大鼠眼内组织对内毒素特异敏感的原因

内毒素诱导的葡萄膜炎(endotoxin-induced uveitis,EIU)是革兰氏阴性菌感染相关的人类内源性葡萄膜炎的常用模型。然而在全身注射内毒素诱导的大鼠EIU中,尽管眼内组织表现出明显炎症,但在其它器官组织观察不到明显的病理组织学变化。大鼠眼内组织选择性被内毒素感染的现象提示,其眼内组织对内毒素可能具有某种未知的特殊敏感性。本文就大鼠眼内组织对内毒素特异敏感的可能原因进行综述分析,以期能为临床治疗葡萄膜炎提供新思路。

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 fibrinogenlike 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 mononuciear 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. House 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.

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 marrowderived 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.

光化学法建立视网膜分支静脉阻塞大鼠模型及相关研究

目的:观察光化学法构建视网膜分支静脉阻塞(branch retinal vein occlusion,BRVO)大鼠模型的自然病程和不良事件。方法:选取30只SD(Sprague Dawley)大鼠尾静脉注射孟加拉红1min后,532nm激光(80mW,100μm,100ms)于视盘颞侧视网膜静脉二级分叉处进行单光点光凝50点建立BRVO模型。分别于1、3、5、7、10、14和21d检测全视野视网膜电图(electroretinogram,ERG)、荧光素眼底血管造影(fundus fluorescein angiography,FFA)和相干光断层扫描(optical coherence tomography,OCT)。于1、5和21d各时间点随机处死2只大鼠行HE病理和血管内皮生长因子-α(vascular endothelial growth factor-α,VEGF-α)免疫组化染色。结果:光凝后,3只大鼠死亡,3只严重出血导致视网膜大部分脱离,1只出现视网膜凹陷,1只白内障。FFA和眼底(荧光)彩照发现BRVO大鼠模型造模成功率为73%(22/30),1d时近端变粗,远端变细,3～7d光凝血管完全再通。ERG示光凝1d后暗适应3.0反应b波降低至正常眼的0.694±0.042倍,5～7d下降至最低点约为正常眼0.487±0.064倍,之后开始上升,21d上升至初始值0.708±0.0465倍。OCT和HE病理切片分别于在体和离体水平发现第1d视网膜节细胞层和外核层水肿,3～5d水肿消失且激光光凝点视网膜附近250μm外核层开始凋亡变薄,到21d外核层变薄只剩3～4层细胞。免疫组化发现激光光凝部位VEGF-α第1d表达水平大于光凝前,第5d光凝处VEGF-α表达量无明显差异,21d光凝处VEGF-α表达略低于光凝前。结论:激光光凝制作BRVO模型是一种切实可行的方法,其疾病的演变和发展可以部分模拟人体BRVO的进程。同时由于造模成功率较低以及激光光凝相关并发症较多,在实际使用中有待进一步改进方法。

Involvement of CXCR3-associated Chemokines in MHV-3 Induced Fulminant Hepatic Failure

The role of chemokines in murine hepatitis virus strain 3 (MHV-3) induced fulminant hepatic failure (FHF) is not well defined. In this study, we investigated the role of the CXC chemokine receptor 3 (CXCR3)- associated chemokine [monokine induced by IFN-gamma (Mig/CXCL9) and interferon-gamma-inducible protein 10 (IP-10/CXCL10)] in the recruitment of intrahepatic lymphocytes and subsequent fulminant hepatic failure induced by MHV-3. Balb/cJ mice (6-8 weeks, female) were intraperitioneally injected with 100 PFU MHV-3.The proportions and numbers of T cells and NK cells as well as the expression of CXCR3 on T cells and NK cells in the liver, spleen and blood were analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines (CXCL9 and CXCL10) was detected by realtime PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes on the splenic lymphocytes. Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. These results suggest that the CXCR3- associated chemokines (CXCL9 and CXCL10) may play animportant role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and hepatic failure in MHV-3 infection.

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 m RNA quantification.Moreover,Sirt1 m RNA 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 m RNA 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 m RNA 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.

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-y （IFN-3,） 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. Key words： fulminant viral hepatitis; murine hepatitis virus strain 3; gamma delta T cell receptors T cells; tumor necrosis factor-a; interferon-α

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-Ⅰmice with the wild-type Pde6b and Vldlr genes(Pde6b~(+/+)-Vldlr~(+/+)),which had normal ocular phenotypes;the F2-Ⅱmice with homozygous mutant Vldlr gene(Pde6b~(+/+)-Vldlr^(-/-)),which exhibited the retinal NV phenotype;the F2-Ⅲmice with homozygous mutant Pde6b gene(Pde6b^(-/-)-Vldlr~(+/+)),which exhibited the RP phenotype.Specifically,the F2-Ⅳmice 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 than 10×109/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.