Gene Rearrangement and Expression of PRKACA and PRKACB Govern Morphobiology of Pancreatobiliary Oncocytic Neoplasms.

Intraductal oncocytic papillary neoplasms (IOPNs) are distinct from intraductal papillary mucinous neoplasms based on characteristic morphologic and genetic features represented by fusion genes involving PRKACA or PRKACB (PRKACA/B). However, pancreatic and biliary tumors with partial oncocytic features are often encountered clinically, and their molecular features are yet to be clarified. This study included 80 intraductal papillary neoplasms: 32 tumors with mature IOPN morphology (typical), 28 with partial or subclonal oncocytic features (atypical), and 20 without oncocytic features (control). We analyzed PRKACA/B fusion genes, including ATP1B1::PRKACA, DNAJB1::PRKACA, and ATP1B1::PRKACB, by reverse-transcription PCR; mRNA expression of fusion genes and nonrearranged PRKACA/B genes by quantitative reverse-transcription PCR; mutations in KRAS, BRAF, and GNAS by targeted sequencing or droplet digital PCR; and the expression of cyclic adenosine monophosphate (cAMP)-dependent protein kinase catalytic subunits α (PRKACA) and ß (PRKACB), phosphorylated cAMP response element-binding protein, and aberrations of p16, p53, SMAD4, STK11, and ß-catenin by immunohistochemistry. PRKACA/B fusion genes were detected in 100% (32/32) of typical, 46% (13/28) of atypical, and 0% (0/20) of control (P < .05). Expression of PRKACA, PRKACB, and phosphorylated cAMP response element-binding protein was upregulated in neoplasms with PRKACA/B fusion genes (P < .05). mRNA expression of the PRKACA/B fusion genes and protein expression of PRKACA or PRKACB tended to be higher in typical than in atypical cases (mRNA, P = .002; protein expression, P = .054). In some atypical neoplasms with mixed subtypes, PRKACA/B fusion genes were superimposed exclusively on oncocytic components. Typical IOPNs harbored fewer KRAS and GNAS mutations than control samples and fewer alterations in p53 and STK11 than atypical samples (P < .05). In conclusion, PRKACA/B fusion genes not only are the characteristic drivers of IOPNs but also play a crucial role in the development of subclonal oncocytic neoplasms. Moreover, oncocytic morphology is strongly associated with upregulation of PRKACA/B, which may provide clues for potential therapeutic options.

Indispensable roles of OX40L-derived signal and epistatic genetic effect in immune-mediated pathogenesis of spontaneous pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) refers to a spectrum of diseases with elevated pulmonary artery pressure. Pulmonary arterial hypertension (PAH) is a disease category that clinically presents with severe PH and that is histopathologically characterized by the occlusion of pulmonary arterioles, medial muscular hypertrophy, and/or intimal fibrosis. PAH occurs with a secondary as well as a primary onset. Secondary PAH is known to be complicated with immunological disorders. The aim of the present study is to histopathologically and genetically characterize a new animal model of PAH and clarify the role of OX40 ligand in the pathogenesis of PAH. RESULTS: Spontaneous onset of PAH was stably identified in mice with immune abnormality because of overexpression of the tumor necrosis factor (TNF) family molecule OX40 ligand (OX40L). Histopathological and physical examinations revealed the onset of PAH-like disorders in the C57BL/6 (B6) strain of OX40L transgenic mice (B6.TgL). Comparative analysis performed using different strains of transgenic mice showed that this onset depends on the presence of OX40L in the B6 genetic background. Genetic analyses demonstrated a susceptibility locus of a B6 allele to this onset on chromosome 5. Immunological analyses revealed that the excessive OX40 signals in TgL mice attenuates expansion of regulatory T cells the B6 genetic background, suggesting an impact of the B6 genetic background on the differentiation of regulatory T cells. CONCLUSION: Present findings suggest a role for the OX40L-derived immune response and epistatic genetic effect in immune-mediated pathogenesis of PAH.

Induction of lipocalin-type prostaglandin D synthase in mouse heart under hypoxemia.

Hypoxemia is a common manifestation of various disorders and generates pressure overload to the heart. Here we analyzed the expression of lipocalin-type prostaglandin D synthase (L-PGDS) in the heart of C57BL/6 mice kept under normobaric hypoxia (10% O2) that generates hemodynamic stress. Northern and Western blot analyses revealed that the expression levels of L-PGDS mRNA and protein were significantly increased (> twofold) after 14 days of hypoxia, compared to the mice kept under normoxia. Immunohistochemical analysis indicated that L-PGDS was increased in the myocardium of auricles and ventricles and the pulmonary venous myocardium at 28 days of hypoxia. Moreover, using C57BL/6 mice lacking heme oxygenase-2 (HO-2(-/-)), a model of chronic hypoxemia, we showed that the expression level of L-PGDS protein was twofold higher in the heart than that of wild-type mouse. L-PGDS expression is induced in the myocardium under hypoxemia, which may reflect the adaptation to the hemodynamic stress.

Interleukin-17 is a critical target for the treatment of ankylosing enthesitis and psoriasis-like dermatitis in mice.

Ankylosis is a major pathological manifestation of spondyloarthropathy. The aim of this study was to evaluate the effects of anti-IL-17 therapy on spontaneous ankylosing enthesitis in mice. In this study, we used male DBA/1 mice as a spontaneous ankylosis model. Serum IL-17 concentrations were determined using enzyme-linked immunosorbent assay. Male DBA/1 mice from different litters were mixed and caged together preceding the treatment at 10 weeks (wk) of age (prophylaxis) or 21 wk of age (intervention). Treatment with anti-IL-17 antibodies or saline was initiated after caging in groups of mice and administered weekly. The onset of tarsal ankylosis was assessed by ankle swelling and histopathological examination. Pathological changes and mRNA expression levels were assessed in joints and ears obtained at the experimental end-point. We found that circulating IL-17 increased with the onset of ankylosis in male DBA/1 mice, coinciding with the onset of dermatitis. The symptoms of dermatitis corresponded to the pathological characteristics of psoriasis: acanthosis with mild hyperkeratosis, scaling, epidermal microabscess formation and augmented expression of K16, S100A8 and S100A9. Prophylactic administration of anti-IL-17 antibodies significantly prevented the development of both ankylosis and dermatitis in male DBA/1 mice caged together. On the other hand, administration of anti-IL-17 antibodies after disease onset had a lesser but significant effect on ankylosis progression but did not affect dermatitis progression. In conclusion, IL-17 is a key mediator in the pathogenic process of tarsal ankylosis and psoriasis-like dermatitis in male DBA/1 mice caged together. Thus, IL-17 is a potential therapeutic target in ankylosing enthesitis and psoriasis in humans.

Epigallocatechin gallate stimulates the neuroreactive salivary secretomotor system in autoimmune sialadenitis of MRL-Fas(lpr) mice via activation of cAMP-dependent protein kinase A and inactivation of nuclear factor κB.

The water channel aquaporin 5 (AQP5) plays a crucial role in regulating salivary flow rates. Xerostomia is often observed in patients with Sjögren's syndrome, and this is attributed to reduced AQP5 expression in the salivary glands. Recently, anti-type 3 muscarinic cholinergic receptors (M3R) autoantibodies and nuclear factor κB (NF-κB) have been found to be negative regulators of AQP5 expression in the salivary gland. Anti-M3R autoantibodies desensitize M3R to salivary secretagogues in Sjögren's syndrome, while activated NF-κB translocates to nuclei and binds to the AQP5 gene promoter, resulting in the suppression of AQP5 expression. We previously documented that epigallocatechin gallate (EGCG), which is a robust antioxidant contained in green tea, ameliorates oxidative stress-induced tissue damage to the salivary glands of MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice, which are widely used as a model of Sjögren's syndrome. Reactive oxygen species (ROS) can activate NF-κB and inactivate protein kinase A (PKA), which is a key driver of AQP5 expression. In this study, we examined the effects of administering EGCG to MRL-Fas(lpr) mice with autoimmune sialadenitis on the levels of AQP5, activated NF-κB p65 subunit, activated PKA, activated c-Jun N-terminal kinase (JNK) (an activator of NF-κB), inhibitor κB (IκB) and histone deacetylase 1 (HDAC1) (an inhibitor of NF-κB). In EGCG-treated mice, intense aster-like immunostaining for AQP5 was observed on the apical plasma membranes (APMs) of submandibular gland acinar cells. Likewise, PKA, IκB and HDAC1 were highly expressed in salivary gland tissues, whereas the expression of JNK and NF-κB p65 was negligible. Rank correlation and partial correlation analyses revealed that treatment with EGCG upregulated AQP5 expression on the APM of acinar cells through activation of PKA and inactivation of NF-κB, while IκB and HDAC1 played a pivotal role in the induction of AQP5 expression by PKA. Our study indicates that EGCG may have therapeutic potential for Sjögren's syndrome patients.

Orexin-A expression in human peripheral tissues.

Orexin-A is a neuropeptide present in the brain and is known to regulate feeding and sleeping. In this study, we examined the systemic distribution of orexin-A in human tissues. Immunoreactivity for orexin-A was detected in ganglion cells of the thoracic sympathetic trunk, myenteric plexuses and endocrine cells of the gastrointestinal tract, islet cells of the pancreas and syncytiotrophoblasts and decidual cells of the placenta. In the gastrointestinal tract, orexin-A immunoreactivity was detected in the myenteric plexuses from 26 gestational weeks to birth. In double immunostaining in the pancreas, a great majority of insulin-positive cells was simultaneously positive for orexin-A. mRNA expression for prepro-orexin was also detected in the kidney, adrenal gland, pancreas, placenta, stomach, ileum, colon and colorectal epithelial cells. These results suggest the production of orexin-A in various human peripheral tissues and orexin-A may also play important roles in some peripheral organs.

Epigallocatechin gallate inhibits oxidative stress-induced DNA damage and apoptosis in MRL-Fas(lpr) mice with autoimmune sialadenitis via upregulation of heme oxygenase-1 and Bcl-2.

Pathogenic effects of reactive oxygen species (ROS) in the salivary glands of patients with Sjögren's syndrome have been demonstrated. Epigallocatechin gallate (EGCG), which is a catechin derivative and exhibits potent antioxidant activity, has been reported to ameliorate autoimmune sialadenitis in a murine model, but the mechanism underlying its protective action remains to be investigated. Herein, we examined the effects of EGCG administration to MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice on disease severity of autoimmune sialadenitis and protein expression levels of 11 sialadenitis-related molecules - heme oxygenase-1 (HO-1) (antioxidant); thymidine glycol (marker of DNA damage); gp91phox/NADPH oxidase 2 (prooxidant); single-stranded DNA (ssDNA) and cleaved caspase 3 (apoptotic cell markers); p53 and Bax (proapoptotic molecules); Bcl-2 (antiapoptotic molecule); SSA/Ro, SSB/La, and Ifi202 (autoantigens). In EGCG-treated mice, the severity of sialadenitis was substantially decreased. Expression levels of thymidine glycol, gp91phox, ssDNA, cleaved caspase 3, p53, Bax, SSA/Ro, SSB/La, and Ifi202 in duct epithelial cells of salivary glands from EGCG-treated mice were reduced, whereas HO-1 and Bcl-2 were overexpressed. Results of correlation analysis among sialadenitis severity and 11 sialadenitis related-molecules, and those of partial correlation analysis between apoptotic related-molecules and sialadenitis severity or HO-1 suggested that the consecutive pathogenic cycle including activated autoimmune reactions, ROS synthesis, DNA damage and p53-dependent apoptosis was associated with the pathogenesis of autoimmune sialadenitis in MRL-Fas(lpr) mice. Overexpression of HO-1 and Bcl-2 mediated by EGCG blocked this pathogenic cycle, subsequently resulting in the inhibition of ROS-mediated DNA damage and apoptosis, and protected salivary gland tissues from oxidative stress. Clinically, green tea catechin may have therapeutic efficacy for Sjögren's syndrome.

Sjögren's syndrome-like autoimmune sialadenitis in MRL-Faslpr mice is associated with expression of glucocorticoid-induced TNF receptor-related protein (GITR) ligand and 4-1BB ligand.

Although costimulatory molecules have been shown to play crucial roles in the immune response, their involvement in the pathogenesis of Sjögren's syndrome is incompletely understood. In this study, we evaluated the relationship between the severity of spontaneous Sjögren's syndrome-like autoimmune sialadenitis in MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice and the expression of 6 costimulatory molecules that play important roles in the immune response: CD80, CD86, OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), glucocorticoid-induced TNF receptor-related protein ligand (GITRL), and B cell-activating factor of the tumor necrosis factor family (BAFF). Expression of the costimulatory molecules in the submandibular salivary glands of age-matched autoimmune MRL-Fas(lpr) mice and non-autoimmune MRL/MpJ-+/+(MRL/+) and C3H/HeJ-lpr/lpr (C3H-Fas(lpr)) mice was examined immunohistochemically and scored on a scale of 0 to 3. The severity of sialadenitis was evaluated histologically and scored on a scale of 0 to 3. We found that all of the costimulatory molecules were expressed in duct epithelial cells of salivary glands from MRL-Fas(lpr) mice, whereas immunoreactivity was absent or weak in the MRL/+ mice. The staining intensity for all 6 costimulatory molecules was significantly higher in the MRL-Fas(lpr) than in the MRL/+ mice. Partial correlation analysis was performed to assess the degree of association between costimulatory molecule staining scores and disease scores, which clearly revealed a significant correlation for only GITRL and 4-1BBL. These molecules showed negligible immunoreactivity in the submandibular glands of C3H-Fas(lpr) mice, suggesting that their expression was independent of the Fas(lpr) mutation. In conclusion, the expression of GITRL and 4-1BBL in salivary gland duct epithelial cells is associated with background genes in the MRL strain, but not with the Fas(lpr) mutation itself, and contributes significantly to the pathogenesis of autoimmune sialadenitis in MRL-Fas(lpr) mice. These results suggest that GITRL and 4-1BBL may be effective targets for the development of therapies for Sjögren's syndrome.

Hypoxemia induces expression of heme oxygenase-1 and heme oxygenase-2 proteins in the mouse myocardium.

Heme oxygenase (HO) catalyzes oxidative breakdown of heme, and constitutes two isozymes, HO-1 and HO-2. Here, we explored the tissue-specific regulation of expression of HO-1 and HO-2 under hypoxemia. There was no significant change in the overall expression levels of HO-1 and HO-2 mRNAs and proteins in the lung during adaptation of C57BL/6 mice to normobaric hypoxia (10% O(2)). However, immunohistochemical analysis revealed the increased expression of HO-1 and HO-2 proteins after 28 days of normobaric hypoxia in the pulmonary venous myocardium that is the extension of the left atrial myocardium into pulmonary venous walls. Moreover, the expression of HO-2 protein was increased in the sub-endocardial myocardium of ventricles under hypoxia, while HO-1 protein level was increased in the full-thickness walls. Thus, hypoxemia induces expression of both HO-1 and HO-2 proteins in the myocardium. Using C57BL/6 mice lacking HO-2 (HO-2(-/-)), which manifest chronic hypoxemia, we also showed that the HO-1 protein level in the lung was similar between HO-2(-/-) mice and wild-type mice. Unexpectedly, HO-1 protein level was lower by 35% in the HO-2(-/-) mouse liver than the wild-type liver. These results indicate that the expression of HO-1 protein is regulated in a tissue-specific manner under hypoxemia.

Characterization of lymphangiogenesis in various stages of idiopathic diffuse alveolar damage.

In pulmonary fibrosis, an abnormal healing process, is believed to be involved in the damage to lung tissue. This process has not been correlated with lymphangiogenesis, which has garnered current interest in relation to wound healing. The aim of the present study was to clarify the characteristics of lymphangiogenesis in pulmonary fibrosis associated with idiopathic diffuse alveolar damage. Formalin-fixed and paraffin-embedded lung tissues from 13 autopsy cases with idiopathic diffuse alveolar damage were used. Antibodies specific to CD34 and D2-40 were used to detect blood vessels and lymphatics, respectively, and immunohistochemical examinations and morphometry analyses were performed. The standardized density of capillaries was increased significantly in the exudative stage of diffuse alveolar damage, whereas that of the lymphatics remained unchanged. In the proliferative stage, new lymphatics emerged, primarily in the intra-alveolar fibrotic lesions where capillaries were absent. In the fibrotic stage, in which the lung was shrunken, as revealed by the elevated density of pulmonary arteries, the standardized density of capillaries was reduced significantly. The standardized area density of the interstitium was elevated in the proliferative stage and subsequently reduced in the fibrotic stage. Three-dimensional reconstruction of images revealed that some new lymphatics lacked connection to existing lymphatics. During the progression of diffuse alveolar damage, lymphangiogenesis occurs independent of capillary angiogenesis.

The definition of fibrogenic processes in fibroblastic foci of idiopathic pulmonary fibrosis based on morphometric quantification of extracellular matrices.

There is limited information regarding the process of tissue remodeling in fibroblastic foci associated with idiopathic pulmonary fibrosis. The aim of this study was to identify the different pathologic stages of tissue remodeling in fibroblastic foci based on the histopathologic differences in the glycosaminoglycan distribution and collagen deposition. In addition, we also aimed at clarifying the stage-specific characteristics by taking into consideration the expression pattern of matrix metalloproteinase and angiogenesis. Lung biopsies of 16 patients with idiopathic pulmonary fibrosis were used. The presence of glycosaminoglycans was detected by Alcian blue staining, and type I collagen was detected by immunohistochemical analysis with a primary antibody specific to the cross-linked carboxyterminal telopeptide of type I collagen. The fibroblastic foci characterized by the expression intensity of Alcian blue and telopeptide of type I collagen were divided into 3 groups, namely, Alcian blue(+)telopeptide of type I collagen(weak), Alcian blue(+)telopeptide of type I collagen(+), and Alcian blue(weak)telopeptide of type I collagen(+); consequently, 3 new stages were defined--stages I, II, and III, respectively. A significant inverse correlation was observed between the area densities of Alcian blue(+) and telopeptide of type I collagen(+) in fibroblastic foci. Stage I was characterized by the expression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloprotease-2 in fibroblasts and the overlying epithelium of fibroblastic foci, and also the absence of capillary angiogenesis. In contrast, the expression of these proteins was attenuated in stage III, except for that of matrix metalloproteinase-2 in fibroblasts. In stages II and III, capillary angiogenesis was observed. Lymphangiogenesis was undetected in all the 3 stages. Thus, pathologic staging helps understand the roles of the factors involved in tissue remodeling in idiopathic pulmonary fibrosis.

Macrophages participate in lymphangiogenesis in idiopathic diffuse alveolar damage through CCL19-CCR7 signal.

An abnormal healing process is believed to be involved in lung tissue damage in pulmonary fibrosis. Lymphangiogenesis has been determined to play a role in structural remodeling in diffuse alveolar damage. However, the mechanism of lymphangiogenesis remains unclear. The aim of this study is to investigate the cellular mechanisms of lymphangiogenesis in diffuse alveolar damage, focusing on the roles of macrophages. Formalin-fixed and paraffin-embedded lung tissues from 13 autopsy cases with idiopathic diffuse alveolar damage were used. Antibodies specific for D2-40 and CD68 were mainly used as the markers for lymphatics and macrophages, respectively, and immunohistochemical examinations and morphometric analyses were performed. Immunohistochemistry showed the aggregation of numerous CD68+ macrophages around newly formed lymphatics in the intraalveolar fibrotic lesions in the proliferative stage, and some of the CD68+ macrophages were colocalized with the lymphatic endothelium. These macrophages were characterized by the expression of vascular endothelial growth factor-C. Among the 3 stages of diffuse alveolar damage, the number of Ki-67+ cells on the lymphatic endothelium tended to increase in newly formed lymphatics of the proliferative stage. In addition, the number of CD68+ macrophages on the lymphatic endothelium was significantly increased in the newly formed lymphatics of the proliferative stage more than those of the fibrotic stage. The CD68+ macrophages around the newly formed lymphatics coexpressed CCR7. Dual immunostaining showed the coexpression of CCL19-a ligand for CCR7-on the lymphatic endothelium. Thus, macrophages may participate in lymphangiogenesis in diffuse alveolar damage, which is facilitated by CCL19 and CCR7.

Mast cell hyperplasia in the skin of Dsg4-deficient hypotrichosis mice, which are long-living mutants of lupus-prone mice.

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)( hage ) mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.

A non-major histocompatibility locus determines tissue specificity in the pathogenic process underlying synovial proliferation in a mouse arthropathy model.

BACKGROUND: The incidence and characteristics of spontaneous ankylosis in the ankle of specific F(1) mice descended from two Fas-deficient strains were reported. Here the coincidence of synovial proliferation and ankylosis in the descendent F(2) mice is reported. AIM: To clarify whether the two distinct manifestations are genetically different. METHODS: An arthropathic group of mice (MCF(2)) were bred by intercrossing MRL/Mp.Fas(lpr)-sap(-)/sap(-) and C3H/He.Fas(lpr) mice. All mice were killed by bleeding under anaesthesia when they were 6 months old. Pathological grades for synovial proliferation were determined by microscopical examination. To obtain a linkage locus, the whole genome of male MCF(2) mice was scanned by using 73 microsatellite markers. RESULTS: Synovial proliferation was equally observed in male and female MCF(2) mice. No correlation was observed between the grades of synovial proliferation and the ankylosis occurring in the MCF(2) mice. A suggestive susceptibility locus was shown in the middle of chromosome 11. This locus was an MRL allele with a recessive inheritance mode. CONCLUSION: The pathogenic mechanisms of synovial proliferation and ankylosis are genetically different. The present locus is overlapped with some loci associated with rheumatoid arthritis and with others associated with experimental arthritides.

Hypoxemia and blunted hypoxic ventilatory responses in mice lacking heme oxygenase-2.

Heme oxygenase (HO) catalyzes physiological heme degradation and consists of two structurally related isozymes, HO-1 and HO-2. Here we show that HO-2-deficient (HO-2(-/-)) mice exhibit hypoxemia and hypertrophy of the pulmonary venous myocardium associated with increased expression of HO-1. The hypertrophied venous myocardium may reflect adaptation to persistent hypoxemia. HO-2(-/-) mice also show attenuated ventilatory responses to hypoxia (10% O2) with normal responses to hypercapnia (10% CO2), suggesting the impaired oxygen sensing. Importantly, HO-2(-/-) mice exhibit normal breathing patterns with normal arterial CO2 tension and retain the intact alveolar architecture, thereby excluding hypoventilation and shunting as causes of hypoxemia. Instead, ventilation-perfusion mismatch is a likely cause of hypoxemia, which may be due to partial impairment of the lung chemoreception probably at pulmonary artery smooth muscle cells. We therefore propose that HO-2 is involved in oxygen sensing and responsible for the ventilation-perfusion matching that optimizes oxygenation of pulmonary blood.