C-C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model.

BACKGROUND: Previous studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure. AIM: This study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis  virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism. RESULTS: By gene array analysis, chemokine (C-C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1ß and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1ß or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model. CONCLUSION: The CCR5-MIP-1ß/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

SOCS3 expression correlates with severity of inflammation in mouse hepatitis virus strain 3-induced acute liver failure and HBV-ACLF.

Recently, suppressor of cytokine signaling-3 (SOCS3) has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure (HBV-ACLF) have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 (MHV-3)-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells (PBMCs) of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immunohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B (CHB). Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.

Fibrinogen-like protein 2 deficiency inhibits virus-induced fulminant hepatitis through abrogating inflammatory macrophage activation.

BACKGROUND: Heterogeneous macrophages play an important role in multiple liver diseases, including viral fulminant hepatitis (VFH). Fibrinogen-like protein 2 (FGL2) is expressed on macrophages and regulates VFH pathogenesis; however, the underlying mechanism remains unclear. AIM: To explore how FGL2 regulates macrophage function and subsequent liver injury during VFH. METHODS: Murine hepatitis virus strain 3 (MHV-3) was used to induce VFH in FGL2-deficient (Fgl2-/-) and wild-type (WT) mice. The dynamic constitution of hepatic macrophages was examined. Adoptive transfer of Fgl2-/- or WT bone marrow-derived macrophages (BMDMs) into WT recipients with macrophages depleted prior to infection was carried out and the consequent degree of liver damage was compared. The signaling cascades that may be regulated by FGL2 were detected in macrophages. RESULTS: Following MHV-3 infection, hepatic macrophages were largely replenished by proinflammatory monocyte-derived macrophages (MoMFs), which expressed high levels of FGL2. In Fgl2-/- mice, the number of infiltrating inflammatory MoMFs was reduced compared with that in WT mice after viral infection. Macrophage depletion ameliorated liver damage in WT mice and further alleviated liver damage in Fgl2-/- mice. Adoptive transfer of Fgl2-/- BMDMs into macrophage-removed recipients significantly reduced the degree of liver damage. Inhibition of monocyte infiltration also significantly ameliorated liver damage. Functionally, Fgl2 deletion impaired macrophage phagocytosis and the antigen presentation potential and attenuated the proinflammatory phenotype. At the molecular level, FGL2 deficiency impaired IRF3, IRF7, and p38 phosphorylation, along with NF-κB activation in BMDMs in response to viral infection. CONCLUSION: Infiltrated MoMFs represent a major source of hepatic inflammation during VFH progression, and FGL2 expression on MoMFs maintains the proinflammatory phenotype via p38-dependent positive feedback, contributing to VFH pathogenesis.

[KCTD9, a novel potassium channel related gene, was highly expressed in hepatic NK cells and T cells of fulminant hepatitis mice induced by MHV-3].

OBJECTIVE: To explore the mechanisms of a novel potassium channel gene named KCTD9 (potassium channel tetramerization domain containing 9) in model of fulminant viral hepatitis induced by murine hepatitis virus 3 (MHV-3). METHODS: 78 BALB/cJ mice(6 male) were randomly and equally assigned to two groups, model group of fulminant viral hepatitis induced by MHV3 and its control. 75 C3H/HeJ female mice were done into two groups, 39 for model group of chronic hepatitis induced by MHV3, 36 for control. Various samples including spleen, liver and lymphocytes from mice of two model groups and the controls were examined for KCTD9 expression by real time quantitative PCR and Immunohistochemistry. Independent-samples T test or one-way ANOVA were carried out in different groups. RESULTS: Increased expressions of KCTD9 mRNA was observed in livers of both model mice of fulminant viral hepatitis and chronic hepatitis. Compared with the control mice, the expressions of KCTD9 mRNA were up-regulated by 577.1-, 8.8-, 59.4- and 10.8-fold in hepatic NK cells, CD4+ T cells, CD8+ T cells and splenic NK cells respectively in model mice of fulminant viral hepatitis 48 hr post MHV-3 infection, whereas down-regulation by 43% and 69% in splenic CD4 + T cells and CD8+ T cells were found respectively. In contrast, in model mice of chronic viral hepatitis the expressions of KCTD9 mRNA were down-regulated by 71% and 51% in hepatic CD4+ T cells and NK cells, respectively. The expression of KCTD9 protein was mainly evidenced in infiltrative mononuclear cells of liver as shown by immunohistochemistry. Basal expression was also investigated and showed constitutive expression of KCTD9 in brain, thymus and other organs in BALB/cJ mice. CONCLUSION: A novel potassium channel gene KCTD9 was highly expressed in hepatic NK cells and T cells of fulminant hepatitis mice induced by MHV-3.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study.

AIM: To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS: The Vldlr -/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS: The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr -/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+ -Vldlr+/+ ), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+ -Vldlr-/- ), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/- -Vldlr+/+ ), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/- -Vldlr-/- ) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION: The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

CAPRL Scoring System for Prediction of 30-day Mortality in 949 Patients with Coronavirus Disease 2019 in Wuhan, China: A Retrospective, Observational Study.

Background: Coronavirus disease 2019 (COVID-19) is a serious and even lethal respiratory illness. The mortality of critically ill patients with COVID-19, especially short term mortality, is considerable. It is crucial and urgent to develop risk models that can predict the mortality risks of patients with COVID-19 at an early stage, which is helpful to guide clinicians in making appropriate decisions and optimizing the allocation of hospital resoureces. Methods: In this retrospective observational study, we enrolled 949 adult patients with laboratory-confirmed COVID-19 admitted to Tongji Hospital in Wuhan between January 28 and February 12, 2020. Demographic, clinical and laboratory data were collected and analyzed. A multivariable Cox proportional hazard regression analysis was performed to calculate hazard ratios and 95% confidence interval for assessing the risk factors for 30-day mortality. Results: The 30-day mortality was 11.8% (112 of 949 patients). Forty-nine point nine percent (474) patients had one or more comorbidities, with hypertension being the most common (359 [37.8%] patients), followed by diabetes (169 [17.8%] patients) and coronary heart disease (89 [9.4%] patients). Age above 50 years, respiratory rate above 30 beats per minute, white blood cell count of more than10 × 109/L, neutrophil count of more than 7 × 109/L, lymphocyte count of less than 0.8 × 109/L, platelet count of less than 100 × 109/L, lactate dehydrogenase of more than 400 U/L and high-sensitivity C-reactive protein of more than 50 mg/L were independent risk factors associated with 30-day mortality in patients with COVID-19. A predictive CAPRL score was proposed integrating independent risk factors. The 30-day mortality were 0% (0 of 156), 1.8% (8 of 434), 12.9% (26 of 201), 43.0% (55 of 128), and 76.7% (23 of 30) for patients with 0, 1, 2, 3, ≥4 points, respectively. Conclusions: We designed an easy-to-use clinically predictive tool for assessing 30-day mortality risk of COVID-19. It can accurately stratify hospitalized patients with COVID-19 into relevant risk categories and could provide guidance to make further clinical decisions.

[Establishment of pig acute liver failure model and the role of pig fibrinogen-like protein 2].

OBJECTIVE: To establish a pig model of fulminant hepatic failure for evaluating the pre-clinical efficacy of drug treatment on severe hepatitis, and to detect the expression of fibrinogen-like protein-2 (fgl2) prothrombinase in the model, so as to provide basis for gene therapy targeting to fgl2 for fulminant hepatic failure. METHOD: D-galactosamine hydrochloride was used to induce pig model of fulminant hepatic failure, and the experiment animals were divided into model group (rapid injection of D-galactosamine hydrochloride by ear vein, a dose of 1.2 g/kg) and negative control group (5% Glucose). Clinical, biochemical and pathological changes of animals were observed. The expression of pigs fgl2 (pfgl2) mRNA in liver tissue was detected by real time RT-PCR, the expression of pfgl2 protein in liver tissue was detected by immunohistochemistry. RESULTS: A pig model of fulminant hepatic failure was successfully established using the D-galactose hydrochloride; Real time RT-PCR of liver fgl2 mRNA showed that fgl2 mRNA expression was increased significantly in liver tissue of fulminant hepatic failure pig model compared with the control group (P = 0.016); Immunohistochemical staining showed that there were fgl2 protein expression in liver tissue of fulminant hepatic failure pig model, mainly in the membrane and cytoplasm of hepatocytes, inflammatory cells, liver sinusoidal endothelial cells and vascular endothelial cells of liver cell necrosis region. However, there are no fgl2 positive staining on negative control. CONCLUSIONS: The pig model of fulminant hepatic failure induced by D-galactosamine hydrochloride is similar to human pathological process and can be used to evaluate the pre-clinical efficacy and safety of drug treatment on fulminant hepatic failure. Abnormal expression of pfgl2 at both mRNA level and protein level in the liver of fulminant hepatic failure pig model shows that pfgl2 induced coagulation pathway is also involved in the development of fulminant hepatic failure. Gene therapy targeting fgl2 genes for fulminant hepatic failure may provide a new means for the treatment of fulminant hepatic failure.

[Role of CD4-CD8- T cells in the murine hepatitis virus type 3 induced chronic viral hepatitis].

OBJECTIVE: To investigate role of CD4-CD8- T cells in murine hepatitis virus type 3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice and to identify their surface markers. METHODS: Thirty C3H/Hej mice received 10 Pfu MHV-3 intraperitoneally, the CD4-CD8- T cells were isolated using magnetic bead sorting on 0, 4, 15, 30, 40 days post MHV-3 infection. The cytotoxic effects of CD4-CD8- T cells on normal and infected hepatocytes, CD8+ T cells and unrelated-virus (murine cytomegalovirus, MCMV) infected CD8+ T cells were examined by non-radioactive cytotoxicity assay. The surface markers of CD4-CD8- T cells were determined by flow cytometry. RESULTS: MHV-3 infected CD4-CD8- T cells showed significant cytotoxic effect on CD8+ T cells, but not on infected hepatocytes or MCMV infected CD8+ T cells. The analysis of cell surface markers demonstrated that the CD4-CD8- T cells are a completely new T cell subset. CONCLUSIONS: CD4-CD8- T cells have significant cytotoxic effect on virus specific CD8+ T cells in MHV-3 infected C3H/Hej mice, which suggests that CD4-CD8- T cells have immune modulatory functions in the development of chronic viral hepatitis. The phenotype of these CD4-CD8- T cells detected by flow cytometry is TCR alpha beta +CD3+CD4- CD8- CD25- CD28- CD30- CD44+.

Fibrinogen-like protein 2/fibroleukin prothrombinase contributes to tumor hypercoagulability via IL-2 and IFN-gamma.

AIM: To examine the role of Fibrinogen-like protein 2 (fgl2)/fibroleukin in tumor development. Fgl2 has been reported to play a vital role in the pathogenesis in MHV-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo- and xeno- graft rejection by mediating "immune coagulation". METHODS: Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (HCC) model on nude mice (established from high metastasis HCC cell line MHCC97LM6) were obtained. RESULTS: Hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human HCC nude mice. Hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, NK cells, and CD8(+) T lymphocytes and vascular endothelial cells. Hfgl2 mRNA was localized in cells that expressed hfgl2 protein. Fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. In vitro, IL-2 and IFN-gamma increased hfgl2 mRNA by 10-100 folds and protein expression in both THP-1 and HUVEC cell lines. One-stage clotting assays demonstrated that THP-1 and HUVEC cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. CONCLUSION: The hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction.

[The functions of liver natural killer cells in murine fulminant hepatitis induced by murine hepatitis virus strain 3].

OBJECTIVES: To investigate the role of liver natural killer cells (NK cells) in murine hepatitis virus strain 3 (MHV-3) induced murine fulminant hepatitis. METHOD: Balb/cJ mice (6-8 weeks, female) were intraperitoneally injected with 100 PFU MHV-3. The numbers of NK cells in their livers, spleens, blood and bone marrow and the expression of CD69 on liver NK cells at 0, 24, 48 and 70 h after MHV-3 infection were analyzed by flow cytometry. The cytotoxic activity of liver NK cells was detected by a non-radioactive cytotoxicity assay. The levels of IFN gamma produced by hepatic NK cells were detected by intracellular cytokine staining. RESULT: Following MHV-3 infection, the proportion of liver NK cells in the mice increased remarkably and reached the peak (43.9%+/-2.3%) at 48 h, then kept a high proportion until the mice were sacrificed. The proportion of NK cells in the peripheral blood also significantly increased and reached the peak (18.0%+/-5.4%) at 48 h. However, there were few NK cells in the peripheral blood at 70 h after infection; the ratio was only 1.3%+/-0.6%. In the spleens and bone marrow, the proportions of NK cells were both significantly decreased from 0 h to 48 h and then slightly increased. The expression of CD69 on liver NK cells was highly up-regulated after the infection and the cytotoxic activity of hepatic NK cells at 48 h was also significantly enhanced. In addition, an increase in IFN gamma production by hepatic NK cells was observed at 48 h. CONCLUSION: After MHV-3 infection, NK cells were recruited to the liver quickly, probably from the spleen and bone marrow. Recruited NK cells remarkably express CD69, enhance cytotoxic activity and IFN gamma production, which correlate with the disease severity of fulminant viral hepatitis. Our results suggest that liver NK cells may play a pivotal role in the pathogenesis of fulminant viral hepatitis.

γδ T Cells Contribute to the Outcome of Murine Fulminant Viral Hepatitis via Effector Cytokines TNF-α and IFN-γ.

The mechanisms involved in virus-induced severe hepatitis have not been fully elucidated. In this study, we investigated the role of gamma delta T cell receptors (γδ) T cells in the pathogenesis of fulminant viral hepatitis (FVH) induced by murine hepatitis virus strain 3 (MHV-3). The model of FVH was established by intraperitoneal injection of MHV-3 into Balb/cJ mice. The survival days of mice, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined. The proportions of γδ T cells in blood, spleen and liver, and cytokines secreted by hepatic γδ T cells were analyzed by flow cytometry. The function of hepatic γδ T cells was examined by cytotoxicity assay. Balb/cJ mice died in 3 to 6 days post MHV-3 infection, with severe hepatic necrosis and significant augmentation of serum ALT and AST levels. The proportions of γδ T cells in blood, spleen and liver were significantly increased post MHV-3 infection, while those of the early activating molecule CD69-expressing γδ T cells and productions of cytokines tumor necrosis factor-alpha (TNF-α) and interferon-γ (IFN-γ) increased remarkably in the liver. These highly activated liver γδ T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect of liver γδ T cells against hepatocytes might involve the TNF-α and IFN-γ pathway. These results demonstrated that γδ T cells might contribute to the pathogenesis of MHV-3-induced FVH through the effector cytokines TNF-α and IFN-γ.

Effects of hydrogen-rich saline on endotoxin-induced uveitis.

The therapeutic effects of hydrogen-rich saline (HRS) have been reported for a wide range of diseases mainly via selectively reducing the amount of reactive oxygen species. Oxidative stress plays an important role in the pathogenesis of uveitis and endotoxin-induced uveitis (EIU). In this study, we investigated whether HRS can mitigate EIU in rats. Sprague-Dawley rats were randomly divided into Norm group, Model group, HRS group, dexamethasone (DEX) group, and rats in the latter three groups were injected with equal amount of lipopolysaccharide (LPS) to induce EIU of different severities (by 1 mg/kg of LPS, or 1/8 mg/kg of LPS). Rats in HRS group were injected with HRS intraperitoneally at three different modes to purse an ameliorating effect of EIU (10 mL/kg of HRS immediately after injection of 1 mg/kg of LPS, 20 mL/kg of HRS once a day for 1 week before injection of 1 mg/kg of LPS and at 0, 0.5, 1, 2, 6, 8, 12 hours after LPS administration, or 20 mL/kg of HRS once a day for 1 week before injection of 1/8 mg/kg of LPS, and at 0, 0.5, 1, 2, 6, 8, 12, 24 hours and once a day for 3 weeks after LPS administration). Rats of DEX group were injected with 1 mL/kg of DEX solution intraperitoneally immediately after LPS administration. Rats in Norm and Model groups did not receive any treatment. All rats were examined under slit lamp microscope and graded according to the clinical signs of uveitis. Electroretinogram, quantitative analysis of protein in aqueous humor (AqH) and histological examination of iris and ciliary body were also carried out. Our results showed that HRS did not obviously ameliorate the signs of uveitis under slit lamp examination and the inflammatory cells infiltration around iris and cilliary body of EIU induced by 1 mg/kg or 1/8 mg/kg of LPS (P > 0.05), while DEX significantly reduced the inflammation reflected by the above two indicators (P < 0.05). The impaired retinal function of mild EIU induced by 1/8 mg/kg of LPS, showed by delay of peak time of b-wave of Dark adapted 3.0 electroretinogram, was not significantly restored by HRS (P > 0.05), while DEX had an obvious therapeutic effect (P < 0.05). However, HRS exerted an inhibition trend on elevation of protein in AqH of EIU induced by 1 mg/kg of LPS, and significantly reduced the increasing amount of protein in AqH of mild EIU induced by 1/8 mg/kg of LPS (P < 0.05). In conclusion, HRS could not obviously mitigate EIU in rats, while it could inhibit the elevation of AqH protein.

The reason for the amelioration of N-methyl-N-nitrosourea-induced retinitis pigmentosa in rats by hydrogen-rich saline.

AIM: To investigate the effects of hydrogen-rich saline (HRS) on microglia activation and Sirtuin type 1 (Sirt1) in rats with N-methyl-N-nitrosourea (MNU)-induced retinitis pigmentosa (RP). METHODS: Rats were divided into norm (N) group, model (M) group and HRS (H) group. Rats in M and H groups were given saline and HRS respectively prior to and after administration of MNU. At one day (d1) and d3 afterwards, electroretinogram and histological examination were performed to confirm the effects of HRS on retinal function and structure of MNU-induced RP. Immunofluorescence staining of anti-ionized calcium-binding adapter molecule 1 (Iba1), a maker of microglia cells, was performed, with quantitative real-time polymerase chain reaction (qRT-PCR) for its mRNA quantification. Moreover, Sirt1 mRNA and protein expression in the retinas were detected by Western blot and qRT-PCR. RESULTS: HRS preserved the retinal function and mitigated the reduction of photoreceptor degeneration in MNU-treated retinas. The presence of microglia cells was somewhat more obvious in H group than that in M group at d1. HRS suppressed the further activation of microglia cells, with the number of microglia cells less than that of M group at d3. Results of qRT-PCR of Iba1 were consistent with those of immunofluorescence staining, with the mRNA expression of Iba1 in H group more intensive than that of M group at d1 (P<0.05), while less than that of M group at d3 (P<0.05). Furthermore, the Sirt1 mRNA and protein expression decreased after MNU administration, while HRS mitigated the MNU-induced downregulation of Sirt1. CONCLUSION: HRS can effectively keep microglia activation induced by MNU to an appropriate extent, while upregulate Sirt1 in MNU-induced RP.

Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats.

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.

[Construction of the p-mfgl2shRNA and its effect on mfgl2 expression in vitro].

OBJECTIVE: To construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro. METHODS: A plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression. RESULTS: Cotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines. CONCLUSIONS: The study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B.

AIM: To evaluate the expression of fibrinogen-like protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury. RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2) was detected in 21 of 23 patients (91.30%) with severe AOC hepatitis B, while only 1 of 13 patients (7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.

[Expression of human fibroleukin gene acute on chronic hepatitis B and its clinical significance].

OBJECTIVE: To investigate the mRNA and protein expressions of human fibroleukin gene (hfg12) in acute on chronic (AOC) hepatitis B and its clinical significance. METHODS: Liver tissues were obtained from 23 patients with AOC hepatitis B, 13 patients with chronic hepatitis, and 14 patients with cirrhosis to be examined histologically. Immunohistochemistry and in situ hybridization were used to detect the mRNA and protein expressions of hfg12 in the liver tissues. Double staining was used to the hfg12 positive samples to examine both the hfg12 and fibrin. Four specimens of liver tissue from normal donors were used as controls. RESULTS: Immunohistochemistry showed that hfg12 was expressed in the liver tissues of 21 out of the 23 patients with AOC hepatitis B (91.30%) and only one out of the 13 patients with chronic hepatitis (7.69%). In situ hybridization showed that hfg12 was expressed in the liver tissues of 13 out of he 23 patients with AOC hepatitis B and in none of the 27 patients with chronic hepatitis or cirrhosis. In patients with AOC hepatitis Kupffer's cell, CD68 positive, was numerous and big, mainly distributed in the necrosis areas. It was identified as the same of hfg12-expressing cells. CONCLUSION: High expression of hfg12 is one of the molecular mechanisms of necrosis of liver cells in AOC hepatitis.

[The study of cis-element HNF4 in the regulation of mfg12 prothrombinase/fibroleukin gene expression in response to nucleocapsid protein of MHV-3].

OBJECTIVE: To identify the transcription factor(s) that is essential for activation of mfgl2 prothrombinase/fibroleukin gene in response to nucleocapsid protein of murine hepatitis virus type 3 (MHV-3). METHODS: Western blotting was performed to investigate whether HNF4 is expressed in macrophages of Ba1b/c mice where mfgl2 is expressed. Confocus microscope immunofluorescence was performed to show whether N protein of MHV enters into the nucleus of infected cells, which is a critical step for the N protein to facilitate its transactivation property. To facilitate the identification of three candidate factor(s) including hepatocyte nuclear factor 4 (HNF4)/liver factor A1 (LF-A1), cytomegalovirus immediate early gene 1.2 (IE1.2) regulatory element and granulocyte- macrophage colony stimulating factor (GM-CSF) in response to mfgl2 activation upon the stimulation of MHV-A59 N protein, gel mobility shift assay (GMSA), competition experiments and site directed mutagenesis were performed. RESULTS: Western blotting displayed that HNF4 was constitutively expressed in macrophages and did not show significant change under the stimulation of different MHV. Confocus microscope immunofluorescence clearly showed that N protein of MHV entered into the nucleus of infected cells. GMSA and competition experiments demonstrated binding to both HNF4 and IE1.2 fragments could be competed with the cold specific oligonucleotides but not with the same amount of non-specific oligos nucleotides. A super shift band was observed when HNF4 antibody was pre-incubated with the nuclear extracts indicating the interaction between the HNF4 element and mfgl2 promoter. Site directed mutagenesis of cis-elements HNF4 (pfgl2HNF4mut) and HNF4/IE1.2 (pfgl2HNF4/IE1.2mut) mutations abolished over 75% of transcription from wild-type mfgl2 promoter. However the pfgl2IE1.2mut displayed almost wild-type promoter activity (75% approximately 80%). CONCLUSIONS: The factor HNF4 binds to mfgl2 promoter and serves as an essential transcription factor for mfgl2/fibroleukin expression in response to MHV-3 N protein.