Introduction

Idiopathic autoimmune hepatitis(AIH)is a chronic in flammatory liver disease characterized by elevated liver enzymes,hypergammaglobulinemia,autoantibodies,intrahepatic lymphoplasmacytic in filtration and high relapse rate after withdrawal of immunosuppressant[1,2].Drug-induced autoimmune hepatitis(DIAIH)is similar to classical AIH.However,remission is maintained in DIAIH after withdrawal of immunosuppressant[3].Currently,the differentiation DIAIH from AIH in early onset patients is still challenging[2–4].

（1）技术措施制定更具科学性。以往井队把作为参考书的技术交底作为辅助内容，靠经验打井，势必出现以往经验老套、新的经验不成熟等问题。技术交底作为指导书以大量的实钻数据和邻井资料为依据，由公司技术专家制定，具有科学性。

Over the last decade,the pathogenesis of AIH received much attention,but it remains to be clari fied.The transcription factor Forkhead box P3(Foxp3)has been recognized as a lineage speci fication factor of regulatory T cells(Tregs),which not only has a highly restricted expression within the CD4+subset of T cells but also regulates their differentiation and function[5].Recently a subset of CD4+Foxp3+T cells expressing CD25(named as CD4+Foxp3+CD25+Tregs),which accounts for 5–10%of the entire CD4+ T cell population,was discovered to play a key role in maintaining immune equilibrium via its potent suppressor activity[6,7].More recently,several studies[8–11]showed that a subset of CD4+Foxp3+CD25−Tregs was augmented in the peripheral blood in T cell-mediated autoimmune diseases including systemic lupus erythematosus,multiple sclerosis,rheumatoid arthritis and type 1 diabetes mellitus.However,the frequencies of CD4+Foxp3+CD25+/−Tregs in DIAIH and AIH remain to be clarified.

In this study,we aimed to differentiate DIAIH from AIH by comparing the biochemical changes,histological features,and frequencies of CD4+Foxp3+CD25+/−Tregs in liver tissues or peripheral blood lymphocytes.

Methods

Patients

We enrolled 15 well-characterized DIAIH patients and 24 AIH patients diagnosed in our Liver Unit between May 2008 and May 2014 and followed them for one year.None of the patients underwent immunosuppressive therapy prior to liver biopsy.Diagnosis was made at initial presentation by exclusion of competing etiologies and assessment of relevant clinical and histological data at the time of diagnosis.The diagnosis of AIH was made according to international AIH group score system in which the cut off score is 16 points[12].All 24 AIH patients were classical AIH with typical histological features including marked portal in flammation,interface hepatitis,and varying degrees of lobular hepatitis[13].Nonclassical phenotypes of AIH at presentation were excluded,which included acute severe AIH(acute presentation with high level bilirubin and signs of acute liver failure with an INR＞1.5 at any time but without histological evidence of cirrhosis)and histological atypical AIH(AIH-PBC or AIH-PSC overlap syndrome)[14–17].The diagnosis of DIAIH was made by the time correlation of the drug intake on the onset of hepatitis and by an aggregate score for each patient of＞16 points according to international AIH group score system and by the exclusion of other causes of hepatitis(i.e.,HBV,HCV,HEV,EBV).The presences of the following two criteria were also necessary for diagnosis of DIAIH:(i)liver injury resolves on withdrawal of medication that triggered the disease,with or without immunosuppressive therapy to induce remission;(ii)no relapse within one year after withdrawal of all immunosuppressants[18].

针对三峡水库蓄水后的不同阶段，以及不同河段的演变特点，对可采区的位置确定实行动态规划。根据已有研究可知，随着三峡水库蓄水进程的不断发展，水沙条件的变化会带来不同河段的局部河势调整，对于堤防安全、防洪形势以及航槽位置均可能造成重要影响。主航道的定线不是一成不变，而必须根据水沙条件和洲滩格局的变化不断有所调整。而可采区位置、范围和开采高程的确定，必须考虑上述影响。此外，对于修订后的《长江中下游干流河道采砂规划》中的保留区，经分析研究当开采条件成熟时可在规划期内改变为可采区。

Histological evaluation

The 39 liver biopsy slides from all of the patients were stained by HE and evaluated using a standardized histological scoring system by three experienced hepatopathologists who were blinded to the clinical information.The following data were collected:the number of lymphocytes,plasmacytes,neutrophils or eosinophils in three lobules and portal tracts,the average number of in flammatory cells per lobule or portal tract.The presences of bile duct proliferation,rosette formation,zone 3 necrosis,and focal necrosis were evaluated and scored as “yes/no”.Cellular edema,ballooning degeneration and acidophilic degeneration were divided into mild,moderate or severe.Interface hepatitis or lobular in flammation was divided into mild,moderate or severe.Hepatic in flammation was assessed by the use of hepatic activity index(HAI)score[19]while fibrosis was staged with Scheuer system(ranging from 0=no fibrosis to 4=cirrhosis)[20].

Immunopathological evaluation

In summary,our study demonstrates that DIAIH at initial presentation has higher levels of serum aminotransferases,higher frequency of zone 3 necrosis and more severe lobular in flammation characterized by lymphocytic and eosinophilic in filtrations compared with AIH.For the first time,we also demonstrate that the number of hepatic CD4+Foxp3+CD25−Tregs is higher in DIAIH than AIH.Additionally,there are positive correlations in DIAIH between the degree of lobular in flammation and either the levels of aminotransferases or the number of lobular CD4+Foxp3+CD25−Tregs.We suggest that clinicians should use the differences identi fied between patients with DIAIH versus AIH to differentiate the two diseases in their early stages.

Flow cytometry analysis

Blood samples were collected from 9 of 15 DIAIH patients,8 of 24 AIH patients,and from 8 healthy controls one day before or on the same day of liver biopsy and before immunosuppressive therapy.Total 25 mL of each blood sample was topped up with MACS buffer(PBS,0.5%BSA,2.5 mmol/L EDTA)to a total volume of 35 mL,layered onto 15 mL of human lymphocyte separation medium(Dakewe,Shenzhen,China)and centrifuged at 400 g for 30 min.The mononuclear cells were collected and washed twice with MACS buffer.To quantify Tregs in blood samples, flow cytometry(BD,FACSCalibur,San Jose,USA)was performed on mononuclear cells stained with mouse anti-human CD4 conjugated to FITC,anti-CD25 conjugated to APC,or anti-Foxp3 conjugated to PE(BD,FACSCalibur,San Jose).The number of CD4+Foxp3+CD25+/−Tregs in a total number of 1×104 CD4+T cells was then analyzed with Flowjo 7.6.1 software.

Statistical analysis

Recent studies demonstrated that defects in immune regulation and a lack of functional Tregs were correlated with the development of AIH,but it was not clear whether the defect in patients with AIH is due to a failure of Treg recruitment or function,or whether the Tregs are simply overwhelmed by dominating effector cell responses[24].Foxp3 is considered a speci fic marker of CD4+CD25+Tregs,but increasing evidence suggests that human CD4+CD25−effector T cells can transiently express Foxp3 upon activation.The CD4+Foxp3+CD25+Tregs are the major population of regulatory cells,which has been recognized as a critical player in maintaining immune equilibrium via its potent suppressor activity,[5,7]but its action has not been described in patients with DIAIH versus AIH.In this study,we found that the number of intrahepatic or peripheral blood CD4+Foxp3+CD25+Tregs was not different between DIAIH and AIH.However,it remains to be further clari fied whether there is a difference in the function of CD4+Foxp3+CD25+Tregs between DIAIH and AIH.

Results

General characteristics of the patients

The present study showed signi ficantly higher levels of serum aminotransferases and more severe degree of lobular in flammation in DIAIH versus AIH patients,and a positive correlation between these two parameters in DIAIH.These data are important because they help the clinician to differentiate patients with DIAIH from those with AIH.

Table 1 Data at entry in patients with DIAIH versus AIH.

AIH:autoimmune hepatitis;DIAIH:drug-induced autoimmune hepatitis;EO:eosinophil;ALT:alanine aminotransferase;AST:aspartate aminotransferase;TBil:total bilirubin;ALP:alkaline phosphatase;GGT:gamma-glutamyltransferase;IgG:immunoglobulin G;PT:prothrombin time.

Variables DIAIH(n=15) AIH(n=24) P value Age(yr) 53.12(46.43–57.18) 54.26(46.53–65.17) 0.212 Female 15(100%) 23(95.8%) 0.431 Duration of drug intake(mon) 11.25(4.13–8.26) – –Duration of symptoms(d) 32.00(17.00–48.50) 63.50(28.75–91.75) 0.066 EO(×109/L) 0.07(0.043–0.127) 0.10(0.050–0.270) 0.222 Platelet(×109/L) 179.00(144.25–220.50) 183.00(136.75–227.00) 0.389 ALT(U/L) 404.50(299.00–734.75) 309.50(158.32–574.75) 0.033 AST(U/L) 454.10(287.00–767.00) 315.00(177.95–396.33) ＜0.001 TBil(μmol/L) 54.68(29.02–88.46) 48.38(18.21–75.09) 0.316 ALP(U/L) 191.00(111.25–275.83) 167.70(114.00–230.00) 0.754 GGT(U/L) 172.00(118.50–256.25) 218.90(139.00–331.00) 0.212 Albumin(g/L) 31.85(27.90–35.85) 33.40(30.38–40.00) 0.345 IgG(g/L) 20.70(18.93–24.10) 20.60(18.40–26.50) 0.881 PT(s) 13.00(10.83–15.28) 11.50(10.80–13.33) 0.141 ANA 1:320(1:320–1:1000) 1:1000(1:320–1:1000) 0.138

Table 2 Comparison of histological features between DIAIH versus AIH.

AIH:autoimmune hepatitis;DIAIH:drug-induced autoimmune hepatitis;HAI:hepatic activity index.

Histological features DIAIH(n=15) AIH(n=24) P value Portal lymphocytes 113.35(98.64–196.75) 98.55(81.26–179.33) 0.676 Lobular lymphocytes 21.82(16.73–33.26) 12.66(9.58–21.67) 0.040 Portal plasma cells 5.67(4.00–8.17) 5.50(3.25–9.50) 0.856 Lobular plasma cells 3.78(1.84–6.00) 3.00(2.25–3.00) 0.703 Portal neutrophils 1.00(0.55–3.33) 2.50(2.02–3.75) 0.182 Lobular neutrophils 1.12(1.00–4.25) 2.00(1.25–2.55) 1.034 Portal eosinophils 3.33(1.84–4.84) 1.50(1.00–3.00) 0.014 Lobular eosinophils 4.33(1.17–6.00) 1.50(1.12–3.00) 0.011 Bile duct proliferation 11(73.3%) 7(29.2%) 0.049 Rosettes formation 4(26.7%) 10(41.7%) 0.667 Zone 3 necrosis 7(46.7%) 3(12.5%) 0.017 Focal necrosis 9(60.0%) 12(50.0%) 0.697 Moderate to severe interface hepatitis 7(46.7%) 14(58.3%) 0.384 Fibrosis score at index biopsy 0.352 F0 2(13.3%) 0 F1 4(26.7%) 9(37.5%)F2 8(53.3%) 9(37.5%)F3 1(6.7%) 6(25.0%)F4 0 0 HAI score 8(7–13) 7(6–10) 0.705 Moderate to severe lobular in flammation 10(66.7%) 3(12.5%) 0.038 Severe cellular edema 5(33.3%) 10(41.7%) 1.000 Severe ballooning degeneration 5(33.3%) 10(41.7%) 1.000 Severe eosinophilic degeneration 4(26.7%) 10(41.7%) 0.667

In this study,the drugs that were suspected due to the induction of DIAIH in 15 cases included:nitrofurantoin(n=4),lovastatin(n=4),droxacin(n=1),amoxicillin/clavulanic acid(n=1),nimesulide(n=1),terbina fine(n=1),or herbal medicine(radixbupleuri,n=3).These were suspected on clinical grounds because of temporal association between the development of liver damage and the intake of pharmaceuticals at recommended doses in each case.The median duration of drug use prior to diagnosis of DIAIH was 11.25 months(4.13–8.26).

The histological comparison of DIAIH with AIH

Overall,the histological characteristics were very similar between DIAIH and AIH patients.However,the number of eosinophilic in filtrations in both the lobules and portal tracts was higher in DIAIH than AIH cases,and the higher number of lymphocytic in filtration in the lobules and higher frequencies of zone 3 necrosis were seen in DIAIH cases(Table 2 and Fig.1)(P＜0.05).There was no clinical immunoallergic sign(i.e.,arthragia,urticaria)in two groups.The degree of lobular in flammation was more severe in DIAIH cases(P＜0.05,Table 2).Correlation analysis revealed positive correlations between the degree of lobular in flammation and the serum levels of ALT or AST in DIAIH patients(r=0.28,P＜0.05;r=0.75,P＜0.01;respectively).Additionally,the presence of bile duct proliferation in portal tracts was more common in DIAIH cases(P＜0.05,Table 2).

Analysis of CD4+Foxp3+CD25+/−Tregs frequency in liver tissues

As shown in Fig.2A–D,the presence of Foxp3+ Tregs revealed a good concordance with CD4+ T cells.As expected,CD25 was presented only in a small number of Foxp3+cells.CD4+Foxp3+CD25+or CD4+Foxp3+CD25−Tregs were individually identi fied and counted.The result showed that the number of CD4+Foxp3+CD25−Tregs in the lobules and portal tracts was signi ficantly higher in DIAIH patients than that in AIH patients(P＜0.001)whereas the number of CD4+Foxp3+CD25+Tregs in the lobules and portal tracts was not different between the two groups(P＞0.05,Fig.2E and F).

Fig.1.HE staining shows histological features in a patient with DIAIH(top row)or AIH(bottom row).The liver architecture is almost complete in DIAIH(A)and AIH(D);interface in flammation and numerous lymphocytic in filtration in the portal tract of DIAIH(B)and AIH(E),numerous lymphocytes in lobule and eosinophils in both portal tract and lobule of DIAIH(B and C);only a few lymphocytes in lobule of AIH(F);zone 3 necrosis in DIAIH(C)while rosettes formation in DIAIH(C)and AIH(F).A,D:original magni fication×40;B,E:original magni fication×200;C,F:original magni fication×400.

The correlation analysis of the degree of lobular in flammation and the frequency of CD4+Foxp3+CD25−Tregs

The frequencies of blood CD4+Foxp3+CD25+/−Tregs were not signi ficantly different in patients with DIAIH or AIH compared with healthy controls(all P＞0.05)and that the frequencies of both cells in the blood were also similar in patients with DIAIH versus AIH(both P＞0.05,Fig.4).

2.4 施工安全与企业信誉的关系信誉是一种隐形效益，是企业多年经营后所获得的社会对企业的评价。施工安全是企业信誉的一个非常重要的方面。一个高信誉的企业必定是个安全工作做得完善的企业。因为信誉高是企业综合素质的反映，当然包括在安全方面的出色管理。建筑企业欲在当今市场中求发展，做好施工安全管理是极其重要的一环。创造信誉得靠人为，人的安全无保证，怎能去创信誉。企业要获得信誉，先要保证开创信誉人的安全。

The frequency analysis of peripheral blood CD4+Foxp3+CD25+/−Tregs

There was a positive correlation between the degree of lobular in flammation and the frequency of CD4+Foxp3+CD25−Tregs in DIAIH(r=0.502,P＜0.01)(Fig.3A),but not in AIH(r=0.069,P＞0.05)(Fig.3B).

药学继续教育面临的机遇与挑战；总结继续教育方案中使用的媒体方法对提高临床社区药学服务的影响；明确全球职业持续发展与继续教育的责任；药学继续教育质量的基础；开展继续教育项目。

Discussion

Clinical characteristics of patients with DIAIH and AIH at initial presentation are summarized in Table 1.All DIAIH patients were female,with a median age of 53.12 years(46.43–57.18).Total 95.8%(23/24)of AIH patients were female,with a median age of 54.26 years(46.53–65.17).The median values of ALT and AST were 404.50 U/L(299.00–734.75)and 454.10 U/L(287.00–767.00)in DIAIH patients and 309.50 U/L(158.32–574.75)and 315.00 U/L(177.95–396.33)in AIH patients,respectively.The ALT and AST values were signi ficantly higher in DIAIH than those in AIH patients(P＜0.05).However,there was no signi ficant difference in other biochemical data between the two groups.All DIAIH and AIH patients were positive for ANA.The median values of ANA titres were 1:320(1:320–1:1000)in DIAIH and 1:1000(1:320–1:1000)in AIH,respectively,without signi ficant difference between two groups.There were positive for SMA in one of the AIH patients and two of the DIAIH patients.But all patients tested negative for anti-LKM-1 as well as SLA/LP antibodies.

Several years ago,a clinical study on DIAIH versus AIH showed that serum aminotransferases at presentation were higher in DIAIH although the differences were not signi ficantly different[21].Recently,Licata et al.[22]reported that drug-induced liver injury could be classi fied into three subgroups according to different immune patterns that included drug-induced liver injury with negative non-organ speci fic antibody,drug-induced liver injury with positive non-organ speci fic antibody,and DIAIH.The authors found that DIAIH patients had signi ficantly higher values of serum aminotransferases and gamma globulins and higher frequencies of interface hepatitis,rosette formation and portal plasmocyte in filtration compared to the other two subgroups.DIAIH is currently thought as a clinical entity in which liver injury is associated with the presence of autoantibodies and histologic features of both autoimmune disease and drug-induced liver injury.Based on this,assessing the presence of DIAIH is practical and should be taken into consideration as a potential diagnostic approach[22,23].

Data are presented as median(interquartile range,IQR)or number(%).The Wilcoxon–Mann–Whiteney test was used to compare continuous variables and the Fisher’s exact test was used to compare categorical data.Wilcoxon rank sum test was used to compare nonparametric variables.All reported P values are two-tailed.A P＜0.05 was considered statistically signi ficant.For correlation analysis,the Pearson’s correlation coefficient was calculated.Statistical analyses were done using SPSS version 18(IBM,Armonk,USA).

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Over the last decade,an increased proportion of blood CD4+Foxp3+CD25−Tregs has been reported in patients with systemic lupus erythematosus,rheumatoid arthritis and type 1 diabetes mellitus as compared with healthy controls[8–11].However,the roles of the blood CD4+Foxp3+CD25−Tregs in these autoimmune diseases are not all the same.Our own results reported here showed an increased frequency of intrahepatic CD4+Foxp3+CD25−Tregs in DIAIH versus AIH patients and a positive correlation between the number of lobular CD4+Foxp3+CD25−Tregs and the degree of lobular in flammation in DIAIH.In contrast,the frequency of peripheral blood CD4+Foxp3+CD25−Tregs were similar between healthy controls and patients with DIAIH and AIH suggesting that the increased number of hepatic CD4+Foxp3+CD25−Tregs in DIAIH patients may be secondary to changes in in flammation status,be a speci fic histological feature,or be effector T cells,the latter may contribute to the lobular injury.This notion was supported by the previous studies which showed that conventional CD4+T cells,after TCR engagement in the presence of STAT5-signaling,can either transiently up-regulate Foxp3 to become T cells or stable express Foxp3 to become induced Treg[25,26].However,the function,origin and clinical signi ficance of the intrahepatic CD4+Foxp3+CD25−Tregs and the correlation between CD4+Foxp3+CD25−Treg and CD4+Foxp3+CD25+Treg need to be further clari fied.

Although the differentiation of DIAIH from classical AIH can be made by comprehensive comparing the clinical,serological,histological and immunocytological differences,several potential pitfalls especially for diagnosing AIH still need to be taken into account.First of all,ANA is a non-AIH-speci fic autoantibody and is also frequently detected in the serum of other chronic liver diseases.Secondly,the level of aminotransferases may not always be paralleled with the severity of interface hepatitis.Zone 3 necrosis is highly suggested as part of the histological spectrum of AIH,even if interface hepatitis is not observed[15,27].However,the presence of zone 3 necrosis also often raises the suspicion of DIAIH because of more frequencies of zone 3 necrosis in DIAIH in our study.Lastly,the most important issue in the diagnosis of AIH is exclusion of other liver diseases,because the laboratory findings and the histological features of AIH may also largely resemble those of other chronic liver diseases,including chronic viral hepatitis,and chronic active Epstein-Barr virus infection[16].

Fig.2.Immunostaining shows Foxp3+CD25−cells(black)and Foxp3+CD25+cells(black plus red)(A and C)which are limited in the locations of CD4+T cells(B and D).The CD4+Foxp3+CD25+/− Tregs can be recognized in DIAIH(A and B)or AIH(C and D).A–D:original magni fication×400.The number of CD4+Foxp3+CD25+/− Tregs were analyzed for comparison between DIAIH and AIH patients(E and F).

The frequency and distribution of Tregs in the liver biopsy sections were evaluated by double immunostaining with mouse anti-human Foxp3 monoclonal(Abcam,Cambridge,USA)and rabbit anti-human polyclonal CD25(MXB,Fuzhou,China)antibodies,and by immunostaining with mouse anti-human CD4 monoclonal antibody(MXB,Fuzhou)in adjacent serial sections.The experimental procedures for recognition of antigen-speci fic Foxp3 and CD25 as well as CD4 were carried out using a DouSPTM KIT(MXB,Fuzhou)according to the manufacturer’s instructions.Development of the chromogenic color reaction was accomplished using 5-Bromo-4-Chloro-3-Indolyl Phosphate/nitroblue tetrazolium(BCIP/NBT)or 3-amino-9-ethylcarbazole(AEC)(MXB,Fuzhou).The average number of CD4+Foxp3+CD25+/−Tregs for each lobule or portal tract was calculated from representative photomicrographs(×400)of single/double immunostaining for the cells in three lobules and three portal tracts from serial sections following single/double immunostaining.The relationship between the number of CD4+Foxp3+CD25−Tregs and the degree of lobular in flammation was analyzed.

当前，国内速冻蔬菜加工的主要问题在于规模不大、档次低、种类少、产供销不匹配等，这些问题制约着速冻蔬菜的发展。通常，外商要求单一品种长时间供应，且种类规格统一、品质高、新鲜度好[4]。因此，从确保原料新鲜与出口货源长久角度着手，速冻蔬菜相较于其他蔬菜生产更需要创建一个运送快捷、种类丰富的绿色蔬菜材料基地，这也迎合了我国国情，既可以提升农民生产运营的组织化水平，并且可以有效实现食品的品质控制及品质标准。在建设原料基地的同时，也要及时调节与完善栽种结构，根据食品等级规定应用或禁用药物、化肥，管理工业“三废”对自然环境的影响，确保速冻蔬菜原料的安全性。

Fig.3.The correlation between the degree of lobular in flammation and the frequency of CD4+Foxp3+CD25− Tregs in DIAIH(r=0.502,P＜0.01)(A)or AIH patients(r=0.069,P＞0.05)(B).

Fig.4.Frequencies of CD4+Foxp3+CD25+/−Tregs in peripheral blood were analyzed for DIAIH,AIH or healthy controls(HC).

Contributors

QLM,WSH,YK and SL performed the experiments and analyzed data.GRP designed the study and wrote the manuscript.BDR coordinated the study and edited the manuscript.GRP is the guarantor.

Box-Behnken响应面法优化荜茇总生物碱的提取工艺研究…………………………………………………… 杨家强等（13）：1802

F unding

This study was supported by a grant from the National Natural Science Foundation of China(81270544).

Ethical approval

The study was approved by the Ethics Committee of First Hospital of Jilin University(2008-0028).

Competing interest

No bene fits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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