Immunohistochemistry For α-SMA Research Articles

Retinoid and carotenoid status in serum and liver among patients at high-risk for liver cancer.

BackgroundApproximately 2.7 million Americans are chronically infected with hepatitis C virus (HCV). HCV patients with cirrhosis form the largest group of persons at high risk for hepatocellular carcinoma (HCC). Increased oxidative stress is regarded as a major mechanism of HCV-related liver disease progression. Deficiencies in retinoid and carotenoid antioxidants may represent a major modifiable risk factor for disease progression. This study aims to identify key predictors of serum antioxidant levels in patients with HCV, to examine the relationship between retinoid/carotenoid concentrations in serum and hepatic tissue, to quantify the association between systemic measures of oxidative stress and antioxidant status, and to examine the relationship between retinoids and stellate cell activation.MethodsPatients undergoing liver biopsy (n = 69) provided fasting blood, fresh tissue, urine and completed a diet history questionnaire. Serum and questionnaire data from healthy volunteers (n = 11), normal liver tissue from public repositories and patients without liver disease (n = 11) were also collected. Urinary isoprostanes, serum and tissue retinoid concentrations were obtained by UHPLC-MS-MS. Immunohistochemistry for αSMA was performed on FFPE sections and subsequently quantified via digital image analysis. Associations between urinary isoprostanes, αSMA levels, and retinoids were assessed using Spearman correlation coefficients and non-parametric tests were utilized to test differences among disease severity groups.ResultsThere was a significant inverse association between serum retinol, lycopene, and RBP4 concentrations with fibrosis stage. Serum β-carotene and lycopene were strongly associated with their respective tissue concentrations. There was a weak downward trend of tissue retinyl palmitate with increasing fibrosis stage. Tissue retinyl palmitate was inversely and significantly correlated with hepatic αSMA expression, a marker for hepatic stellate cell activation (r = −0.31, P < 0.02). Urinary isoprostanes levels were inversely correlated with serum retinol, β-carotene, and RBP4.ConclusionsA decrease in serum retinol, β-carotene, and RBP4 is associated with early stage HCV. Retinoid and carotenoid levels decline as disease progresses, and our data suggest that this decline occurs early in the disease process, even before fibrosis is apparent. Measures of oxidative stress are associated with fibrosis stage and concurrent antioxidant depletion. Vitamin A loss is accompanied by stellate cell activation in hepatic tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-016-0432-5) contains supplementary material, which is available to authorized users.

Peroxisome proliferator-activated receptor-γ agonist pioglitazone fails to attenuate renal fibrosis caused by unilateral ureteral obstruction in mice.

Renal tubulointerstitial fibrosis is the common ending of progressive renal disease. It is worth developing new ways to stop the progress of renal fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been studied to treat diabetic nephropathy, cisplatin-induced acute renal injury, ischemia reperfusion injury and adriamycin nephropathy. In this study, unilateral ureteral obstruction (UUO) was used to establish a different renal fibrosis model. PPAR? agonist pioglitazone was administrated by oral gavage and saline was used as control. At 7th and 14th day after the operation, mice were sacrificed for fibrosis test and T lymphocytes subsets test. Unexpectedly, through MASSON staining, immunohistochemistry for α-SMA, and Western blotting for a-SMA and PDGFR-β, we found that pioglitazone failed to attenuate renal fibrosis in UUO mice. However, flow cytometry showed that pioglitazone down-regulated Th1 cells, and up-regulated Th2 cells, Th17 cells and Treg cells. But the Th17/Treg ratio had no significant change by pioglitazone. Real-time PCR results showed that TGF-β and MCP-1 had no significant changes, at the same time, CD4(+) T cells associated cytokines were partially regulated by pioglitazone pretreatment. Taken together, pioglitazone failed to suppress renal fibrosis progression caused by UUO.

Abstract 2821: Joint relationship between iron and retinoids in patients at high-risk for liver cancer

Abstract Approximately 3.2 million Americans are chronically infected with Hepatitis C, forming the largest group of persons at high risk for HCC, with an annual incidence of 3-4%. Increased oxidative stress is regarded as the major mechanism of progression in chronic liver disease (CLD) and subsequent HCC. Hepatic iron overload facilitates the production of reactive oxygen species (ROS), thereby inducing liver damage and fibrosis. We have previously shown that retinoid and carotenoid depletion occurs early in disease and may represent a major modifiable risk. This study aims to determine, in CLD patients and controls, whether retinoid levels modify the association between serum iron concentrations and: (a) oxidative stress biomarkers in urine (b) tissue changes associated with hepatic stellate cell (HSC) activation, and (c) CLD progression. Patients with HCV undergoing liver biopsy (n = 69) provided fasting blood, fresh tissue, urine and completed a diet history questionnaire (DHQ). Serum and questionnaire data from healthy volunteers (n = 11), normal liver tissue from public repositories and patients without liver disease (n = 11) were also collected. Urinary isoprostanes, serum and tissue retinoid concentrations were obtained by LC-MS-MS. Serum iron, ferritin and transferrin were quantitated. Immunohistochemistry for αSMA was performed on FFPE sections and subsequently quantified via digital image analysis. Associations between urinary isoprostanes, αSMA levels, serum iron, serum and tissue retinoids were assessed using Spearman correlation coefficients and non-parametric tests were utilized to test differences among disease severity groups. There was a significant increasing trend for serum transferrin levels with progressive fibrosis, but not for serum iron and ferritin concentrations. All subjects had transferrin saturation within the normal range. Serum β-carotene and lycopene were inversely associated with serum ferritin concentrations (r = -0.29, P &amp;lt; 0.01; r = -0.29, P &amp;lt; 0.01) however, this relationship was not observed for other serum iron measurements. Serum iron measurements were not associated with hepatic retinoids/carotenoids, αSMA expression or urinary isoprostanes. Tissue retinyl palmitate was inversely and significantly correlated with hepatic αSMA expression (r = -0.3, P &amp;lt; 0.02). Serum iron overload was not evident in this population even though a depletion of serum retinoids/carotenoids is observed. The inverse correlation observed between serum ferritin and retinoids/carotenoids suggests a possible joint effect in CLD patients. Our data provide confirmation that hepatic retinoid loss is accompanied by HSC activation, a key player in fibrosis. Further evaluation of hepatic iron status is warranted to assess the relationships between iron and retinoids in HCV. Citation Format: Yachana Kataria, Ryan Deaton, Erika Enk, Milita Petrauskaite, Scott Cotler, Donald Jensen, Peter Gann. Joint relationship between iron and retinoids in patients at high-risk for liver cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2015-2821

Abstract 4021: Cyclooxygenase-2 downregulation reduces activated fibroblasts and modifies the extracellular matrix in MDA-MB-231 breast cancer xenograft

Abstract COX-2 is an important mediator of inflammation that significantly influences tumor angiogenesis, invasion and metastasis. Here, we have investigated the role of COX-2 in modifying the number of activated cancer associated fibroblasts (CAFs) and in altering the extracellular matrix (ECM) in a breast cancer model. To investigate the role of COX-2 in modulating the ECM, we used an MDA-MB-231 cell clone (Clone 13) expressing a short hairpin RNA (shRNA) to downregulate COX-2 [1]. Clone 13 cells were characterized for significantly lower basal and TPA-induced COX-2 and PGE2 expression compared to parental MDA-MB-231 cells using ELISA (PGE2), western blot (COX-2 protein) and q-RT-PCR (COX-2 mRNA). Tumors were derived from parental (n = 5) and Clone 13 (n = 6) MDA-MB-231 cells following inoculation in the mammary fat pad in SCID mice. Tumors were excised at ∼ 500 mm3 and immunohistochemically stained to quantify vessel density (CD31) and activated CAFs (α-smooth muscle actin (SMA)) in 5 μm thick formalin fixed sections. Stained sections were digitally scanned and positive staining quantified using manufacturer supplied software (Aperio Technologies, CA). Clone 13 tumors showed delayed tumor growth compared to parental MDA-MB-231 tumors. We have previously observed that collagen 1 (Col1) fiber density and fiber volume were significantly lower in COX-2 reduced Clone 13 tumors compared to parental tumors [2]. While cancer cells shape Col1 fiber patterns through the secretion of various enzymes, Col1 fiber is laid down by activated CAFs within or around the tumor. Quantification of activated CAFs by immunohistochemistry for α-SMA in the tumors, and immunoblotting for α-SMA of crude protein extracted from the tumors, revealed significantly fewer CAFs and significantly reduced levels of α-SMA protein in Clone 13 tumors compared to parental MDA-MB-231 tumors. We previously observed a significant decrease in permeability as well as reduced influx and efflux of macromolecular transport in Clone 13 tumors compared to parental tumors, but no difference in vascular volume [2]. Immunohistochemistry for CD31 staining of endothelial cells did not detect a significant difference in CD31 density between Clone 13 and parental tumors further confirming our previous observations about vascular volume. These data reveal the multi-faceted effects of COX-2 in modifying the structure and function of the ECM, and identify the ability to attract and activate fibroblasts as one mechanism by which COX-2 modifies the ECM. Acknowledgements: We thank Mr. Gary Cromwell for technical assistance. This work was supported by NIH R01CA82337 and P50 CA103175.

Pituitary tumor transforming gene as a novel regulatory factor of liver fibrosis

Pituitary tumor-transforming gene (PTTG) is involved in multiple cellular pathways. We studied the development of liver fibrosis induced by thioacetamide (TAA) in knockout (PTTG-/-) and wildtype (PTTG+/+) mice. Liver fibrosis in PTTG+/+ and PTTG-/- mice was induced by escalating dose TAA treatment (50-400mg/kg, i.p.) for 12 weeks and assessed by histochemistry, immunohistochemistry, liver hydroxyproline, serum fibrosis markers and fibrosis-related mRNA expression by real-time PCR determination. Both PTTG+/+ and PTTG-/- mice treated with TAA developed signs of fibrosis and inflammatory cell infiltration. However, histological signs of bridging fibrosis and connective tissue square morphometry were significantly attenuated in mice lacking PTTG. α-SMA immunohistochemistry revealed that hepatic stellate cell activation was markedly reduced in PTTG-/- mice compared to wildtype controls. Hepatic hydroxyproline levels were significantly lower in fibrotic PTTG-/- group. The serum TNFα and hepatic TNFα mRNA expression were significantly lower in fibrotic PTTG-/- animals, as well as hepatic TGFβ and VEGF mRNA levels compared to TAA-treated wildtype controls. Serum hyaluronate and TGFβ levels were markedly elevated in fibrotic mice of both genotypes, but were not altered by the absence of PTTG. TAA-induced fibrosis development is significantly ameliorated in PTTG-/- mice. These animals demonstrated diminished stellate cell activation, suppressed circulating serum markers of inflammation, fibrogenesis and angiogenesis. The presented findings suggest that PTTG is functionally required for hepatic fibrosis progression in an animal model of chronic liver injury. PTTG can be considered as a new important target for prevention and treatment of liver fibrosis/cirrhosis.

The Effect of Local Rho-Kinase Inhibition on Murine Wound Healing

The process of cutaneous wound healing and contraction is a complex orchestra of temporally and spatially controlled signaling moieties and pathways. Rho-kinase (ROCK) has been implicated as a key downstream effector of transforming growth factor β1 (TGF-β1) signal transduction, which ultimately coordinates α-smooth muscle actin (α-SMA)-mediated wound contraction. Previous in vitro studies of ROCK inhibition have demonstrated decreased fibroblast contraction and epithelial advancement after wounding. In vivo systemic ROCK inhibition revealed modest late wound healing attenuation in contrast to α-SMA knock-out rodents. The goal of this investigation was to explore the effect of local delivery of a ROCK inhibitor on an in vivo murine model of wound healing. Full-thickness cutaneous wounds were created in 24 white mice and were treated daily with local topical delivery of 100 μL of 10(-4) M ROCK inhibitor (Y27632) or saline control. Percent wound surface area over time and compensatory epithelial advancement was quantified. After 12 days, tissue was harvested for assessment of inflammatory cellular density, immunohistochemistry for α-SMA, and TGF-β protein expression. Wounds subjected to local ROCK inhibition were significantly larger than controls at early time points, and this effect of delayed wound contraction in ROCK-inhibited wounds persisted for 7 days after wounding (P < 0.05). The effect of ROCK inhibition on delay of wound contraction was normalized at day 12. There was no significant difference between control and ROCK-inhibited groups in the area of the nascent epithelium beyond the wound margin, wound inflammatory cellular density, and TGF-β1 expression. However, ROCK inhibition led to as statistically significant decrease in α-SMA stress fiber formation compared to controls [32.5% (3.5%) vs 17.4% (2.6%); P < 0.05]. Local delivery of a ROCK inhibitor delayed the time course of murine wound contraction in a manner similar to that previously reported in α-SMA knock-out rodents, thus demonstrating an effective method for in vivo ROCK inhibition. Accordingly, α-SMA stress fiber formation was inhibited in treated wound beds without altering local inflammation, TGF-β1 expression, or epithelial wound edge advancement.

Hepatic toll‐like receptor 4 expression is associated with portal inflammation and fibrosis in patients with NAFLD

Notwithstanding evidences implicating the lipopolysaccharides (LPS)/toll-like receptor-4 (TLR4) axis in the pathogenesis of NAFLD, there are no studies aimed to characterize hepatic TLR4 expression in NAFLD patients. We aimed to analyse hepatic TLR4 expression and to verify its relationship with disease activity/evolution in NAFLD patients. Liver tissue from 74 patients with NAFLD and 12 controls was analysed by immunohistochemistry (IHC) for TLR4, α-smooth muscle actin (α-SMA) and cytokeratin-7. IHC for α-SMA was used to evaluate activation of fibrogenic cells (hepatic stellate cells and portal/septal myofibroblasts), that for cytokeratin-7 to count hepatic progenitor cells and bile ducts/ductules, and that for CD68, in a subgroup of 27 patients, for detecting macrophages. Serum LPS-binding protein (LBP), a sensitive marker of LPS activity, was determined in 36 patients and 32 controls. As confirmed by double-labelling experiments, the highest level of TLR4 expression was observed in hepatic progenitor cells, biliary cells and portal/septal macrophages. TLR4-positive hepatic progenitor cells and bile ducts/ductules correlated with portal/interface inflammation, activity of fibrogenic cells and fibrosis (P<0.001). Also the score of TLR4 positivity of porto-septal inflammatory infiltrate correlated with number of hepatic progenitor cells and bile ducts/ductules, activity of fibrogenic cells and fibrosis (P<0.01). Serum LBP was increased in patients compared to controls (P<0.001), and correlated with portal/interface inflammation, activity of portal/septal myofibroblasts and fibrosis (all P<0.05). TLR4 expression by regenerating and inflammatory cells at the porto-septal and interface level, favoured by increased LPS activity, is associated with activation of fibrogenic cells and the degree of fibrosis.

Portal and interface chronic inflammation are associated with the progenitor cell compartment activation during NAFLD

Background and aim: During nonalcoholic fatty liver disease (NAFLD), portal and interface chronic inflammation (PCI and ICI) are strongly associated with fibrosis by activation of hepatic stellate cell (HSC)s (Brunt et al., 2009; Vespasiani-Gentilucci et al., 2014). However, the determinants of PCI and ICI observed in NAFLD remain to be elucidated. Since portal and periportal ductular reaction is related to disease progression, we aimed to investigate if PCI and ICI are associated with hepatic progenitor cell (HPC) compartment activation. Methods: Fifty-two NAFLD patients were studied. NAFLD activity score, fibrosis, PCI and ICI were histologically evaluated. HPCs, intermediate hepatobiliary cells and bile ductules/interlobular bile ducts were evaluated by immunohistochemistry for CK-7, CK-19 and EpCAM. HSC and myofibroblast (MF) activity were determined by immunohistochemistry for α-SMA. Results: PCI and ICI strongly correlated with HPC compartment activation and with the activity of MFs (p≤0.001). Lobular inflammation, ballooning and HPC compartment activation were all associated with both PCI (p<0.01) and ICI (p<0.05) by univariate analysis. In the multivariate models, HPC compartment activation was independently associated with PCI and ICI (OR 4.4, 1.7-11.5; OR 3.4, 1.5-7.9, respectively). Conclusions: During NAFLD, PCI and ICI are strongly associated with HPC compartment activation and this association is likely one determinant subtending the strong association between PCI/ICI and fibrosis.

Inhibitory effects of doxycycline on argyrophilic nucleolar organizing regions and a-smooth muscle actin expression in proliferative bovine corneal myofibroblasts in vitro

To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms. Cell culture and identification: bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type I collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10% of BSA RPMI-1640. Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. The cells staining positive for Vimentin and α-SMA indicated the presence of corneal myofibroblasts. Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10, 20, 40, 60, 80 mg/L) , a bland control group and the dexamethasone group (120 mg/L) were set up, each group had 30 cases. The argyrophilic nucleolar organizing regions (AgNOR) staining and the immunohistochemistry for α-SMA were performed when the cells were treated for 24 hours and 48 hours. The AgNOR count (Ag-c), AgNOR area (Ag-a) and the expression of α-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA, further pairwise comparisons using Independent-Samples t test. Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts. Microscopic examination and immunohistochemical staining confirmed that the cells cultured were bovine corneal myofibroblasts. The Ag-c and Ag-a of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increase. 24 h:bland control group Ag-c was 6.40 ± 0.6, 60 mg/L doxycycline group Ag-c was 2.23 ± 0.43;bland control group Ag-a was (34.80 ± 2.36) µm(2), 60 mg/L doxycycline hormone group Ag-a was (19.91 ± 2.15) µm(2). 48 h: bland control group Ag-c was 7.27 ± 0.6,60 mg/L doxycycline hormone group Ag-c was 2.80 ± 0.76, bland control group Ag-a was (36.27 ± 1.99) µm(2), 60 mg/L doxycycline group Ag-a was (13.75 ± 2.09) µm(2). The differences were statistically significant: in the same time intervention (FAg-c 24 h = 252.55, FAg-a 24 h = 202.16, P < 0.05, FAg-c 48 h = 169.38, FAg-a 48 h = 853.23, P < 0.05), in the same concentrations intervention (tAg-c = 6.98, tAg-a = 11.62, P < 0.05). And 60 mg/L of doxycycline had an obviously inhibitory action as 120 mg/L dexamethasone in the same treated hours (dexamethasone group Ag-a 24 h = 30.56 ± 3.66, dexamethasone group Ag-a 48 h = 28.35 ± 1.23 ),the differences were not statistically significant (tAg-a 24h = 1.182, P = 0.242,tAg-a 48 h = 0.21, P = 0.832). As the concentrations investigated, doxycycline can inhibit the expression of α-SMA in the bovine corneal myofibroblasts (189.90 ± 7.48, 140.20 ± 7.79, 113.20 ± 8.98, 98.00 ± 3.50, 85.50 ± 4.99), the difference was statistically significant (F = 761.79, P = 0.00). While dexamethasone had no significant role in the expression of α-SMA (bland control group was 225.10 ± 6.74, the dexamethasone group was 228.50 ± 7.12), and the statistically difference was not obvious (t = 1.096, P = 0.287). As the concentrations of doxycycline was increased from 10 mg/L to 80 mg/L, the AgNOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro. Compared with dexamethasone, doxycycline significantly suppressed the expression of α-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.

Lack of the Matricellular Protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) Attenuates Liver Fibrogenesis in Mice

IntroductionSecreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC.MethodsTwo in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC+/+) and knock-out (SPARC−/−) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC−/− and SPARC+/+ mice using Affymetrix Mouse Gene ST 1.0 array.ResultsSPARC expression was found induced in fibrotic livers of mouse and human. SPARC−/− mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC−/− mice when compared to SPARC+/+ mice; in addition, MMP-2 expression was increased in SPARC−/− mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli.ConclusionsOverall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.

Homemade lyophilized cross linking amniotic sustained-release drug membrane with anti-scarring role after filtering surgery in rabbit eyes.

To investigate the antifibrotic effect of the freeze-dried bilayered fibrin-binding amniotic membrane as a drug delivery system on glaucoma surgery in rabbit model. The aim of this study was to prepare a novel local delivery system for the sustained and controllable release of 5-Fu. Twenty-four Japanese white rabbits were randomized into three groups: the experimental group (ocular trabeculectomy in combination with 5-Fu loaded freeze-dried bilayered fibrin-binding amniotic membrane transplantation), the control group (ocular trabeculectomy in combination with 5-Fu) and the blank group (single trabeculectomy). HE staining, massion staining and immunohistochemistry for α-SMA were performed on days 7, 14, 21 and 30 following surgery. The concentration of 5-Fu in rabbit aqueous humor was examined by high performance liquid chromatography (HPLC) 3 days after the surgery. Statistical differences were noted in intraocular pressure among groups on day 7, 14, 21 and 30 following surgery. Histology further demonstrated that trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane yielded well wound healing and no scar formation and was beneficial for long term effect. HPLC showed a good slow-release effect with freeze-dried bilayered fibrin-binding amniotic membrane.

The effects of (±)-Praeruptorin A on airway inflammation, remodeling and transforming growth factor-β1/Smad signaling pathway in a murine model of allergic asthma

(±)-Praeruptorin A (PA) is a pair of coumarin enantiomers isolated from the root of Peucedanum praeruptorum Dunn (PPD), a common Chinese herbal medicine for the treatment of asthma. Considering its anti-inflammatory, anti-contractile and anti-hyperplasia activities, the effects of PA on airway inflammation and airway remodeling were investigated using a murine model of chronic asthma. Ovalbumin-sensitized BALB/c mice were challenged with ovalbumin to induce asthma every other day on eight successive weeks. PA was administered intragastrically before every ovalbumin challenge. Airway responsiveness was evaluated by a lung function analysis system 48h after the last ovalbumin challenge. The total and differential leukocytes in bronchoalveolar lavage fluid (BALF) were counted using a hemocytometer and Diff-Quick-stained smears. Lung tissue samples were used for hematoxylin and eosin, periodic acid Schiff, Masson's trichrome and α-SMA immunohistochemistry staining. Levels of cytokines in BALF, immunoglobulin (Ig) E in serum as well as expression of TGF-β1 and Smad proteins in lung tissue were measured by enzyme-linked immunosorbent assay, immunohistochemistry or western blot analysis. Compared with the model group, PA suppressed airway inflammation, airway hyperresponsive and remodeling, reduced levels of IL-4 and IL-13 in BALF, and IgE in serum, inhibited expression of TGF-β1 and pSmad2/3, up-regulated the expression of Smad7 in lung tissue, and also increased the levels of INF-γ in BALF. These results suggested that PA significantly suppressed airway inflammation and airway remodeling induced by ovalbumin challenge, and is a potential candidate for the treatment of asthma.

Identification of robust and disease-specific stromal alterations in spondyloarthritis synovitis

Backgroundand objectivesThe cellular and molecular pathways driving synovial inflammation and stromal remodeling in spondyloarthritis (SpA) remain largely unknown. As SpA and rheumatoid arthritis (RA) show clearly distinct patterns of structural...

Deletion of clock gene Per2 exacerbates cholestatic liver injury and fibrosis in mice

The Period 2 (Per2) gene is an important component of the circadian system and is thought to modulate many physiological and pathological processes in mammals. In the previous study, we have disclosed the protective role of Per2 against carbon tetrachloride induced liver injury and fibrosis. Here we further assess the effect of Per2 deficiency on cholestatic hepatic injury and fibrosis. Cholestasis was induced by bile duct ligation (BDL) for 10 days in wild-type (WT) and Per2(-/-) mice. Masson trichrome staining and analysis of α-SMA immunohistochemistry were performed to show the collagen accumulation and the HSC activation, respectively. The mRNA levels of fibrosis-related genes were monitored by quantitative real-time PCR. Following BDL, livers from Per2(-/-) mice exhibited markedly increased extent of bile infarct and extracellular matrix (ECM) deposition compared with WT mice. Furthermore, the expressions of fibrosis-related genes like TNF-α, TGF-β1, Col1α1 and TIMP-1 were dramatically elevated in Per2(-/-) cholestatic liver. Our observations indicated that clock gene Per2 plays a protective role in mediating liver injury and fibrosis during cholestasis.

Study of self-made freeze-dried bilayered fibrin-binding amniotic membrane in ocular trabeculectomy in rabbits.

To investigate the antifibrotic effect of freeze-dried bilayered fibrin-binding amniotic membrane on trabeculectomy in a rabbit model. Twenty-four Japanese white rabbits were randomized into three groups: the experimental group (ocular trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane transplantation), the control group (ocular trabeculectomy in combination with natural bilayered fibrin-binding amniotic membrane) and the blank group (single trabeculectomy). Clinical observation, hematoxylin-eosin staining, Massion staining, real-time PCR and immunohistochemistry for α-SMA were performed on days 7, 14, 21 and 30 following surgery. Statistical differences were noted in survival analysis and intraocular pressure (IOP) among groups on days 7, 14, 21 and 30 following surgery. Histology, immunohistochemistry and real-time PCR further demonstrated that trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane resulted in good wound healing and no scar formation. Self-made freeze-dried bilayered fibrin-binding amniotic membrane may inhibit the formation of scarring in glaucoma after trabeculectomy.

Toll-like receptor-4 expression by hepatic progenitor cells and biliary epithelial cells in HCV-related chronic liver desease

Background . Toll-like receptor-4 (TLR4) is a transmembrane pattern recognition receptor that plays a key role in innate immunity by triggering inflammatory responses to Gram negative bacteria lipopolysaccharide (LPS) (1). Since liver is the main clearance organ for LPS, which is excreted in large amounts in the bile (2), it is not surprising that TLR4 has been involved in the pathogenesis of most liver diseases (3). Numerous evidences suggest a role for TLR4 in the pathogenesis of chronic hepatitis C virus (HCV) infection (4) and hepatic fibrosis (5), but the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. Aim and methods. We aimed to evaluate, by means of immunohistochemistry (IHC) and real-time polymerase chain reaction (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Sixty-one patients with chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by IHC for TLR4, α–smooth muscle actin (αSMA) and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. A score of activation of portal/septal myofibroblasts and lobular hepatic stellate cells (HSCs) was evaluated by IHC for α-SMA, whereas IHC for CK-7 was analysed in order to count hepatic progenitor cells (HPCs), interlobular bile ducts and intermediate hepatocytes. Results . The parenchimal elements responsible for the highest TLR4 level of expression were HPCs and biliary epithelial cells (BECs) of interlobular bile ducts in the infected group. Double-labeling experiments with anti-TLR4, anti-CK7 and anti-CD133 confirmed this finding. TLR4-positive HPCs and interlobular bile ducts were significantly correlated with the stage of liver disease (p<0.001), the grade of inflammation (p<0.001), and with the activity of portal/septal myofibroblasts (p<0.001). Rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (p<0.001). TLR4 expression positively correlated with fibrosis (p<0.05) and inflammation (p<0.05). Conclusions . The expression of TLR4 in HPCs and BECs in HCV-related liver damage significantly correlates with inflammation, activation of portal/septal myofibroblasts and fibrosis. 1) Beutler. Nature 2004;430:257-63; 2) Van Bossuyt et al. J Hepatol 1988;7:325-37; 3) Seki et al. Hepatology 2008;48:322-35; 4) Machida et al. J Virol 2006;80:866-74. 5) Seki et al. Nat Med 2007;13:1324-32.

TNF-α plays a role in hepatocyte apoptosis in Niemann-Pick type C liver disease

Niemann-Pick type C (NPC) is a fatal autosomal recessive lysosomal storage disease clinically characterized by neurodegeneration and liver disease. Heterogeneous mutations in the NPC1 and NPC2 genes cause impaired egress of free cholesterol from lysosomes, leading to accumulation of cholesterol and glycosphingolipids. Key features of NPC liver disease include hepatic apoptosis, inflammation, and fibrosis. It is unclear what signaling events regulate these disease processes in NPC. We hypothesize that tumor necrosis factor alpha (TNF-alpha), which is involved in both proinflammatory and apoptotic signaling cascades, is a key mediator of inflammation, apoptosis, and fibrosis in NPC liver disease. In this study, we evaluated the role of TNF-alpha signaling in NPC liver disease by utilizing NPC1-specific antisense oligonucleotides to knock down NPC1 expression in control and TNF-alpha knockout mice. In the absence of TNF-alpha, NPC1 knockdown produced liver disease with significantly less inflammation, apoptosis, and fibrosis.

Polypoid Uterine Leiomyosarcomas in a Sheep

Leiomyosarcoma was diagnosed in the uterus surgically removed from a 3-year-old pet Suffolk ewe with a history of bleeding from the vulva, spontaneous lactation, and nursing behavior. The uterus contained multiple well-circumscribed, soft, intraluminal polypoid masses of variable sizes (0.5-4 cm). The masses were red, with white, smooth, and glistening cut surfaces. Histologically they comprised variably dense sheets of moderately pleomorphic, plump spindle cells embedded in richly vascularized stroma. The mitotic index was usually low (0-1/high-power field), but in some polyps there were up to 10 mitoses/high-power field. Neoplastic cells stained positive for alpha smooth muscle actin (alpha-SMA) by immunohistochemistry. Ultrastructural features of neoplastic cells included the presence of basal lamina, scant microfilaments, contracted nuclei with blunt ends, and flat intercellular junctions. Uterine leiomyosarcoma was diagnosed based on cellular morphology and atypia and positive immunohistochemistry for alpha-SMA.

Glomerular Epithelial Cell Injury Accelerates the Progression of Antibody-Induced Mesangial Proliferative Nephritis

Background: Anti-Thy-1.1 antibody induces mesangioproliferative glomerulonephritis; however, the mesangial lesion is spontaneously recovered to the normal feature. Glomerular epithelial cells (GECs) play a crucial role in the glomerular function. Very little is known about the involvement of GECs in this disease model. This study is designed to investigate whether GEC injury prolongs the mesangial lesion. Methods: The effects of GEC damage on mesangioproliferative nephritis were studied with combined treatment using puromycin aminonucleoside (PAN) and monoclonal antibody (MAb) against rat mesangial Thy-1.1. Urinary protein, BUN, Pcr and Ccr were measured. To clarify the underlying mechanisms, morphological study and immunohistochemistry for α-SMA, FGF-2 and PCNA were carried out. Results: Simultaneous administration of PAN plus MAb induced progressive mesangioproliferative nephritis compared to PAN or MAb alone. Rats with combined treatment displayed renal dysfunction with massive proteinuria. Morphological studies showed that the glomeruli in combined group had features resembling those of progressive mesangial proliferative glomerulonephritis in humans. Morphologic lesions of GECs in acute nephritic phase were severer than those in other groups. Immunohistochemistry revealed that glomeruli of combined treatment exhibited persistent overexpression of α-SMA and FGF-2. Conclusions: Simultaneous dysfunction of GECs and mesangial cells can lead to persistent glomerular perturbations with prolonged phenotypic change of mesangial cells, resulting in end-stage renal deficiency. GEC damage during the acute nephritic phase contributes to the progression of irreversible renal disease.

Changes in cellular motility and cytoskeletal actin in fibroblasts from patients with chronic venous insufficiency and in neonatal fibroblasts in the presence of chronic wound fluid

Purpose: Fibroblasts (fb) play an important role in wound healing involving motility, contraction, fibrosis, and expression of the cytoskeletal protein α-smooth muscle actin (α-sma). Patients with chronic venous insufficiency (CVI) are known to have dermal changes and impaired venous ulcer healing. To investigate whether these dermal-fb have an altered ability to migrate and whether chronic wound fluid from venous ulcers alters neonatal fb motility, we examined cell migration and α-sma. Methods: Fibroblasts were cultured from the margin of venous ulcers (du-fb, n = 4, CEAP 6), from patients with venous reflux without ulcer (dr-fb, n = 5, CEAP 2), and from the ipsilateral thigh of the same patients with (pu-fb) and without (pr-fb) ulcer, respectively. The abbreviations used are p and d, which represent proximal and distal, respectively; u and r represent ulcer and reflux, respectively. Neonatal foreskin fibroblasts (nf-fb) were exposed to chronic venous ulcer wound fluid (CVUWF, 300 μg protein/mL, n = 3) or bovine serum albumin (BSA, control). Fibroblast motility was determined by means of time-lapse photo-images, and the rate (micrometer per hour) was calculated. Immunohistochemistry for α-sma was analyzed with confocal laser microscopy. Results: The rate of motility (micrometer per hour ± SEM) was decreased for both du-fb (11.4 ± 0.7) and dr-fb (13.8 ± 0.6), when compared with pu-fb (21.9 ± 0.9) and pr-fb (24.7 ± 1.1), respectively. The motility rate for nf-fb was lower in CVUWF (24.7 ± 2.0) than in BSA (37.1 ± 6.7). An elevated level of microfilament bundles of α-sma for both du-fb and dr-fb, compared with those of pu-fb and pr-fb, and also in nf-fb treated with CVUWF was demonstrated by means of immunohistochemistry. Conclusion: These data demonstrate a reduced motility in the dermal fb of patients with CVI. Patients with reflux disease without ulcer are predisposed to these changes. Furthermore, it appears that CVUWF causes changes in motility and α-sma expression in nf-fb as demonstrated in du-fb. These findings suggest that reduced motility and CVUWF, representing the microenvironment of venous ulcers, play a significant role in impaired wound healing. (J Vasc Surg 2001;33:1233-41.)