Distinct role of ShcC docking protein in the differentiation of neuroblastoma.

The biological and clinical heterogeneity of neuroblastoma is closely associated with signaling pathways that control cellular characteristics such as proliferation, survival and differentiation. The Shc family of docking proteins is important in these pathways by mediating cellular signaling. In this study, we analysed the expression levels of ShcA and ShcC proteins in 46 neuroblastoma samples and showed that a significantly higher level of ShcC protein is observed in neuroblastomas with poor prognostic factors such as advanced stage and MYCN amplification (P<0.005), whereas the expression level of ShcA showed no significant association with these factors. Using TNB1 cells that express a high level of ShcC protein, it was demonstrated that knockdown of ShcC by RNAi caused elevation in the phosphorylation of ShcA, which resulted in sustained extracellular signal-regulated kinase activation and neurite outgrowth. The neurites induced by ShcC knockdown expressed several markers of neuronal differentiation suggesting that the expression of ShcC potentially has a function in inhibiting the differentiation of neuroblastoma cells. In addition, marked suppression of in vivo tumorigenicity of TNB1 cells in nude mice was observed by stable knockdown of ShcC protein. These findings indicate that ShcC is a therapeutic target that might induce differentiation in the aggressive type of neuroblastomas.

IL-18 gene polymorphism confers susceptibility to the development of anti-GAD65 antibody in Graves' disease.

AIMS: This study aimed to investigate whether interleukin-18 (IL-18) gene polymorphisms are associated with the development of antibody against the 65-kDa isoform of recombinant human glutamic acid decarboxylase (GAD65Ab) in patients with Graves' disease. METHODS: A total of 398 unrelated Japanese patients with Graves' disease, with and without GAD65Ab, were recruited. Three single nucleotide polymorphisms in the IL-18 gene were examined and the polymorphic allele and the genotype and haplotype frequencies calculated. RESULTS: The frequency of the GG genotype at position -4675 of the IL-18 gene was significantly lower in Graves' disease patients with GAD65Ab than those without (4% vs. 24%, P = 0.0126). The -4675C allele frequency was significantly greater in patients with GAD65Ab than those without (69% vs. 53%, P = 0.0168). The homozygous -4675G/-607A/-137G haplotype was less common in Graves' disease patients with GAD65Ab than those without (4% vs. 23%, P = 0.0144). CONCLUSIONS: These findings in a Japanese population indicate that Graves' disease patients carrying the GG genotype at position -4657 of the promoter of the IL-18 gene or a gene in linkage disequilibrium with the -4675G/-607A/-137G haplotype have a low risk for the development of GAD65Ab in Graves' disease.

Reproducible biphasic cutaneous edema induced by topical and repeated application of antigen in sensitized mice.

An allergic dermatitis model was developed by repeated sensitization and challenge with antigen (ovalbumin, OA) over 7 months in mice. ddY mice were sensitized by i.p. injection of OA adsorbed on Al(OH)3 (1 microg OA/2 mg Al(OH)3/animal) once every 3 weeks. Antigen challenge was conducted by injection of OA solution (0.1, 1 and 10 microg/site) into the skin of the hind paw instep 10 d after the respective sensitizations. At the 1st challenge, all the 3 groups showed an immediate edematous response with the peak at 30 min or 1 h after the challenge. The group challenged with the highest dose (10 microg/site) of the antigen developed a clear late-phase edema, which was observed at the 2nd challenge, increasing until the 3rd challenge, reaching a plateau at further challenges. On the other hand, such late phase edema scarcely developed in the group challenged with the lowest dose (0.1 microg/site) of the antigen. The amount of circulating specific IgE antibody increased following repeated sensitizations and challenges in all groups, but there were no significant differences in the levels among them. Mepyramine suppressed the early edema by approximately 50%, yet the late phase edema was unaffected. In conclusion, using Al(OH)3+antigen for sensitization and an appropriate amount of antigen for challenge, reproducible biphasic edematous responses were observed long-term without desensitization. This model may be classified as an acute allergic dermatitis and can be useful for quantitatively evaluating the effects of anti-allergic drugs.

Cytokine profiles in eye muscle tissue and orbital fat tissue from patients with thyroid-associated ophthalmopathy.

Eye muscle (EM) and retroorbital fat tissue are two major sites of involvement in thyroid-associated ophthalmopathy (TAO). Lymphocytic infiltration in these tissues is a prominent histological feature of TAO. We have investigated the cytokine gene profiles in EM and orbital fat (OF) tissues from patients with TAO. Total RNA was isolated from EM tissue of 14 patients and from OF tissues of 29 patients with TAO. Cytokine gene expression was assessed by RT-PCR using paired primers for interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and glyceraldehyde-3-phosphate dehydrogenase. IFNgamma, TNFalpha, IL-1beta, and IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient. On the other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and 38% of the patients, respectively, and IFNgamma, IL-1beta, and IL-6 mRNA were less often detected compared with EM tissue. The enlargement of EM tissue as assessed by computed tomography correlated significantly with TNFalpha mRNA expression in EM tissue. The orbital volume was positively correlated with IL-6 mRNA expression and negatively correlated with IL-4 mRNA and IL-10 mRNA expression in OF tissue. These results suggest that T helper (Th) 1-like cytokines predominate in EM tissue in most patients and that the predominant cytokine profile in OF tissue varies from patient to patient. Both Th1-like and Th2-like immune responses may play roles in the development of two components of ophthalmopathy.

Technetium-99m tetrofosmin parathyroid imaging in patients with primary hyperparathyroidism.

OBJECTIVE: To confirm the clinical significance of 99mTc-tetrofosmin imaging for the localization of hyperfunctioning parathyroid glands in patients with primary hyperparathyroidism. METHODS: All patients were imaged with 99mTc-tetrofosmin at 10 minutes and 2 hours after radiotracer injection, and with ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI). The parathyroid/thyroid uptake ratio of 99mTc-tetrofosmin (P/T uptake ratio) was calculated. PATIENTS: Twenty patients with primary hyperparathyroidism were referred to our clinic, underwent surgical neck exploration or mediastinotomy and were diagnosed as having parathyroid adenoma. These patients were investigated for the preoperative localization by 99mTc-tetrofosmin scintigraphy. RESULTS: 99mTc-tetrofosmin imaging demonstrated focal uptake in 19 out of 20 patients with parathyroid adenoma. Two of the lesions were ectopic. US identified 17 parathyroid glands. CT and MRI initially detected 17 parathyroid glands. However, two additional parathyroid glands were localized on repeated CT and MRI in tandem with the results of the 99mTc-tetrofosmin imaging. Thus, the sensitivity and specificity of tetrofosmin imaging were 95% (19/20) and 95% (19/20); US, 85% (17/20) and 94% (16/17); initial CT, 85% (17/20) and 94% (16/17); and initial MRI, 88% (17/20) and 94% (16/17), respectively. The P/T uptake ratio at 2 hours after tetrofosmin injection was correlated with the serum concentration of intact PTH (rs=0.47, p<0.05) and the resected tumor weight (rs=0.53, p<0.05). CONCLUSION: 99mTc-tetrofosmin scintigraphy is useful for localization of parathyroid adenoma. Tetrofosmin uptake depends on the tumor weight and serum intact PTH levels.

Percutaneous microwave coagulation therapy for unresectable hepatocellular carcinoma.

BACKGROUND/AIMS: Percutaneous microwave coagulation therapy (PMCT) has recently been introduced as a new treatment for hepatocellular carcinoma (HCC) in Japan. This study was performed to evaluate its efficacy and safety. METHODOLOGY: Thirteen patients with 17 nodules of unresectable HCC were subjected to PMCT under ultrasonic guidance. The tumors ranged from 1.2-4.4 cm in size. Assessment of the efficacy of PMCT was made by follow-up with dynamic computed tomography (CT). RESULTS: In the patients with small HCC (< or = 2.0 cm), 8 of 10 nodules (80%) showed complete remission after PMCT. In small nodules located on the liver surface, 3 out of 4 nodules (75%) showed complete remission. However, in the patients with larger HCC (> or = 2.1 cm), 5 out of 7 nodules developed local recurrence after PMCT. Regarding assessment of the necrotic area after PMCT, dynamic CT revealed enhancement that was possibly caused by congestion of the liver parenchyma surrounding the area of necrosis due to PMCT in the early phase of the treatment. Therefore, the necrotic area must be assessed carefully. Although a slight heat sensation and/or pain during microwave irradiation (a common effect of PMCT) occurred in all patients, there were no serious adverse effects. CONCLUSIONS: Complete remission of small HCC (< or = 2 cm in diameter) can be achieved with PMCT alone, but there seem to be limitations to its effectiveness with larger HCC (> or = 2.1 cm). There were no serious adverse effects from PMCT and the therapy can be safely carried out even in patients with poor liver function.

Severe acute pancreatitis associated with hyperlipidemia: report of two cases and review of the literature in Japan.

Two cases of severe acute pancreatitis associated with type V hyperlipoproteinemia are reported. A 39-year-old obese woman was hospitalized with continuous severe abdominal pain. The diagnosis was made on the day of admission to our hospital, and treatment using continuous regional arterial infusion of a protease inhibitor and an antibiotic was performed with good results. The other patient was a 35 year-old woman in the 35th week of pregnancy, and a diagnosis of gestational hyperlipidemic pancreatitis was made on the day of onset. She was treated supportively using intravenous hyperalimentation, protease inhibitors, and antibiotics. She recovered from the acute pancreatitis and delivered a healthy term infant. It is difficult to diagnose acute pancreatitis in patients with type V hyperlipoproteinemia, because even when serum amylase levels are high, the value is reduced by high serum triglycerides. Early diagnosis was achieved in both of the present cases, and early intensive therapy was performed, which may be of the utmost importance in saving the life of a patient.

Percutaneous microwave coagulation therapy for superficial hepatocellular carcinoma.

We report a 67 year-old man with residual hepatocellular carcinoma after arterial embolization therapy, which was located on both the anterior and inferior surfaces of segment 6 of the liver. Percutaneous microwave coagulation therapy could be performed safely and the treated tumor became non-enhancing on contrast computed tomography. Two years after treatment, the tumor remains non-enhancing on contrast computed tomography and has decreased in size. Percutaneous microwave coagulation therapy appears to be useful even in patients who have superficial liver tumors associated with cirrhosis.

Control of solitary gastric fundal varices and portosystemic encephalopathy accompanying liver cirrhosis by balloon-occluded retrograde transvenous obliteration (B-RTO): a case report.

In a patient with liver cirrhosis complicated by solitary gastric fundal varices and portosystemic encephalopathy, Balloon-occluded retrograde transvenous obliteration (B-RTO) of the varices was performed. The gastric varices were decreased in size 2 weeks after treatment and had not recurred after 1 year. B-RTO successfully occluded the portosystemic shunt (gastrorenal shunt). Accordingly, the patient's blood ammonia levels, total bile acid level, and 15 min retention rate of indocyanine green decreased, and his hepatic encephalopathy improved. However, since consecutive increase in blood flow through the portal collateral vessels except for gastrorenal shunt vessel at 6 months and 1 year after B-RTO was noted, further careful follow-up may be required.

Increased serum soluble Fas in patients with Graves' disease.

We addressed the role of soluble Fas (sFas), which suppresses Fas-mediated apoptosis, in the pathogenesis of Graves' disease (GD). The serum concentration of sFas was measured by enzyme-linked immunosorbent assay and the expression of sFas mRNA in thyroid tissues by reverse transcriptase-polymerase chain reaction. The serum concentration of sFas was significantly increased in untreated GD (mean+/-SD: 1.57+/-0.48 ng/mL) compared to age-matched control subjects (0.77+/-0.46 ng/mL). The serum sFas level tended to decrease after the medication of antithyroid drugs for 6 to 8 weeks and was significantly decreased in patients who were euthyroid for more than 3 years (0.98+/-0.23 ng/mL), compared to that in untreated GD. The concentration of serum sFas was significantly correlated with anti-thyrotropin (TSH) receptor antibody titers, but not with the other clinical parameters (free triiodothyronine [FT3], free thyroxine [FT4], TSH, antithyroglobulin antibody titer, antimicrosomal antibody titer, or 123I uptake). The sFas mRNA was detected in thyroid tissue, cultured thyrocytes, and intrathyroidal lymphocytes. sFas was detected in supernatant of cultured thyrocytes from patients with GD. Its production by thyrocytes was induced by culture with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). The present study confirms serum sFas increases in GD and provides evidence of local production of sFas by thyrocytes and its regulation by cytokines. These data suggest that sFas may play a role in the pathogenesis of GD.

The effect of tulobuterol tape on histamine-induced bronchoconstriction in conscious guinea pigs: long duration of action.

The objective of the present study was to determine the action duration of tulobuterol tape within a 24-hr period in conscious guinea pigs. The bronchoconstriction induced by histamine-inhalation was significantly inhibited by tulobuterol tape in comparison with its placebo tape 8 and 12 hr after binding, and the inhibitory rate was 50+/-11% and 35+/-13%, respectively. Twenty-four hours after binding, the inhibitory effect of tulobuterol tape gradually diminished, but the inhibitory rate was maintained at 30+/-14%. These results suggest that tulobuterol tape has a long lasting bronchodilatory action.

Color Doppler ultrasonography in patients with subacute thyroiditis.

We studied the utility of color Doppler ultrasonography in patients with subacute thyroiditis. Eighteen patients with subacute thyroiditis (SAT) with painful goiter and thyrotoxicosis underwent color Doppler ultrasonography during the acute and recovery stages of the disease. Thyroid vascularization in these patients was compared with that of 15 untreated patients with Graves' disease and 17 control subjects. During the acute stage of subacute thyroiditis, color Doppler ultrasonography showed low echogenicity without increased tissue vascularity in the affected swollen thyroid. In the recovery stage, color Doppler ultrasonography showed isoechogenicity with slightly increased vascularization. Vascularization became normal at 1 year follow-up time. In contrast, marked by increased vascularization was observed in patients with untreated Graves' disease. Color Doppler ultrasonography showed clear differences between SAT and Graves' disease patients. Vascularity was significantly correlated with serum free thyroxine (FT4) and thyrotropin (TSH) concentrations in the recovery stage (3 months after the initial ultrasonography). Color Doppler ultrasonography accurately visualized lesions without increased vascularity in the acute stage of SAT and lesions of slightly increased vascularity in the recovery stage. Color Doppler ultrasonography may be a useful, noninvasive, and rapid method for differentiating SAT from Graves' disease and for evaluating and monitoring the location and activity of lesions in SAT.

Technetium-99m tetrofosmin imaging in patients with subacute thyroiditis.

We studied the significance of technetium-99m tetrofosmin scintigraphy in patients with subacute thyroiditis (SAT). Six patients with SAT, who had painful goitre with thyrotoxicosis, underwent 99mTc-pertechnetate scintigraphy and 99mTc-tetrofosmin imaging during the acute and recovery stages of SAT. The thyroid uptake ratio of tetrofosmin was compared with the clinical parameters associated with SAT. 99mTc-pertechnetate scintigraphy showed markedly reduced uptake in the thyroid during the acute stage of SAT, suggesting that the appropriate metabolic pathway is not functioning. Conversely, 99mTc-tetrofosmin images showed diffuse increased uptake in the thyroid region on early and delayed imaging. Tetrofosmin images in the acute stage and in the recovery stage of SAT showed different clearance curves for tetrofosmin uptake. The uptake ratio assessed as thyroid uptake/background (T/B) correlated with the serum C-reactive protein concentration. In conclusion, 99mTc-tetrofosmin uptake may reflect the inflammatory process associated with SAT, and thus this tracer may have potential as a marker of disease activity and severity.

[Percutaneous ethanol injection for unresectable pancreatic cancer-report of two cases].

Two cases of unresectable pancreatic cancer underwent percutaneous ethanol injection (PEI). The first was a 65-year-old man with a 43 x 39 mm mass in the head of the pancreas. He received PEI (total 38.5 ml of ethanol) 6 times. Serum CA19-9 decreased from 3,800 U/ml to 1,700 U/ml. The patient was dead 6 months after PEI. Autopsy revealed coagulative necrosis at the site of ethanol injection. The second case was a 66-year-old man with a 70 x 50 mm mass in the body of the pancreas. He was treated by PEI (total 10 ml). After PEI, his abdominal pain was relieved considerably. He died 4 months after PEI treatment. These findings suggest that PEI may be useful not only for pain control but also for tumor reduction of unresectable pancreatic cancer.

Nicotinamide decreases cytokine-induced activation of orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

We used flow cytometry to investigate the effects of nicotinamide, an inhibitor of poly (ADP ribose) synthetase, on the cell-surface expression of cytokine-induced human leukocyte antigen (HLA)-A,B,C antigen, HLA-DR antigen, intercellular adhesion molecule 1, CD44, and Fas expression in cultured orbital fibroblasts from patients with thyroid-associated ophthalmopathy. After two to seven passages, cultured orbital fibroblasts were incubated for 3 days with interferon gamma or tumor necrosis factor alpha in the presence of nicotinamide. Nicotinamide inhibited the induction of both HLA-DR and intercellular adhesion molecule 1 expression by cytokines on fibroblasts but did not interfere with induction of HLA-A,B,C, or CD44 expression. Nicotinamide also inhibited the proliferation of orbital fibroblasts, as assessed by a [3H]-thymidine incorporation assay and cell counts. Nicotinamide also enhanced the expression of the apoptosis-mediating protein Fas on fibroblasts. Our data suggest that nicotinamide inhibits cytokine-induced activation of fibroblasts and thus may decrease the autoimmune injury to the orbit in thyroid-associated ophthalmopathy.

Localization and clinical significance of thyrotropin receptor mRNA expression in orbital fat and eye muscle tissues from patients with thyroid-associated ophthalmopathy.

We have studied the cellular localization of thyrotropin receptor (TSH-R) mRNA in orbital fat and extraocular muscle tissues from patients with thyroid-associated ophthalmopathy (TAO) using Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization, and we correlated the findings with clinical estimates of ophthalmopathy. Although we failed to detect TSH-R mRNA in orbital tissues by Northern blot, TSH-R cDNA was amplified in orbital fat tissue from 13 of 25 patients with TAO and from 2 of 4 control subjects, in eye muscle tissue from 2 out of 7 patients with TAO, and in cultured orbital fibroblasts and subcutaneous fibroblasts from TAO patients. In situ hybridization showed that TSH-R mRNA was detected in cultured orbital fibroblasts as well as skin fibroblasts obtained from the patient. Furthermore, the expression of TSH-R mRNA in orbital fat tissue from patients with TAO significantly correlated with the orbital fat volume and the severity of ophthalmopathy, especially the extent of eye muscle dysfunction. These results suggest that the expression of TSH-R in the orbit, especially fibroblasts, may play a role in the pathogenesis and clinical manifestations of the ophthalmopathy in patients with TAO, although a secondary effect, involving fibroblasts in TAO is also possible.