardial damage in experimental autoimmune myocarditis in rats through suppression of iNOS. METHODS: Myocarditis was induced in 30 Lewis rats by injection of porcine cardiac myosin and vesnarinone was orally administered to 20 of the 30 rats. On day 21 after immunization (the climax of inflammation), the hemodynamics were examined and the severity of myocarditis was evaluated by determining the area ratio (%) [affected/entire area] of myocardial lesions in histological sections. Levels of serum CK-MB, NOx (NO2(-)+NO3-), TNF-alpha and IL-1 beta, and cyclic GMP, iNOS mRNA, TNF-alpha and IL-1 beta in heart tissues were determined. Expression of iNOS and TNF-alpha protein were examined by immunohistochemical methods. RESULTS: Histopathological examination revealed extensive myocardial destruction and massive infiltration of inflammatory cells in the vesnarinone-untreated rats. The area ratio of the lesions in the treated rats was significantly lower than that in the untreated ones. Levels of CK-MB, NOx, cyclic GMP, cytokines and iNOS mRNA were significantly lower in the vesnarinone-treated rats. Infiltrating macrophages and cardiomyocytes in the untreated rats showed much higher levels of expression of iNOS and TNF-alpha than those in the vesnarinone-treated rats. CONCLUSIONS: Vesnarinone may prove to be useful in the treatment of myocarditis by attenuating NO production through suppression of iNOS induced by cytokines.

Levels of soluble Fas in patients with myocarditis, heart failure of unknown origin, and in healthy volunteers.

This study showed that serum levels of sFas were elevated in patients with myocarditis, and that this elevation was correlated with sIL-2R level as a marker of T-cell activation. Therefore, sFas levels may be associated with T-cell activation in patients with myocarditis, and elevation of sFas may inhibit apoptosis in activated T cells, leading to persistent cell-mediated destruction of myocytes in myocarditis.

Proliferation of smooth muscle cells in acute allograft vascular rejection.

OBJECTIVES: To determine whether immune injury during acute cardiac rejection induces phenotypic modulation of arterial smooth muscle cells and lesion formation, we studied the expression of embryonic myosin heavy-chain isoform and degrees of intimal proliferation in aortas and coronary arteries of allografted rabbit hearts. Modulation of phenotype in arterial smooth muscle cells during acute vascular injury is a widely reported phenomenon, and proliferation and migration of medial smooth muscle cells contribute to development of intimal hyperplasia of arteries in response to immune injury. METHODS: Rabbit hearts were heterotopically transplanted to the neck without immunosuppression. Hearts were harvested at 2, 5, 7, and 10 days after transplantation. Proliferation of smooth muscle cells was assessed by bromodeoxyuridine labeling. Staining for immunohistochemical indicators was done with use of monoclonal antibodies that recognize T lymphocytes and all types of smooth muscle cells (SM1), adult type of smooth muscle cells (SM2), and embryonic myosin heavy-chain isoform. Intimal thickening and luminal narrowing were assessed with a computer-assisted video image analysis system. RESULTS: Intimal thickening and luminal narrowing in aortas and coronary arteries gradually increased in a time-dependent manner. The neointima thus formed consisted of proliferating smooth muscle cells positive for both SM1 and embryonic myosin heavy-chain isoform and massive T lymphocyte accumulation. Intimal proliferation was more prominent in aortas and large epicardial coronary arteries than in the intramyocardial small coronary arteries. CONCLUSIONS: These findings suggest that allogeneic immune injury facilitates phenotypic modulation of smooth muscle cells, which may contribute to subsequent transplantation-associated atherosclerosis.

Uptake of indium-111-anti-intercellular adhesion molecule-1 monoclonal antibody in the allografted rat lung during acute rejection.

BACKGROUND: Although many methods for detection and quantification of allograft rejection of the lung have been explored, only histologic diagnosis by lung biopsies has gained widespread acceptance. To examine whether indium-111-anti-intercellular adhesion molecule-1 monoclonal antibody imaging can noninvasively detect acute lung rejection, we measured the uptake of this radiopharmaceutical in lung tissue and with scintigraphy in orthotopically transplanted rat lungs. METHODS: The left lung transplant model was used with Lewis- to Wistar-King rat allografts and Lewis isograft controls. Lungs were harvested 2,3,4,5,6, and 7 days after transplantation. The transplanted and native lungs were removed 24 hours after injection of the radiotracer, weighed, and counted in a gamma well counter; uptake ratios of the transplanted or native lungs were then calculated, and scintigraphy was performed. RESULTS: Histologic rejection scores by the grading system of the International Society for Heart and Lung Transplantation at 2,3,4,5,6, and 7 in the allografts were 1.2 +/- 0.2, 2.3 +/- 0.6, 3.0 +/- 0.5, 3.7 +/- 0.4, and 4, and 4, respectively. The uptake ratios of the allografts 3,4, and 5 days after transplantation were significantly higher than the values of the respective isografts and correlated with histologic rejection grades. However, 6 days after transplantation, uptake ratios of allografts decreased and did not correlate with histologic grades. On days 3,4, and 5 after transplantation, the tracer uptake within the allografts was visualized by means of scintigraphy. CONCLUSIONS: We conclude that indium-111-anti-intercellular adhesion molecule-1 monoclonal antibody increased during mild to moderate acute lung rejection. An abnormal scintigram with this radiotracer suggests that lung biopsy should be performed to exclude lung rejection.

Participation of nitric oxide and peroxynitrite in the development of myocardial tissue damage in acute myocardial infarction.

Many factors, including superoxides, contribute to tissue damage in acute myocardial infarction (AMI). Excess nitric oxide (NO) produced by inducible NO synthase (iNOS) has also been reported to participate in myocardial injury associated with AMI, but its exact role remains unclear. To elucidate the role of NO and peroxynitrite in the pathogenesis of myocardial injury associated with AMI, we examined the expression of iNOS in the autopsied specimens of the left ventricle obtained from 15 patients with AMI and five with old MI by immunohistochemistry using an anti-iNOS polyclonal antibody. The distribution of nitrotyrosine was also examined immunohistochemically. In patients who died from 12 hours to 3 weeks after the infarction, positive immunoreactivity for iNOS was observed in residual myocytes, macrophages, and vascular endothelial cells in the peri-infarcted area. Degenerating myocytes in that area in all of that group showing positive staining for iNOS were also stained positive for anti-nitrotyrosine antibody selfsame. These findings were not observed in the myocardial specimens obtained from patients who died within 12 hours after the onset of AMI, showing a minimal number of inflammatory cells, or in the specimens from patients with an old myocardial infarction, which showed scar tissue and no cellular infiltration. Inducible NOS and nitrotyrosine were expressed in damaged myocardium from patients with AMI, suggesting that the NO radical and peroxynitrite are involved in the pathogenesis of myocardial damage.

Programmed cell death in the myocardium of arrhythmogenic right ventricular cardiomyopathy in children and adults.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty replacement of the right ventricular myocardium. Recently, the myocardial loss in ARVC has been suggested to be related to apoptosis. However, it is still unknown whether this phenomenon is already established in the myocardium of pediatric cases with this disease. We examined the histopathologic characteristics of the ventricular myocardium in specimens obtained from 10 patients, including 3 children with ARVC, and investigated the occurrence of apoptosis in the myocardium by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay and agarose-gel electrophoresis of DNA. Endomyocardial biopsy specimens from the 10 cases and a necropsy sample from one adult case with ARVC were examined. Histopathologic examination of biopsy specimens from the pediatric cases revealed extensive fibrosis. Typical fatty infiltration was demonstrated in one of the 3 pediatric cases. These findings were similar to those in adult cases; the histopathologic index based on the severity of myocardial damage, including myocyte degeneration and fibrosis, was not significantly different from that in adult cases. TUNEL assay revealed positive reactivity of the myocardial cells. The apoptotic index was 1.4 +/- 0.4% in children and 1.6 +/- 0.5% in adults (difference not statistically significant). Agarose-gel electrophoresis of a DNA extract of the myocardial tissue of the autopsy case revealed DNA fragmentation. Cases with idiopathic ventricular tachycardia and control cases with a cardiac transplant (with no rejection) had minimal histopathologic findings and negative reactivity in the TUNEL assay. These results indicate that myocardial damage is already established in cases diagnosed as ARVC in childhood, and suggest that the myocardial damage is closely related to apoptosis in children, as well as in adults, in this disease.

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses.

Beta 3-adrenergic receptor gene polymorphism is not a major genetic determinant of obesity and diabetes in Japanese general population.

To assess the contribution of a replacement of Trp at codon 64 of beta 3-adrenergic receptor by Arg to fat distribution and metabolic disturbances in Japanese general population, we examined the missense mutation in 1122 persons consisting of 817 men aged 50.0 +/- 8.9 years and 305 women aged 50.8 +/- 8.5 years in Kyushu, Japan. The incidence of Arg64 allele was 0.21; no age-dependent decrease of the allele frequency was observed, suggesting that the mutation was not associated with early mortality. The genotype was not significantly correlated with body mass index or the thickness of visceral fat estimated by ultrasonography. Glucose tolerance and glucose-induced insulin secretion were not significantly different among subjects with Trp/Trp, Trp/Arg and Arg/Arg at codon 64. Although in obese persons the ratio of heterozygotes for the mutation tended to be higher in subjects with impaired glucose tolerance than in subjects with normal glucose tolerance, the tendency was not observed in non-obese persons. Furthermore none of 39 non-obese individuals homozygous for the mutation was diabetic, whereas two out of six obese homozygous persons were diabetic. These observations suggest that the missense mutation may not be a main determinant of obesity in populations taking low fat/low energy Japanese-style diet and it may not be deleterious at least in non-obese individuals.

Thermal preconditioning protects rat cardiac muscle cells from doxorubicin-induced apoptosis.

Doxorubicin (DOX=adriamycine), an effective chemotherapeutic agents for cancers, has severe cardiotoxicity. In the paresent study, we examined the protective effect of thermal preconditioning (TP) against apoptosis of rat cardiac muscle cells induced by DOX. Treatment with DOX (10 microM) for 24 hrs resulted in apoptosis of cardiac muscle cells, which was evaluated by examining "DNA ladder" formation and TUNEL staining. The number of TUNEL-positive cells was significantly decreased in cells subjected to TP by incubation at 42 degrees C for 30 min, 24 hrs prior to DOX-treatment. Antisense oligonucleotides of the heat shock protein (HSP) 70 blunted this effect. These results indicate that DOX-induced apoptosis in cardiac muscle cells is prevented by TP, at least in part, via a HSP70-mediated mechanism.

The effect of forskolin on the teratogenicity of methylxanthines in the chick embryo heart.

Interactions between forskolin and methylxanthines, including caffeine and isobutylmethylxanthine (IBMX), in the developing chick embryo heart were investigated. Forskolin, a potent activator of adenylate cyclase, was administered to young chick embryos (Hamburger-Hamilton stage 24) together with caffeine or IBMX at doses where each agent alone caused minimal embryotoxicity. The incidence of malformation in the embryonic chick heart or aorta induced by caffeine (5 x 10(-7) or 5 x 10(-6) mol) and IBMX (1 or 2.5 x 10(-6) mol) significantly increased with coadministration of forskolin (1 x 10(-9) mol). Cardiovascular malformations included ventricular septal defect, double outlet right ventricle, and aortic arch anomalies. These results indicate that forskolin potentiates the teratogenicity of caffeine or IBMX on the cardiovascular system in the chick embryo and suggest that this potentiation may be related to increase intracellular cAMP due to stimulation of adenylate cyclase (forskolin) and inhibition of phosphodiesterase (methylxanthines).