clinical observation of immunity for severe acute pancreatitis

Clinical Observation of Immunity for Severe Acute PancreatitisZhiMin Liu,1YinFeng Shen,2NaiQiang Cui,1,4and Jing Yang3Abstract—The aim of our study was to observe the dynamic changes of immunity for patients withsevere acute pancreatitis(SAP)and intervention by traditional Chinese medicine.Twenty-threepatients who met the inclusion criteria were randomized to combined treatment of traditionalChinese medicine and Western medicine(TCM)or conventional western medicine treatment(WM)groups.The clinical data for all patients were collected.Peripheral venous blood sampleswere obtained from patients on days1,7,14,and28after admission.Biochemical data including thepercentage of CD4+/CD8+/natural killer(NK)cells/B lymphocytes/HLA-DR and CD4+/CD8+ratioin serum were determined byflow cytometer.Patients’characteristics and immunity at admissionwere similar between the two groups.The secondary infection was different.The levels of T-lymphocyte subsets in the TCM group were quite different from the WM group,with muchmore the percentage of CD4+and the CD4+/CD8+ratio on days7,14,and28and much lessthe percentage of CD8+on days14and28.On days14and28,the levels of NK cells and Blymphocytes were significantly higher in the TCM group compared with the paredwith the TCM group,the levels of HLA-DR were significantly decreased in the WM group ondays7,14,and28.The immune dysregulation exists in the development and progression ofSAP.The combined treatment of traditional Chinese medicine and western medicine can upregulatethe patient’s immune and maintain the immune balance.KEY WORDS:severe acute pancreatitis;immunity;treatment;traditional Chinese medicine.Severe acute pancreatitis(SAP)is a special emergency abdominal surgery which has caught close attention by surgical clinicians and gastrointestinal experts.It is characterized by the severity,rapid progress,multisystem damage,and long course[1].Over decades,with the development of the pathogenesis of SAP and clinical treatment technology,the mortality is still more than10% [2].The imbalance of immunity was caused by con-sumption of antibody and complement which involved in anti-inflammatory and immunomodulation and activation of reticuloendothelial system by trypsin,endotoxin,and bacteria in circulation[3].Recent researches suggested that immune dysregulation played an important role in the development of SAP[4].In recent years,the cytokine network in the immune dysfunction is widespread concerned in the pathological mechanism of acute pancreatitis[5,6]. However,there were few studies to investigate the immunity in the course of SAP in clinic.Cell-mediated immunity and humoral immune are the important components of immune system,the percentage of CD4+/CD8+/natural killer(NK)cells/B lymphocytes/ HLA-DR and CD4+/CD8+ratio in peripheral blood mononuclear cells(PBMC)can reflect immune func-tion[7].In the occurrence and development of SAP, there is a marked change of the immune response which is thought to have a central role in the development of systemic inflammatory response syn-drome(SIRS),multiple organ dysfunctions,secondary infection,and subsequent septic complications[8–10].1Nankai Hospital,Nankai Clinical School,Tianjin Medical University,Tianjin,300010,People’s Republic of China2Surgery,Chinese Medicine Hospital of Hubei Province,HubeiUniversity of Chinese Medicine,Wuhan,People’s Republic of China3Department of Pharmacological,Tianjin Institute of Acute AbdominalDiseases,Tianjin,People’s Republic of China4To whom correspondence should be addressed at Nankai Hospital,Nankai Clinical School,Tianjin Medical University,Tianjin,300010,People’s Republic of China.E-mail:tiramisu_yang@0360-3997/10/0000-0001/0#2010Springer Science+Business Media,LLC Inflammation(#2010)DOI:10.1007/s10753-010-9249-5In this study,we aimed to observe the immunity for patients with SAP,who were randomly treated by conventional western medicine treatment or combined treatment of traditional Chinese medicine and Western medicine and evaluate the dynamic features of immunity in PBMC for patients with SAP.MATERIALS AND METHODSPatientsAll patients with AP admitted to the National Medical Center of Biliopancreatic Diseases at Nankai Clinical School of Tianjin Medical University between April1,2009and December31,2009were recruited. All patients met the criteria for severe disease proposed at the International Symposium on Acute Pancreatitis (AP)[11],and the diagnosis of AP was based on typical clinicalfindings associated with3-fold increases or more in serum amylase and/or serum lipase activities and/or morphologic abnormalities compatible with AP seen on contrast-enhanced computed tomography[12].Initial severity of pancreatitis was assessed by using the Acute Physiology and Chronic Health Evaluation II(APACHE II)[13],Ranson scores,Balthazar scores[14],and SAP was judged by an inclusion criterion with an APACHE II score of8or higher,a Ranson score of3or higher,and Balthazar scores of5or higher.Inclusion and Exclusion CriteriaWe did the randomized clinical trial for patients with SAP at our department.Inclusion criteria were age between18and70years and signed informed consent. Exclusion criteria were:(1)patients diagnosed with concurrent pancreatic or peripancreatic infection;(2) pregnant or lactating females;(3)patient with cancer;(4) patient in dying condition;(5)patient with serious organ dysfunction;(6)patient with immunodeficiency disease or autoimmune disease;and(7)patients in another research. TreatmentAll patients were treated in our institution according to a standardized interdisciplinary management protocol, such as gastrointestinal decompression,prophylactic anti-biotics(ciprofloxacin/metronidazole or imipenem/cilasta-tin),intravenousfluids,nutritional support and organ system support.Study DesignIt was double-blind to doctors and patients.The patients were randomly divided into the two groups and allocation concealment was by computer through the third party.Chinese Materia Medica was given to patients in combined treatment of traditional Chinese medicine and Western medicine group(TCM).The placebo was administrated to conventional western medicine treatment group(WM).We used drug for intragastric administration and coloclysis four times every day after admission.If the patient did not need nasogastric tube,the drug was administered by mouth.EthicsThe hospital ethics committee approved the study. Patients or their relatives were provided with printed information and signed consent before entry into the study. Data CollectionThe following data were collected prospectively from all patients:(1)demographic data including gender, age,and etiology of pancreatitis;(2)clinical data including score of severity,secondary infection and mortality;(3)biochemical data including the percentage of CD4+/CD8+/NK cells/B lymphocytes/HLA-DR and CD4+/CD8+ratio in PBMC.Peripheral venous blood samples(2mL)were obtained from patients on days1, 7,14,and28after admission using a strict aseptic technique.Biochemical data were determined using BD Multitest IMK kit(BD Corporation,USA),FITC anti-human CD14(ebioscience Corporation,USA),PE anti-human HLA-DR(ebioscience Corporation,USA),PE Mouse lgG2b lsotype control(ebioscience Corporation, USA),FACSortflow cytometer(BD Corporation,USA), and CellQuest software.Statistical AnalysisThe one-way ANOV A test was performed by the software package SPSS11(Chicago,IL).Continuous data are presented as the mean±standard error of the parisons of continuous and categorical variables between two groups used the nonparametric Mann–Whitney U test and theχ2test with Yates correction,respectively.Correlations were evaluated with the Spearman rank test.A P<0.05was considered statistically significant.Liu,Shen,Cui,and YangRESULTSAfter exposing blind,23patients with SAP were recruited:11in TCM group and12in WM group.For patients in two groups,data on gender,age,etiology, Ranson score,APACHE II score,Balthazar scores, surgical treatment rate,and mortality are given in Table1.Patient characteristics at admission were similar between the two groups.The secondary infection was different between the TCM and WM group.In the TCM group,a female patient with SAP and early multiple organ failure(renal,pulmonary,andcardiocirculatory),she died on day3of persistent early organ failure(renal and pulmonary).Biochemical DataFor patients with SAP in the two groups,the percentage of CD4+significantly increased on7days during all time points(P<0.05,Table2);compared with TCM group,it was markedly depleted at14and28days (P<0.05,Table2)in WM group;the percentage of CD4+ was similar at1day between the two groups(P>0.05, Table2),and it was obvious change at other points(P< 0.05,Table2).The measured the percentages of CD8+ was significantly higher in WM patients on14and28days compared with the TCM group(P<0.05,Table3);The percentage of CD8+significantly increased at14and 28days for patients with SAP in the WM group(P<0.05, Table3);in contrast,there was no serious change in the TCM group(Table3).The CD4+/CD8+ratios were significantly higher in the TCM patients on7,14,and 28days compared with the WM group(P<0.05,Table4); the ratio was markedly depleted on28days(P<0.05,Table4)in the WM group.The percentage of NK cells significantly decreased on7days for patients with SAP in the two groups(P<0.05,Table5),and it was maintained at a low level on7and14days in the WM group;compared with the WM group,the percentage of NK cells levels were significantly increased in the TCM group on days14 and28(P<0.05,Table5).The percentages of B lymphocytes progressively decreased in the WM group (P<0.05,Table6);in contrast,it was maintained at a relatively high and stable level in the TCM group; compared with the WM group,the percentage of B lymphocytes levels were significantly increased in the TCM group on days14and28(P<0.05,Table6). Compared with the TCM group,the percentage of HLA-DR levels were significantly more than the WM group on days7,14,and28(P<0.05,Table7);there was markedly depleted on28days(P<0.05,Table7)in the WM groups; the percentages of HLA-DR were maintained at a relatively high and stable level in the TCM group(Table7). DISCUSSIONSAP is one of the most serious pancreatic diseases and SAP patients tend to suffer from SIRS,multipleTable1.Characteristics of SAP Patients Between TCM and WM GroupTCM(n=11)WM(n=12)P value Age(year)53.5±14.650.2±11.8>0.05 Gebder(M/F),n(%)5/65/7>0.05 Biliary cause,n(%)45–Alcohol cause,n(%)11–Hyperlipoidemia cause,n(%)34–Unknown cause,n(%)32>0.05 APACHE II score a9.2±7.78.8±7.2>0.05 Ranson score a 5.5±1.9 4.9±1.3>0.05 Balthazar scores a 4.5±1.1 4.3±1.3>0.05 Infection,n(%)1(9.1)5(41.7)<0.05 Surgical treatment,n(%)00>0.05 Mortality,n(%)1(9.1)0>0.05 a Patients at admissionparison of Percentages Changes in CD4+Day1Day7Day14Day28 TCM group(n=10)35.8±7.846.2±9.4a40.8±9.0a41.8±10.0WM group(n=12)33.9±9.240.0±8.4b37.7±11.8b21.8±5.0b P P>0.05P<0.05P<0.05P<0.05a In the TCM group,the comparisons of1versus7days,7versus 14days were significantly different,P<0.05b In the WM group,the comparisons of1versus7days,7versus 14days,14versus28days were different significantly,P<0.05parison of Percentages Changes in CD8+Day1Day7Day14Day28 TCM group(n=10)23.7±11.723.2±8.719.8±4.1a18.8±5.1WM group(n=12)25.2±7.925.5±6.730.6±10.6b47.3±10.5b P P>0.05P>0.05P<0.05P<0.05a In the TCM group,the comparison of7versus14days was different significantly,P<0.05b In the WM group,the comparisons of7versus14days,14versus 28days were significantly different,P<0.05Clinical Observation of Immunity for Severe Acute Pancreatitisorgan dysfunction syndrome(MODS),secondary infec-tion,and subsequent septic complications[15].There is no effective treatment for the deleterious impact of multiple organ dysfunctions trigged by SIRS,and death is often the outcome.SAP is a serious pathological condition which can lead to SIRS caused by local inflammation of the pancreas[16].In the early stage of SAP,the hypercytokinemia plays a dominant role in the development of SIRS,a pathological condition in which inflammatory cytokines increase substantially and induce a cascade reaction,is released from immuno-competent cells,thereby triggering excessive immune response[17,18].In the late stage of SAP after1to 3weeks,evidence accumulated that the hyperinflamma-tory response is counteracted by the systemic release of anti-inflammatory mediators,and there is an immuno-suppression which is thought to contribute to the development of secondary infections increasing the risk of MODS[19–21].The excessive release of inflamma-tory mediators during SAP is the main cause of the intestinal mucosa injury[22];the proinflammatory cytokines can increase the permeability of intestinal mucosa and aggravate the body invasion of intestinal bacteria continuously,which leads to bacterial trans-location during inflammatory responses[23,24].Infec-tion of pancreatic necrosis and subsequent septic complications damage the cellular immunity exacerbated the impaired immune system[25].In this report,we describe a randomized clinical trial for SAP patients by different treatments in our institution. The gender,age,etiology,severity(based on scoring systems,APACHE II scores specifically),surgical treat-ment rate,and mortality were similar in the two groups; however,the rates of secondary infection were different. Our outcomes suggested that the use of Chinese Materia Medica for patients with SAP was associated with bacterial contamination of pancreatic necrosis[26].The Chinese Materia Medica may prevent morphologic changes in the intestine,eliminate pathogens in the intestinalflora,and reduce bacterial translocation.Immunological events are believed to be involved in the pathogenesis of SAP,which immune injury in AP is essentially unclear[27].A number of immunologic parameters have been shown to be changed in the course of SAP,for example the density of CD4+/CD8+/NK cells/B lymphocytes/HLA-DR and the ratio of CD4+/ CD8+in the periphery.In our study,the patient’s immune status in the two groups was similar at admission.After a period of time after treatment,the levels of T-lymphocyte subsets in the TCM group wereparison of Percentages Changes in CD4+/CD8+RatioDay1Day7Day14Day28 TCM group(n=10)2.03±1.14 2.49±1.95a 2.12±0.34a 2.15±0.31WM group(n=12)2.17±0.32 1.71±0.69b 1.67±0.870.47±0.08b P P>0.05P<0.05P<0.05P<0.05a In the TCM group,the comparisons of1versus7days,7versus 14days were significantly different,P<0.05b In the WM group,the comparisons of1versus7days,14versus 28days were significantly different,P<0.05parison of Percentages Changes in NK CellsDay1Day7Day14Day28 TCM group(n=10)12.3±10.09.0±10.1a19.2±15.0a19.0±13.0 WM group(n=12)12.5±8.29.7±4.2b9.8±3.715.8±8.3b P P>0.05P>0.05P<0.05P<0.05a In the TCM group,the comparisons of1versus7days,7versus 14days,14versus28days were significantly different,P<0.05b In the WM group,the comparisons of1versus7days,14versus 28days were different significantly,P<0.05parison of Percentages Changes in B LymphocytesDay1Day7Day14Day28 TCM group(n=10)20.6±11.118.8±7.9a17.4±10.3a17.0±7.3WM group(n=12)23.8±10.519.2±7.2b12.8±5.3b7.3±3.6b P P>0.05P>0.05P<0.05P<0.05 a In the TCM group,the comparisons of1versus7days,7versus14days were significantly different,P<0.05b In the WM group,the comparisons of1versus7days,7versus 14days,14versus28days were different significantly,P<0.05parison of Percentages Changes in HLA-DRDay1Day7Day14Day28 TCM group(n=10)84.07±34.9885.80±38.6088.50±58.6a88.41±57.1 WM group(n=12)81.69±36.2077.17±14.62b71.31±58.0b44.06±26.35b P P>0.05P<0.05P<0.05P<0.05a In the TCM group,the comparisons of7versus14days was significantly different,P<0.05b In the WM group,the comparisons of1versus7days,7versus 14days,14versus28days were different significantly,P<0.05Liu,Shen,Cui,and Yangquite different from the WM group,with much more the percentage of CD4+and the CD4+/CD8+ratio on days7,14,and28(P<0.05)and much less the percentage of CD8+on days14and28(P<0.05).On days14and28,the levels of NK cells and B lymphocytes were significantly higher in the TCM group compared with the pared with the TCM group,the levels of HLA-DR were significantly decreased in the WM group on days7,14,and28(P< 0.05).By observation,we could conclude that combined treatment of traditional Chinese medicine and western medicine would improve the level of the patient’s immune system,with maintaining the immune balance at a better level.In contrast,the bad level of immunity in the WM group may be relevant to more patients with infection.Clinical and experimental studies support the theory on the gut fuelling the process of SAP,including leaded to pancreatic or systemic infection by bacterial translocation from the gut[26],stimulated and strengthened SIRS and MODS[28],enhanced lung injury[29],increased pancreatic necrosis,and deteriorated the disease[30].We used Chinese Materia Medica as a special kind of the selective gut decontamination,which can restore gastro-intestinal function by facilitating bowel movement and relieving enteroparalysis[31],maintain morphologic changes in the intestine[26],prevent translocation for bacteria and endotoxin[32],counteract with endotoxin and regulate Ca2+-Mg2+-ATPase in the pancreatic acinar cells[33].The gut appears to play a key role in the development of SAP’s complications,and recovery of intestinal function in patients with SAP would moderate the disease and restore the damage of immune system [34].Considering that immune dysregulation plays an important role in the course of SAP,some researchers have adopted immunotherapy to treat this disease by using immunosuppressant and Immunomodulator[4]. Curley et al.[35]reported that a reduction in circulating CD4+T cells in SAP could decrease levels of antiendotoxin core antibody and interleukin-6,mitigate disease severity.Yao et al.[36]found that TA1can improve tissue injury and reduce the mortality rate through modulating CD3+/CD4+T cell ratio and reducing the release of inflammatory factors and cytokines.HLA-DR which is a MHC class2protein has a central role in the development of the immune response[37]and is closely related to secondary infection for patients with SAP[38].Kylanpaa ML et al.[39]reported that the low proportion of HLA-DR-positive monocytes in AP can reflect the degree ofimmune suppression and be reversed in vitro by GM-CSF and/or IFN-g.Our present data clearly indicate thatthe levels of T lymphocytes,B lymphocytes,NK cells,and HLA-DR were significantly higher in the TCMgroup compared with the controls.Thisfinding con-firmed that the development of SAP had a close relationship with the imbalance of immune system,withexcessive immune response in the early stage andimmunosuppression in the late[40,41].In conclusion,the immune dysregulation exists inthe development and progression of SAP.The combinedtreatment of traditional Chinese medicine and westernmedicine can upregulate the patient’s immune systemand maintain the immune balance.The question on thepharmacokinetics of traditional Chinese medicine,onhow to be effective in SAP,must be clear in the future. 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