Lipopolysaccharide-Induced Lung Injury Is Independent of Serum Vitamin D Concentration

Vitamin D deficiency is increasing in incidence around the world. Vitamin D, a fat-soluble vitamin, has documented effects on the innate and adaptive immune system, including macrophage and T regulatory (Treg) cell function. Since Treg cells are important in acute lung injury resolution, we hypothesized that vitamin D deficiency increases the severity of injury and delays injury resolution in lipopolysaccharide (LPS) induced acute lung injury. Vitamin D deficient mice were generated, using C57BL/6 mice, through diet modification and limited exposure to ultraviolet light. At 8 weeks of age, vitamin D deficient and sufficient mice received 2.5 g/kg of LPS or saline intratracheal. At 1 day, 3 days and 10 days, mice were anesthetized and lung elastance measured. Mice were euthanized and bronchoalveolar lavage fluid, lungs and serum were collected. Ex vivo neutrophil chemotaxis was evaluated, using neutrophils from vitamin D sufficient and deficient mice exposed to the chemoattractants, KC/CXCL1 and C5a, and to bronchoalveolar lavage fluid from LPS-exposed mice. We found no difference in the degree of lung injury. Leukocytes were mildly decreased in the bronchoalveolar fluid of vitamin D deficient mice at 1 day. Ex-vivo, neutrophils from vitamin D deficient mice showed impaired chemotaxis to KC but not to C5a. Vitamin D deficiency modestly impairs neutrophil chemotaxis; however, it does not affect lung injury or its resolution in an LPS model of acute lung injury.     

Renal IL-18 Production Is Macrophage Independent During Obstructive Injury

Background

Interleukin 18 (IL-18) is a pro-inflammatory cytokine that mediates fibrotic renal injury during obstruction. Macrophages are a well-known source of IL-18; however, renal tubular epithelial cells are also a potential source of this cytokine. We hypothesized that IL-18 is predominantly a renal tubular cell product and is produced during renal obstruction independent of macrophage infiltration.

Methods

To study this, male C57BL6 mice were subjected to unilateral ureteral obstruction (UUO) vs. sham operation in the presence or absence of macrophage depletion (liposomal clodronate (1 ml/100 g body weight i.v.)). The animals were sacrificed 1 week after surgery and renal cortical tissue harvested. Tissue levels of active IL-18 (ELISA), IL-18 receptor mRNA expression (real time PCR), and active caspase-1 expression (western blot) were measured. The cellular localization of IL-18 and IL-18R was assessed using dual labeling immunofluorescent staining (IFS).

Results

Immunohistochemical staining of renal tissue sections confirmed macrophage depletion by liposomal clodronate. IL-18 production, IL-18R expression, and active caspase 1 expression were elevated in response to renal obstruction and did not decline to a significant degree in the presence of macrophage depletion. Obstruction-induced IL-18 and IL-18R production localized predominantly to tubular epithelial cells (TEC) during obstruction despite macrophage depletion.

Conclusion

These results demonstrate that renal tubular epithelial cells are the primary source of IL-18 production during obstructive injury, and that tubular cell production of IL-18 occurs independent of macrophage infiltration.     

Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus

The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.     

Sake Yeast Suppresses Acute Alcohol-Induced Liver Injury in Mice

Brewer’s and baker’s yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methonine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.     

Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl₄)-induced acute and chronic liver injury in Rgs5^LacZ/LacZ reporter mice. Furthermore, CCl₄ treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl₄ and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.     

The Liver Cells in Acute Starvation and Refeeding

A series of 52 young male white laboratory mice were starved for a maximum of five days, and 45 normally fed animals were used as controls. A group of experimental animals and a corresponding control group were sacrificed after two, three, and four days' starvation and after six, 12 and 17 days' refeeding. Even after two days the animals had lost weight, and the liver cells had diminished highly significantly. A return to the initial values was noted after 12 days' refeeding. In the experimental animals a strong negative correlation was noted between the cell count per field and body weight (r =–0.712, b =–7.520), while no such correlation was observed for the controls. The volume of the liver cells was reduced by 61.7 %, the volume of their nuclei by 16.2 % after four days' starvation. After two to three days' starvation the liver cells often showed a slight accumulation of fat. Mitoses were frequent in the liver cells during the stage of regeneration, when the cells had attained the same size as in the controls.     

Oroxylin A Accelerates Liver Regeneration in CCI4-Induced Acute Liver Injury Mice

Introduction

Based on the previous research that oroxylin A can suppress inflammation, we investigated the hepatoprotective role of oroxylin A against CCl₄-induced liver damage in mice and then studied the possible alteration of the activities of cytokine signaling participating in liver regeneration. Wild type (WT) mice were orally administrated with oroxylin A (60 mg/kg) for 4 days after CCl₄ injection, the anti-inflammatory effects of oroxylin A were assessed directly by hepatic histology and indirectly by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin. Proliferating cell nuclear antigen (PCNA) staining was performed to evaluate the role of oroxylin A in promoting hepatocyte proliferation. Serum IL-1β, TNF-α, IL-6 and IL-1Ra levels were measured by enzyme-linked immunosorbent assay (ELISA) and liver HGF, EGF, TNF-α, IL-6, IL-1Ra and IL-1β gene expression was determined by quantitative real-time PCR. The data indicated that the IL-6 and TNF-α mRNA of oroxylin A administered group significantly increased higher than the control within 12 hours after CCl₄ treatment. Meanwhile, oroxylin A significantly enhanced the expression of IL-1Ra at the early phase, which indicated that oroxylin A could facilitate the initiating events in liver regeneration by increasing IL-1Ra which acts as an Acute-Phase Protein (APP). In addition, a lethal CCl₄-induced acute liver failure model offers a survival benefit in oroxylin A treated WT mice. However, oroxylin A could not significantly improve the percent survival of IL-1RI^−/− mice with a lethal CCl₄-induced acute liver failure.

Conclusions

Our study confirmed that oroxylin A could strongly promote liver structural remodeling and functional recovery through IL-1Ra/IL-1RI signaling pathway. All these results support the possibility of oroxylin A being a therapeutic candidate for acute liver injury.     

急性药物性肝损伤65例临床分析

目的:探讨急性药物性肝损伤的致病药物、临床特点及预后.方法:回顾性分析近年来本院确诊的急性药物性肝损伤患者的临床情况.结果:本组引起急性药物性肝损伤的最常见药物为抗结核药,占47.69％,其次为中草药、抗肿瘤药、滋补药、抗真菌药,分别占16.92％、9.23％、7.69％、3.61％.引起肝损伤发生时间因所用药物不同而差异较大,用药后出现药物性肝损伤的时间分别为用药2周内(36.92％)、2-4周内(32.31％)、4周-12周内(23.08％)、大于12周(7.69％),大多数病例出现于服药后12周内(92.31％.本组急性药物性肝损伤的临床表现主要为消化道症状明显,乏力、食欲下降,占89.23％;巩膜黄染、尿黄,占43.08％;皮肤瘙痒38.46％;恶心、呕吐,占35.38％;药物性肝损伤临床分型以肝细胞损伤型多见,占61.54％,其次为胆汁淤积型(29.23％),混合型较少(9.23％).结论:引起急性药物性肝损伤的药物种类繁多,很多临床常见的药物可引起肝损伤,早期诊断并停止用药、积极治疗是阻止疾病进展和改善预后的关键.     

The Ratio of Circulating Regulatory T Cells (Tregs)/Th17 Cells Is Associated with Acute Allograft Rejection in Liver Transplantation

CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR) (n = 16) and stable allograft liver function group (Gr-SF) (n = 22). The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI) after liver transplantation. In addition, the percentages of CTLA-4⁺, HLA-DR⁺, Ki67⁺, and IL-10⁺ Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.     

Ischemic Preconditioning in the Liver Is Independent of Regulatory T Cell Activity

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI.     

对乙酰氨基酚诱导的小鼠药物性肝损伤的模型研究

改良对乙酰氨基酚（acetaminophen,APAP）单独诱导小鼠急性肝损伤的模型和致死模型。随机将小鼠分为4组：空白对照组、APAP3h组、APAP6h组和APAP12h组,每组5只。饥饿15h后用对乙酰氨基酚诱发小鼠肝损伤。测定各组血清ALT、AST及胆红素含量,HE染色观察各组肝组织损伤情况。观察生存率时,小鼠随机分为对照组、禁食＋APAP（500mg／kg）组、禁食＋APAP（300mg／kg）组和不禁食＋APAP（500mg／kg）组,四组同时给药,然后记录各组小鼠的生存情况,绘制四组小鼠的生存曲线。小鼠注射APAP后,随时间的延长,ALT、AST水平逐渐升高,均明显高于空白对照组（P〈0．05）。小鼠肝脏HE染色可见,APAP中毒组小鼠肝细胞坏死及炎性细胞浸润。禁食＋APAP（500mg／kg）组小鼠自16h开始出现死亡,72h时全部死亡,死亡率明显高于不禁食组和禁食＋APAP（300mg/kg）组小鼠。该研究对APAPI起的C57／BL6小鼠药物性肝损伤模型进行改良,使其更加稳定和便于研究,为进一步探究APAP诱导肝毒性的机制及防治措施奠定了基础。     

Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity

Background

Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization.

Methodology/Principal Findings

We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the α_2A-adrenoceptor (α_2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14''304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of α_2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation.

Conclusions/Significance

Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

Melatonin-Mediated Cytoprotection against Hyperglycemic Injury in M��ller Cells

Oxidative stress is a contributing factor to the development and progression of diabetic retinopathy, a leading cause of blindness in people at working age worldwide. Recent studies showed that Müller cells play key roles in diabetic retinopathy and produce vascular endothelial growth factor (VEGF) that regulates retinal vascular leakage and proliferation. Melatonin is a potent antioxidant capable of protecting variety of retinal cells from oxidative damage. In addition to the pineal gland, the retina produces melatonin. In the current study, we investigated whether melatonin protects against hyperglycemia-induced oxidative injury to Müller cells and explored the potential underlying mechanisms. Our results show that both melatonin membrane receptors, MT1 and MT2, are expressed in cultured primary Müller cells and are upregulated by elevated glucose levels. Both basal and high glucose-induced VEGF production was attenuated by melatonin treatment in a dose-dependent manner. Furthermore, we found that melatonin is a potent activator of Akt in Müller cells. Our findings suggest that in addition to functioning as a direct free radical scavenger, melatonin can elicit cellular signaling pathways that are protective against retinal injury during diabetic retinopathy.     

Downregulation of Connexin 32 Attenuates Hypoxia/Reoxygenation Injury in Liver Cells

Gap junction intercellular communication is involved in ischemia–reperfusion (IR) injury of organs. Connexins are proteins that are critical to the function of gap junctions. To clarify the role of gap junctions in IR injury in liver cells, the function of gap junctions was modulated in an in vitro hypoxia/reoxygenation (H/R) model. BRL‐3A rat liver cells, endogenously expressing connexins Cx32 and Cx43, were used to model the process of hepatic IR injury. Suppression of gap junction activity was achieved genetically, using Cx32‐specific small interfering RNA (siRNA), or chemically, with pharmacological inhibitors, oleamide, and 18‐α‐GA. BRL‐3A cells subjected to H/R exhibited reduced cell survival and pathologies indicative of IR injury. Cx32‐specific siRNA, oleamide, and 18‐α‐GA, respectively, decreased gap junction permeability, as assessed by the parachute assay. Pretreatment with Cx32‐specific siRNA increased cell survival. Pretreatment with oleamide or 18‐α‐GA did not improve cell survival. Modulating gap junction by Cx32 gene silencing protected BRL‐3A liver cells from H/R.     

Metabolomic Characterizations of Liver Injury Caused by Acute Arsenic Toxicity in Zebrafish

Arsenic is one of the most common metalloid contaminants in groundwater and it has both acute and chronic toxicity affecting multiple organs. Details of the mechanism of arsenic toxicity are still lacking and profile studies at metabolic level are very limited. Using gas chromatography coupled with mass spectroscopy (GC/MS), we first generated metabolomic profiles from the livers of arsenic-treated zebrafish and identified 34 significantly altered metabolite peaks as potential markers, including four prominent ones: cholic acid, glycylglycine, glycine and hypotaurine. Combined results from GC/MS, histological examination and pathway analyses suggested a series of alterations, including apoptosis, glycogenolysis, changes in amino acid metabolism and fatty acid composition, accumulation of bile acids and fats, and disturbance in glycolysis related energy metabolism. The alterations in glycolysis partially resemble Warburg effect commonly observed in many cancer cells. However, cellular damages were not reflected in two conventional liver function tests performed, Bilirubin assay and alanine aminotransferase (ALT) assay, probably because the short arsenate exposure was insufficient to induce detectable damage. This study demonstrated that metabolic changes could reflect mild liver impairments induced by arsenic exposure, which underscored their potential in reporting early liver injury.