Renoprotection From Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age‐matched OVE diabetic mice to produce bi‐transgenic OVE‐JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE‐JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day‐old OVE‐JTMT mice, blood glucose and HbA1c were indistinguishable from age‐matched OVE mice. However, endothelial‐specific MT overexpression in OVE‐JTMT mice mitigated several DN complications including significantly increased non‐fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE‐JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560–576, 2017. © 2016 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Ultrastructural morphometry of capillary basement membrane thickness in normal and transgenic diabetic mice

Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300-350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (+87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues.     

Impaired baroreflex control of renal sympathetic nerve activity in type 1 diabetic mice (OVE26)

To investigate the effects of chronic diabetes on baroreflex control of renal sympathetic nerve activity (RSNA), OVE26 diabetic (transgenic mouse line which develops hyperglycemia within the first 3 weeks after birth) and FVB control mice 5–6 months old were studied. Under anesthesia, RSNA in response to sodium nitroprusside (SNP)– and phenylephrine (PE)-induced mean arterial pressure changes (ΔMAP) were measured. Baroreflex-induced inhibition of RSNA during PE infusion was characterized using the sigmoid logistic function curve. Baroreflex-induced excitation of RSNA during SNP infusion was characterized by the RSNA vs. ΔMAP relationship. Mean arterial pressure (MAP) responses to the left aortic depressor nerve (ADN) stimulation were evaluated. Compared to FVB control, we found in OVE26 mice that (1) RSNA in response to MAP increase during PE infusion was dramatically reduced, as characterized by the maximal gain of the RSNA sigmoid logistic function curve (FVB: −20.0±5.1; OVE26: −7.6±0.8%/mm Hg, P<0.05); (2) RSNA in response to MAP decrease during SNP infusion was also attenuated (P<0.05); (3) MAP responses to ADN stimulation were reduced (P<0.05). We concluded that chronic diabetes impairs baroreflex control of RSNA in OVE26 diabetic mice. The use of the transgenic OVE26 diabetic mouse model may underlie a foundation for the further understanding of diabetes-induced autonomic neuropathy.     

Role of metallothioneins as danger signals in the pathogenesis of colitis

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress‐responsive proteins with immune‐modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti‐MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT‐positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)‐induced colitis compared to congenic wild‐type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS‐colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80‐positive macrophage infiltration in DSS‐ and trinitrobenzene sulphonic acid‐colitis, with a tendency towards a better outcome. Whole‐body single‐photon emission computer tomography of mice injected with radioactive anti‐MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti‐MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT‐positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Functional changes in baroreceptor afferent, central and efferent components of the baroreflex circuitry in type 1 diabetic mice (OVE26)

Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes mellitus induced functional changes of neural components at multiple sites within the baroreflex arc. Type 1 diabetic mice (OVE26) and FVB control mice were anesthetized. Baroreflex-mediated HR responses to sodium nitroprusside- (SNP) and phenylephrine- (PE) induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor function was characterized by measuring the percent (%) change of baseline integrated aortic depressor nerve activity (Int ADNA) in response to SNP- and PE-induced MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve (ADN) and the right vagus nerve were assessed. Compared with FVB control mice, we found in OVE26 mice that (1) baroreflex-mediated bradycardia and tachycardia were significantly reduced. (2) The baroreceptor afferent function in response to MAP increase did not differ, as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was significantly attenuated. (3) The maximum amplitude of bradycardic responses to right vagal efferent stimulation was augmented. (4) In contrast, the maximum amplitude of bradycardic responses to left ADN stimulation was decreased. Since Int ADNA was preserved in response to MAP increase and HR responses to vagal efferent stimulation were augmented, we conclude that a deficit of the central mediation of baroreflex HR contributes to the overall attenuation of baroreflex sensitivity in OVE26 mice. The successful conduction of physiological experiments on the ADN in OVE26 mice may provide a foundation for the understanding of cellular and molecular mechanisms of diabetes-induced cardiac neuropathy.     

Reactive oxygen species promote angiogenesis in the infarcted rat heart

The purpose of this study was to determine whether reactive oxygen species (ROS) promote cardiac angiogenesis following myocardial infarction (MI) and contribute to cardiac repair. Rats with MI were treated with or without antioxidants, tempol and apocynin. Hearts of these rats were collected at days 2, 4, 7 and 14 post‐MI. We examined the spatial and temporal relationship between oxidative stress and angiogenesis as well as the potential regulation of ROS in cardiac angiogenesis. We found: (i) following MI, gp91^phox, a subunit of NADPH oxidase, a key enzyme for ROS production, was significantly increased in the border zone at day 2, followed by the infarcted myocardium at day 4, peaked at day 7 and declined at day 14, while superoxide dismutase was significantly reduced; (ii) malondialdehyde, a marker of oxidative stress, was significantly increased in the infarcted myocardium at day 7; (iii) pre‐existing blood vessels in the infarcted myocardium underwent necrosis post‐MI, whereas newly formed vessels appeared at the border zone at day 4, and then extended into the infarcted myocardium, where microvascular density peaked at day 7 and (iv) antioxidant treatment significantly reduced microvascular density in the infarcted myocardium at day 7. These observations suggest that following MI, angiogenesis is mostly active in the infarcted myocardium in the first week, which is temporally and spatially coincident with enhanced ROS. Suppression of angiogenesis by antioxidants indicates that ROS promote angiogenesis in the infarcted myocardium and contribute to cardiac repair. Further studies are required to determine the mechanisms responsible for ROS‐mediated cardiac angiogenesis.     

Antiapoptotic effect and inhibition of ischemia/reperfusion-induced myocardial injury in metallothionein-overexpressing transgenic mice

Previous studies using a cardiac-specific metallothionein (MT)-overexpressing transgenic mouse model have demonstrated that MT inhibits ischemia/reperfusion-induced myocardial injury. The present study was undertaken to test the hypothesis that the MT inhibition is associated with suppression of apoptosis mediated by mitochondrial cytochrome c release and caspase-3 activation. An open-chest coronary artery occlusion and reperfusion procedure to produce ischemia/reperfusion-induced left ventricle infarction was used in MT-overexpressing transgenic mice and non-transgenic controls. After 30 minutes of ischemia, the left ventricle was reperfused to allow blood flow through the previously occluded coronary artery bed. Myocardial infarction produced after reperfusion for 4 hours was significantly reduced in the MT transgenic mice. This inhibition correlated with the antiapoptotic effect of MT, as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay, mitochondrial cytochrome c release and caspase-3 activation. Ischemia/reperfusion-induced lipid peroxidation was also significantly inhibited in the MT-transgenic heart. Dimethylsulfoxide, a chemical scavenger for reactive oxygen species, was used to confirm the antioxidant effect of MT and found to suppress myocardial infarction and lipid peroxidation just as MT did. This study thus demonstrates that MT suppresses ischemia/reperfusion-induced myocardial apoptosis through, at least in part, the inhibition of cytochrome c-mediated caspase-3 activation pathway. The antiapoptotic effect of MT likely results from the suppression of oxidative stress and correlates with the inhibition of myocardial infarction.     

Over‐expression of metallothionein predicts chemoresistance in breast cancer

Metallothionein (MT) plays a role in fundamental cellular processes such as proliferation, apoptosis and differentiation. We examined MT expression in women with invasive breast ductal carcinoma who underwent mastectomy/lumpectomy without neo‐adjuvant treatment. We showed that MT was over‐expressed in 87.9% of breast cancer tissues examined, with the mean percentage of positive cells at 30%. There were two patterns of MT expression: predominantly cytoplasmic in 75.9% and nuclear in 24.1% of MT‐positive cases. Higher MT scores were associated with poorer histological grade (p = 0.009) but were independent of age, tumour size and oestrogen receptor status. For patients who were treated with adjuvant chemotherapy (cyclophosphamide/methotrexate/5 fluorouracil‐ or doxorubicin‐based regimes), those with high MT expression had a significantly lower recurrence‐free survival (p = 0.048), suggesting a role of MT in predicting disease recurrence. Down‐regulation of MT in MCF‐7 cells by silencing the MT‐2A gene (the most abundantly expressed of the 10 known functional MT isoforms) increased chemosensitivity of the cells to doxorubicin. To examine the mechanisms underlying these clinical data, we used siRNAs to decrease MT‐2A mRNA expression and protein expression. In MT down‐regulated cells challenged with the IC₅₀ concentration of doxorubicin, we observed a significant reduction in cell viability. Cell cycle analysis also revealed a corresponding increase in apoptosis in the MT down‐regulated cells following doxorubicin exposure, showing that down‐regulation of MT increased susceptibility to doxorubicin cytotoxicity. The data suggest that MT could be a potential marker of chemoresistance and a molecular therapeutic target. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Hypoinnervation is an early event in experimental myocardial remodelling induced by pressure overload

Structural and functional remodelling of cardiomyocytes, capillaries and cardiac innervation occurs in left ventricular hypertrophy (LVH) and heart failure (HF) in response to pressure‐induced overload. However, the onset, time course and the extent of these morphological alterations remain controversial. In the present study, we tested the hypothesis that the progression from hypertrophy to HF is accompanied by changes in the innervation (hyper‐ or hypoinnervation). Left ventricles of wild‐type murine hearts subjected to pressure overload‐induced hypertrophy by transverse aortic constriction (TAC) were investigated by morphometric and design‐based stereological methods at 1 and 4 weeks after TAC and compared with sham‐operated mice. Mice developed compensated LVH at 1 week and typical signs of HF, such as left ventricular dilation, reduced ejection fraction and increased relative lung weight at 4 weeks post‐TAC. At the (sub‐)cellular level, cardiomyocyte myofibrillar and mitochondrial volume increased progressively in response to mechanical overload. The total length of capillaries was not significantly increased after TAC, indicating a misrelationship between the cardiomyocyte and the capillary compartment. The myocardial innervation decreased already during the development of LVH and did not significantly decrease further during the progression to HF. In conclusion, our study suggests that early loss of myocardial innervation density and increased heterogeneity occur during pressure overload‐induced hypertrophy, and that these changes appear to be independent of cardiomyocyte and capillary remodelling.     

Tissue‐ and column‐specific measurements from multi‐parameter mapping of the human cervical spinal cord at 3 T

The aim of this study was to quantify a range of MR parameters [apparent proton density, longitudinal relaxation time T₁, magnetisation transfer (MT) ratio, MT saturation (which represents the additional percentage MT saturation of the longitudinal magnetisation caused by a single MT pulse) and apparent transverse relaxation rate R₂*] in the white matter columns and grey matter of the healthy cervical spinal cord. The cervical cords of 13 healthy volunteers were scanned at 3 T using a protocol optimised for multi‐parameter mapping. Intra‐subject co‐registration was performed using linear registration, and tissue‐ and column‐specific parameter values were calculated. Cervical cord parameter values measured from levels C1–C5 in 13 subjects are: apparent proton density, 4822 ± 718 a.u.; MT ratio, 40.4 ± 1.53 p.u.; MT saturation, 1.40 ± 0.12 p.u.; T₁ = 1848 ± 143 ms; R₂* = 22.6 ± 1.53 s^–1. Inter‐subject coefficients of variation were low in both the cervical cord and tissue‐ and column‐specific measurements, illustrating the potential of this method for the investigation of changes in these parameters caused by pathology. In summary, an optimised cervical cord multi‐parameter mapping protocol was developed, enabling tissue‐ and column‐specific measurements to be made. This technique has the potential to provide insight into the pathological processes occurring in the cervical cord affected by neurological disorders. © 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.     

Interleukin‐37 ameliorates myocardial ischaemia/reperfusion injury in mice

Innate immune and inflammatory responses are involved in myocardial ischaemia/reperfusion (I/R) injury. Interleukin (IL)‐37 is a newly identified member of the IL‐1 family, and functions as a fundamental inhibitor of innate immunity and inflammation. However, its role in myocardial I/R injury remains unknown. I/R or sham operations were performed on male C57BL/6J mice. I/R mice received an injection of recombinant human IL‐37 or vehicle, immediately before reperfusion. Compared with vehicle treatment, mice treated with IL‐37 showed an obvious amelioration of the I/R injury, as demonstrated by reduced infarct size, decreased cardiac troponin T level and improved cardiac function. This protective effect was associated with the ability of IL‐37 to suppress production of proinflammatory cytokines, chemokines and neutrophil infiltration, which together contributed to a decrease in cardiomyocyte apoptosis and reactive oxygen species (ROS) generation. In addition, we found that IL‐37 inhibited the up‐regulation of Toll‐like receptor (TLR)‐4 expression and nuclear factor kappa B (NF‐kB) activation after I/R, while increasing the anti‐inflammatory IL‐10 level. Moreover, the administration of anti‐IL‐10R antibody abolished the protective effects of IL‐37 in I/R injury. In‐vitro experiments further demonstrated that IL‐37 protected cardiomyocytes from apoptosis under I/R condition, and suppressed the migration ability of neutrophils towards the chemokine LIX. In conclusion, IL‐37 plays a protective role against mouse myocardial I/R injury, offering a promising therapeutic medium for myocardial I/R injury.     

Interleukin‐2 treatment reverses effects of cAMP‐responsive element modulator α‐over‐expressing T cells in autoimmune‐prone mice

Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self‐tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)‐2 critically maintains homeostasis of regulatory T cells (T_reg) and effector T cells in the periphery. Previously, we identified the cAMP‐responsive element modulator α (CREMα) as a major factor responsible for decreased IL‐2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over‐expresses CREMα in T cells (CD2CREMαtg), we provided in‐vivo evidence that CREMα indeed suppresses IL‐2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas^–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas^–/– mice. This was accompanied by a massive expansion of double‐negative (DN) T cells, enhanced numbers of interferon (IFN)‐γ‐producing T cells and reduced percentages of T_regs. Treatment of FVB/Fas^–/–CD2CREMαtg mice with IL‐2 restored the percentage of T_regs and reversed increased IFN‐γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL‐2 in vivo .     

Macrophage‐specific NOX2 contributes to the development of lung emphysema through modulation of SIRT1/MMP‐9 pathways

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS‐producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase‐induced emphysema using NOX2‐ and NOX1‐deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2‐positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase‐induced alveolar airspace enlargement and elastin degradation were prevented in NOX2‐deficient mice, but not in NOX1‐deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase‐9 (MMP‐9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage‐restricted functional NOX2 in elastase‐induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68‐positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase‐induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage‐specific NOX2 participates in elastase‐induced emphysema through the involvement of SIRT1/MMP‐9 pathways in mice. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

细胞间旁分泌在牵张刺激心肌细胞肥大中的作用

目的:探讨细胞间旁分泌在牵张刺激心肌细胞肥大中的作用。方法:用牵张刺激培养成纤维细胞、微血管内皮细胞的条件培养基,刺激心肌细胞,观察[³H]-Leu掺入的变化。结果:牵张培养成纤维细胞、微血管内皮细胞的条件培养基均可显著刺激心肌细胞[³H]-Leu掺入。条件培养基中血管紧张素II、内皮素含量显著升高。用特异的血管紧张素II、内皮素受体拮抗剂可明显抑制心肌细胞[³H]-Leu掺入率,联合应用时抑制率可达80%以上。结论:牵张刺激可通过诱导心肌间质细胞内分泌活化启动心肌肥大反应。     

Non‐structural 5A protein of hepatitis C virus induces a range of liver pathology in transgenic mice

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). However, the mechanism of HCV pathogenesis is not well understood. Our previous in vitro studies suggested that non‐structural 5A (NS5A) protein may play an important role in liver pathogenesis. To elucidate the mechanism of HCV‐induced liver pathogenesis, we investigated the histopathological changes of liver in transgenic mice harbouring the NS5A gene. We generated transgenic mice harbouring HCV NS5A gene under the control of hepatitis B virus (HBV) enhancer. Pathological changes were analysed by immunohistochemical staining and western blot analysis. Lipid composition and reactive oxygen species (ROS) production in NS5A transgenic mice were analysed. HCV NS5A transgenic mice developed extraordinary steatosis over 6 months old and induced HCC in some mice. NS5A was co‐localized with apolipoprotein A‐I in fatty hepatocytes. In addition, the extraordinarily high levels of ROS, NF‐κB and STAT3 were detected in hepatocytes of NS5A transgenic mice. These data suggest that NS5A, independent of other HCV viral proteins, may play an important role in the development of hepatic pathologies, including steatosis and hepatoceullular carcinoma in transgenic mice. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Effect of natural full weight‐bearing during standing on the rotation of the first metatarsal bone

To evaluate the rotational change in the first metatarsal bone (1MT) of the foot during natural standing using an upright computed tomography (CT) scanner with 320‐detector rows. A total of 52 feet of 28 asymptomatic subjects (aged 23–39 years) were evaluated in the natural standing position with or without weight‐bearing. A foot pressure plate was used to determine the non‐weight‐bearing (NWB) or single leg full‐weight‐bearing (s‐FWB) conditions. CT examinations were performed using a noise index of 15 for a slice thickness of 5 mm, rotation speed of 0.5 sec, and slice thickness of 0.5 mm. The rotation of the 1MT was measured on the coronal CT image, which cut the sesamoids' bellies in the frontal slide of the first metatarsal and sesamoids perpendicular to the longitudinal bisection of the third metatarsal, and compared between the weight‐bearing conditions. Intra‐ and inter‐observer reliabilities of the rotation angle were also evaluated. The intra‐ and inter‐observer correlation coefficients were 0.961 and 0.934, respectively. The 1MT pronation angle was significantly greater in the s‐FWB condition than in the NWB condition (15.2° ± 5.4° vs. 12.5° ± 5.3°, P < 0.01). No sex difference was found in the magnitude of the 1MT pronation angle as a result of weight‐bearing. This study first demonstrated that pronation of 1MT occurs due to natural full‐weight‐bearing in asymptomatic feet. The 1MT's rotational movement under weight‐bearing conditions may relate to the onset and pathogenesis of the hallux valgus. Clin. Anat. 32:715–721, 2019. © 2019 Wiley Periodicals, Inc.     

Neurokinin-1 receptor activation induces reactive oxygen species and epithelial damage in allergic airway inflammation

Background An induction of reactive oxygen species (ROS) is characteristic for inflammation but the exact pathways have not been identified for allergic airway diseases so far. Objective The aim of this study was to characterize the role of the tachykinin NK‐1 receptor on ROS production during allergen challenge and subsequent inflammation and remodelling. Methods Precision‐cut lung slices of ovalbumin (OVA)‐sensitized mice were cultivated and ROS‐generation in response to OVA challenge (10 μg/mL) was examined by the 2′,7′‐dichloroflourescein‐diacetate method. Long‐term ROS effects on epithelial proliferation were investigated by 5‐bromo‐2′‐deoxyuridine incorporation (72 h). In vivo, the results were validated in OVA‐sensitized animals which were treated intra‐nasally with either placebo, the tachykinin neurokinin 1 (NK‐1) receptor antagonist SR 140333 or the anti‐oxidant N‐acetylcystein (NAC) before allergen challenge. Inflammatory infiltration and remodelling were assessed 48 h after allergen challenge. Results ROS generation was increased by 3.7‐fold, which was inhibited by SR 140333. [Sar⁹,Met¹¹(O₂)]‐Substance P (5 nm ) caused a tachykinin NK‐1 receptor‐dependent fourfold increase in ROS generation. Epithelial proliferation was decreased by 68% by incubation with [Sar⁹,Met¹¹(O₂)]‐SP over 72 h. In‐vivo, treatment with SR 140333 and NAC reduced epithelial damage (91.4% and 76.8% vs. placebo, respectively, P<0.01) and goblet cell hyperplasia (67.4% and 50.1% vs. placebo, respectively, P<0.05), and decreased inflammatory cell influx (65.3% and 45.3% vs. placebo, respectively, P<0.01). Conclusion Allergen challenge induces ROS in a tachykinin NK‐1 receptor‐dependent manner. Inhibition of the tachykinin NK‐1 receptor reduces epithelial damage and subsequent remodelling in vivo. Therefore, patients may possibly benefit from treatment regime that includes radical scavengers or tachykinin NK‐1 receptor antagonists.     

Metallothionein expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patient survival

Theocharis S, Klijanienko J, Giaginis C, Rodriguez J, Jouffroy T, Girod A, Point D, Tsourouflis G & Sastre‐Garau X
(2011) Histopathology 59 , 514–525 Metallothionein expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patient survival Aims: Metallothionein (MT) has been implicated in several aspects of cancer pathobiology, such as differentiation, proliferation, apoptosis and invasion. The aim of the present study was to evaluate the clinical significance of MT expression in mobile tongue squamous cell carcinoma (SCC). Methods and results: MT protein expression was assessed immunohistochemically on 49 mobile tongue SCC specimens, and was analysed in relation to clinicopathological characteristics, and overall and disease‐free patient survival. All of the examined mobile tongue SCC cases showed MT positivity in tumour cells; however, neither MT overexpression nor staining intensity was significantly associated with clinicopathological parameters. MT cellular distribution was significantly associated with histopathological grade of differentiation and depth of invasion (P = 0.0188 and P = 0.0484, respectively). MT staining intensity was identified as a significant predictor of overall patient survival at both univariate (P = 0.0377) and multivariate (P = 0.0472) levels. Twenty‐seven (55.10%) of the examined SCC cases showed MT positivity in squamous tongue epithelium adjacent to the tumour, the MT positivity being correlated with depth of invasion (P = 0.0281), vascular invasion (P = 0.0194), and the existence of lymph node metastases (P = 0.0194). Conclusions: MT may be implicated in the development and progression of mobile tongue SCC and could be considered as a useful clinical marker for patient management and prognosis.