Different Molecular Expressions In Thymi From Ocular And Generalized Myasthenia Gravis Patients

Myasthenia gravis (MG) is an acquired autoimmune disease which failure ofneuromuscular transmission. Anti-acetylcholine receptor (AChR) T cells play acrucial role in the pathogenesis of MG, for they allow and regulate the synthesisof the high affinity antibodies that lead to AChR loss which destroy postsynapticmembrane of the neuromuscular junction (NMJ). Disease is common of thenervous system disease that serious harm to the patient’s physical and mentalhealth. MG is one of ideal animal models to study the pathogenesis and treatmentand prevention of autoimmune disease, for the clear antigen-AChR, antibodies-AChR antibody (AChRAb) and target of pathological effects–NMJ. The mainclinical manifestation of MG is fluctuating contraction weakness of skeletalmuscle. Initial symptoms of MG usually present as ocular misalignment. SevereMG patients will involve bulbar muscle and lead to the respiratory muscleparalysis and crisis of myasthenia gravis and death. According to differentinvolved muscles and different symptoms, MG is divided into ocular MG (OMG)and generalized MG (GMG). OMG is named as type Ⅰfor the only involvedextraocular muscles and always present as fluctuating ptosis and diplopia. GMGis named as typeⅡ~Ⅴ involved not only the skeletal muscle of limbs, but alsobulbar muscular and manifestation with paralysis of the limbs and trunk muscle,even of respiratory muscles, myasthenic crisis and death. The pathogenesis of MG has been the focus of attention in neurology and immunology in more thanhalf a century.Thymi of MG patients are in a state of chronic inflammation and about80~90%patients complicated with pathologic thymus, according to CT scan. Allthe pathologic thymus could present as hyperplasia, cysts, necrosis, hypertrophyand thymoma. The clinical manifestation will be relieved by thymectomy inpatients with pathologic thymus. For refractory MG patients, removal of theseemingly normal but in fact may be chronic inflammation thymus always couldreach a satisfied outcome. The cellular and humoral immunity are subject toinhibition in post-operation MG patients and the AChRAb was reduced, whichindicated thymus play a very important role in the induction and maintenance ofthe occurrence and development process of MG. Previous studies presumed thatthe initial site where induced abnormal immune response of MG was thymus. Butit is unclear what type immune response occurred in thymus. It is essential toclarify the correlation between the immune response of thymus and the differentclinical manifestation of OMG and GMG, which is crucial to unveil thepathogenesis of MG. And also this perhaps could guide the treatment andprevention of other autoimmune disease. Using the immunohistochemicalstaining (IHC), immunofluorescence staining (IF), real-time polymerase chainreaction (R-T PCR), single nucleotide polymorphism (SNP) and western-blot(WB), we will analyze the different molecular expression in thymus from OMGand GMG patients to elaborate the relation between immune response in thymusand different clinical symptoms between OMG and GMG. Part One Different molecular expression of OMG andGMG patients with thymomaAim1. Analyze different onset age, sexual and duration of OMG and GMG withthymoma.2. Analyze different Foxp3and CXCL13expression in OMG and GMG withthymoma.MethodWe undertook this study that enrolled all thymoma patients with or withoutMG in Tangdu Hospital from2003to2010. A total of311thymoma patientsunderwent thymectomy and116patients with MG. A total of58of the116MGpatients and73thymoma patients without MG (Non-MG) were eventuallyenrolled. Of the58MG with thymoma patients,35patients were identified asOMG, and others as GMG.73thymoma patients with Non-MG were identified ascontrol group.Using SPSS13.0, we analyzed the clinical data of MG patients withthymoma. Then, we analyzed different expression of Foxp3and CXCL13in MGpatients with various types thymoma.ResultCompared with GMG thymoma patients, the onset age of OMG malepatients was almost in late-onset and their onset age was later than that GMGmale thymoma patients (P=0.037). The onset age of female patients betweenOMG and GMG patients with thymoma was shown little difference and did notreach significance (P=0.126). Compared to patients with OMG, a decreasedFoxp3expression was seen in types AB, B1and B2thymoma with GMG, with the decrease in the former two types reaching significance (P=0.001,0.043).However, a significantly increased expression of CXCL13was seen in types B1and B2thymoma patients with GMG (P=0.027,0.048), compared to those withOMG. Furthermore, the CXCL13expression in type AB thymoma patients withGMG was higher than those with Non-MG (P=0.003).ConclusionThere was significant difference of onset age in different gender of GMGand OMG patients with thymoma. Furthermore, different molecular expression inGMG and OMG with distinct histological thymoma implied that the distincthistological of thymoma and molecular expression participated the mechanism ofdifferent clinical symptoms of MG. Part Two Different molecular expression of OMG andGMG in thymic hyperplasiaAim1. Analyze different CD4, CD25, Foxp3and CXCL13expression in thymusof infant, adult and hyperplasia with Non-MG by IHC and R-T PCR.2. Analyze different CD4, CD25, Foxp3and CXCL13expression in OMGand GMG with thymic hyperplasia by IHC and R-T PCR.3. Analyze the relation between different CD4, CD25, Foxp3and CXCL13expression and OMG and GMG with thymic hyperplasia and the severity of MG.MethodWe undertook this study that enrolled all thymic hyperplasia patients with orwithout MG in Tangdu Hospital from2008to2011. A total of34of52MGpatients with thymic hyperplasia were eventually enrolled. Of the34MG patientswith thymic hyperplasia,14patients were identified as OMG, and others as GMG.And we also enrolled6thymic hyperplasia patients with Non-MG as our firstcontrol group,6adult with lung cancer but atrophic thymi as the second controlgroup,12infants thymi with congenital heart disease as the third control group.ResultThere was no significant difference of CD4and Foxp3expression in OMG,GMG and the three control groups. But the expression of CD25and CXCL13was significantly difference among groups. A higher CD25expression was foundin infant thymi, decreased in atrophic thymi, and a lowest expression in Non-MGhyperplastic thymi. However, a significant increased expression of CD25wasfound in OMG patients, and higher in GMG. In contrast to CD25expression, weobserved a low expression of CXCL13among three control groups and no difference was found. CXCL13expression was significantly higher inhyperplastic thymi with GMG or with OMG than those with Non-MG. Resultsfrom RT PCR was similar to IHC. No significant difference of CXCL13expression was found between GMG and OMG. Regression analysis showed amoderate positive correlation between thymic CD25level and MG symptomseverity. Similarly, a positive correlation was found between thymic CXCL13expression and severity of MG.ConclusionThere was difference molecular expression among infant, adult, thymichyperplasia with Non-MG, GMG and OMG. Our results suggested that thedecreased CD25expression related with aging. The positive relationship betweenCD25and CXCL13expression and the severity of MG hinted that these twomolecular was a marker of the severity of MG. Part three Expression of Tfh cells in thymus with OMGand GMGAim1. Analyze different CD4+CXCR5+T cells expression in thymi with OMG andGMG.2. Analyze different CXCR5, Bcl-6, ICOS and IL-21expression in thymi withOMG and GMG by IHC, IF, WB, SNP, R-T PCR.MethodWe undertook this study that enrolled all thymectomy patients with orwithout MG in Tangdu Hospital from2009to2011. We eventually enrolled21OMG,18GMG and19Non-MG patients in our study. Using IHC, IF, WB, SNP,R-T PCR, we analyzed different Tfh cells expression in OMG and GMG patients.We detected the expression of CD4+CXCR5+cells to reflect Tfh cells expressionwith laser scanning confocal microscope (LSCM) of OMG and GMG in thymi.SNP was used to observe the nucleotide mutation, WB was used to quantitativemolecular protein level, R-T PCR was used to quantitative molecular RNA levels.ResultOur study demonstrated that a highest expression of CD4+CXCR5+T cells inGMG patients, then a higher expression in OMG and a low expression inNon-MG. WB and IHC revealed a high expression of ICOS and Bcl-6in GMGgroup, from quantitative, or positioned, respectively. SNP discovered nucleotidemutations of ICOS in MG patients. R-T PCR results showed that a remarkableincreased expression of CXCR5, ICOS, Bcl-6in GMG, and an increased in OMG, compared with adult atrophic thymi. There was no significant difference ofCXCR5, ICOS, Bcl-6expression between Non-MG and OMG patients. Amonggroups, highest expression of IL-21was found in GMG patients that indicate anactivation of B cells leaded to more antibody secretion. Similarly, higher IL-21expression in thymic hyperplasia with Non-MG patients hinted an increase ofMG susceptibility. High expression of IL-21may be risk factors initiated MG.ConclusionHigher expression of ICOS, Bcl-6and IL-21in thymoma of GMG patientswas verified by IHC, IF, WB, SNP and R-T PCR. All these findings implied thatTfh cells played an important role in different clinical symptoms of MG.