Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Sensitization of TRPV1 by protein kinase C in rats with mono-iodoacetate-induced joint pain.

OBJECTIVE: To assess the functional changes of Transient receptor potential vanilloid 1 (TRPV1) receptor and to clarify its mechanism in a rat mono-iodoacetate (MIA)-induced joint pain model (MIA rats), which has joint degeneration with cartilage loss similar to osteoarthritis. METHODS: Sensitization of TRPV1 in MIA rats was assessed by transient spontaneous pain behavior induced by capsaicin injection in knee joints and electrophysiological changes of dorsal root ganglion (DRG) neurons innervating knee joints in response to capsaicin. Mechanisms of TRPV1 sensitization were analyzed by a newly developed sandwich enzyme-linked immunosorbent assay that detects phosphorylated TRPV1, followed by functional and expression analyses of protein kinase C (PKC) in vivo and in vitro, which involves TRPV1 phosphorylation. RESULTS: Pain-related behavior induced by intra-articular injection of capsaicin was significantly increased in MIA rats compared with sham rats. In addition, capsaicin sensitivity, evaluated by capsaicin-induced inward currents, was significantly increased in DRG neurons of MIA rats. Protein levels of TRPV1 remained unchanged, but phosphorylated TRPV1 at Ser800 increased in DRG neurons of MIA rats. Phosphorylated-PKCɛ (p-PKCɛ) increased and co-localized with TRPV1 in DRG neurons of MIA rats. Capsaicin-induced pain-related behavior in MIA rats was inhibited by intra-articular pretreatment of the PKC inhibitor bisindolylmaleimide I. In addition, intra-articular injection of the PKC activator phorbol 12-myristate 13-acetate increased capsaicin-induced pain-related behavior in normal rats. CONCLUSION: TRPV1 was sensitized at the knee joint and at DRG neurons of MIA rats through PKC activation. Thus, TRPV1 sensitization might be involved in chronic pain caused by osteoarthritis.

[Three primary cancers of pulmonary cancer, malignant melanoma and esophageal cancer; report of a case].

A 61-year-old man, who had medical history of hepatitis type C, surgery for malignant melanoma of the lower limb, endoscopic mucosal resection for esophageal cancer, was pointed out a pulmonary nodule in the right middle lobe by surveillance computed tomography after 5 years of surgery for melanoma. Pathology of esophageal cancer was squamous cell carcinoma limited in mucosa without lymphatic nor venous invasion. The nodule gradually enlarged and respiratory endoscopic examination could not establish pathological diagnosis. Thoracoscopy-assisted pulmonary biopsy revealed squamous cell carcinoma, and right middle lobectomy with mediastinal node dissection was performed. Histological examination showed moderately differentiated squamous cell carcinoma without lymph node involvement. The stage of lung cancer was T1N0M0, stage IA. Although 9 months have passed since surgery for lung cancer, recurrence of each malignancy has not been detected.

[Assessment of surgical treatment for lung cancer with pericardial or left atrial invasion].

We evaluated surgical results for the patients who underwent pulmonary resection combined with pericardial or left atrial resection due to locally advanced non-small cell lung cancer (NSCLC). Seven patients who underwent pericardial resection (T3 group) and 4 patients who underwent resection of the left atrium (T4 group) were included in this study, and clinical findings and prognosis were evaluated. Eight patients underwent pneumonectomy, and others underwent lobectomy or bilobectomy. Histology of the cancer was squamous cell carcinoma in all patinets. As for pathological node involvement, N0/N1 disease was 72.7% and N2 disease was 27.3%. Induction chemotherapy was performed in 75.0% of T4 group. Adjuvant chemotheraphy was performed in 71.4% of T3 group and 75.0% of T4 group. Five-year-survival was 57.1% in T3 group and 25.0% in T4 group. Five-year-survival was 62.5% in N0/N1 disease and 0% in N2 disease. Statistical significance in prognosis was seen in lymph node status (p = 0.0317). Extended resection of pericardium or left atrium for patients with N2 disease of NSCLC is not recommended. When invasion to pericardium or left atrium is diagnosed during surgery, extended resection should be indicated in patient without N2 metastasis.

[Pleuroperitoneal communication at the beginning of continuous ambulatory peritoneal dialysis; report of a case].

A 46-years-old woman admitted for induction of continuous ambulatory peritoneal dialysis (CAPD). When peritoneal functional test was performed, dyspnea was occurred. Chest X-ray and chest computed tomography (CT) scan revealed massive right hydrothorax. Technetium-99m macroaggregated albumin scintigraphy showed communication between abdominal cavity and thoracic cavity. The thoracoscopic diaphragmal repair was performed. After CAPD was started, right hydrothorax occurred again. Re-repair of the diaphragm was performed in small thoracotomy and small hole was revealed. The hole was sutured and diaphragm was coverd by fibrin glue and polyglycolacid (PGA) felt all over. Since then, CAPD was continued successfully. Thoracoscopic surgery is less invasive and appropriate therapy for this case. It is important that the diaphragm will be covered all over by fibrin glue and PGA sheet because even pin-hole makes recurrence. For detect of the communicative portion, use of indigocarmin and examination of glucose concentration in the pleural effusion were effective.

[Surgical treatment of aortic root disease].

We reviewed aortic root disease and operative procedures. Between January 1982 and December 2008, aortic root operation was performed for 58 patients with various aortic root disease. We chose Bentall type operations in extensive root destructive cases and urgent or reoperative cases. Overall in-hospital mortality was 8.6% (5/58). Four patients (7.5% of survivors) died during the period of followup. Actuarial survival at 15 years was 92%. Freedom from cardiovascular event at 10 and 15 years was 81% and 27%, respectively. Of 5 reoperations in 5 patients, only 1 was required due to complications of the initial Bentall type operation. The Bentall type operations resulted in a durable result. Although, in Marfan syndrome, freedom from cardiovascular event was lower than that in non-Marfan syndrome, actuarial survival rate was equal with non-Marfan syndrome. Close observation is necessary for detecting cardiovascular event, especially in Marfan syndrome.

[Improved cardiac function after inhalation of nitric oxide and tricuspid annuloplasty in dilated cardiomyopathy patient on left ventricular assist system].

A 17-year-old man with severe cardiac failure due to dilated cardiomyopathy was treated by medication. As he did not improve, he had indication for transplantation of the heart and was put on a ventricular assist system as a bridge until he could be transplanted. He also had severe tricuspid regurgitation and tricuspid annuloplasty was performed. Inhalation of nitric oxide was used to decrease pulmonary vascular resistance. Although cerebral infarction was present postoperatively, he is waiting for transplant without paralysis. The ventricular assist system proved to be useful therapy for serious cardiac failure due to terminal dilated cardiomyopathy.

[Cardiovascular surgery for patients with chronic respiratory failure and respiratory dysfunction].

Chronic obstructive pulmonary disease is one of the major comorbidities in elderly patients with heart disease and thoracic aneurysm because of an overlap of risk factors. Although postoperative ventilator dependency is a major concern, recent study has suggested chronic obstructive pulmonary disease (COPD) was not a risk factor for postoperative prolonged mechanical ventilator support for off-pump coronary artery bypass grafting. Cardiopulmonary bypass may induce acute lung injury, but short-term cardiopulmonary bypass alone does not give adverse effect in clinical practice. Perioperative airway management including quitting smoking, use of bronchodilator, avoiding excessive tracheal suction, and early extubation may contribute satisfactory outcome. However, a stage IV COPD patient is contraindicated for cardiac surgery. Patients with interstitial lung disease can undergo cardiac surgery safely if they have moderate performance status even when patients are under home oxygen therapy.

[Interrupted aortic arch type B survived after single stage repair and continuous hemodiafiltration; report of a case].

A 14-day-old, 2,042 g neonate with interrupted aortic arch (IAA) type B was admitted to our hospital because of respiratory failure. Although alprostadil and alprostadil alfadex were used to keep the ductus arteriosus open, her congestive heart failure became worse and anuria and metabolic acidosis started to appear. We performed single-stage repair for her at 14 days old. Continuous hemodiafiltration (CHDF) was initiated immediately after the operation and peritoneal dialysis was started after paralytic ileus improved. CHDF improved her renal function and she was discharged at postoperative day 84. We conclude that CHDF is effective for neonates with acute renal failure although circulation management is rather difficult.

[Thymic carcinoid tumor with Cushing syndrome].

A case of a 71-year-old male with ectopic adrenocorticotropic polypeptide (ACTH)-producing thymic carcinoid tumor presenting Cushing's syndrome was reported. This patient had symptoms of fatigue and a polyposia for 2 years before a mediastinal tumor was detected. Chest computed tomography (CT) scan demonstrated an anterior mediastinal mass, and serum ACTH and cortisol level revealed very high. Secretion of cortisol was not inhibited in an 8-mg dexamethazone suppression test. We diagnosed ectopic ACTH-producing tumor, and performed complete excision of the thymus including thymic tumor. Histologically, the tumor demonstrated typical carcinoid with the positivity of ACTH immunostaining. After the operation, ACTH and cortisol levels were reduced and the clinical symptoms were improved rapidly. We have concluded that it is important to control serum perioperative cortisol level for the prevension of morbidity.

[Palliative right ventricular outflow reconstruction in tetralogy of Fallot with pulmonary atresia and aberrant right subclavian artery; report of a case].

A 1-month-old girl weighting 3.1 kg was diagnosed as tetralogy of Fallot (TOF) with pulmonary artery atresia (PA) and aberrant right subclavian artery. Before the operation, pulmonary blood flow from a ducus arteriosus was maintained by lipo prostaglandin E1 (PGE1). The patient underwent palliative right ventricular outflow tract reconstruction (pRVOTR) because the proximal aberrant right subclavian artery was stenotic and the ductus arteriosus and branch of the left pulmonary artery were so close. Postoperative course was uneventful and pulmonary artery showed good growth. The pRVOTR as 1st procedure is a useful method for hypoplastic pulmonary artery to get equal and good growth. Although there are controversies about the size of right ventricular outflow tract (RVOT), 5 or 6 mm diameter of RVOT is recommended for the operative repair of hypoplastic left heart syndrome. We concluded that the pRVOTR should be one of the options as 1st palliative procedure for TOF with PA and diminutive pulmonary artery.

[Left ventricular-right atrial communication resulting from infective endocarditis].

We report a rare case of acquired left ventricular-right atrial communication resulting from infective endocarditis. A 57-year-old male with aortic regurgitation due to infective endocarditis was referred to our hospital because of severe congestive heart failure. Preoperative transthoracic echocardiography showed aortic, mitral and tricuspid severe regurgitations. Intraoperative transesophageal echocardiography revealed left ventricular-right atrial shunt. The fistula was located at the atrioventricular membranous septum. The communication site from the left view was below the commissure between the right coronary cusp and non-coronary cusp, and from the right view was just above the tricuspid annulus of the septal leaflet. The fistula was closed directly with mattress suture and aortic valve replacement and both mitral and tricuspid ring annuloplasty were carried out simultaneously. The postoperative course was uneventful. It is important to inspect shunts carefully in echocardiography of infective endocarditis with massive regurgitations.

[Emergency small-sized stent placement following aortic true lumen obliteration of Stanford type B acute aortic dissection].

Acute aortic obstruction induced by acute aortic dissection frequently causes life-threatening organ ischemia. Although early reperfusion of lower extremities, renal and mesenteric artery is necessary, surgical treatment such as graft replacement is invasive and may result in myonephropathic-metabolic syndrome (MNMS), which leads to loss of limb and life. We herein report a case of stent placement as a less invasive treatment for true lumen obliteration associated with Stanford type B aortic dissection in a patient with chronic renal failure on hemodialysis. Ten mm metallic stents were placed in the obliterated true lumen of the descending aorta in order to perfuse peripheral organs within 5 hours after occurrence. It relieved symptoms of visceral organ and leg ischemia. On the postoperative day 14, abdominal discomfort after meal, hypertension in the upper extremity and headache appeared. Chest and abdominal computed tomography (CT) revealed stenosis of the true lumen proximal to the stents. On the other hand, the diameter of the true lumen was inversely dilated distal to the stents. Bilateral axillo-femoral artery bypass was performed with relief of upper extremity hypertension and visceral organ ischemia. The patient otherwise had an uneventful course and was discharged on the postoperative day 37. Our experience suggests that emergency stent placement can provide an option that is less invasive, more effective and prompt treatment for patients with visceral organ and leg ischemia resulting from acute aortic dissection.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

The relationship between bone turnover and body weight, serum insulin-like growth factor (IGF) I, and serum IGF-binding protein levels in patients with anorexia nervosa.

Malnutrition is one of the risk factors for bone loss in patients with anorexia nervosa (AN). To clarify the effects of nutritional status on bone metabolism, we examined the relationship between serum levels of nutritional indicators [insulin-like growth factor I (IGF-I), IGF-binding protein-2 (IGFBP-2), and IGFBP-3] and markers for bone metabolism [serum osteocalcin and urinary excretion of C-terminal telopeptide of collagen type I (CrossLaps)] in 45 AN out-patients, including 8 severely malnourished patients who required hospitalization and iv hyperalimentation (IVH). Compared to healthy subjects, serum IGF-I and IGFBP-3 were lower, whereas IGFBP-2 was higher in out-patients who had a body mass index (BMI) less than 16.5 kg/m2. In these patients, urinary excretion of CrossLaps, a marker of bone resorption, was higher, whereas serum osteocalcin, a marker of bone formation, was lower than those in control subjects. All of these parameters were normal in patients whose BMI ranged from 16.5-18.5 kg/m2. Serum levels of osteocalcin correlated positively with BMI (r = 0.512; P<0.0001), IGF-I (r = 0.558; P<0.0001), and IGFBP-3 (r = 0.369; P<0.001) in AN out-patients. In the 8 severely malnourished AN patients, serum levels of IGF-I and osteocalcin significantly increased 3 and 7 days, respectively, after the start of a 5-week IVH therapy regimen and reached normal levels within 5 weeks, accompanied by still elevated urinary excretion of CrossLaps. The present study demonstrates that an improvement in nutritional status in AN patients during IVH therapy rapidly increases the serum IGF-I levels, followed by a progressive increase in osteocalcin, suggesting immediate start of bone formation. However, increased bone resorption appears to continue for at least 5 weeks.

Serum high molecular weight form of insulin-like growth factor II from patients with non-islet cell tumor hypoglycemia is O-glycosylated.

Non-islet cell tumor hypoglycemia (NICTH) is one of major causes of fasting hypoglycemia. In some patients with NICTH, insulin-like growth factor II (IGF-II) produced by and secreted from the tumors is thought to be a hypoglycemic agent. In patients with NICTH, the major form of IGF-II is high molecular weight form of IGF-II, designated as big IGF-II. The generation of big IGF-II in the NICTH syndrome is unclear. It has been reported that in the patients with NICTH big IGF-II lacks normal E-domain O-linked glycosylation, suggesting that the patient's big IGF-II might be generated by abnormal processing of pro-IGF-II. However, we have found that the apparent size of big IGF-II varies in sera from the patients with NICTH, and that there is a possibility that slower migration pattern of IGF-II might be because of a different size of sugar moiety attached to pro-IGF-II. In the present study using the sera from 10 patients with NICTH, we investigated the effect of O-glycosidase digestion on migration of IGF-II and analyzed the results by Western immunoblot. By Western immunoblot analysis the big IGF-II was reduced in size to 9.5 kDa in the enzyme-treated sera of the 10 patients with NICTH. The migration pattern is similar to that observed in sera of normal subjects after O-glycosidase digestion. These data indicate that big IGF-II from patients with NICTH is O-glycosylated, and the sizes of the sugar moiety are larger than those from normal subjects suggesting abnormal glycosylation in NICTH.

Characterization of new monoclonal antibodies to human insulin-like growth factor-II and their application in western immunoblot analysis.

Four immunoglobulin G1 class monoclonal antibodies (mAbs; 1D5, 1D9, 2B11, and 2H11) were produced against recombinant human insulin-like growth factor-II (rhIGF-II). Enzyme-linked immunosorbent assay established that these four mAbs specifically recognized rhIGF-II and hIGF-II, but not rhIGF-I. mAbs 1D5, 1D9, and 2H11 did not cross-react with mouse rIGF-II, although there are only six amino acid differences between mouse IGF-II and human IGF-II. The epitope for each mAb was partially defined by enzyme-linked immunosorbent assay using mouse-human chimera IGF-II mutants and other IGF-II mutants that were prepared by site-directed mutagenic procedures. These results indicated that the epitopes of mAbs 1D5, 1D9, and 2H11 are in the C-domain of hIGF-II around Ala32 to Ser36, and that of 2B11 is in the carboxyl-terminal end of the B- and A-domains of hIGF-II. A specific and sensitive RIA was developed using mAb 2H11. In this RIA, IGF-II variant ([RLPG/S29]IGF-II) and rhIGF-II competed equally with [125I]IGF-II for binding to mAb 2H11. Similar results were produced when mAbs 1D5, 1D9, and 2B11 substituted for 2H11. The potential usefulness of mAb 2H11 in an immunoblot procedure to characterize the heterogeneity of IGF-II in the sera and tumor tissues of patients with nonislet cell tumor hypoglycemia was evaluated. A procedure that combined acid-ethanol extraction of serum or tumor tissues and immunoaffinity concentration of the extracted IGF-II with mAb 2H11-immobilized resin was found to be an effective way to prepare the samples. In Western immunoblots, a quantity of rhIGF-II as low as 3 ng could be identified, whereas 200 ng rhIGF-I or rat IGF-II were not recognized. The levels of IGF-II in the sera of 12 patients with nonislet cell tumor hypoglycemia varied from normal to about twice normal. The mol wt (M(r)) of this IGF-II was between 10-17K. There was little of the processed 7.5K M(r) IGF-II in the sera of these patients. Finally, the source of the high M(r) forms of IGF-II was the tumor, because the ratios of high M(r) forms of IGF-II to 7.5K IGF-II changed dramatically from 99:1 and 91:9 to 4:96 and 32:68 in two patients after successful excision of their tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased serum levels of acid-labile subunit in patients with anorexia nervosa.

One of the observations in malnutrition is that serum insulin-like growth factor (IGF)-I levels are decreased, and this decrease is associated with an altered profile of IGF binding proteins (IGFBPs). In human circulation, IGFs are mostly present as an approximately 150-kDa ternary protein complex consisting of IGFs, IGFBP-3, and acid-labile subunit (ALS). In the present study, to clarify the effect of nutrition on serum ALS levels, we investigated 33 patients with anorexia nervosa. Serum levels of ALS were measured by RIA. Furthermore, we measured serum IGF-I, IGF-II, IGFBP-2, and IGFBP-3 levels in the patients. From these data, we investigated which was the best predictor of body mass index (BMI) as a nutritional status marker. In the patients with anorexia nervosa, the serum ALS levels ranged from 0.7-16.9, with a mean of 10.6 +/- 0.7 mg/L, and the levels were significantly lower than those of normal subjects (13.8 +/- 0.8 mg/L, P < 0.05). Serum ALS levels positively correlated with BMI (r = 0.41, P < 0.05), and the levels increased during treatment. The serum IGFBP-2 levels in the patients were increased (871 +/- 91 microg/L), and the levels inversely correlated with BMI (r = -0.52, P < 0.01). The serum IGF-I and IGFBP-3 levels were low (152 +/- 14 microg/L and 2.56 +/- 0.12 mg/L, respectively), and the levels positively correlated with BMI (r = 0.46, P < 0.01; and r = 0.39, P < 0.05, respectively). The serum IGFBP-2, IGF-I, and IGFBP-3 levels returned toward normal ranges as BMI in the patients improved during treatment. Serum IGF-II levels did not correlate with BMI (r = 0.24, P = 0.17). Stepwise regression analysis revealed that serum IGFBP-2 was the best marker of BMI among these variables. The present study suggested that ALS was regulated by nutritional status, the same as IGF-I, IGFBP-2 and IGFBP-3; but the serum IGFBP-2 was the best predictor of BMI as nutritional status marker among the parameters in patients with anorexia nervosa.

Flavones and flavonols at dietary levels inhibit a transformation of aryl hydrocarbon receptor induced by dioxin.

Dioxins invade the body mainly through the diet, and produce toxicity through the transformation of aryl hydrocarbon receptor (AhR). An inhibitor of the transformation should therefore protect against the toxicity and ideally be part of the diet. We examined flavonoids ubiquitously expressed in plant foods as one of the best candidates, and found that the subclasses flavones and flavonols suppressed antagonistically the transformation of AhR induced by 1 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin, without exhibiting agonistic effects that transform AhR. The antagonistic IC(50) values ranged from 0.14 to 10 microM, close to the physiological levels in human.

Purification by affinity chromatography and physicochemical properties of the guanine-specific ribonuclease of Fusarium moniliforme.

Ribonuclease F1, the guanine-specific ribonuclease of Fusarium moniliforme, was purified to homogeneity by a combination of ethanol fractionation, affinity chromatography and DEAE-cellulose column chromatography. The adsorbent for the affinity chromatography was synthesized by the coupling of periodate-oxidized guanosine 5'-monophosphate to aminohexyl agarose followed by sodium borohydride reduction. Ribonuclease F2, the minor component, was also purified to near homogeneity by the same procedure. Ribonucleases F1 and F2 had the same molecular weight (about 11,000) as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They also showed the same amino acid composition and differed only in the isoelectric point: 4.10 for F1 and 3.96 for F2.