Study On Immune Characteristics Of Praziquantel-resistant Isolates Of Schistosoma Japonicum

Schistosome-a trematode parasites of the genus Schistosoma live within the veinsof human or mammal, Cause zoonotic parasitic diseases that harmful to human healthand draining socio-economic development. Schistosomiasis-also known asbilharzia-is prevalent wide range in some tropical or subtropical countries, of78countries considered endemic for schistosomiasis,52countries have populationsrequiring preventive chemotherapy. According to WHO, estimated7millionindividuals at risk of infection, more than2million individual dead due to infection,the total number of people in need of preventive chemotherapy globally for2012was2.5million. In China, Schistosoma Japonicum is the only species that mainly endemicin the provinces along the middle and lower reaches of the Yangtze River, and now isa primary parasitic disease in China. Since1950the nation schistosomiasis controlprogram was initiated, three phases control strategy has been involved in the program,the prevalence of schistosomiasis in China has been effectively controlled, shanghai,zhejiang, fujian, guangdong, guangxi province reach the criterion of transmissionblocking. But By the end of2012, the total numbers of schistosomiasis japonica caseswere estimated to be0.24million, around0.37million hm2were found to be infestedwith Oncomelania hupensis snails, moreover, new areas were reported to be infestedevery year. So schistosomiasis japonica still is a big public health problem in China.Praziquantel which was invented for tapeworm in1970s, found has curativeeffect for all human schistosoma species, made a breakthrough period of the therapyfor the disease, then replaced other drugs and became the first and virtually the onlychoice of drug for treatment of human schistosomiasis recommend by WHO. Orallyadministered praziquantel is rapidly absorbed, small intestine is the main absorptionpart, measurable amounts appearing in the blood as early as5min after dosing andpeak levels occuring after1-1.5h, the half-life generally ranging between1and1.5h, elimination occurs essentially through the urine. Although massive researches havedone for the mechanism of praziquantel, it is still not fully clear. But it can be roughlydivided into two points, direct effect to schistosome include muscular contraction,tegument damage, change metabolisms. Another is of immune-dependence andimmune-synergy with host.At least250million people required treatment for schistosomiasis every year,while the actual number of people treated in2012was only42.1million. Periodic,targeted treatment with praziquantel is the main strategy of control schistosomiasis.However, repeated treatment for re-infection and the characteristics of concomitantimmunity of schistosome infect, implies that the parasites may develop resistance tothe drug under repeated large-scale chemotherapy. In1994, the praziquantel resistantstrains in schistosoma mansoni have induced in laboratory by Doenhoff et al.Furthermore, decreased sensitivity of S. mansoni to praziquantel has been also foundin some endemic areas ofAfrica and SouthAmerica. There are also some casesreporting failure in treatment of S. haematobium infection using routine treatmentprotocols. In2011, the praziquantel-resistant strains in schistosoma japonicum alsohas been established in mice with sub-curative doses of praziquantel by Liang YS etal. Up to now, there are no available antischistosomicide and vaccine that can replacepraziquantel. Therefore, research in characteristics of praziquantel-resistant,comprehend the biologic and immune feature is given high priority. It can offerinformation to control resistant isolates diffuse and new pathway for vaccine research.Studies have confirmed that the sensitivities of S. mansoni resistant-isolates topraziquantel are significantly lower than susceptible-isolates exhibit in the stages ofadult worms, cercariae and miracidia in vitro. These results also appeared in the stagesof adult worms and egg in vivo.Another research found that there is a significantdifference in proteomics between resistant-and susceptible-isolates, suggesting thatpraziquantel can increase or inhabit the expression of specific genes, such as ABCmultidrug transporters and cytochrome-c oxidase. But there is few informationspertaining to S. japonicum. And there are no reports about immune characteristics. Inthis study, the fractions of soluble antigens of S. japonicum were prepared and host reactions after infection were studied. The main content of the present thesis is listedas following.Part1Study on worm and egg antigen fractions between praziquantel-resistantand-susceptible Schistosoma japonicumMice infected with quantitative cercariae of schistosoma japonicum. Adult wormwere collected from infected mice at42days post-challenge by perfusion, Andseparated the paired worms into females and males under anatomical lens. The livertissue was collected use scissors from mice for eggs. Adult worms and eggs were thentransferred in Cell lysis buffer for Western and IP, The supernatant was collected asAWAand SEAafter centrifugation. AWAand SEAwere fractionated byelectrophoresis using12%sodium dodecyl sulfate-polyacrylamide gel(SDS-PAGE),after electrophoresis the gel was stained and bleached sufficiently, and analyzed useImage Lab software, then compare the fractions of antigen and molecular weights.Result show: The adult worm soluble antigen and soluble egg antigen are complex.Based on molecular weight, AWA-f was separated into21-23different fractions,AWA-m was separated into26-36different fractions, SEAwas separated into22–31different fractions. BetweenAWA-f and AWA-m there are many different fractions,special between40-50kDa and80kDa. Meanwhile, SEAand AWAare different rangefrom40kDa to50kDa. Particularly, SEAand AWA-m different at80kDa, but nothappened on SEAcompare with AWA-f. Comparison of praziquantel resistant-isolatesand susceptible-isolates antigen fractions show, no obvious difference between them.But the results of band density found that under the same concentration, there is asignificant difference in band density between resistant-isolates andsusceptible-isolates, especially obvious appear in SEAand AWA-f.Part2Adult worm tegumental damage in praziquantel-resistant and-susceptibleSchistosoma japonicum treated in vivoThe mice infected with quantitative cercariae of Schistosoma japonicum resistantisolates or susceptible isolates were administred with praziquantel at a dose of75mg/kg from35th day to37th day after infection, at10mins and12h post treatment,mice were killed by blood letting, another group mice were administred with praziquantel at a dose of300mg/kg. Male and female schistosoms were collectedusing a routine perfusion technique, then fixed in buffered4%glutaraldehyde-phosphate, after post-fixation and dehydration, the adult schistosomespecimens were examined by field emission scanning electron microscope. Resultsshow: the tegument surface of adult female worms abtained from untreated controlgroup was smooth, and can see spines and sensory papillae on the back. Tegument ofmale worms was arranged spongy, and can obvious see invagination and crimple, thespines and sensory papillae on the back. Female, after administration of PZQ75mg/kg,susceptible isolates showed blebbing of parts of the dorsal tegument, but resistantisolates didn’t have this appearance emerge. At12h, susceptible isolates tegumentrevealed extensive swelling with various degree, and erosion of the blebbling wasobserved, the resistant isolates, only begin revealed focal swelling of the worm bodyaccompanied by swollen tegument. Under administration of PZQ300mg/kg, at10minsome disarray to the usually regularly arranged spined tubercles both appeared onsusceptible and resistant isolates tegument, but susceptible isolates were more severethan resistant isolates, also, both of them retained the tegumental surface pits. At12h,susceptible isolates tegument revealed extensive swelling with various degree, anderosion of the blebbling was observed, the resistant isolates, only begin revealed focalswelling of the worm body accompanied by swollen tegument. Male, underadministration of PZQ75mg/kg, at10min the amount of susceptible isolates surfaceblebbing was extensive, some areas of gross swelling of the regions were eroded, butresistant isolates just had some blebbings and focal swellings appeared, and surfaceblebbing was also not as appeared. At12h, the amount of susceptible isolates surfaceblebbing was extensive, and was pronounced with surface lesions of varying sizescommon, the resistant isolates showed signs of surface blebbing and slight swellingonly. Under administration of PZQ75mg/kg, at10min both of them showed signs ofsurface wrinkling and pits, at12h, The susceptible isolates were composed ofextensively damaged worms, the majority of which had lost either the whole or thegreater part of the outer tegument exposing the underlying tissues, the resistantisolates, disruption of the parenchymal tissue was pronounced, but still can see surface drape structure.Part3Effet of praziquantel-resistant and-susceptible Schistosoma japonicum onT helper cell immune responsesⅠ）Effet of Schistosoma japonicum praziquantel-resistant and-susceptibleinfection and PZQ treatment on T helper cell sunsetMice infected with quantitative cercariae of schistosoma japonicum. Splenocyteswere prepared from spleens of mice with S. japonicum infection at different stages (2w,5w,7w,9w), Spleen lymphocytes were stimulated with PMAand Ionomycin for6hat37℃in vitro, and the proportions ofTh1(IFN-γproducing cells) and Th2(IL-4producing cells) cells in T cells were detected by intracellular cytokine staining andflow cytometry respectively. The aim of this research was to elucidate the kinetic Thelper lymphocyte-mediated responses, result show：after infected by resistant orsusceptible isolates, Proportion of Th1cell had the highest proportion at2w, thenreduce; at first3stages Th2cell proportion was low, and change was not obvious,reach the highest at9w, compare resistant and susceptible isolates, there are nostatistical differences. After administration of PZQ, Th2cell percent of Jiangsuresistant treated group and untreated group had significant difference at7w;nevertheless, the Jiangsu susceptible had resistant treated group and untreated grouphad significant difference at9w. The hunan isolates had no statistical differences.Resistant isolates compare with susceptible isolates of Th cell proportion, there are nostatistical differences at any stages.Ⅱ）Effet of Schistosoma japonicum praziquantel-resistant and-susceptibleinfection and PZQ treatment on T helper cell cytokine profilesMice infected with quantitative cercariae of schistosoma japonicum. Thesamples of peripheral blood of mice were collected at different stages (2w,5w,7w,9w) for serum preparation. Then the levels of Th1speeific cytokines(IFN-γ, IL-2,TNF) and Th2specific cytokines(IL-4, IL-5) were tested using BD Cytometric BeadArray Mouse Th1/Th2cytokine kit and flow cytometry machine. Result show: afterinfected by cercariae, IL-2displayed no significant change, the level peaked at week5 and7, level of IFN-γpeaked at week5and declined sharply after week7, level ofTNF were elevated through whole process, IL-4、IL-5peaked at week5and displayedno significant change at other stages. Comparison performed between jiangsuresistant and susceptible isolates show, IL-2、IFN-γ、IL-4、IL-5of susceptible isolatewas higher than resistant isolate, but TNF was lower than resistant isolate. However,only IL-4remained significantly difference at week5. Comparison performedbetween hunan resistant and susceptible isolates show, IFN-γ、IL-4、IL-5at week5and TNF at week7of resistant isolates were higher than susceptible isolates, IL-5atweel2of resistant isolates were lower than susceptible isolates. After administrationof PZQ, levels of IL-2、IFN-γ、IL-4、IL-5at week7and9of Jiangsu resistant treatedgroup were higher than untreated group, TNF was lower than untreated group.However, levels of IL-2、IFN-γ、IL-4、IL-5at week7of Jiangsu susceptible treatedgroup were lower than untreated group, TNF was lower than untreated group. Levelsof IL-2、IL-4、IL-5at week7and9of hunan resistant treated group were higher thanuntreated group, IFN-γ、TNF was lower than untreated group. Levels IL-2、IFN-γ、TNF、IL-4、IL-5at week7and9of hunan resistant treated group were lower thanuntreated group. Comparison performed between hunan resistant and susceptibleisolates after PZQ treated show, levels of cytokines of resistant isolates were lowerthan susceptible isolates after PZQ treated. Thereinto, at week2after treatment IFN-γ of Jiangsu isolates and IL-4、IL-5of hunan isolates remained signifiecantlydifference. Conclusions:This is the first time analyze the immune characteristics of S. japonicumpraziquantel resistant isolates and susceptible isolates, we analyzed the alterations ofantigens and host reactions under host be treatment or absence of treatment. Resultshow that in the absence of treatment, there have differences of AWA and SEAbetween susceptible isolates and resistant isolates, which cause the differentexpression of serum cytokines in mice. With PZQ-treated, there are significantdifferences in the effect with praziquantel on the tegument of adult S.japonicumworms between praziquantel resistant and susceptible isolates in vivo, have thecorollary that exposed hidden antigens are slso not the same, course different host Threaction.