Esophageal IgG4 levels correlate with histopathologic and transcriptomic features in eosinophilic esophagitis.

BACKGROUND: Recent data associate eosinophilic esophagitis (EoE) with IgG4 rather than IgE, but its significance and function have not been determined. Our aims were to measure esophageal IgG4 levels and to determine functional correlations as assessed by histologic and transcriptome analyses. METHODS: This case-control study included pediatric subjects with EoE (≥15 eosinophils/HPF) and non-EoE controls. Protein lysates were analyzed for IgA, IgM, and IgG1-IgG4 using the Luminex 100 system; IgE was quantified by ELISA. Esophageal biopsies were scored using the EoE histology scoring system. Transcripts were probed by the EoE diagnostic panel, designed to examine the expression of 96 esophageal transcripts. RESULTS: Esophageal IgG subclasses, IgA, and IgM, but not IgE, were increased in subjects with EoE relative to controls. The greatest change between groups was seen in IgG4 (4.2 mg/g protein [interquartile range: 1.0-13.1 mg/g protein] vs 0.2 mg/g protein [0.1-0.9]; P < .0001). Tissue IgG4 levels correlated with esophageal eosinophil counts (P = .0006); histologic grade (P = .0011) and stage (P = .0112) scores; and IL4, IL10, IL13, but not TGFB1, expression and had strong associations with a subset of the EoE transcriptome. Esophageal IgG4 transcript expression was increased and correlated with IgG4 protein levels and IL10 expression. CONCLUSION: These findings extend prior studies on IgG4 in adult EoE to the pediatric population and provide deeper understanding of the potential significance and regulation of IgG4, demonstrating that IgG4 is a relevant feature of the disease; is closely related to esophageal eosinophil levels, type 2 immunity and T regulatory cytokines; and is likely produced locally.

Cell type-dependent effects of corticosteroid on periostin production by primary human tissue cells.

Overproduction of periostin, an IL-13-inducible matricellular protein, despite corticosteroid treatment is thought to be involved in the chronicity of allergic inflammation seen in corticosteroid-refractory tissue fibrosis. Therefore, we hypothesized that some tissue cells must produce periostin in a corticosteroid-insensitive manner. Here, we show that IL-4 and IL-13 each induced comparable levels of periostin production by primary normal human fibroblasts and microvascular endothelial cells derived from lung and skin. Dexamethasone, a corticosteroid, completely inhibited IL-4/13-induced, but did not affect TGF-ß-induced, periostin production by fibroblasts. In contrast, dexamethasone synergistically enhanced IL-4/13-induced periostin production by microvascular endothelial cells. TGF-ß did not induce periostin production by microvascular endothelial cells. Our novel findings suggest that IL-4/13-induced microvascular endothelium-derived and/or TGF-ß-induced fibroblast-derived periostin might play a pivotal role in corticosteroid-refractory tissue fibrosis, leading to chronic allergic inflammation in the lung and/or skin.

Placement of two types of spiral Z-stents at the bronchial carina for the treatment of terminal lung cancer--a new method.

Dumon Y-stents and Dynamic stents are used to treat carinal stenosis, but their placement severely impairs the expectoration of secretions, making frequent bronchoscopic aspiration necessary. We report here five patients with terminal lung cancer who had stenosis of the lower trachea and main bronchi treated using spiral Z-stents. A long tapered spiral Z-stent was placed in the lower trachea and one main bronchus, and a short straight spiral Z-stent in the contralateral main bronchus. No patients required bronchoscopic aspiration of secretions after stenting. Before stenting, all of the patients were severely dyspnoeic, requiring oxygen and having to sit in the orthopnoeic position. After stenting, the patients' dyspnoea improved, with one patient becoming ambulant without the need for oxygen support. These results suggest that the use of spiral Z-stents in stenosis of the tracheal carina in advanced lung cancer is effective in reducing the need for bronchoscopic aspiration and enhancing quality of life.

Activation of mitogen-activated protein kinase by the nociceptin receptor expressed in Chinese hamster ovary cells.

Activation of the nociceptin receptor stably expressed in Chinese hamster ovary cells induced a transient mitogen-activated protein kinase (MAPK) activation, via pertussis toxin-sensitive G-proteins. The nociceptin receptor-mediated MAPK activation was partially blocked by down-regulation or inhibition of protein kinase C, and suppressed by pretreatment with a phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, a tyrosine protein kinase inhibitor, genistein, and phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, affected the nociceptin-induced MAPK activity. The nociceptin-induced MAPK activation may lead to activation of phospholipase A2 and induce changes in gene expression.

Desensitization and resensitization of delta-opioid receptor-mediated Ca2+ channel inhibition in NG108-15 cells.

1. To approach the mechanisms underlying desensitization of the opioid receptor-mediated Ca2+ channel inhibition, the effects of prolonged application of [D-Ala2, D-Leu5]enkephalin (DADLE) on Ba2+ currents (I(Ba)) through Ca2+ channels were analysed in NG108-15 neuroblastoma x glioma hybrid cells. 2. Inhibition of I(Ba) by 100 nM DADLE desensitized by 57% with a time constant of 4.4 min. 3. Maximal desensitization of the delta-opioid receptor-Ca2+ channel coupling was attained by 1 microM DADLE. The EC50 value for desensitization was estimated to be 78 nM. 4. RNA blot hybridization analysis and immunoblot analysis revealed the expression of beta-adrenoceptor kinase-1 (betaARK1) in NG108-15 cells. 5. Heparin, an inhibitor of betaARK, significantly reduced the magnitude and rate of desensitization, whereas Rp-cyclic AMPS and PKI (14-24)amide, inhibitors of cyclic AMP-dependent protein kinase (PKA), or long-term treatment with phorbol 12-myristate 13-acetate to induce down-regulation of protein kinase C (PKC) had no significant effect. 6. Recovery from desensitization (resensitization) proceeded with a time constant of 6.7 min. Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, significantly attenuated the degree of resensitization. 7. In summary, we have characterized the time course and concentration-dependence of the desensitization of DADLE-induced I(Ba) inhibition in NG108-15 cells. This desensitization was reversible after removal of DADLE. It is suggested that betaARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.

Tumor-promoting activities of hydroquinone and 1,1-dimethylhydrazine after initiation of newborn mice with 1-methyl-1-nitrosourea.

To clarify the suitability of a newborn-mouse carcinogenesis assay to detect tumor-promoting activities of carcinogens, the non-genotoxic hydroquinone (HQ) and genotoxic 1,1-dimethylhydrazine (UDMH) were administered to mice during the promotion stage after treatment with 1-methyl-1-nitrosourea (MNU) (20 mg/kg body wt, single intraperitoneal injection) at day 9 after birth. Initiated males and females thus received either HQ at 0.8% in basal diet, or UDMH, at 20 mg/kg body wt once weekly by subcutaneous injection, from day 14 until the end of the experiment at 30 weeks of age. Uninitiated newborn mice, given an injection of the vehicle (0.01 M citrate buffer (pH 5.5), 20 mg/kg body wt), also received HQ or UDMH in the same way. Histopathologically, focal proliferative lesions were found in the livers of male mice and in the lungs of both male and female mice in the MNU-treated groups. HQ significantly increased the incidence and multiplicity of altered hepatocellular foci, the combined incidence of hepatocellular adenomas and carcinomas in males and the incidence and multiplicity of lung adenomas and the combined incidence of lung adenomas and carcinomas in female mice. In addition, four out of eleven MNU + HQ-treated male mice developed lung carcinomas, showing a significant elevation in multiplicity. UDMH also exhibited a tendency to increase the incidence and multiplicity of lung adenomas in female mice. Thus tumor-promoting effects of HQ or UDMH were apparently exerted in the target organs and the MNU-initiated two-stage newborn-mouse carcinogenesis assay may be useful for detection of genotoxic or non-genotoxic carcinogenicity.

The multiple untranslated first exons system of the human estrogen receptor beta (ER beta) gene.

In order to investigate the regulatory mechanism of the expression of the human estrogen receptor beta (ER beta) gene, we have analyzed the structure of the 5'-untranslated region of the ER beta mRNA in the normal uterine endometrium and liver using the 5'-rapid amplification of the cDNA ends method. The sequence analysis revealed the presence of the two isoforms of the ER beta mRNA containing the distinct 5'-untranslated regions. The genomic analysis revealed that the two isoforms of the message originated from the two distinct untranslated first exons, termed the exon 0K and exon 0N, which were spliced to the exon 1. We termed the two isoforms of the message the ER beta mRNA (0K-1) and ER beta mRNA (0N-1). We further analyzed the distribution of the ER beta mRNA (0K-1) and ER beta mRNA (0N-1) in the ejaculated spermatozoa, liver, uterine endometrium and myometrium, and peripheral leukocytes using the reverse transcription-polymerase chain reaction. The distributions of the two mRNA isoforms were different from each other. From these results, it is indicated for the first time that the expression of the human estrogen receptor beta (ER beta) gene is regulated, at least in part, by the multiple untranslated first exons and promoters system.

Nociceptin receptor-mediated Ca2+ channel inhibition and its desensitization in NG108-15 cells.

It has been shown that the membrane of hybrid NG108-15 neuroblastoma x glioma cells contains a high-affinity binding site for nociceptin. In the present study, we first demonstrated the expression of nociceptin receptor mRNA in NG108-15 cells. Application of nociceptin to NG108-15 cells produced a concentration-dependent (EC50 = 29 nM) inhibition of Ca2+ channel currents in a pertussis toxin-sensitive fashion. This nociceptin-induced inhibition of Ca2+ channel currents was prevented in the presence of omega-conotoxin GVIA, a blocker of the N-type Ca2+ channel, and had both voltage-dependent and -independent components. Prolonged application of nociceptin elicited homologous desensitization of the inhibition with a time constant of 5.3 min. These results indicate that the nociceptin receptor is coupled to the N-type Ca2+ channel via pertussis toxin-sensitive G proteins in NG108-15 cells and that this coupling is associated with rapid and homologous desensitization.

Adaptations to chronic agonist exposure of mu-opioid receptor-expressing Chinese hamster ovary cells.

To investigate cellular adaptation responses induced by chronic agonist treatment of the mu-opioid receptor, Chinese hamster ovary (CHO) cells were stably transfected with the rat mu-opioid receptor cDNA. Chronic treatment with agonists selective for the mu-opioid receptor, [D-Ala2, N-MePhe4, Gy-ol5]enkephalin (DAMGO), morphine and fentanyl, time- and dose-dependently induced down-regulation of the mu-opioid receptor. The down-regulation was not significantly affected by pretreatment with pertussis toxin, but was completely blocked by treatment with hypertonic sucrose, suggesting that receptor internalization mediated by clathrin-coated vesicles is an essential step in the mu-opioid receptor down-regulation. On the other hand, forskolin-stimulated cyclic AMP formation was increased by chronic DAMGO treatment, which was inhibited by pertussis toxin pretreatment. These results indicate that two adaptation responses induced by chronic agonist treatment of the mu-opioid receptor-expressing CHO cells, down-regulation of the mu-opioid receptor and supersensitization of adenylate cyclase, are mediated by distinct mechanisms.

Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

A case of multiple sclerosis with multi-ring-like and butterfly-like enhancement on computerized tomography.

We report a case of multiple sclerosis in which CT showed multiple ring-like enhancement and butterfly-like distribution of a low density area with marginal enhancement. The latter finding is found in other demyelinating disorders but is less common in tumors or abscesses. Therefore, it seems to have some diagnostic value in multiple sclerosis.

Carcinogenicity study of beta-cyclodextrin in F344 rats.

The carcinogenicity of beta-cyclodextrin, a cyclic, water-soluble carbohydrate comprising seven glucose units, was examined in Fischer 344 (F344) rats. Groups of 50 males and 50 females were given the compound in their diet at concentrations of 0 (control), 2.5 or 5% for 104 wk. Surviving rats were then given a basal diet for a further 5 wk and killed at 109 wk. The dose levels were selected from the results of a 13-wk subchronic toxicity study. Dose-dependent inhibitory effects of beta-cyclodextrin on growth were observed in both sexes of the treated groups. The survival rates, mean survival times and range, however, demonstrated no significant differences between the control and treated groups. A variety of tumours developed in all groups, including the control group, but all the neoplastic lesions were histologically similar to those known to occur spontaneously in this strain of rat, and no statistically significant increase in the incidence of any tumour was found for either sex of the treated groups. Thus, it is concluded that under the present experimental conditions, the high dose, about 340-400 times higher than the current daily human intake from ingestion as a food additive and from pharmaceutical use, does not have any carcinogenic potential in F344 rats.

Lack of carcinogenicity of medium-viscosity liquid paraffin given in the diet to F344 rats.

The carcinogenicity of medium-viscosity liquid paraffin was examined in Fischer 344 rats. Groups of 50 males and 50 females were given the material at dietary doses of 0 (control), 2.5 or 5% for 104 wk. Slight increases in food consumption and body weight were observed in both sexes of the 5% group. However, no significant differences between the control and treated groups were noted with regard to clinical signs, mortality and haematology findings. A variety of tumours developed in all groups, including the control group, but all the neoplastic lesions were histologically similar to those known to occur spontaneously in F344 rats, and no statistically significant increase in the incidence of any tumour type was found for either sex in the treated groups. Granulomatous inflammation in the mesenteric lymph nodes, considered to be a reaction to paraffin absorption, was observed with similar incidence and severity in both sexes of the 2.5 and 5% groups. Thus, it is concluded that under the present experimental conditions, the high dose, about 2000-200,000 times higher than the current temporary acceptable daily intake, does not have any carcinogenic potential in F344 rats. Furthermore, granulomatous inflammation observed in mesenteric lymph nodes were not associated with any development of neoplastic lesions.

Assessment of the carcinogenicity of stevioside in F344 rats.

The carcinogenic potential of stevioside, a compound that is used as a sweetener for food and drink, was examined in F344 rats of both sexes. Stevioside was added to powdered diet at concentrations of 0 (control), 2.5 and 5%. The doses were selected on the basis of results from a 13-wk subchronic toxicity study and administered to groups of 50 male and 50 female rats ad lib. for 104 wk. All surviving rats were killed at wk 108. Body weight gains were slightly depressed in line with the dose of stevioside, in both sexes, and a significant decrease in the final survival rate was observed for the 5% treated males. Histopathologically, however, there was no significantly altered development of neoplastic or non-neoplastic lesions attributable to the stevioside treatment in any organ or tissue, except for a decreased incidence of mammary adenomas in females and a reduced severity of chronic nephropathy in males. It is concluded that stevioside is not carcinogenic in F344 rats under the experimental conditions described.

Liver tumour-promoting effects of oxfendazole in rats.

To examine whether oxfendazole has tumour-promoting activity, a total of 100 male Fisher 344 rats were initiated with a single ip injection of 100 mg/kg of diethylnitrosamine (DEN) or given saline vehicle alone and starting 1 wk later given diet containing 500, 250, 100, 10 or 0 ppm of oxfendazole for 8 wk. Sub-groups of five rats each from the DEN plus 250 and 0 ppm groups were killed after wk 1 of oxfendazole treatment and the remaining animals at wk 8. At the termination relative liver weights were significantly increased in the DEN-initiated and non-initiated groups treated with 250 ppm and 100 ppm or more, respectively, compared with the corresponding controls values. Light microscopical examination showed centrilobular hepatocellular hypertrophy in all animals receiving 100 ppm or more. Electron microscopy also revealed marked increases in smooth endoplasmic reticulum in hepatocytes of the DEN plus 500 ppm group. Furthermore, induction of cytochrome P-450 (CYP) 1A1/2, 2B1/2 or 4A1 was observed in the DEN plus 100 ppm group, that of CYP 1A1/2 being most marked. A similar change in CYP 1A1/2 was seen in the DEN plus 10 ppm group. The numbers and areas of connexin 32 (Cx32)-positive spots per hepatocyte were also significantly decreased in a dose-dependent manner. Similar changes in liver weights, P-450 isozymes and Cx32 immunohistochemistry were already evident in the DEN plus 250 ppm group at wk 1. The number of placental form glutathione S-transferase positive single cells was significantly increased in the DEN-initiated groups treated with 250 ppm or more. The results therefore strongly suggest that oxfendazole exerts liver tumour promotion potential.

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats.

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.

[A 13-week subchronic oral toxicity study of carob germ colour in F344 rats].

A13-week subchronic oral toxicity study of carob germ colour, one of natural colour additives was carried out in F344 rats at dose levels of 5.0, 1.7, 0.6, 0.2 and 0% in the powdered diet. Rats were randomly allocated to 5 groups, each consisting of 10 males and 10 females. No animals died during the experiment and no changes in body weights and food intakes were observed in any dosed groups. Changes indicating obvious toxicity of carob germ colour were not observed in the organ weights, hematological, serum biochemical and histopathological examinations. These findings indicate that the treatment of 5% carob germ colour in diet for 13 weeks did not cause any significant toxicity in rat.

[A case of holoprosencephaly with a flat electroencephalogram].

A case of alobar type of holoprosencephaly, a severe impairment of the development of central nervous system, is presented. This is a case report of 2-month-old boy who had a peculiar physiognomy with a microcephalus and an undeveloped forehead. Echoencephalography showed a defect of almost all cerebellum and a singular ventricle accompanied with severe hydrocephalus. However, his brain stem and cerebellum could be confirmed. His electroencephalogram showed complete flat waves and failed to respond to visual evoked potential test (VEP) and auditory brain stem response test (ABR). Any chromosomal abnormalities were not found.

[Identification of two different genetic mutation associated with silent phenotypes for human serum cholinesterase in Japanese].

Two different gene mutations associated with the silent phenotype for human serum cholinesterase were demonstrated. DNA from five individuals with silent gene phenotype of three unrelated Japanese families was amplified by the polymerase chain reaction (PCR) and analyzed by direct sequencing. The first instance demonstrated a G----C transversion at codon 365 from GGA (Gly) to CGA (Arg), which was seen in three individuals of the two families. This mutation was resulted to create a new Taq 1 restriction site (TCGA). The second mutation was shown by a double heterozygous condition with two different silent gene mutations in two members of remaining one family. These mutations were as follows: 1) one type was a frameshift mutation, in which an extra A was inserted in codon 315 (ACC----AACC) to create a new stop codon at position 322 and 2) the other was the same point mutation at codon 365 as seen in the first instance. These results indicated that many silent variants can be distinguished by direct sequence analyses of genomic DNA.

[A case of ovarian cancer with metastatic tumor in the liver with paclitaxel and carboplatin systemic chemotherapy was very effective].

An 82-year-old woman was admitted to the Dept. of Obstetrics and Gynecology, Yamanashi Medical University to try to identify the origin of a liver metastatic tumor. CT examination revealed a small tumor located adjacent to the uterine cervix in a cul-de-sac. With biopsy using MR, it was clearly shown histologically that the origin of the tumor was the ovary. Systemic chemotherapy with paclitaxel and carboplatin was selected as the most reasonable treatment for this case because of the patient's age. After 6 courses of this chemotherapy, the tumor in the cul-de-sac disappeared and the tumor in the liver decreased markedly. Furthermore, no severe side effects were seen during this treatment. This result indicated that systemic chemotherapy with paclitaxel and carboplatin is effective and safe in cases of advanced ovarian cancer.