Ulcerative-Colitis上课讲义

疾病名：溃疡性结肠炎英文名：ulcerative colitis缩写：UC别名：colitis gravis；colitis ulcerativa；溃结；慢性非特异性溃疡性结肠炎ICD号：K51.9分类：消化科概述：溃疡性结肠炎(ulcerative colitis，UC)，简称溃结，1875年首先由Willks和Moxon描述，1903年Willks和Boas将其命名为溃疡性结肠炎，1973年世界卫生组织(WHO)医学科学国际组织委员会正式命名为慢性非特异性溃疡性结肠炎。

病因尚未完全阐明，主要是侵及结肠黏膜的慢性非特异性炎性疾病，常始自左半结肠，可向结肠近端乃至全结肠，以连续方式逐渐进展。

临床症状轻重不一，可有缓解与发作相交替，患者可仅有结肠症状，也可伴发全身症状。

流行病学：溃疡性结肠炎的发病率有明显的地理分布特征，它的发生与该地区的经济发达程度密切相关。

发病率最高的地区是斯堪地那维亚国家和苏格兰，其次为英格兰和北美国家。

本病在这些国家中的发病率为(2～10)人/10万人口，女性比男性1∶0.8，高发年龄为30～60岁，而在亚洲及发展中国家，本病的发病率较低。

我国目前尚无大宗有关发病情况的统计资料。

据1986年在全国慢性腹泻学术会议上的报告，全国12所大医院在20年内确诊的溃疡性结肠炎病例仅581例，少于美国1家医院同期内的病例数。

发病率之低可见一般。

另据北京协和医院的报告从1974～1984年间经纤维结肠镜检查1428例病人中，检查出80例溃疡性结肠炎，检出率为5.6%。

且77.5%的病人为21～50岁的中青年人。

随着诊断技术设备的改进，本病的发病率近年来有升高趋势。

病因：溃疡性结肠炎确切病因还不明确。

目前关于本病的病因学有以下几个学说。

1.感染学说 已经证明某些细菌和病毒在溃疡性结肠炎的发病过程中起重要作用。

因本病的病理变化和临床表现与细菌痢疾非常相似，某些病例粪便中培养出细菌，部分病例应用抗生素治疗有效，似乎提示细菌性感染与本病有关。

O RIGINAL A RTICLEDecreased Mucosal Sulfide Detoxification Is Related to an Impaired Butyrate Oxidation in Ulcerative ColitisVicky De Preter,PhD,*Ingrid Arijs,PhD,*Karen Windey,MSc,*Wiebe Vanhove,BSc,*Severine Vermeire,MD, PhD,*Frans Schuit,PhD,†Paul Rutgeerts,MD,PhD,*and Kristin Verbeke,PhD*Background:Defective detoxification of sulfides leads to damage to the mucosa and may play a role in the etiology of ulcerative colitis(UC). The colonic mucosal thiosulfate sulfurtransferase(TST)enzyme removes H2S by conversion to the less toxic thiocyanate.In this study we meas-ured colonic mucosal TST enzyme activity and gene expression in UC and controls.In addition,the influence of sulfides on butyrate oxidation was evaluated.Methods:Colonic mucosal biopsies were collected from92UC patients and24controls.TST activity was measured spectrophotometrically. To assess gene expression,total RNA from biopsies was used for quantitative reverse-transcription polymerase chain reaction(RT-PCR).In20 UC patients,gene expression was reassessed after theirfirst treatment with infliximab.To evaluate the effect of sulfides on butyrate oxidation, biopsies were incubated with1.5mM NaHS.Results:TST enzyme activity and gene expression were significantly decreased in UC patients vs.controls(P<0.001).UC patients,classified into disease activity subgroups,showed a significantly decreased TST activity and gene expression in the subgroups as compared to healthy sub-jects(P<0.05for all).In20patients,gene expression was reassessed after theirfirst infliximab therapy.In responders to infliximab,a signifi-cant increase in TST gene expression was observed.However,TST mRNA levels did not return to control values after therapy in the responders. In controls,but not in UC,sulfide significantly decreased butyrate oxidation.Conclusions:We found an impaired detoxification mechanism of sulfide at TST protein and RNA level in UC.Inflammation was clearly associated with the observed TST deficiency.(Inflamm Bowel Dis2012;18:2371–2380)Key Words:ulcerative colitis,sulfide,butyrate,thiosulfate sulfurtransferase,gene expressionS hort-chain fatty acids(SCFAs)are the end-products of luminal microbial fermentation of undigested dietary carbohydrates and to a lesser extent of dietary and endoge-nous proteins.1The amount and type of these SCFAs depends largely on the structures available for fermentation and the composition of the colonic microbiota.With regard to the maintenance of colonic health and barrier function, butyrate has drawn the most attention,as it considered the major energy source for the colonic mucosa.2Inhibition of the oxidation of butyrate and other SCFAs induces an energy-deficient state ultimately resulting in reduced absorption of sodium,reduced secretion of mucin,and a shorter life of the colonocytes.3–5The capacity of the intestinal mucosa to oxidize bu-tyrate is decreased in patients with ulcerative colitis (UC).6–11UC is an inflammatory bowel disease(IBD)that likely results from an inadequate response of the mucosal immune system to the commensal intestinal microbiota in genetically susceptible subjects12and is characterized by alternating periods of relapses and quiescent disease.13 Recently,we confirmed a deficient butyrate metabolism in UC that was related to disease activity.10In addition,we showed that this butyrate oxidation deficiency was due to a combination of reduced butyrate transport and a defect in the butyrate oxidation pathway.11Biochemical,microbiological,and epidemiological findings implicate sulfide toxicity in the initiation of UC.14–16Additional Supporting Information may be found in the online version ofthis article.Received for publication February8,2012;Accepted February21,2012.From the Translational Research Center for Gastrointestinal Disorders(TARGID)and Leuven Food Science and Nutrition Research Centre(LFoRCe),University Hospital Gasthuisberg,KULeuven,Leuven,Belgium;and†Gene Expression Unit,Department of Molecular Cell Biology,KULeuven,Leuven,Belgium.Supported by a grant from the Fund for Scientific Research-Flanders(F.W.O.Vlaanderen)Belgium(FWO project no.G.0600.09);V.D.P.and I.A.are postdoctoral fellows and S.V.is a clinical researcher from the Fund forScientific Research–Flanders.Reprints:Kristin Verbeke,PhD,Translational Research Center forGastrointestinal Disorders(TARGID),O&N1,box701,Herestraat49,3000Leuven,Belgium(e-mail:Kristin.Verbeke@med.kuleuven.be).Copyright V C2012Crohn’s&Colitis Foundation of America,Inc.DOI10.1002/ibd.22949Published online20March2012in Wiley Online Library(wileyonlinelibrary.com).Inflamm Bowel Dis Volume18,Number12,December20122371Reducing sulfur compounds,and more particular hydrogen sulfide (H 2S),inhibit the oxidation of butyrate and other SCFAs.These events have been identified in in vitro 15,17and in vivo studies.18H 2S is an extremely toxic agent 19and is produced in the colonic lumen by the action of sulfate-reducing bacteria (SRB)on dietary and mucinous sulfate and sulfur amino acids such as methionine,cystine,cysteine,and taurine.20,21Increased fecal levels of H 2S and a higher metabolic activity of SRB have been reported in UC patients as compared to controls,22–24although normal fecal H 2S concentrations were found in another study.25To prevent the deleterious effect of sul-fide on the colonic epithelium,the large intestine disposes of two enzymes responsible for the clearance of H 2S.H 2S can be methylated to methanethiol by thiol S-methyltrans-ferase (TMT).26However,Levitt et al 27showed that the detoxification of H 2S proceeds mainly via oxidation to thiosulfate and further conversion to the less toxic thio-cyanate by thiosulfate sulfurtransferase (TST,also known as rhodanese)in the presence of cyanide.In the present study we explored whether the activity of TST was related to UC disease activity and we corre-lated the enzyme activity to the expression of the corre-sponding gene in the mucosa of UC patients.In addition,we explored the correlation between sulfide detoxification and butyrate metabolism.In a subset of patients,mucosal gene expression of the TST gene was reassessed after their first treatment with infliximab (Remicade;Centocor,Mal-vern,PA)to evaluate whether reduced TST activity was secondary to inflammation.MATERIALS AND METHODSEthics StatementThe study was carried out at the University Hospital Gasthuisberg (Leuven,Belgium)and the trial was registered at (NCT 01282905).The study was approved by the University Hospital Ethics Committee and written con-sent was obtained from all participating subjects.SubstratesKrebs-Henseleit buffer,sodium butyrate,sodium chlo-ride,glycine,sodium hydrogen sulfide,potassium cyanide,po-tassium thiocyanate,and Triton X-100were all obtained from Sigma-Aldrich (St.Louis,MO).Ferric nitrate,formaldehyde (37%),dipotassium phosphate,and monopotassium phosphate were from Merck (Darmstadt,Germany),and sodium thiosul-phate and EDTA from Acros Organics (Geel,Belgium).Nitric acid (65%)was from Chem-Lab (Zedelgem,Belgium),perchloric acid from Fisher Scientific (Leicestershire,UK),Na-[1-14C]-butyrate from Moravek (Brea,CA),and hyamine-hydroxide from Perkin-Elmer (Waltham,MA).Study DesignCohort 1:Kinetics of the TST EnzymeRectal biopsy specimens were obtained from four UC patients and four controls who underwent colonoscopy for the investigation of irritable bowel syndrome or screening for adenomas (characteristics:Table 1).From each patient,two biopsy specimens were taken in adjacent sites of affected parts of the rectum that were directly frozen in liquid nitrogen forTABLE 1.Baseline Characteristics of the UC Patients and Healthy Controls (HC)in the Different Cohorts (Median (25th –75th Percentile))Cohort 1Cohort 2Cohort 3Cohort 4UCHCUCHCUCHCUC Responder UCNonresponderMale/female 3m/1f 2m/2f 35m/33f 14m/6f 9m/7f 10m/14f 2m/4f 9m/5f Median age (years)45(43-50)72(60-83)49(35-57)61(57-66)37(33-52)57(43-67)31(28-39)49(40-63)Median age at diagnosis (years)35(30-44)—36(25-46)—31(25-51)—21(14-24)35(29-47)Median disease duration (years)9(6-13)—8(4-15)—5(2-8)—20(11-22)10(6-15)Disease location(Montreal classification)Pancolitis 2—23—6—410Left-sided colitis 2—41—9—24Proctitis ——4—1———Medication None 1—3—————5-Aminosalicylates ——34—6———Corticosteroids ——9—4———Immunosuppressants 1—7—5———Anti-TNF 2—15—1—614Inflamm Bowel Dis Volume 18,Number 12,December 2012De Preter et al 2372measurement of TST activity.Disease activity was assessed endoscopically using the Mayo Endoscopic Score proposed by Schroeder et al.28Score0was considered inactive disease, score1mild,score2moderate,and score3severe disease.The biopsy specimens were incubated with different concentrations of Na2S2O3(0,10,25,50,75,100mM). Enzyme kinetics as a function of the substrate concentration was assessed using the Michaelis–Menten equation.The appa-rent values of V max(maximum rate of metabolism)and K m (the substrate concentration at which the reaction rate is half the maximum value)were calculated byfitting the data to a Lineweaver–Burke plot.29Cohort2:Case–Control StudyRectal biopsy specimens were obtained prospectively from68UC patients and from20controls who underwent colonoscopy for the investigation of irritable bowel syndrome or screening for adenomas(characteristics:Table1).From most patients,9to10biopsy specimens were taken in adja-cent sites of affected parts of the rectum.The endoscopic evaluation of disease activity was assessed as described above. Two biopsies were directly frozen in liquid nitrogen for mea-surement of TST activity and two biopsies were immersed in RNAlater(Applied Biosystems,Lennik,Belgium)for RNA isolation.All biopsies were stored atÀ80 C until analysis. Four biopsies were transported immediately to the laboratory in ice-cold,pregassed(95%O2/5%CO2)Krebs–Henseleit buffer,pH7.4,containing11mM glucose for measurement of butyrate oxidation.Results of the latter experiment have previ-ously been published.11The residual biopsies of the UC patients werefixed in Carnoy’sfixative for5hours,dehydrated, cleared,and paraffin-embedded for histological examination. Cohort3:Influence of NaHS on ButyrateOxidation RateThe influence of1.5mM NaHS on the butyrate oxida-tion rate was evaluated in24biopsies obtained prospectively from controls and16from UC patients(characteristics:Table 1).Four rectal biopsy specimens were obtained as described above.The biopsies were transported immediately to the labo-ratory in ice-cold,pregassed(95%O2/5%CO2)Krebs–Hen-seleit,pH7.4,containing11mM glucose for measurement of butyrate oxidation.The NaHS solutions was freshly prepared on the day of the experiment and added to the incubation vials just before the addition of Na-[1-14C]-butyrate.Cohort4:Retrospective Study of the Influence of Infliximab Treatment on TST Gene Expression From20UC patients(characteristics:Table1)with moderate to severe UC(endoscopic Mayo score of at least2), refractory to corticosteroids and/or immunosuppression,biopsy specimens were obtained within a week prior to thefirst infu-sion of infliximab.Infliximab was administered as a single infusion(5mg/kg body weight)or as a repeated dose at weeks 0and2(each time5mg/kg body weight).The patients under-went a second colonoscopy with rectal biopsies4weeks after thefirst infliximab infusion in the case of a single infusion or 6weeks after thefirst administration when they received a loading dose of infliximab.The biopsies were taken at sites of active inflammation but at a distance of ulcerations.In the case of healing at control endoscopy,the biopsies were obtained in the areas where lesions were present before ther-apy.The response to infliximab was assessed at the time of the second endoscopy.Response was defined as a complete mucosal healing with a Mayo endoscopic subscore of0or130 and a grade0or1on the histological score for UC.31,32 Patients who did not achieve complete healing were consid-ered nonresponders,although some of them showed endo-scopic and/or histological improvement.Half of the biopsies were directly frozen in liquid nitrogen and stored atÀ80 C for isolation of RNA.The residual biopsies werefixed in Car-noy’sfixative for5hours,dehydrated,cleared,and paraffin-embedded for histological examination as described above. Histological AssessmentHistological disease activity was assessed on biopsies taken at the same endoscopic examination.In UC,the grading scale for histological assessment of inflammation developed by Geboes et al was used.31Shortly thereafter,hematoxylin/ eosin-stained sections from biopsies were evaluated for archi-tectural change(grade0),chronic inflammatory infiltrate (grade1),lamina propria neutrophils and eosinophils(grade 2),neutrophils in the epithelium(grade3),crypt destruction (grade4),and erosion or ulceration(grade5).TST AssayTST activity was measured using sodium thiosulphate as substrate based on the work of Aminlari and Gilanpour33with minor adaptations.Immediately prior to the enzyme assay,a bi-opsy specimen was homogenized with a hand homogenizer (Pellet mixer,VWR,UK)and suspended in200l L of0.1M potassium phosphate buffer with1mM EDTA and0.5%Triton X-100(pH7.9).Ten l L of tissue homogenate was incubated with15l L of a known concentration of sodium thiosulfate and 125l L60mM potassium cyanide for10minutes at37 C. Reactions were terminated on ice by the addition of50l L 37%formaldehyde.The reaction product,thiocyanate,immedi-ately forms a red precipitate upon addition of100l L25mM ferric nitrate containing20%nitric acid and was detected spec-trophotometrically at450nm(iEMS Reader MF,Labsystems, Helsinki,Finland).Each sample was assayed in triplicate and a sample in which the enzyme was inactivated by heat(5minutes at100 C),served as control.The protein concentration of the biopsy specimens was measured using the BSA protein assay (Pierce,Rockford,IL).An equal volume of0,1M potassium phosphate buffer containing1mM EDTA and0.5%Triton X-100was used as the control for background absorbance. Quantification of Butyrate OxidationThe procedure for measurement of the butyrate oxida-tion rate in biopsy specimens has been described previously.10Inflamm Bowel Dis Volume18,Number12,December2012TST Activity and Butyrate Metabolism in UCBriefly,each specimen was placed in a 10-mL conical incuba-tion flask,together with 1mL sterile Krebs–Henseleit buffer (pH 7.4)containing 11mM glucose and 1mM Na-butyrate (Sigma-Aldrich).Two vials prepared without biopsies served as blanks.After addition of 37kBq Na-[1-14C]butyrate (Sigma-Aldrich)the vials were gassed with 95%O 2/5%CO 2for 30seconds and immediately sealed with a gas-tight rubber stopper equipped with a polypropylene center well.The vials were incubated for 2hours in a shaking water bath (37 C,120oscillations/min).The reaction was stopped by injecting 0.25mL 10%perchloric acid through the stopper into the incuba-tion medium.To trap the released 14CO 2,0.3mL 0.5M hya-minehydroxide was injected into the center wells.After an equilibration period of 90minutes at 4 C,the center wells were transferred into scintillation vials together with 10mL scintillation liquid (Hionic Fluor,Packard,Downers Grove,IL).Radioactivity was measured in a liquid scintillation coun-ter (Packard,Tri-card 2100TR).Isolation of RNATotal RNA was extracted from the biopsy specimens using the RNeasy Mini Kit (Qiagen,Valencia,CA)according to the manufacturer’s instructions.The integrity and quantity of total RNA were assessed with a 2100Bioanalyzer (Agilent,Waldbronn,Germany)and a Nanodrop ND-1000spectropho-tometer (Nanodrop Technologies,Wilmington,DE),respec-tively.The extracted RNA was used for quantitative real-time reverse-transcription polymerase chain reaction (qPCR)analysis.qPCR AnalysisqPCR was performed to quantify the gene expression of TST and b -actin was used as the endogenous reference gene.In addition,the expression of interleukin 8(IL-8)was meas-ured as marker for inflammatory activity.34cDNA was synthe-sized from 0.5l g of total RNA using the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas,St.Leon-Rot,Germany),following the manufacturer’s protocol.Primers and dual-labeled probes were designed using Oligoanalyzer 3.1software and synthesized by Sigma-Genosys (Haverhill,UK).The oligonucleotide sequences are shown in Supporting Table S1.qPCR was performed in a final reaction volume of 25l L on a Rotor-Gene 3000instrument (Corbett Research,Mor-tlake,Australia),using QuantiTect Multiplex PCR NoROX Kit (Qiagen,Venlo,The Netherlands),according to the manu-facturer’s instructions.Cycle threshold values were determined by Rotor-Gene 6.0.16software.All samples were amplified in duplicate reactions.The relative expression of target mRNA levels was calculated as a ratio relative to the b -actin refer-ence mRNA.35StatisticsStatistical analysis was performed with SPSS software (SPSS 17.0for Windows;Chicago,IL).As the data were not normally distributed (Shapiro–Wilks),they were presented as median (25th–75th percentile).Data were compared by non-parametric tests (Friedman analysis of variance [ANOVA]plus Wilcoxon test or Kruskal–Wallis plus Mann–Whitney U -test,with Bonferroni correction for multiple testing).The level for statistical significance was set at P <0.05.For correlation analysis,Spearman’s rank correlation coefficient was used.Kendall Tau rank correlation coefficient was applied to corre-late mRNA levels to endoscopic/histological evaluation.RESULTSTST Activity Rate at Different ConcentrationsAt all five concentrations of thiosulfate (10,25,50,75,and 100mM),the TST enzyme activity was signifi-cantly lower in biopsies from UC patients (two mild UC,one moderate UC,and one severe UC)as compared to con-trol subjects (P ¼0.021for all)(Table 2).Apparent Km and V max values were determined using a Lineweaver–Burk plot (Fig.1).The V max value was significantly higher in control subjects than in UC patients (P ¼0.043),whereas Km values were not different between the two groups (Table 2).The TST activity was evaluated in the presence of 50mM Na 2S 2O 3in the case–control study.TABLE 2.TST Enzyme Activity (nmol/mg protein.min)at Different Concentrations of Sodium Thiosulfate and Kinetic Constants (V max (nmol/mg protein.min)and K m (Mm))for the TST Activity in UC and Controls (Median (25th –75th percentile);a ¼0.05)Controls (n ¼4)UC (n ¼4)P -value TST enzyme activity10mM 211(176-260)87(73-91)0.02125mM 354(263-429)134(107-150)0.02150mM 441(338-540)231(194-251)0.02175mM 447(328-549)226(206-272)0.021100mM 430(328-528)227(194-241)0.021KineticsV max 512(415-512)291(237-291)0.043K m26(23-28)25(23-26)NSInflamm Bowel Dis Volume 18,Number 12,December 2012De Preter et al 2374Case–Control Study TST Enzyme ActivityThe TST enzyme activity was statistically signifi-cantly decreased in UC patients as compared to the con-trols (171.7[102.4–221.2]vs.393.9[283.3–609.6]nmol/mg protein.min;P <0.0001).Classification of the UC patients according to disease activity using endoscopic cri-teria demonstrated a significantly decreased TST activity in inactive (217.8[185.5–267.3]nmol/mg protein.min;P ¼0.001),mild (219.9[210.4–267.1]nmol/mg protein.min;P ¼0.03),moderate (103.8[82.0–152.1]nmol/mg protein.min;P <0.001)and severe (100.3[63.2–139.9]nmol/mg protein.min;P <0.001)as compared to normal colon (Fig.2).Furthermore,the group with moderate and severe UC differed significantly from the groups with inactive (P <0.001for both)and mild disease (P ¼0.004and P ¼0.002,respectively).TST activity was not different between inactive and mild UC or between moderate and severe UC.Similar results were obtained when UC patients were categorized based on histological criteria (Table 3).TST enzyme activity was inversely correlated with the endoscopic evaluation of disease activity (Kendall’s s ¼À0.478,P <0.001)and with the histological assessment of disease activity (Kendall’s s ¼À0.359,P <0.001).mRNA ExpressionqRT-PCR analysis showed a significant decrease in TST expression in UC as compared with normal tissue (rel-ative TST mRNA expression [corrected for b -actin]of 0.74[0.39–1.22]vs.1.86[1.55–2.16];P <0.001).Subsequent division of the UC samples into disease activity groups according to endoscopic criteria demonstrated a signifi-cantly decreased TST expression in inactive (1.45[1.25–1.50]),mild (0.94[0.51–1.12]),moderate (0.56[0.37–0.59])and active (0.35[0.29–0.44])disease as compared to controls (P <0.001for all)(Fig.3).TST mRNA levelswere also significantly lower in mild,moderate,and severe disease than in inactive disease (P ¼0.003for mild and P <0.001for moderate and severe).TST mRNA levels in severe disease were also significantly lower than in mild disease (P ¼0.02).TST expression was not different between mild and moderate,or between moderateandFIGURE 1.Kinetic analysis of the SCN Àproduction from thiosulfate in biopsies isolated from control subjects and patients with UC using a Lineweaver–Burkplot.FIGURE 2.TST enzyme activity in control colon (n ¼20)and inac-tive (n ¼27),mild (n ¼9),moderate (n ¼14),and severe (n ¼18)UC (horizontal bar,median).a–c Medians not sharing the same superscript letter within a column are significantly different (P <0.001).TABLE 3.TST Enzyme Activity and Gene Expression UC Compared to Control Colon with Disease Activity Based on Histological Criteria (Median (25th –75th percentile))Enzyme ActivityGene Expression Controls (n ¼20)393.9(283.3-609.6)a 1.86(1.55-2.10)a UC grade 0(n ¼10)200.6(163.2-254.9)b 1.40(1.08-1.64)b UC grade 1(n ¼21)215.8(196.8-257.6)b 1.24(1.13-1.45)b UC grade 2(n ¼6)105.5(92.9-240.9)b,c 0.73(0.60-0.87)c UC grade 3(n ¼13)166.3(90.7-238.1)b,c 0.54(0.31-0.73)c,d UC grade 4(n ¼6)116.5(101.2-149.3)c 0.34(0.30-0.41)c,d UC grade 5(n ¼12)105.7(79.1-144.3)c0.36(0.33-0.50)ddMedians not sharing the same superscript letter within a column are sig-nificantly different (P <0.05).Inflamm Bowel Dis Volume 18,Number 12,December 2012TST Activity and Butyrate Metabolism in UCsevere disease.Similar results were obtained when UC patients were categorized based on histological criteria (Ta-ble 3).The mRNA level of TST correlated positively with the TST enzyme activity (Spearman’s q ¼0.403,P <0.001),whereas an inverse relationship was found with Mayo score (Kendall’s s ¼À0.650[P <0.001])and the histological score (Kendall’s Tau s ¼À0.631[P <0.001]).Additionally,we measured IL-8mRNA levels,as a marker of inflammatory activity,which were significantly higher in inflamed (mild,moderate,and severe disease)and noninflamed biopsy specimens (inactive disease)than in control biopsy specimens (P <0.001for all)(Fig.3).IL-8mRNA levels were strongly correlated with the endo-scopic scores of inflammation (Kendall’s s ¼0.734[P <0.001])and the histological score (Kendall’s s ¼0.609[P <0.001]),but were negatively correlated with the TST mRNA levels (Spearman’s q ¼À0.764[P <0.001]).Correlation of TST Activity and Butyrate Oxidation DataPrevious evaluation of the butyrate oxidation rate in biopsy specimens of the same UC patients and controls showed a significantly lower oxidation rate in the group of UC patients as compared to the control group (P <0.0001)and the impaired butyrate oxidation rate in UC correlated with disease activity.11Combination of the TST activity and butyrate oxidation results demonstrated a sig-nificant relationship between both in UC and controls (Spearman’s q ¼0.699,P <0.001;Fig.4).Influence of NaHS on the Butyrate Oxidation RateThe presence of 1.5mM NaHS in the incubation me-dium significantly reduced the butyrate oxidation rate in bi-opsy specimens of control patients (P ¼0.003)(Table 4),yet the oxidation rate remained significantly higher than in UC patients (P <0.001).In UC patients the butyrate me-tabolism rate was not influenced by addition of NaHS.Influence of Infliximab on TST Gene ExpressionOf the 20patients studied before and after their first infliximab treatment,six (two male;four female)showed complete endoscopic and histological healing andwereFIGURE 3.TST mRNA expression in control colon (n ¼20)and inactive (n ¼27),mild (n ¼9),moderate (n ¼14),and severe (n ¼18)UC (horizontal bar,median).a–d Medians not sharing the same superscript letter within a column are significantly different (P <0.05).FIGURE 4.Relation between TST enzyme activity and butyrate oxi-dation rate in UC (^)and controls (~)combined (q ¼Spearman’s correlation coefficient).Inflamm Bowel Dis Volume 18,Number 12,December 2012De Preter et al 2376defined as responders,whereas 14were considered nonres-ponders (nine male;five female).Responders were signifi-cantly younger than nonresponders (31[28–39]vs.49[40–63]years;P ¼0.02).In all patients,responders and nonresponders to-gether,the mRNA levels of TST were significantly lower before and after therapy than in controls of cohort 2(P <0.001;Fig.5).In contrast,IL-8mRNA levels were signifi-cantly higher both before and after therapy as compared to controls (P <0.001;Fig.5).Infliximab treatment significantly increased the rela-tive TST mRNA levels (corrected for b -actin)in the res-ponders group from 0.49(0.44–0.57)to 1.03(0.82–1.37)(P ¼0.028),whereas the TST mRNA levels remained unchanged in the nonresponders (0.36[0.31–0.52]vs.0.51[0.42–0.74],P ¼NS).Although TST expression increased after infliximab treatment,the levels did not reach the val-ues observed in controls (1.86[1.55–2.16];P <0.001).The levels of the inflammation marker IL-8significantly decreased after treatment in the responders from 8.13(4.34–10.27)to 1.15(0.86–1.72)(P ¼0.028).IL-8levels tended to decrease in the nonresponders:37.15(27.36–64.69)vs.17.05(5.29–35.26)(P ¼0.056).DISCUSSIONH 2S is the principle product of colonic SRB and a fermentation product of dietary and mucinous sulfate and sulfur-containing amino acids.The effects of H 2S on colo-nic cells are well documented.H 2S inhibits cellular respira-tion by inhibiting oxygen binding to mitochondrial cyto-chrome-c-oxidase.This inhibition causes a reduction in the production of cellular adenosine triphosphate (ATP).36H 2S also affects different cellular pathways (cell cycle,inflam-mation,cell adhesion)at concentrations similar to those found in the colon.37Roediger et al 14showed that H 2S impedes butyrate oxidation,the principal energy source for colonocytes.Several lines of experimental evidence impli-cate hydrogen sulfide as a damaging agent in the pathoge-nesis of UC.Experiments in several animal models suggest that bacteria are essential in the development of colitis,with an-tibiotic treatment or rearing of animals in germ-free condi-tions leading to amelioration of colonic inflammation.38In UC,a dysbiosis in the gut microbiota composition has been demonstrated as compared to healthy control sub-jects.39The activity of SRB in UC patients is higher than in healthy controls,23,40although cultures obtained from mucosal biopsies have not shown such clear differences.41As a consequence,metabolites of the intestinal bacteria also differ in fecal samples of IBD patients as compared to healthy controls.42However,some groups challenge the notion that bac-terially produced H 2S contributes to the pathogenesis of UC.Moore et al 25found no significant difference in lumi-nal sulfide (free and total)levels between healthy control patients and patients with UC.Jorgensen and Mortensen 43differentiated between the effect of free and bound fecal sulfide on the butyrate oxidation capacity in rat colono-cytes.Their results indicated that the majority of fecal sul-fide is bound and metabolically inert.These results do not support a primary etiological role for luminal sulfide in UC.Questions have been raised as to the accuracy of the estimates of H 2S in the lumen.44Fecal samples are the end products of colonic fermentation,so their analysis probably only reflects the metabolic processes occurring in the distal colon rather than in the more proximal parts.Also,most of the H 2S that is produced in the lumen of the intestine is absorbed and metabolized.45Indeed,the mucosal detoxifi-cation of sulfides occurs via a number of enzymes.The colonic mucosa can clear hydrogen sulfide from the colonic lumen by methylation to methanethiol.How-ever,Levitt et al 46showed that the oxidation rate of H 2S by the colonic mucosa was 105times higher than the meth-ylation rate.Recent data shows that TST is not involved inTABLE 4.Influence of NaHS on Butyrate Oxidation Rate (nmol/mg protein.h)in UC and Controls (median (25th –75th percentile))Baseline1.5mM NaHSP -valueControls (n ¼24)39.75(33.99-42.79)32.84(25.74-37.70)P ¼0.003UC (n ¼16)10.92(4.98-23.47)8.66(3.70-22.17)NSFIGURE 5.qRT-PCR analysis of TST (A)and IL-8(B)in controls (n ¼20)(~),infliximab UC responders (n ¼6)(^),and infliximab UC nonresponders (n ¼14)().Lines between two points represent the change in expression before and after treatment.Inflamm Bowel Dis Volume 18,Number 12,December 2012TST Activity and Butyrate Metabolism in UC。

【英文名称】Ulcerative Colitis[编辑本段]【基本概述】是一种局限于结肠粘膜及粘膜下层的炎症过程。

病得多位于乙状结肠和直肠，也可延伸至降结肠，甚至整个结肠。

病理漫长，常反复发作。

本病见于任何，但20－30岁最多见。

[编辑本段]【病理】溃疡性结肠炎-病因溃疡性结肠炎的病因至今仍不明。

虽有多种学说，但目前还没有肯定的结论。

细菌的原因已经排除，病毒的原因也不象，因为疾病不会传染，病毒颗粒也未能证实。

克隆病患者血清溶酶体升高，溃疡性结肠炎患者则为正常。

基因因素可能具有一定地位，因为白人中犹太人为非犹太人的2～4倍，而非白人比白人约少50%，Gilat等对特拉维也夫的犹太人的研究中报道溃疡性结肠炎的发病率明显降低，为3.8/10万，而丹麦哥本哈根为7.3/10万，英国牛津7.3/10万和美国明尼苏达州7.2/10万。

此外，女性与男性比例也仅0.8而其他报道为1.3。

显然地理上和种族上的差异影响本疾病的发生。

心理因素在疾病恶化中具有重要地位，现在已明确溃疡性结肠炎患者与配对对照病例相比并无异常的诱因。

再者，原来存在的病态精神如抑郁或社会距离在结肠切除术后明显改善。

有人认为溃疡性结肠炎是一种自身免疫性疾病，许多病人血中具有对正常结肠上皮与特异的肠细菌脂多糖抗原起交叉反应的抗体。

再者，淋巴细胞经结肠炎病员的血清培养可变为对结肠上皮有细胞毒性。

此外在结肠炎病人的T和B淋巴细胞群中发现有改变。

但以后认识到这些异常并非疾病发生所必须，而是疾病活动的结果。

事实上，Brandtzueg等清楚地证明并非溃疡性结肠炎病人残留腺体中组织水平免疫球蛋白活动有缺陷，IgA运输正常，而IgG 免疫细胞反应为对照病员的5倍。

因此，有可能IgG在疾病的慢性过程中具有作用，但与疾病发生则无关。

总之，目前认为炎性肠道疾病的发病是外源物质引起宿主反应，基因和免疫影响三者相互作用的结果。

根据这一见解，慢性溃疡性结肠炎与克隆病是一个疾病过程的不同表现。

白细胞净化——溃疡性结肠炎的新疗法 Leukocytapheresis with Leukocyte Removal Filter as NewTherapy for Ulcerative ColitisKoji Sawada, Kuniki ohnishi, Tadashi Kosaka, Shinji Chikano,Yoshiro Yokota, et alTherapeutic apheresis 1997;1(3): 207-211用白细胞除去滤器的白细胞净化疗法（leukocytapheresis, LCAP ）用于多种疾病治疗的有效性已被确定，包括免疫应答紊乱性疾病如类风湿性关节炎、突眼性甲状腺功能亢进症（Graves 病）、白塞病及寻常性天疱疮等。

溃疡性结肠炎（ulcerative colitis, UC ）是以来源于循环的炎性细胞，包括单核细胞、淋巴细胞和中性细胞的浸润为特征。

若干因素导致炎性细胞在病变局部聚集；这些因素包括在微环境中细胞因子的释放及循环炎性细胞表达的活性表面标志与其在局部靶细胞表达的活性表面标志之间的相互作用。

近来研究显示，致炎细胞因子包括IL-1、IL-6、IL-8、TNF-α和IFN-γ分泌增加，诱导在UC 肠部炎症的发生，可能起着重要作用。

这些细胞因子强力诱导炎症反应和在炎症UC 肠黏膜表达单核细胞数量与同外周血一样增多。

在最近2年来，我们已经很方便地将LCAP 用于治疗难治性UC ，现在报告我们的临床研究结果，包括部分已经发表过的资料。

观察在LCAP 实施之前、实施过程中和实施之后外周血细胞的变化及白细胞的除去率。

这个报告客观的评价在LCAP 治疗前、治疗过程中和治疗后白细胞（WBC ）、红细胞（RBC ）、血小板数（PLT ）和白细胞分类百分数的变化，以及在LCAP 治疗期间经过白细胞滤器柱前后，人类白细胞抗原HLA-DR + 细胞和粘附分子阳性细胞的除去率。