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@#Objective To identify the genotype of Toxoplasma gondii isolated strains from a congenital teras（KS strain）and an HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province. Methods T. gondii DNA of tachyzoites of a isolate from a congenital teras（KS strain）and blood DNA of an HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province were extracted，and 11 loci were identified for the genotype by polymerase chain reaction restriction fragment length polymorphism（PCR⁃RFLP）. Results The complete bands were obtained from the congenital teras（KS strain）and HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province，and identified as T. gondii gene type I. Conclusion T. gondii gene type I may be the dominant genotype strain of T. gondii among the women who have the abnormal pregnant outcomes and HIV⁃Toxoplasma co⁃infected patients in Jiangsu Province.
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Objective To clone and express a high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Main-land strain)and analyze its function. Methods The DNA fragment of open reading frame encoding Sj HMGB1 protein was ampli-fied by RT-PCR from the mRNA of S. japonicum worms,then it was subcloned into the expression vector pET28a(+)to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3),and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombi-nant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding ca- pacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 pro-tein and infected with 45±2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection,the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue,respectively. The worm and egg reduction rates were calculat-ed respectively. Results A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR,which was the open reading frame(ORF)encoding SjHMGB1protein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA,and the recombinant protein immu-nized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abun-dantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effec-tive immune protection against S. japonicum. Conclusion The gene encoding HMGB1 from S. japonicum and the soluble recombi-nant SjHMGB1 protein with natural functional activity are obtained,and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.
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Objective To study the effect of praziquantel on cells within sehistosomal ovum granuloma in the lung of mice.Methods Forty-eight mice were divided into 4 groups.Group A:first,the mice were injected with sehistosomal ova hypodermically in abdomen,and 10 days later,injected with schistosomal ova intravenously in the cauda;Group B:in addition to the injection of schistosomal ova as the same of Group A,the mice were administered with praziquantel[300 mg/(kg·d)]for 3 days,one day before the intravenous injection of the ova;Group C:in addition to the injection of schistosomal ova as the same of Group A,the mice were administered with praziquantel(75 mg/kg,B.i.d.) for 5 days weekly until the mice were sacrificed;Group D:the same as Group C but praziquantel was given to the mice from the 29th after the intravenous injection of the ova.Three mice of each group were sacrificed on the 7th,14th,28th,56th day after the intravenous injection of the ova and the lung tissues were fixed with formalin and the slices were HE stained.Twenty-five to thirty pieces of schistosomal ovum granuloma were examined and the neutrophilic granulocytes,eosinocytes,lymphocytes,fibroblasts and macrophages within the ovum granulomas were counted and the mean numbers of them of each group were calculated and compared.Results Compared with Group A,the mean numbers of neutrophilic granulocytes,eosinocytes and macrophages within the ovum granulomas were decreased significantly,and the extend of the increase of fibroblasts reduced significantly in the three groups administered with praziquantel,and especially in Group C.On the 56th day after the intravenous injection of the ova,the mean numbers of neutrophilic granulocytes,eosinocytes and macrophages decreased by 54.4%、87.0% and 23.1%,and the extend of the increase of fibroblasts reduce by 59.4%,respectively in Group C,compared with Group A.The numbers of lymphocytes did not change very much in 4 groups.Conclusion Praziquantel can restrain inflammatory cells and the hyperplasia of fibroblasts within schistosomal ovum granulomas.
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Objective To study the effects of lipopolysaccharide(LPS) on the expression of toll like receptor 4 (TLR4), reactive oxygen species(ROS) and on the proliferation of cells as well as secretion of six proinflammmatory cytokines including TNF-α, IL-1, IL-6, IL-8, IL-10, IL-12 levels in SKOV3 cells. And to explore the mechanism of SKOV3 cells in regulation. Methods Cultured primary SKOV3 cells were stimulated with different concentrations of LPS (0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml and 20 μg/ml) for 4 h, the TLR4 expression in SKOV3 cells were examined by flow cytometry;1 μg/ml LPS stimulated SKOV3 for 4 h, 8 h, 12 h, 24 h respectively, the TLR4 expression and cell cycle in SKOV3, cell proliferation, ROS level as well as cells and TNF-α and IL-1, IL-6, IL-8, IL-10, IL-12 levels in the culture medium were assayed by flow cytometry, MTT, CBA assay respectively. Results LPS with different concentrations of LPS stimulation in-duced an increased TLR4 expression, however, the expression was reduced when LPS concentration up to 10 μg/ml. LPS stimulation for 4 h, 8 h induced an increased TLR4 expression and cell proliferation. Stimulated for 24 h, however, the TLR4 expression and cell growth were inhibited in S period. Meanwhile, LPS stimulation for 4 h, 8 h, 12 h, 24 h induced a higher ROS secretion in comparison with control group. LPS stimulation induced a stronger cytokine response in comparison with control group, as demonstrated by the production of TNF-α, IL-1, IL-6, IL-8 secretion in cultured SKOV3 cells, while IL-10 and IL-12 with low expression have no obvious difference in the all medium samples. Conclusion TLR4 expression, cell proliferation, ROS and proin-flammmatory cytokine secretion could be induced in SKOV3 through LPS stimulation. The study provide new ex-periment evidences for human ovarian cells SKOV3 immunity regulation and inflammation reaction to promote cells inhibition after LPS stimulation.
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Objective To explore the impact of acute Toxoplasma gondii infection on cerebral proteins and nerve growth in mice by 2D electrophoresis. Methods The cerebral proteins from C57BL/6J mice infected with Toxoplasma gondii and normal paired mice were extracted. The discrepant proteins were checked by 2D electrophoresis. Isoelectric focusing was determined as the first direction (immobilized pH gradient gel 3-10) ,SDS-PAGE as the second direction to execute 2D electrophoresis, and PDQuest 1.0 software was used to analyze 2D electrophoretogram. Results The protein spots in Toxoplasma gondii infected mice and normal paired mice were (132 ±10) and (170 ± 13) , respectively. After the analysis by PDQuest 1. 0 software, only 19 protein spots were found to express in infected mice and only 37 protein spots were found to express in normal paired mice. Additionally, the obvious quantitative changes in a part of proteins of the cerebrum in the both group occurred. Conclusion There are obvious changes in cerebral proteins from mice with acute Toxoplasma gondii infection, which provids useful clues for studying the cerebral proteins injury in acute Toxoplasma gondii infected mice and the new cure drug.
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To investigate the effect of Toxoplasma gondii infection upon the expression of brain-derived neurotrophic factor (BDNF) mRNA and N-methyl-D-aspirate receptor (NMDA) subunits NR2A and NR2B,Wistar rats of 4 weeks old were randomly divided into 3 groups with 10 rats in each group in which 2 mL suspensions of T.gondii tachyzoits in the concentrations of 2×10~7/mL and 2×10~5/mL were injected intra-peritoneally to rats in group A and group B respectively, serving as the experimental groups, while 2 mL of sterile physiologic saline was injected intra-peritoneally in group C serving as the control group. Four weeks after injection, the expressions of BDNF mRNA and BDNF protein in the brain tissues were detected by in situ hybridization and immunohistochemical assay and the expressions of NR2A and NR2B immune activity in the hippocampal CA1,CA3 and DG were investigated by using computer-assisted image analysis system. Compared with the control group, the expression of BDNF protein in the hippocampus of the experimental groups was significantly enhanced [(64.27±23.18), (50.39±19.34) vs (44.68±22.74)/mm~2,P＜0.05]. In addition, the increased expressions of BDNF mRNA in the hippocampus of the experimental groups were also demonstrated [(0.13±0.02), (0.12±0.02) vs (0.09±0.01); P＜0.05]. In the expression of the NR2A protein, their expressions in group A and B of rats were significantly lower than that of group C in CA3 (P＜0.05),but there was no significant change in CA1 and DG. In the expression of NR2B protein, the expressions in group A and B were also lower than that of group C in CA1 and CA3, and had no significant change in DG. It is evident that the expressions of BDNF mRNA and BDNF protein in hippocampal tissues were significantly increased following chronic infection with T.gondii, supporting the hypothesis that BDNF may be involved in the intrinsic neuro-protective mechanism.
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Objective To optimize the conditions of the expression of fusion protein GST-SjMP10 and to evaluate the value of fusion protein GST-SjMP10 for diagnosis of schistosomiasis.Methods The optimal concentration of IPTG for the expression of fusion protein GST-SjMP10 was chosen in inducing the expression of GST-SjMP10 with different concentration of IPTG,and the soluble fusion protein GST-SjMP10 was identified by SDS-PAGE.The fusion protein GST-SjMP10 was purified by chromatographic affinity with glutathione Sepharose 4B gel.The sensitivity and specificity of purified fusion protein GST-SjMP10 for diagnosis of schistosomiasis were determined by enzyme linked immunosorbent assay(ELISA)to detect the IgG antibody in sera from the patients with acute schistosomiasis,advanced schistosomiasis and clonorchiasis as well as healthy subjects.Results Most of the expressed fusion protein GST-SjMP10 was in soluble status when the concentration of IPTG was reduced to 0.1 mmol/L and the fusion protein GST-SjMP10 could be purified by chromatographic affinity.The positive rate of anti-GST-SjMP10 antibody in the sera from the patients with acute and chronic schistosomiasis japonica was 97.5% and 96.7% respectively.No cross reactivity of the fusion protein GST-SjMP10 was found in the detection for the sera from clonorchiasis patients,and no false positive was found in the detection for the sera of healthy subjects.Conclusion The fusion protein GST-SjMP10 was expressed successfully and showed high sensitivity and specificity for the diagnosis of schistosomiasis japonicum.
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The specialization of medical science determines the objective existence of asymmetric information in medical and health industry.These different understandings in the psychological and behavioral area influenced the development of physician-patient relationship.Medical personnel should look squarely at the professional attributes and dare to play a leading role.Meanwhile,the exchange of the role of patient and medical personnel,understanding patient,clearing of communication channels and enlarging patients′ means of communication will promote harmonious relations between doctors and patients.
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The basic nature of medical ethics education is the cultivation of morality for medical students,in which humanistic spirit is the fundamental element.Humanistic spirit is in great need for the development of medical ethics,the transformation of medical mode,the harmonious development of physician-patient relationship,and the healthy growth of medical students.Based on present realistic conditions,humanistic spirit should be developed in the fields of classroom training,medical professional education,and other aspects of campus culture.It would be necessary to find multi-path training modes of humanistic spirit.
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This study reported the primary application of FA-ELISA from 107-121kDa fration antibody of Schistosoma Japonicum for detection of the short-term antibody in patients with schistosomiasis. The result showed that this method provided high sensitivity (with positive rate of 93. 33% with 30 cases of schistosomiasis) and good specificity (no false postive in 60 health objects). When one group of schistosome patients were examined with FA -ELISA and with the routine SEA-ELISA before chemotherapy and at 8 months,1 year and 1. 5 year after treatment,it showed good property for evaluation of chemotherapy efficacy with FA -ELISA,which was much better than the routine method.
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Objective To explore the Schistosoma japonicum cercariae collected method and the quantity of cercariae obtained from a great quantity artificial infected snails. Methods In laboratory condition, Oncomelania snails were infected with schistosome miracidia. Sixty days post-infection all snails were divided into 7 shares. Cercariae shed from 1 share snails every day and the number of all shedding days were 40. Cercariae shed from snails were collected with low-velocity centrifuge and the cercariae sediment were weighted. Results One thousand and nine hundred g snails bred for 120 days post-infection, the infection and survival rates were 36. 00% and 51. 58%. Cercariae col-lected were 10. 5 g from 40 days collection. Cercariae quantity of shedding from 1 000 positive snails per day was 0. 257 4 g.
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Objective To observe the influence of suspension concentrate of niclosamide on the enzyme activity of Oncomelania hupensis in order to explore its molluscicidal mechanism. Methods Oncomelania hupensis snails were collected in the habitates of river marshland in Dantu District, Zhenjiang City, Jiangsu Provence and were divided into 2 groups. The snails of the treated group were sprinkled with 25% suspension concentrate of niclosamide. The snails of the control group were sprinkled with distilled water. The soft body tissue of the snail was separated and the sections of snail tissue were made in the Cryostat Microtome. The stain of enzyme-histochemistry showed CCO, LDH, SDH, CHE and NOS had been done, and then the staining block was made by routine method. The staining reaction in the snail tissue and the average gray density were observed with the image analysis system of biomicroscope. Results The enzyme activity of CCO, LDH, SDH, CHE and NOS located in the mouth, muscle fiber, tegumentary membrane, ganglia, liver and pharyngeal cavity of Oncomelania hupensis snails. The enzyme activities of CCO, LDH, SDH, CHE and NOS in the treated group were significantly lower than that in the control group. Conclusions Niclosamide can affect the transmitting of neurohypophysis and obstruct the energy and result in the disorder of the physiological functions in Oncomelania hupensis. It is one of the reasons of Oncomelania hupensis death.
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Objective To investigate the value of mix recombinant antigen in schistosomiasis diagnosis. Methods The recombinant antigens of SjC23 (HD),SjC21.7 and SjCMP10 were expressed in vitro and purified by the affinity chromatography method. The efficacies of soluble egg antigen (SEA),single recombinant antigen and mix recombinant antigen for schistosomiasis diagnosis by Enzyme-linked Immunosorbent Assay were compared. Results The diagnostic efficacy was the same when the antibody IgG of the same group sera of schistosomiasis was detected by different quantities of 2.5 ?g/ml and 7.5 ?g/ml of SEA immobilized on microplate, and their absorbency A was the same, but there was a significant difference in the diagnostic efficacies between single recombinant antigen and mix recombinant antigen when the antibody IgG of the same group sera of schistosomiasis was detected by the same quantity of single recombinant antigen or mix recombinant antigen immobilized on microplate, the absorbency A of mix antigen reacted with the sera of schistosomiasis was significant higher than that of the single recombinant. The positive rates were very similar when 39 sera of acute schistosomiasis,80 sera of chronic schistosomiasis and 27 sera of advanced schistosomiasis were detected by SEA or mix recombinant antigen by ELISA in the same time. No cross-reaction presented when 20 clonorchiasis sera were detected by the mix recombinant antigen and no false positive presented when 40 of healthy sera were detected by the mix recombinant antigen. Conclusion The schistosomiasis diagnostic method by using the mix recombinant antigen has been established, which is helpful for improving the efficacy of schistosomiasis diagnosis.
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Objective To evaluate the molluscicidal effect of colistin E on Oncomelania hupensis. Methods The molluscicidal effect of colistin E on O. hupensis was checked by using the immersion method and spraying method. The toxicity of colistin E on fish was observed by using the toxic test. Results The snail mortality of each group was 100% when the snails were immersed in the colistin E solutions at a concentration of 5. 0, 1. 0 g/L for 24, 48 h and 72 h separately. When the snails were immersed in the colistin E solution at a concentration of 0. 5 g/L for 24, 48 h and 72 h, the death rates were 95% , 100% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 1 g/L for 24, 48 h and 72 h, the mortality was 90%, 95% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 01 g/L for 24, 48 h and 72 h, the mortality was 70%, 86% and 100% respectively. The snail mortality by the spraying method in a dose of 35, 70, 140 mg/m2 of colistin E was 60%, 100% and 100%. The result of toxic test showed that the toxicity of colistin E on fish was low. Conclusion Colistin E is an effective molluscicide, and is worthy of investigation further.
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Objective To search for a biomarker from the serum of Schistosoma japonicum infected rabbits for early diagnosis of schistosomiasis. Methods The sera were obtained from different periods of the infected rabbits. The serum proteins were generated by WCX kit (Bruker Daltonics GmBH) and analyzed by the technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results In the mass range from 1 000 to 12 000 Da, sixty-three proteins were captured by WCX kit. ClinProTools software was used to find the differential expressed proteins. The result revealed 7 distinct proteins compared with normal serum. Among them,1 787 Da protein expression was increased (P
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Objective To explore the effect of praziquantel on schistosomal ovum granuloma in the lung of mice.Methods Forty-eight mice were divided into 4 groups.Group A:first,the mice were injected with schistosomal ova hypodermicly in abdomen and 10 days later,injected with schistosomal ova intravenously in the cauda;Group B:in addition to the injection of schistosomal ova as the same of Group A,the mice were administered with praziquantel [300 mg/(kg?d)] for 3 days from the last day of the intravenous injection of the ova;Group C:in addition to the injection of schistosomal ova as the same of Group A,the mice were administered with praziquantel(75 mg/kg,B.i.d.)for 5 days weekly until the mice were sacrificed;Group D:the same as Group C but praziquantel was given to the mice from the 29th after the intravenous injection of the ova.Three mice of each group were sacrificed on the 7th,14th,28th,56th day after the intravenous injection of the ova and the lung tissues were fixed with formalin and the slices were HE stained.Fifteen-thirty pieces of schistosomal ovum granuloma were examined and their areas were measured and the mean areas of each group were calculated and compared.Results On the 7th,14th and 28th days after the intravenous injection of the ova,the mean area of schistosomal ovum granuloma in Group C was significantly less than that in Group A,and there was a significant difference between the two groups,P 0.05.On the 56th day,the mean areas of schistosomal ovum granuloma in Group B,C,D were significantly less than that in Group A,all P
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Objective To find out the valuable early diagnostic antigen of Schistosoma japonicum. Methods The sera of rabbits were collected at different time after the rabbits were infected with cercariae of Schitosoma japonicum. The fractions of the soluble cercaria antigen (SCA), soluble adult worm antigen (AWA) and soluble egg antigen (SEA) were separated by SDS-PAGE and recognized by Western blotting with rabbits' sera of different time of post-infection. Results In Western blotting, the bands of 94, 48, 41, 40 kDa and 38 kDa of SCA appeared the earliest and were recognized by the rabbits sera of 2-week post-infection, the bands of 71 kDa and 23 kDa of SCA reacted with the rabbits sera of 3-week post-infection strongly. The bands of 71 kDa and 58 kDa of AWA appeared the earliest and were recognized by rabbits sera of 3-week post-infection. The bands of SEA reacted earliestly to the rabbits sera of 4-week post-infection were 270, 151, 73, 69, 50 kDa and 24 kDa. Conclusion The fraction antigens of 94, 71, 48, 41, 40, 38 kDa and 23 kDa of SCA, the fraction antigens of 71 kDa and 58 kDa of AWA and the fraction antigens of 270, 151, 73, 69, 50 kDa and 24 kDa of SEA could be recognized by sera of acute infected rabbits and might have potential early immuno-diagnosis value for schistosomiasis.
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Objective To observe the impact of water temperature on shedding and infectivity of the cercariea of Schistosoma japonicum under laboratory condition. Methods Infected snails were exposed to 1, 5, 10, 15, 20, 25, 30, 35℃ and 40℃ water for 4 hours for shedding of cercariae respectively, the shedding rates and total numbers of cercariae shed were investigated. The mice were infected with cercariae in 1, 10, 20, 30℃ and 40℃ water for 30 minutes respectively, the infection rates and mean worm burden of mice were investigated. Results The cercariae were shed from infected snails with Jiangsu or Yunnan isolate of [WT5”BX]S. japonicum in water ranged from 1℃ to 40℃, but the fittest water temperatures range for shedding of cercariae of Yunnan isolate of S.japonicum[WT5”BZ] is from 20℃ to 35℃, while fittest range for Jiangsu isolate is from 15℃ to 35℃. All of the infection rates of mice infected with cercariae of Jiangsu isolate of S.japonicum in the water ranged from 1℃ to 40℃ were 100.0%. Infection rates of mice infected with cercariae of Yunnan isolate of S. japonicum in the water ranged from 1℃ to 40℃ were more than 83.3%. Conclusions When infected snails are exposed to the water with temperature ranged from 1℃ to 40℃ it is possible for cercariae to shed and infect the final hosts . It is suggested that there is a possibility of infection with S.japonicum when contacting the water near marshlands with infected snails in winter.
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Objective To develop a fast, simple immunodiagnosis assay for early diagnosis of schistosomiasis. Methods The soluble cercariae antigen(SCA) labeled colloidal dye was used as the detecting antigen for schistosomiasis. A dipstick dye immunoassay(SCA-DDIA) for early diagnosis of schistosomiasis was established. The antibodies in sera of infected rabbits in early stage of infection by SCA-DDIA were detected and compared with SEA-DDIA. The sera from people with acute and chronic schistosomiasis and other parasitic diseases and from healthy people were tested by SCA-DDIA and SEA-DDIA. Results In infected rabbits during early stage of infection, the average time of antibody detected by SCA-DDIA was 22 d , at day 30 post-infection all experimental rabbits were positive with SCA-DDIA, the detected time was earlier than that with SEA-DDIA. The sensitivity of SCA-DDIA for acute, chronic schistosomiasis japonica were 100.0% and 93.3% respectively. The specificity for healthy persons was 99.0%. The cross reaction rates with paragonimiasis westermani, clonorchiasis sinensis and fasciolopsiasis buski were 26.3%, 0 and 0 respectively. The results were similar to that by SEA-DDIA. Conclusion The SCA-DDIA is more useful for early diagnosis of schistosomiasis.