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.

Infliximab, a TNF-α inhibitor, reduces 24-h ambulatory blood pressure in rheumatoid arthritis patients.

Tumour necrosis factor-alpha (TNF-α) is an important mediator in the pathogenesis of rheumatoid arthritis (RA) and hypertension. TNF-α inhibitors improve clinical symptoms and inhibit joint destruction in RA, but their effect on blood pressure (BP) has not been fully investigated. We measured 24-h BP using an ambulatory BP monitor in 16 RA patients treated with a TNF-α inhibitor, infliximab, to investigate its influence on BP and its association with the regulatory factors of BP and renin-angiotensin-aldosterone and sympathetic nervous systems. Infliximab significantly reduced the 24-h systolic BP (SBP) from 127.4±21.8 to 120.1±23.4 mm Hg (P<0.0001). Particularly, morning BP (0600-0800 h) decreased from 129.7±19.7 to 116.9±13.4 mm Hg (P<0.0001), and daytime BP decreased from 131.8±15.1 to 122.5±13.7 mm Hg (P<0.0001). Infliximab significantly reduced the plasma level of norepinephrine and plasma renin activity (PRA) (from 347.5±180.7 to 283.0±181.8 pg ml(-1) and 2.6±2.7 to 2.1±2.9 ng ml(-1) h(-1), respectively) but did not significantly reduce the plasma levels of dopamine and epinephrine. The reduction in morning SBP correlated with the reduction in the norepinephrine level (P<0.05) but not with that in PRA and inflammatory parameters related to RA. This study shows the effect of infliximab on ambulatory BP, especially daytime BP, which may be partly accounted for by the reduction of sympathetic nerve activity after infliximab treatment.

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.

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 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.

Activation of mu-opioid receptor induces expression of c-fos and junB via mitogen-activated protein kinase cascade.

BACKGROUND: Opioid-induced long-term functional alterations of the nervous system, such as tolerance, addiction, and dependence, conceivably involve changes in gene expression. The authors have previously reported that opioid receptors are functionally coupled to extracellular signal-regulated kinase, a class of the mitogen-activated protein kinase. To address whether activation of the opioid receptor induces changes in gene expression through the activation of extracellular signal-regulated kinase, the authors examined mu-opioid receptor (MOR)-induced immediate early gene expression. METHODS: Chinese hamster ovary cells stably expressing MOR were used. Cells were stimulated by MOR agonists after 24-h serum starvation. Expression of c-fos and junB genes was analyzed by RNA blot hybridization. To explore the mechanism of MOR-mediated c-fos and junB expression, activity of a transcription factor, Elk-1, was assessed by reporter assay. Furthermore, to investigate the functional consequences of c-fos and junB induction, MOR-mediated formation of the functional transcription factor complex AP-1 was examined by reporter assay and electrophoretic mobility shift assay. RESULTS: Mu-opioid receptor activation induced c-fos and junB messenger RNAs, which were inhibited by pretreatment of the cells with pertussis toxin and PD98059, an inhibitor of extracellular signal-regulated kinase cascade. MOR stimulation elevated Elk-1-mediated transcriptional activity by about 10-fold. AP-1-mediated transcriptional activity was stimulated by MOR agonists by about twofold. Electrophoretic mobility shift assay revealed that AP-1 binding activity in the nuclear extract was elevated by MOR activation and further showed that products of c-fos and junB genes are involved in formation of AP-1 complex. CONCLUSIONS: Mu-opioid receptor activation induces c-fos and junB expression and elevates AP-1-mediated transcriptional activities via the mitogen-activated protein kinase cascade.

The novel untranslated exon "exon 0T" encoded between the exon 0 and exon 1 of the rat estrogen receptor alpha (ER alpha) gene.

We have recently isolated two untranslated first exons, exon 0N and exon 0S, of rat estrogen receptor alpha (ER alpha) gene from the liver by use of 5'-rapid amplification of the cDNA ends (5'-RACE) method. In this communication, we further analyzed the 5'-untranslated region (UTR) of the ER alpha mRNA in rat anterior hypophysis in order to investigate the existence of the other 5'-untranslated exon(s) of rat ER alpha gene. Total RNA from the anterior hypophysis of 8-week-old female Wistar strain male rats was subjected to 5'-RACE with antisense primers located in exon 1 of the rat ER alpha gene and one of the positive clones (clone 35) was sequenced. The nucleotide sequence of clone 35 revealed the insertion of a previously unidentified exon (which we termed "exon 0T") between exon 0 (the first reported 5'-UTR form of rat ER alpha mRNA) and exon 1 of rat ER alpha mRNA. Analysis of rat genomic DNA indicated that exon 0T was located between exon 0 and exon 1 of rat ER alpha gene. We further investigated the distribution of ER alpha mRNA containing exon 0T in several brain regions and various peripheral tissues of 8-week-old male and female Wistar strain rats by use of reverse transcription-polymerase chain reaction (RT-PCR). The distribution of the ER alpha mRNA (0T-1) was essentially similar to that of ER alpha mRNA in which exon 0 was spliced onto exon 1 reported previously. These results indicate that (1) exon 0T is a novel untranslated exon of rat ER alpha gene which is located between exon 0 and exon 1 on rat genomic DNA, (2) exon 0T is inserted between exon 0 and exon 1 of ER alpha mRNA by alternative splicing, and (3) this alternative splicing may occur in tissues where the transcription of ER alpha gene is initiated from exon 0.

Testicular toxicity of nitrofurazone causing germ cell apoptosis in rats.

In order to clarify the mechanism underlying testicular toxicity of nitrofurazone (NF), two experiments were performed. In experiment 1, sequential histopathological examination of testes after a single oral administration of 100 or 300 mg/kg NF to male rats demonstrated that degeneration of pachytene spermatocytes with an eosinophilic, shrunken appearance in stages VII-VIII and vacuolation of Sertoli cells were first observed 12 h after treatment. By 24 h, degeneration of pachytene spermatocytes in stages VII-XII and diplotene spermatocytes were observed. On post-treatment day 4, neither spermatocytes nor spermatids located inside the pachytene spermatocytes in stage VII were seen anywhere. Generation of seminiferous epithelium progressed with recovery to almost normal morphology after 12 weeks, although some morphological changes were still present. No lesions were apparent in spermatogonia, preleptotene spermatocytes, leptotene spermatocytes, zygotene spermatocytes or Leydig cells. Degenerate pachytene spermatocytes and some round spermatids seen after 24 h showed positive TdT-mediated dUTP-biotin nick end labeling (TUNEL). In addition, DNA laddering patterns were detected with agarose gel electrophoresis, and increased electron density of nuclei and cytoplasm of degenerating spermatocytes with nuclear chromatin focal aggregations were observed by electron microscopy, indicating that cell death was attributable to apoptosis. In experiment 2, sequential serum sex-related hormone levels were assayed after a single oral administration of 300 mg/kg NF to male rats and revealed a significant increase of testosterone and a decrease of progesterone at 6 h, and decreases of luteinizing hormone at 12 h and testosterone at 24 h. Prolactin tended to decrease from 12 h after treatment and the decrease was significant at 48 h. No significant changes were observed in levels of follicle-stimulating hormone or estradiol. The probability that NF damages germ cells by causing a hormonal imbalance is extremely low, since no pattern of hormonal imbalance that could be regarded as the cause of the testicular degeneration was observed until 12 h after NF treatment when pachytene spermatocytes began to degenerate. The present experiments suggest that NF damages Sertoli cells and pachytene spermatocytes in stages VII-XII directly.

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.

The novel isoform of the progesterone receptor cDNA in the human testis and detection of its mRNA in the human uterine endometrium.

A novel isoform (termed isoform S) of the progesterone receptor (PR) cDNA (PR isoform S cDNA) which consists of a previously unidentified 5' sequence and exons 4-8 of the intracellular PR gene has been cloned from the human testicular cDNA library. The 5' sequence of the message was confirmed to be derived from a novel exon (termed exon S) by genomic cloning. The expression level of the PR isoform S mRNA was higher in the spermatozoon than in the uterine endometrium with a lower expression level of the PR isoforms B and A mRNAs in the spermatozoon than in the endometrium. These results implied that the PR isoform S which was possibly translated from the PR isoform S mRNA in the spermatozoon might be related to the cell surface membrane PR. Moreover, the PR isoform S in the uterine endometrium might play some physiological and/or pathogenic roles.

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat.

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.

A new spontaneously diabetic non-obese Torii rat strain with severe ocular complications.

A new spontaneously diabetic strain of the Sprague-Dawley rat was established in 1997 and named the SDT (Spontaneously Diabetic Torii) rat. In this research, we investigated the characteristics of the disease condition in the SDT rats. The time of onset of glucosuria was different between male and female SDT rats; glucosuria appeared at approximately 20 weeks of age in male rats and at approximately 45 weeks of age in female rats. A cumulative incidence of diabetes of 100% was noted by 40 weeks of age in male rats, while it was only 33.3% even by 65 weeks of age in female rats. The survival rate up to 65 weeks of age was 92.9% in male rats and 97.4% in female rats. Glucose intolerance was observed in male rats from 16 weeks of age. The clinical characteristics of the male SDT rats were (1) hyperglycemia and hypoinsulinemia (from 25 weeks of age); (2) long-term survival without insulin treatment; (3) hypertriglyceridemia (by 35 weeks of age); however, no obesity was noted in any of the male rats. The histopathological characteristics of the male rats with diabetes mellitus (DM) were (1) fibrosis of the pancreatic islets (by 25 weeks of age); (2) cataract (by 40 weeks of age); (3) tractional retinal detachment with fibrous proliferation (by 70 weeks of age) and (4) massive hemorrhaging in the anterior chamber (by 77 weeks of age). These clinical and histopathological characteristics of the disease in SDT rats resemble those of human Type 2 diabetes with insulin hyposecretion. In conclusion, SDT rat is considered to be a potentially useful model for studies of diabetic retinopathy encountered in humans.

The relationship between decrease in Cx32 and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis in the rat.

To examine the relationship between the decrease in connexin 32 (Cx32) and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis, a total of 20 male F344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or given the saline vehicle alone and starting 2 weeks later given diet containing 0.18, 0.09, and 0% clofibrate for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Absolute and relative (ratios to body weight) liver weights were significantly increased in the DEN + clofibrate groups compared with the DEN-alone group. Diffuse hepatocellular hypertrophy with granular cytoplasmic eosinophilia characterized by a marked increase in peroxisomes and smooth endoplasmic reticulum, was observed in the clofibrate treated rats. Induction of cytochrome P450 (CYP) 4A1 and 2B1/2 was noted in the DEN + clofibrate groups, this being most marked in the CYP 2B1 case. Immunohistochemically, positive immunostaining for anti-CYP 4A1 and CYP 2B1 were observed diffusely and centrilobularly, respectively. The numbers and areas of Cx32-positive spots per hepatocyte in the centrilobular areas in the treated rats were significantly decreased in an essentially dose-dependent manner, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form (GST-P) were decreased in a dose dependent manner in the clofibrate treated groups. These results suggest that the CYP 2B1/2 induction and Cx32 decrease in centrilobular hepatocytes, similarly to those thought to be involved in the hepatic promotion mechanism of phenobarbital, may also play important roles in clofibrate actions in the liver, in addition to its causation of oxidative DNA injury.

Liver tumor promoting effects of fenbendazole in rats.

In order to examine whether fenbendazole has tumor-promoting activity, a total of 70 male Fischer 344 rats were initiated with a single intraperitoneal injection of 100 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone; beginning 1 wk later, rats were given a diet containing 3,600; 1,800; 600; 200; 70; or 0 ppm of fenbendazole for 8 wk. Subgroups of 5 rats each from the DEN+ 1,800; DEN+0; 1,800; and 0 ppm groups were euthanatized after 1 wk of fenbendazole treatment, and the remaining animals were euthanatized at 8 wk. After 1 wk, relative liver weights (ratios to body weights) were significantly increased in the DEN+ 1,800 and 1,800 ppm groups, and based on light microscopy, periportal hepatocellular hypertrophy was evident in these groups. After 8 wk, relative liver weights were significantly increased in the groups given > or =600 ppm with or without DEN initiation. Periportal hepatocellular hypertrophy, characterized by a marked increase in smooth endoplasmic reticulum, was observed in the groups given > or =600 ppm with or without DEN initiation. Induction of cytochrome P-450 (CYP) 1A2, 2B1, or 4A1 was noted in the fenbendazole-treated groups with or without DEN initiation; that associated with CYP 1A2 was most marked. Positive immunostaining for anti-CYP 1A1/2 or CYP 2B1/2 was observed diffusely in the livers of animals in the DEN+1,800 and DEN+3,600 ppm groups. The numbers and areas of connexin 32 (Cx32)-positive spots per square centimeter in centrilobular hepatocytes were significantly decreased in an almost dose-dependent manner with fenbendazole treatment after DEN initiation. In situ hybridization for Cx32 mRNA revealed a remarkable decrease in its expression in the centrilobular hepatocytes in the DEN+70 ppm group. The numbers of glutathione S-transferase placental-form positive single cells (plus mini foci) were significantly increased in the DEN+ 1,800 and DEN+3,600 ppm groups. Since those agents that induce CYP 2B1/2 isozymes and reduce Cx32 in centrilobular hepatocytes have been suggested to be liver tumor promoters, the present results indicate that fenbendazole may be a liver tumor promoter.

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.