Study On Body Fluid To Seek Potential Diagnositic Biomarker In Tuberculous Mengitis

BackgroundTuberculous meningitis (TBM) is an infectious disease of centralnervous system caused by Mycobacterium tuberculonsis (MTB), whichmay account for7-10%of TB patients, mainly resulting in non-suppurativeinflammation among meninges, also expanded to brain parenchyma and thespinal cordm, meninges. Recently, in terms of people’s frequent migration,immunosuppressants widely used, emergence of drug-resistant TB strainsas well as epidemic immune deficiency virus, which gradually facilitatesTBM incidence. In the TBM prevention and treatment process, early andaccurate diagnosis is crucial for the medication, therefore, the precise andefficient diagnostic method to TBM is still an important problem to besettled.The latest diagnosis of TBM mainly relies on cerebrospinal fluid (CSF)cytology test, biochemical tests and so on. However in the early stage ofTBM, CSF cytology, and biochemical changes are atypical, which makesTBM diagnosis very difficult. In the study, using iTRAQ labeling couplingwith liquid chromatography tandem mass spectrometry (iTRAQ-LCMS/MS) strategy to compare CSF protein phenotypes between TBMpatients and healthy controls(HC), aiming to identifying TBM-related differential proteins, the results could provide insight into the pathogenesisand potential diagnostic biomarker for TBM.Additionally, giving the evidence that damage located at blood-brainbarrier (BBB) from meningitis which enables tuberculous meningitispatient to be detected of metabolic molecules in the peripheral blood. Thissuggests there may be tuberculous meningitis related metabolic moleculecould be found in the blood. Here, plasma metabolomics of TBM patientaims to screening the diagnostic marker of TBM.Objective1. Comparing CSF differential proteins between TBM patients andHC using absolute quantification (iTRAQ) proteomics, global screeningand validation of differential protein of TBM, then capturing the diseaserelated differential proteins, by which, it could uncover the potentialdiagnostic biomarker of TBM.2. NMR based metabonomic method was used to compare plasmametabolic profiling of TBM patients and HC. This was aimed toidentifying the differentially expressed metabolites using as potentialdiagnostic biomarker.MethodsSection1includes: To determine such biomarkers, a quantitativeproteomic study was performed here to identify differential proteins in thecerebrospinal fluid (CSF) obtained from TBM patients (n=12) and HC(n=12). Using LC-MS/MS coupling with bioinformatics analysis toidentifying the differential proteins, after further validation by WB,Meanwhile, in receiver–operator curve analyses, it could assess the validityof the diagnostic biomarker. Section2includes: To rule out the disease related differential plasmametabolite, applying Nuclear Magnetic Resonance (NMR) metabolomicsbased strategy globally profile of plasma metabolites in TBM patientsparalleled with HC, prelimilary analysis of differential metabolites in itsfunctions，which reveals its potential role in the underlying pathogenesis ofTBM.Results1. Using quatitity iTRAQ Proteomic strategy,which reveals81differential proteins, based on bioinformatics analysis, the results suggestthat coagulation cascades, inflammation response and cell adhesionmolecules is mainly related. Western blotting showed significantlydecreased NELL2levels in TBM subjects relative to healthy controls, andarea under the receiver operating characteristic curve analysis demonstratedNELL2could distinguish TBM subjects from healthy controls with83.3%sensitivity and75%specificity. In conclusion, CSF NELL2shows promiseas a diagnostic biomarker for TBM.2. NMR coupling with OPLS-DA based metabolomic trategyseparates TBM patient from HC,24differential metabolites were ruled outbetween the two groups(VIP>1.0), which includes N-acetylglycoprotein,pyruvate, glucose, nicotinamide,1-methylhistidine, glucono-delta-lactone.The Expression aforementioned in tuberculous meningitis has beenincreased; LDL, VLDL, lipids, isoleucine, leucine, valine,glutamicacid(Glutamine), methionine, histidine,3-hydroxybutyrate,Acetate, acetone, ethanol, phosphorylcholine(PC),glycerophosphorylcholine, acetoacetate, glutamine, N-methylnicotinamideis down regulated in the TBM. In the identified differential metabolites, N–acetyl-glycoprotein, Nicotinamide, Glucose (alpha Glucose, betaGlucose) is well capable of separating the TBM group and HC group. Byestablishing ROC curve analysis, AUC value was0.810and0.782respectively. In the test set, these four markers among12TBM and13HC,the AUC value was0.827, while in the training set, between virusmeningitis group and TBM group, the AUC value was0.782.Conclusions1. NELL2could be a potential diagnostic biomarker of TBM.2. Using the NMR based metabonomics analysis of plasma, wesuccessfully identified a panel of differential metabolites of TBM in plasma.By analyzing the underlying the molecular function of these differentialmetabolites, we provided valuable cures for uncovering the underlyingmechanisms of TBM. In addition, we performed the independent tests tovalidate the diagnostic performance of some differential metabolites, whichfacilitated to develop an objective diagnostic test for TBM.