Diet‐induced obesity increases the frequency of Pig‐a mutant erythrocytes in male C57BL/6J mice

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig‐a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30‐week old male mice reared on either a high‐fat diet (60% calories from fat) that exhibit an obese phenotype or a normal‐fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N‐ethyl‐N‐nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig‐a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non‐obese mice with respect to Pig‐a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig‐a mutant frequencies (increased 2.5‐3.7‐fold, p < 0.02) in erythrocytes as compared to non‐obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668–677, 2016. © 2016 Wiley Periodicals, Inc.     

Stem cell marker prominin‐1 regulates branching morphogenesis,but not regenerative capacity,in the mammary gland

Prominin‐1 (Prom1) is recognized as a stem cell marker in several tissues, including blood, neuroepithelium, and gut, and in human and mouse embryos and many cancers. Although Prom1 is routinely used as a marker for isolating stem cells, its biological function remains unclear. Here we use a knockout model to investigate the role of Prom1 in the mammary gland. We demonstrate that complete loss of Prom1 does not affect the regenerative capacity of the mammary epithelium. Surprisingly, we also show that in the absence of Prom1, mammary glands have reduced ductal branching, and an increased ratio of luminal to basal cells. The effects of Prom1 loss in the mammary gland are associated with decreased expression of prolactin receptor and matrix metalloproteinase‐3. These experiments reveal a novel, functional role for Prom1 that is not related to stem cell activity, and demonstrate the importance of tissue‐specific characterization of putative stem cell markers. Developmental Dynamics 240:674–681, 2011. © 2011 Wiley‐Liss, Inc.     

Blockade of interleukin‐6 receptor enhances the anti‐arthritic effect of glucocorticoids without decreasing bone mineral density in mice with collagen‐induced arthritis

In a mouse arthritis model, we investigated whether interleukin‐6 receptor (IL‐6R) blockade would enhance the anti‐arthritic effect of glucocorticoids (GCs). DBA/1J mice were immunized with type II collagen (CII), and were treated with prednisolone (PSL) and/or anti‐mouse IL‐6R antibody (MR16‐1). Also, the effects of IL‐6 on gene expression and the nuclear translocation of glucocorticoid receptors (GRs) were examined in cultured cells treated with dexamethasone (DEX). PSL reduced the arthritis score dose‐dependently in the collagen‐induced arthritis (CIA) mouse model. The arthritis score in the PSL (3 mg/kg) + MR16‐1 group was lower than in the PSL (3 mg/kg) group, and at the same level as in the PSL (6 mg/kg) group. Lumbar vertebra bone mineral density (BMD) was decreased significantly in CIA mice and was higher in the PSL (3 mg/kg) + MR16‐1 group than in the PSL (6 mg/kg) group. In the in‐vitro synovial cells, IL‐6 pretreatment attenuated the inhibitory effect of DEX on cyclooxygenase (COX)‐2 expression and inhibited the nuclear translocation of GR induced by DEX. In contrast, in MC3T3‐E1 osteoblastic cells, IL‐6 pretreatment exacerbated the decrease in expression of osteocalcin and the increase in expression of receptor activator of nuclear factor kappa‐B ligand (RANKL) by DEX. We demonstrated that IL‐6 signalling blockade by an anti‐IL‐6R antibody can augment the anti‐arthritic effect of GCs and inhibit the bone loss they cause.     

An obesogenic diet started before puberty leads to abnormal mammary gland development during pregnancy in the rabbit

Alterations to the metabolic environment during puberty can impact future lactation efficiency and mammary tumorigenesis. During this study, we used a model of rabbits receiving an obesogenic diet (OD), starting before puberty and extending until mid‐pregnancy. Three months later, the body weight of OD animals was significantly higher than that of controls and their mammary glands displayed a precocious and abnormal development at mid‐pregnancy. OD mammary ducts were filled with dense products, while alveolar structures invaded most of the fat pad. The proportion of secretory epithelium was significantly higher in OD mammary tissue, which contained an abundant accumulation of milk proteins and lipids. In conclusion, an obesogenic diet started before puberty induced an accelerated development of the rabbit mammary gland, leading to an accumulation of secretory products at mid‐pregnancy. These results support the critical influence of nutrition on mammary growth and differentiation, which may be deleterious to mammary development and subsequent lactation. Developmental Dynamics 240:347–356, 2011. © 2011 Wiley‐Liss, Inc.     

High incidence of mammary intraepithelial neoplasia development in Men1‐disrupted murine mammary glands

Mutations of the MEN1 tumour suppressor gene predispose patients to the development of multiple endocrine neoplasia type 1 (MEN1) syndrome, which is characterized by multiple endocrine tumours, including prolactinomas. The recent findings of the interaction between menin, encoded by the MEN1 gene, and the oestrogen receptor, as well as the observation of rare cases of mammary carcinomas in our heterozygous Men1 mutant mice, led us to investigate a putative tumour suppressor function of the Men1 gene in mouse mammary cells by disrupting the gene in luminal epithelial cells. A significantly higher incidence of mammary intraepithelial neoplasia (MIN) was observed in mutant WapCre‐Men1^F/F mice (51.5%) than in WapCre‐Men1^+/+ (0%) or Men1^F/F (7.1%) control mice. The majority of MIN observed in the mutant mice displayed complete menin inactivation. Because of the leakage of WapCre transgene expression, prolactinomas were observed in 83.3% of mutant mice, leading to premature death. As there was no correlation between MIN development and elevated serum prolactin levels, and phospho‐STAT5 expression was decreased in mammary lesions, the increased incidence of MIN lesions was most likely due to Men1 disruption rather than to prolactinoma development. Interestingly, in MIN lesions, we found a decrease in membrane‐associated E‐cadherin and beta‐catenin expression, the latter of which is a menin partner. Finally, reduced menin expression was found in a large proportion of two independent cohorts of patients with breast carcinomas. Taken together, the current work indicates a role of Men1 inactivation in the development of mammary pre‐cancerous lesions in mice and a potential role in human mammary cancer.     

Sinus‐like dilatations of the mammary milk ducts,Ki67 expression,and CD3‐positive T lymphocyte infiltration,in the mammary gland of wild European rabbits during pregnancy and lactation

Sinus‐like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E‐cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14‐positive cells, supported by a thin layer of fibrous tissue. Multi‐segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3‐positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus‐like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus‐like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune cells.     

Obesity promotes prolonged ovalbumin‐induced airway inflammation modulating T helper type 1 (Th1), Th2 and Th17 immune responses in BALB/c mice

Clinical and epidemiological studies indicate that obesity affects the development and phenotype of asthma by inducing inflammatory mechanisms in addition to eosinophilic inflammation. The aim of this study was to assess the effect of obesity on allergic airway inflammation and T helper type 2 (Th2) immune responses using an experimental model of asthma in BALB/c mice. Mice fed a high‐fat diet (HFD) for 10 weeks were sensitized and challenged with ovalbumin (OVA), and analyses were performed at 24 and 48 h after the last OVA challenge. Obesity induced an increase of inducible nitric oxide synthase (iNOS)‐expressing macrophages and neutrophils which peaked at 48 h after the last OVA challenge, and was associated with higher levels of interleukin (IL)‐4, IL‐9, IL‐17A, leptin and interferon (IFN)‐γ in the lungs. Higher goblet cell hyperplasia was associated with elevated mast cell influx into the lungs and trachea in the obese allergic mice. In contrast, early eosinophil influx and lower levels of IL‐25, thymic stromal lymphopoietin (TSLP), CCL11 and OVA‐specific immunoglobulin (IgE) were observed in the obese allergic mice in comparison to non‐obese allergic mice. Moreover, obese mice showed higher numbers of mast cells regardless of OVA challenge. These results indicate that obesity affects allergic airway inflammation through mechanisms involving mast cell influx and the release of TSLP and IL‐25, which favoured a delayed immune response with an exacerbated Th1, Th2 and Th17 profile. In this scenario, an intense mixed inflammatory granulocyte influx, classically activated macrophage accumulation and intense mucus production may contribute to a refractory therapeutic response and exacerbate asthma severity.     

Aspiration biopsy of mammary analogue secretory carcinoma of accessory parotid gland: Another diagnostic dilemma in matrix‐containing tumors of the salivary glands

Mammary analogue secretory carcinoma (MASC) is a newly described rare salivary gland tumor, which shares morphologic features with acinic cell carcinoma, low‐grade cystadenocarcinoma, and secretory carcinoma of the breast. This is the first reported case of MASC of an accessory parotid gland detected by aspiration biopsy with radiologic and histologic correlation in a 34‐year‐old patient. Sonographically‐guided aspiration biopsy showed cytologic features mimicking those of low‐grade mucoepidermoid carcinoma, including sheets of bland epithelial cells, dissociated histiocytoid cells with intracytoplasmic mucinous material, and spindle cells lying in a web‐like matrix. Histologic sections showed a circumscribed tumor with microcystic spaces lined by bland uniform epithelial cells and containing secretory material. The tumor cells expressed mammaglobin and BRST‐2. The cytologic features, differential diagnosis, and pitfalls are discussed. The pathologic stage was pT1N0. The patient showed no evidence of disease at 1 year follow‐up. Diagn. Cytopathol. 2014;42:49–53. © 2012 Wiley Periodicals, Inc.     

Over‐expression of the LTC4 synthase gene in mice reproduces human aspirin‐induced asthma

Background The pathogenesis of aspirin‐induced asthma (AIA) is presumed to involve the aspirin/non‐steroidal anti‐inflammatory drug (NSAID)‐induced abnormal metabolism of arachidonic acid, resulting in an increase in 5‐lipoxygenase (5‐LO) metabolites, particularly leukotriene C₄ (LTC₄). However, the role of LTC₄ in the development of AIA has yet to be conclusively demonstrated. Objective The aim of this study was to evaluate the contribution of the lipid product LTC₄ secreted by the 5‐LO pathway to the pathogenesis of AIA. Methods To evaluate antigen‐induced airway inflammation, the concentrations of T‐helper type 2 cytokine in bronchoalveolar lavage fluid (BALF) obtained from LTC₄ synthase‐transgenic (Tg) and wild‐type (WT) mice after challenge with ovalbumin were measured. Subsequently, the ex vivo and in vivo effects of the NSAID sulpyrine were investigated in these Tg and WT mice by measuring the secretion of LTC₄ from sulpyrine‐treated BAL cells and the levels of LTC₄ in BALF following challenge with sulpyrine. Finally, the sulpyrine‐induced airway response by the administration of pranlukast, an antagonist of the cysteinyl (cs)‐LT1 receptor, was analysed. Results The concentrations of IL‐4, ‐5, and ‐13 in BALF from Tg mice were significantly higher than those in WT mice. In addition, sulpyrine augmented the secretion of LTC₄ in BALF and by BAL cells in Tg mice, but not in WT mice. Additionally, the increased airway resistance induced by sulpyrine could be reduced by treatment with pranlukast. Furthermore, the secretion of LTC₄ from mast cells, eosinophils, and macrophages was increased in the allergen‐stimulated LTC₄ synthase gene Tg mice, even in the absence of sulpyrine, as well as in BAL cells after sulpyrine. Conclusion and clinical relevance The over‐expression of the LTC₄ synthase in a mouse asthma model also replicates the key features of AIA. And our study supports that cys‐LTs play a major role in the pathogenesis of AIA in patients with chronic asthma. Cite this as: H. Hirata, M. Arima, Y. Fukushima, K. Honda, K. Sugiyama, T. Tokuhisa and T. Fukuda, Clinical & Experimental Allergy, 2011 (41) 1133–1142.     

Anti‐interleukin‐6 receptor antibody prevents systemic bone mass loss via reducing the number of osteoclast precursors in bone marrow in a collagen‐induced arthritis model

Systemic bone loss is a hallmark of rheumatoid arthritis (RA). Inflammatory cytokines such as interleukin (IL)‐6 promote bone resorption by osteoclasts. Sphingosine‐1‐phosphate (S1P) controls the migration of osteoclast precursor cells (OCPs) between the blood and bone marrow, in part via S1P receptors (S1PR1 and S1PR2) expressed on the surface of OCPs. OCPs (CD11b⁺Gr‐1^low+med) isolated from bone marrow of DBA/1J mice were stimulated with IL‐6. S1P‐directed chemotaxis of OCPs was evaluated using a transwell plate. mRNA expression of S1PR1 and S1PR2 was measured. DBA/1J mice were immunized with bovine type II collagen (days 0 and 21) and anti‐mouse IL‐6 receptor antibody (MR16‐1) was administered on days 0 and/or 21. Trabecular bone volume was analysed using micro‐computed tomography. The percentage of OCPs in tibial bone marrow and S1PR1 and S1PR2 mRNA expression in OCPs were measured. IL‐6 stimulation significantly decreased S1P‐directed chemotaxis of OCPs. IL‐6 induced S1PR2 mRNA expression, but not S1PR1 mRNA expression, in OCPs. Bone volume was significantly lower in arthritic mice than in non‐arthritic control mice on day 35. Treatment of immunized mice with MR16‐1 significantly inhibited bone loss. In MR16‐1‐treated mice, the percentage of OCPs and expression of S1PR2 mRNA was each decreased compared with arthritic mice on day 14, but not on day 35. IL‐6 increased the number of OCPs in tibial bone marrow via up‐regulating S1PR2, thus playing a crucial role in systemic bone loss induced by inflammation.     

Histology of the pouch epithelium and the mammary glands during chemically induced oestrus in the brushtail possum (Trichosurus vulpecula)

Changes in the epithelium of the maternal pouch and the mammary gland of brushtail possums (Trichosurus vulpecula) were examined after animals were treated to induce ovulation with follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mares' serum gonadotrophin (PMSG) and oestradiol. The mammary glands were similar in appearance to those described in eutherian mammals and in previous studies on other marsupials. Exposure of possums to these compounds, particularly PMSG, appeared to result in changes in the mammary glands that could be associated with milk/secretion production. In contrast, the pouch epithelium had a similar histological appearance to that of epithelium from other parts of the body regardless of whether the animal was exposed to stimulants. These preliminary observations are discussed in the context of the purported role of the pouch epithelium and the mammary gland in production of secretions at oestrus and provision of immunological protection to the neonatal marsupial.     

Analysis of gene expression in PTHrP−/− mammary buds supports a role for BMP signaling and MMP2 in the initiation of ductal morphogenesis

Parathyroid hormone–related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP^?/? mammary buds, and in WT and K14‐PTHrP ventral skin at E15.5. By cross‐referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds. Developmental Dynamics 238:2713–2724, 2009. © 2009 Wiley‐Liss, Inc.     

Japanese herbal medicines shosaikoto,inchinkoto, and juzentaihoto inhibit high‐fat diet‐induced nonalcoholic steatohepatitis in db/db mice

Few studies have investigated the effects of Japanese herbal medicines on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). To the best of our knowledge, only one study has examined whether high‐fat (HF) diet‐fed db/db mice are appropriate animal models of NASH. We investigated the effects of four types of Japanese herbal medicines (shosaikoto (TJ‐9), inchinkoto (TJ‐135), juzentaihoto (TJ‐48), and keishibukuryogan (TJ‐25)) on hepatic lesions of HF diet‐fed db/db mice. Db/db mice were divided into six groups: control diet (control); HF diet (HF); and HF diet supplemented with TJ‐9, TJ‐135, TJ‐48, or TJ‐25 (TJ‐9, TJ‐135, TJ‐48, and TJ‐25, respectively). Mice were killed after 6 weeks of treatment, and biochemical and pathological analyses were performed. Mice in the HF group consistently developed histopathological features consistent with definite NASH, and marked necroinflammation occurred. Serum alanine aminotransferase levels in the TJ‐9, TJ‐135, and TJ‐48 groups were significantly improved compared with those in the HF group. With regard to liver histology, TJ‐9 and TJ‐48 significantly improved lobular inflammation, and TJ‐135 significantly improved ballooning degeneration. We have shown that HF diet‐fed db/db mice are animal models that correctly recapitulate the histopathology of human NASH and that TJ‐9, TJ‐135, and TJ‐48 inhibit necroinflammatory activity in this model.