The Diagnosis And Treatment Of TREM-1 In Secondary Infection Of Severe Acute Pancreatitis

Severe acute pancreatitis(SAP) is a common emergency in clinic,which is very dangerous and difficult to treat with some unknown characters such as etiology and pathogenesis.The incidence of SAP has the trend of rising in recent years.The principle of individual treatment of SAP has been widely accepted,which markedly enhance the clinical efficacy.However,the mortality rate is still as high as 20%-30%.There are two deaths peaks in the duration of SAP.The first one caused by SIRS occurs in the course of the first week,and the second is infection,which occurs in 1-3 weeks.Most of the death(80-90%) is caused by infection.The early diagnosis of infection has become the focus of controversy.The most caution in clinic is that non-selective and long time application of broad-spectrum antibiotics will increase the probability of double infection and extend the ICU duration,even increase mortality.Pancreatic tissue necrosis is not necessarily infection,and the surgery is not an absolute indication.The delay caused by the failure of non-surgical treatment in pancreatic infection is fatal.The high false positive rate(FPR) and a long time(about two weeks) of the bacteriological examination of the specimens from the fine-needle aspiration is also dangerous.It is difficult to clinical satisfaction with this method of identification the infected pancreatic necrosis.The majority process SAP is often not gradual and changes in a short time,which always result in multiple organ dysfunction.Hence,Prof.Sheng-dao Zhang,Qun-hua Zhang and Yu-Pei Zhao pointed out that early diagnosis SAP infection is critical in the treatment of SAP. How to identify early diagnosis of secondary infection is the most controversy in the SAP treatment.C-reactive protein and procalcitonin has been used to predict infection and prognosis in clinic,but the value is very limited.One of the main problems is that there is no single indicator in serum can accurately predict early infection in pancreatic necrosis.A sensitive and specific indicator is required to be found in monitoring disease progression,guiding the use of antibiotics and showing the time of surgery.The triggering receptor expressed on myeloid cells(TREM) family is a member of the immunoglobulin superfamily and includes at least two activating receptors,namely TREM-1 and TREM-2.The understanding of the process of inflammatory response, included in its start and development,is deeply known since the first discovery of TREM-I in 2001.TREM-1 is mainly distributed in the peripheral blood neutrophil leukocyte and monocytes subsets,which are natural immune response cells.The expression of TREM-1, identifying specifically the surface receptor of pathogens,is also selectively found in the phagocytes in the pulmonary,intestinal fluid and other body fluids.The TREM-1,include extracellular,transmembrane domain and a short cytoplasmic tail section of the conservative structure,is a transmembrane glycoprotein.Engagement of TREMs,after association with the adapter protein DAP12(which contains an immunoreceptor tyrosine-based activation motif),This residue allows pairing with transmembrane adapter proteins,which contain a negatively charged amino acid in the transmembrane domain and a cytoplasmic immunoreceptor tyrosine-based activation motif(ITAM).Bouchon reported that the TREM-1 expression was significantly increased in neutrophilic leukocytes and monocytes surface in infection due to Gram-positive bacteria,fungi or negative bacteria, which caused acute inflammation and granulomatous inflammation,as well as endotoxin (LPS) or other organisms resulting in septic shock.However,in the non-infected immune complex inflammatory response,such as scales psoriasis and ulcerative colitis,it will not appear TREM-1 expression.In vitro,the combined culture with living bacteria or its cell wall composition and neutrophils and monocytes can increased the surface expression of TREM-1.The diagnostic value of TREM-1 in inflammatory response is receiving increasing attention.TREM-1 expanded the inflammatory response effect caused by bacterial and fungal infections,.There is an autocrine loop of positive feedback formed by downstream cytokines and TREM-1,which will influence the natural immunity and enhance the inflammatory reaction amplification.TREM-1-mediated signal transduction pathway plays a crucial role in the cascade of inflammatory reaction amplification and the incidence of sepsis.Gibot demonstrated that the rapid detection of the soluble TREM-1(sTREM-1) in bronchoalveolar lavage fluid with Western methods,would confirm the judgment of bacterial or fungal pneumonia.In patients with septic shock,the level of TREM-1 expression in monocytes is also high.TREM- 1 is the potential molecular target in the treatment of inflammation.According the knowledge that the TREM included in IgSF and TREM-1 and IgG had some homology, we demonstrate the possibility of the TREM-1 fusion protein IgG integrated with the two immunoglobulin characteristics.This fusion protein with the surface of immune cells TREM-1 competitive with TREM-1 ligand to inhibit activation of the role of signal.The research in TREM is currently a hot topic.The diagnostic value of TREM-1 on the bacterial / fungal infection has been increasingly recognized,and the exploration as a therapeutic target continues.In China,the research of TREM-1 is almost blank.There is still in dispute about the pathogenesis of SAP.The newly propose that inflammatory mediators involved in the Waterfall reaction induced systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction,enriched the SAP pathogenesis. A research demonstrated that there is obvious difference of the expression of TREM-1mRNA in interleukin in mild acute pancreatitis(MAP) and severe acute pancreatitis(SAP).The result suggested that the TREM-1 will play an important role in acute pancreatitis.Firstly,the study is to reveal the importance position of TREM-1 in secondary infection of SAP,through analyzing the mRNA expression level of TREM-1 in SAP rats and pigs.Second,assess the value of TREM-1 as a diagnosis indicator of intra-abdominal secondary infection in SAP patients,examining the TREM-1 expression in peripheral blood,comparing with bacterial culture as the gold standard.Third,respectively in human being and rats,construct TREM1-IgG fusion protein and the protein eukaryotic expression vector,and in CHO cells stability expression is achieved.Fourth,the expression and purify of rat TREM-1/ human IgG1 fusion protein in Escherichia coli are achieved.Last, intervention treatment in SAP rats with decoy receptors was to certify its therapeutic targets value.1.Detection of TREM-1 expression in pancreatic tissue of SAP ratObjective:To detect TREM-1 expression in pancreatic tissue of SAP rats and sham-operated ratsMethods:18 male SD rats were randomly divided into sham-operated group,the severe acute pancreatitis(SAP) group and secondary infection group,6 respectively.The construction of the SAP model was successfully achieved through retrograde injection of 3%sodium taurocholate in pancreatic duct of rats.After 6h,the Escherichia coli suspension Construction was injected in abdominal cavity for INP rat model.The histological changes in the pancreas were observed,and the levels of amylase were detected using automatic biochemical analyzer.The expression of TREM-1 mRNA in pancreatic tissue was detected through real-time PCR.Results:The SAP models were successfully constructed.The rats in three groups were sacrificed in 24 hours.The 0.5 g pancreatic tissue was removed and the total mRNA was extracted.The rats TREM-1 was amplified through the reverse transcription-polymerase chain reaction(RT-PCR).The recovery and purification of the PCR products with the expected length was made,and the same sequence was clarified with the announced GenBank sequence.With the real-time PCR,the mRNA expression of TREM-1 in INP group was significantly higher than that in SAP group(P＜0.05).And the mRNA expression in sham-operated group was lower than that in SAP group.Conclusion:The TREM-1 expression was significantly increased in SAP and the secondary infection models in rat.These data suggested that TREM-1 might play an important role in the process of severe acute pancreatitis.2.The construction of secondary infection with severe acute pancreatitis in pig model and the detection of TREM-1 gene expression in peripheral bloodObjective:To construct the model of secondary infection with severe acute pancreatitis in pig and detect the TREM-1 expression in peripheral blood before and after secondary infection.Methods:3%sodium pentobarbital was injected in abdominal cavity in 6 Pigs.The SAP models were successfully constructed,using injection of the mixture of sodium taurocholate and trypsin in pancreatic duct with duodenal endoscopy.After 24 hours of the procedure,pancreatitis was confirmed with CT scan.Then the blood samples were collected.Immediately the activated E.coil bacterium was injected in necrosis area of the pancreas by the CT-guided needle.7 days later,the local area and the surrounding circumstances were observed with pancreatic CT.According to the scans,weather the injection of the activated strain of E.coli standard suspension was judged.14 days later, the animals were killed.The 5ml blood sample was executed before death.The pancreatic tissue and the pathological changes were observed.The relative quantitative level of TREM-1 mRNA expression in the peripheral blood was detected by real-time PCR before and after secondary infection in pigs.Results:The constructions of secondary infection model with severe acute pancreatitis in pigs were successful,confirmed by imaging and histological changes.The pig TREM-1 was amplified through the reverse transcription-polymerase chain reaction(RT-PCR).The recovery and purification of the PCR products with the expected length was made,and the same sequence was clarified with the announced GenBank sequence.The relative TREM-1 expression before and after secondary infection with SAP in pig were 1.51±1.38 and 4.99±3.28,respectively.Paired t-test showed that the blood level of TREM-1mRNA after the infection was significantly higher than that before the infection(P＜0.05). Conclusion:There is a significant increase of TREM-1 in secondary infection with SAP in pig.It is suggested that TREM-1 involved in the pathogenesis of the entire process of SAP.3.The diagnostic value of TREM-1 as an indicator in patients with intra-abdominal secondary infectionObjective:Cloned human TREM-1 gene,and discuss the presence of soluble TREM-1(sTREM-1) in peripheral blood from patients may be an indicator of secondary infection.Methods:34 cases of patients after 1-2 weeks admission with SAP(2003 Guideline in China) were selected from 2006 November to 2007 December.All the patients highly suspected abdominal secondary infection,excluding the infection from respiratory tract, urinary tract and skin.The mRNA was extracted in the 5 ml peripheral blood,RT-PCR amplification of TREM-1,the recovery and purification,sequencing results correctly.The level of C-reactive protein(CRP) in serum was tested.The TREM-1 expression was detected with real-time PCR in the peripheral blood.The bacterial culture results of the samples,from B ultrasound / CT guided puncture of the cyst or surgery,was treated as gold standard.Then the patients were divided into infected and non-infected group. Through ROC curve analysis,the diagnosis value of TREM-1 and CRP as indicators to judge the secondary infection were analyzed and the threshold was calculated.Results:In 34 patients,there was 18 men and 16 women(male:female = 1.125:1), and the average age was 48.82±18.08.Twelve cases were diagnosed as secondary intra-abdominal infection.Two patients died,three cases received surgery.TREM-1 expression in infected and uninfected group had a significant difference(P＜0.05).The area under the ROC curve(AUC) of TREM-1 was 0.795.The sensitivity and specificity were 83.8%and 81.8%.And the AUC of CRP was 0.795,the sensitivity and specificity were 50.0%and 84.6%.Conclusion:The diagnosis value of TREM-1 expression in the peripheral blood of SAP patients was better than CRP.The combined of these two indictors,can improve the diagnostic efficiency.4.The constructor of eukaryotic expression vector and expression of TREM1 - IgG fusion protein in human and ratObjective:To construct the eukaryotic expression vector of TREM1-IgG fusion protein in human and rat and to achieve stable expression in CHO cells. Methods:Firstly,clone the TREM-1 extracellular region fragment of human,the extracellular domain fragments of rat and the constant region of heavy chain in IgG1-Fc of human,respectively.Second,The gene encoding the mTREM-1/IgGlFc fusion protein were constructed in eukaryotic expression vector pcDNA3.1(+)by means of T-A cloning and subcloning techniques,then was transfected into CHO cells for stable expression. Third,the cell supernatant was collected.Protein A-Sepharose CL-4B-immune affinity purification,and rapid accessed to crude protein.The expression of the fusion protein was detected by SDS-PAGE and Western Blot.Results:The TREM-1 extracellular region fragment of human,the extracellular domain fragments of rat and the constant region of heavy chain in IgGl-Fc of human gene fragment were synthesized and amplified from the total RNA by RT-PCR.The resulting products were cloned into pMD-18T vector.The pMD-18T/human TREM-1,the pMD-18T/rat TREM-1 and the pMD-18T/human IgGl construct were then transformed into E.coli DH5α,and the gene was sequenced.The eukaryotic expression vector pcDNA3.1(+) colonies were selected and were respectively cut by EcoR / XhoI and EcoRI / XhoI.T4 DNA ligase connects three fragments were constructed.Human and rat TREM1-IgGlFc/pcDNA3.1(+) recombinant plasmid were constructed,sequencing confirmed that sequence,promoter,termination of location and ORF entirely correct.Two recombinant plasmids were stably transfected into CHO cells with lipo2000 and selected with G418.RT-PCR showed that expression of the target gene was found in stably transfected CHO cells lines.The supernatants were harvested,and TREM-IgG fusion protein was purified by protocols including protein A affinity chromatography.In SDS-PAGE assay,about 66 KD bovine serum albumin and 39 KD specific protein were detected.In Western Blot assay,this product could be specifically detected by anti-human IgGl Fc antibody,which confirm it is an IgG fusion protein.Conclusion:Construction of the Human and rats TREM1-IgGlFc fusion protein eukaryotic expression vector,and stably expressedin CHO cells.5.Rat TREM1-IgGl fusion protein prokaryotic expression vector construction, expression and identificationObjective:To construct ratTREM1-IgGl fusion protein prokaryotic expression vector, expression and purification of recombinant rat TREM1- IgGl fusion protein.Methods:Rat TREM1-IgGl fusion gene was cut down from rat TREM1-IgGlFc pcDNA3.1(+) recombinant plasmid by restriction enzymes,EcoRI and XhoI,and inserted into the expression vector corresponding pGEX4T-1 site,and then was transformed into E.coli BL21 where it was induced to express proteins by IPTG(isopropyl-1-thio-b Dgalactopyranoside).The expressed proteins were analyzed through SDS-PAGE,purified through Glutathione Sepharose 4B.Determine the fusion protein concentration.The expression of the fusion protein was detected by SDS-PAGE,Western Blot and PMF.Results:Rat TREM1-IgGlFc pcDNA3.1(+) recombinant plasmid by restriction enzymes,EcoRI and XhoI.Electrophoresis confirmed that the fusion gene rat TREM1-IgGl was cut down.and inserted into the expression vector corresponding pGEX4T-1 site,and then was transformed into E.coli BL21 where it was induced to express proteins by IPTG.In SDS-PAGE assay,about 66 KD specific protein was detected. In Western Blot assay,this product could be specifically detected by anti-human IgGl Fc antibody,which confirmed it was an IgG fusion protein.17 matching peptide were measured by peptide mass fingerprint PMF,which confirmed that samples were the purpose proteins.And the concentration of the purified fusion protein was 1 mg / ml.Conclusion:Construction rat TREM1- IgC.l fusion protein prokaryotic expression vector,expression and purification of recombinant rat TREM1- IgG.l fusion protein. Laying the foundation for further study of its as a therapeutic target molecule SAP.6.The efficacy study of the fusion TREM1-IgG protein in the treatment of SAP ratsObjective:To observe the effect of the fusion TREM1-IgG protein in the treatment of SAP rats.Methods:20 male SD rats were randomly divided into SAP group(group A),the control treatment(GST) group(group B) an,the fusion protein therapy group(group C) and the control group(n=5).The SAP models in rats were successfully constructed by intraperitoneal arginine injection.After six hours,the treatment was given.The GST protein and fusion protein were respectively injected in tail vein in Group B and C.The concentration was 4mg/kg.In group A,500μl saline was injected.After 24 hours,all the rats were killed.And the levels of amylase,aminopherase and creatinine in serum were detected.The histological changes were observed and scored in pancreas,liver and lung.Results:24 hours after the treatment,there was no significant difference in the level of aminotransferase and creatinine among GST group,TREM1-IgG fusion protein group and SAP group,The level of serum amylase in TREM1-IgG fusion protein group had a downward trend.In TREM1-IgG fusion protein group,the pathology scores in pancreatic inflammation,bleeding,necrosis,lung inflammation and total organ damage had significantly improved than the other two groups(P＜0.05).Conclusion:The fusion TREM1-IgG protein can effectively alleviate the inflammatory response and reduce damage to pancreas and lung.In SAP rats,it was proved that TREM-1 as a therapeutic target molecule in SAP is possible.Through this study,the subjects come to the following conclusions:1.TREM-1 involved in severe acute pancreatitis at the "excessive" inflammatory response,the expression level of TREM-1 was obviously high when the secondary infection happened.2.The TREM-1 gene expression in the peripheral blood was a non-invasive,rapid and economic indictor in intra-abdominal secondary infection of SAP patients.The use of TREM-1 was prospected to judge the secondary infection and the time of surgery intervene.3.Successfully constructed the eukaryotic expression vector of human TREM1-IgG fusion protein and achieved stable expression in CHO cells.4.Successfully constructed the prokaryotic expression vector of rat TREM1-IgG fusion protein and achieved stable expression and purification.5.The fusion TREM1-IgG protein can effectively alleviate the inflammatory response and reduce damage to pancreas and lung.In SAP rats,it was proved that TREM-1 as a therapeutic target molecule in SAP is possible.Summary:The subject demonstrated that TREM-1 played an important role in the process of severe acute pancreatitis and had the diagnosis value in the intra-abdominal secondary infection.Construction and expression of TREM-1 / IgGl fusion protein eukaryotic and prokaryotic expression vector,through the intervention of SAP rats proved TREM-1 as a therapeutic target molecule SAP value.To laying the foundation for further study of follow-up search the natural ligand of TREM-1,and providing new ideas of SAP pathogenesis and comprehensive treatment.