Congenital infection of rabbits with Schistosoma japonicum and protective immunity of offspring

Background Recently congenital infection with Schistosoma japonicum (S. japonicum) has been domonstrated in pigs, rabbits, mice and dogs. We explored the rabbit as an animal model for the congenital infection of schistosomiasis japonica and assessed the effect of a congenital S. japonicum infection on the resistance of rabbit kittens to a postnatal challenge infection.Methods Sixteen pregnant New Zealand white rabbits were infected with a single dose of S. japonicum cercariae. The exposed animals were divided into three groups according to the gestation age at the time of infection. Diagnosis of prenatally acquired S. japonicum infection in the rabbit kittens was primarily based on serological tests in combination with parasitological and histopathological findings. Congenitally infected kittens were challenged percutaneously with 100 S. japonicum cercariae to assess the effect of a congenital S. japonicum infection on kitten resistance to a postnatal challenge infection.Results The overall prevalence of congenital infection in offspring of infected mothers was 20% (12/60). The congenital infection rate in group L (late gestation) was much higher than in group E (early gestation) and group M (mid-gestation) (P<0.05). After a postnatal challenge infection, prenatally infected kittens had a 54.66% worm reduction rate, 41.45% egg reduction rate, and 51.76% granuloma size reduction rate compared to nave kittens.Conclusions This study demonstrates the possibility of congenital infection of S. japonicum in rabbits and the resistance of congenitally infected kittens to a postnatal challenge infection. These results have important implications not only for epidemiological investigations, but also in designing government control programs for schistosomiasis.     

Vertical Transmission of Schistosoma Jkaponicum in the Rabbit

The aim of the present study was to confirm observations on the vertical transmission of Schistosoma japonicum in the rabbit.S.japonicum-infected pregnant rabbits were used in this study.Perfusion of mother rabbits was done 9 weeks after infection in order to obtain worm burdens in relation to their initial cercarial dose.Anti-schistosoma specific IgM antibodies in serum samples collected from rabbit kittens were detected by ELISA.Our results showed that gestation period lasted the normal 29-31 days.All the exposed mother rabbits became infected with S.japonicum.Positive IgM antibody OD values were detected in 12 out of the 60 kittens examined (20.0%).In group C and A,40.0% and 17.9% of the kitten were congenitally infected,respectively.18.1% of the kittens born to mothers infected with a single dose of 200 cercariae perrabbit were positives;this is not significantly different from that obtained for the 600 dose group (22.2%).Three randomly selected IgM^ kittens harbored between one and two adult worms.The livers of these kittens displayed granulomatous lesions.It is concluded that congenital S.japonicum infection does occur in the rabbit and is affected by the mother stage of pregnancy and to a lesser extent by its infection load.     

Schistosomiasis in China: acute infections during 2005–2008

Background Significant progress has taken place over the past 50 years in the control of schistosomiasis japonica in China. However, the available data suggested that schistosomiasis has re-emerged shortly after the World Bank Loan Project which was conducted from 1992 to 2001. The national control program with a revised strategy to control schistosomiasis by using integrated measures has been implemented since 2005. In this study, we aimed to evaluate the effect of the national program on schistosomiasis control from 2005 to 2008.
Methods A retrospective study was carried out to analyze the epidemic patterns of acute infections with Schistosoma japonicum （S. japonicum）, based on the number of acute cases annually collected from the web-based national communicable diseases reporting system from 2005 to 2008.
Results A total of 564, 207, 83 and 57 acute cases infected with S. japonicum were reported nationwide in 2005, 2006, 2007 and 2008, respectively, with an average annual reduction rate of 46.35% during last four years. Six outbreaks of acute infection with S. japonicurn were reported in 2005 but none in the period of 2006 to 2008. All acute cases that were reported mainly came from the lake regions and became infected during the higher risk periods from the 27th to 43rd weeks of the year. Most of these cases are students （44.87%）, farmers （31.51 %） and fishermen （7.79%） who got the infection by water contact mainly through swimming （41.49%） and production activities （40.25%）. With time, the proportion of imported cases among all acute cases increased due to more frequent movement of people that has occurred with a more mobile population.
Conclusions The national control program on schistosomiasis aliened with the revised control strategy has been effectively brought into effect. However, there is still a significant risk of infection among students, farmers and fishermen living in the lake regions. Therefore, it is important to strengthen control measures among risk populations in the high risk areas of transmission, or the lake regions.     

Expression and Significance of NF-κB, IL-1β and COX-2 in the Murine Model of Estrogen-dependent Experimental Vulvovaginal Candidiasis

Objective To investigate the possible role of estrogen in the pathogenesis of vulvovaginal candidiasis(VVC). Methods Estrogen-dependent experimental murine model of C. albicans vaginal infection was established by injecting subcutaneously with estradiol benzoate and then 5×106 stationary-phase C. albicans blastoconidia was inoculated intravaginally to mice (group EI), and other 3 groups were set up: estrogen-treated but not infected (group E); estrogen-untreated but infected (group I); normal control (group C). The dynamic change of colony-forming unit (CFU) of cervivovaginal lavage fluid was observed. Vaginal tissues at different time points (d 2, d 4, d 7 and d 14) after inoculation of C. albicans were obtained. In situ hybridization staining was used to detect expression of cyclooxygenase-2 (COX-2) mRNA and expression of nuclear factor-κB (NF-κB) was examined by immuohistochemistry. ELISA was applied to determine the interlenkin-1β (IL-1β) level. Results The constitutional high level expression of COX-2 mRNA in the vaginal tissue of group E was significantly higher than that in group C on d 4 and d 7 (P<0.01), and the optical density (OD) in group EI was higher than that in the other 3 groups (P<0.05). There were higher IL-1a levels in vaginal tissues from d 4 to d 7 postin- oculation in group EI and group I than group C (P<0.01). Furthermore, IL-1β in group EI was markedly elevated on d 4 and d 7 compared with group I (P<0.01). Compared with group C, the expression of NF-κB in group E was increased obviously on d 4 (P<0.01), and there was significant difference between group EI and group Ion d 4 and d 7 (P<0.01). Conclusions In the murine model of estrogen-dependent experimental VVC, estrogen promotes the infection establishment by up-regulating expression of COX-2 via activating NF-κB signal pathway, and the high expression of COX-2 promoted by the interaction of IL-1β and NF-êB after infection formation was involved in persistence of infection.     

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12

Background The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum ( S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S.japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses.Methods The plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-γ and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM).Results The plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45. 53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-γ but decreases in IL-4.No significant differences in CD4^ and CD8^ subgroup ratios were observed after the challenges.Conclusions The multivalent DNA vaccine pVIVO2-1L12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Thl responses and, hence, the protective immunity.     

Local Expression of Vaginal Th1 and Th2 Cytokines in Murine Vaginal Candidiasis under Different Immunity Conditions

To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 （IL-2）/Th2 （IL-4, IL-10, TGF-β1） cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-β1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-β1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans （C. albicans）, and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.     

Effect of Tumor Necrosis Factor-α on Acyl Coenzyme A： Cholesteryl Acyltransferase Activity and ACAT1 Gene Expression in THP-1 Macrophages

In order to explore the effect and mechanisms of tumor necrosis factor-α (TNF-α) on the activity of the acyl coenzyme A: cholesteryl acyltransferase (ACAT), THP-1 monocytes were cul- tured and induced to differentiate into macrophages with phorbol ester. TNF-α (60 ng/mL) was added at different time points into the macrophage-containing medium and the ACAT enzyme activity was measured by quantifying the incorporation of 1-14C oleoyl CoA into cholesteryl esters. The expres- sion of ACAT-1 protein and mRNA was respectively detected by Western blotting and RT-PCR in THP-1 macrophages 24 h after treatment with TNF-α (60 ng/mL). The results indicated that ACAT activity in THP-1 macrophages treated with TNF-α was increased in a time-dependent manner. The expression levels of ACAT-1 protein and mRNA were significantly increased in THP-1 macrophages after treatment with TNF-α (P<0.05). It was suggested that TNF-α could increase the activity of ACAT in THP-1 macrophages by up-regulating the expression of ACAT-1 gene.     

Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccinecandidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3‘ RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi) , Drosophila melanogaster (D.melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24. 921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E coil JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunitwere firstly sequenced, cloned, and expressed in E coil.     

Activation of nuclear factor κB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans

Background This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans. Methods Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor KB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting. Results M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression. Conclusion This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.     

EOTAXIN AND EOTAXIN-2 EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELL

Objective To study the role of eotaxin and eotaxin-2 expression by Th2 cytokine and analyze their relationship in normal human bronchial epithelial cell line-BEAS-2B cell. Methods Levels of eotaxin mRNA and protein expression in the bronchial epithelial cell line BEAS-2B cell were determined with RT-PCR and ELISA. We also used RT-PCR to evaluate eotaxin-2 expression under the regulation of Th2 cytokine IL-4 and IL-13 as well as proinflammatory agent-TNFα. Results Eotaxin mRNA expression was the highest at the time point of 12h under the stimulation of TNF-α. While Th2 cytokine IL-4 and IL-13 had the amplification effect on the expression. Eotaxin protein was also elevated with the combination stimulation of proinflammatory agent TNF-α and IL-4 in dose and time dependent manner（ P 〈 0. 01 ）. These results were also seen when the cells were stimulated by TNF-α and 1L- 13. Eotaxin-2 mRNA expression was the highest at the time point of 8h. The expression evaluated by semi-quantitative RT-PCR also elevated under the co-stimulation of TNF-α and IL-4 or TNF-α and IL-13 and it should significantly correlate with Eotaxin （ P 〈 0. 05 ）. Conclusion This study demonstrated that Th2 cytokine like IL-4 and IL-13 enhances eotaxin and eotaxin-2 expression when co-stimulated with proinflammatory agent TNF-α. These results showed that Th2 cytokines existence is the strong evidence for bronchial epithelial cells taking part in the allergic inflammation especially in eosinophils recruitment.     

Effect of antimicrobial agents on the toll-like receptors and inflammatory cytokines in liver tissue of the alcohol-induced liver disease in rats with Vibrio vulnificus sepsis

Background Septicemia and inflammation-mediated septic shock caused by Vibrio vulnificus （VV） is strongly associated with chronic liver disease. This study examined the effects of antimicrobial therapy on expression of hepatic toll-like receptors and inflammatory cytokines in rats with alcohol-induced liver disease complicated by VV sepsis. Methods Male Sprague-Dawley rats were assigned to the following treatment groups： normal control （N）, alcoholic liver disease control （A）, antimicrobial-treated alcoholic liver disease control （AA）, alcoholic liver disease with VV sepsis （AV）, and antimicrobial-treated alcoholic liver disease with VV sepsis （AVA）. Alcohol-induced liver disease was observed in all groups except N. Expression of mRNAs encoding hepatic toll-like receptors 2 and 4, myeloid differentiation protein-2, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and IL-10 was determined by RT-PCR. Results mRNAs encoding toll-like receptors 2 and 4 and myeloid differentiation protein-2 were significantly up-regulated in group AV as compared to control groups at 2-24 hours of sepsis; peak expression occurred at 12 hours. These mRNAs were also up-regulated in group AVA but to lesser degrees than in group AV at comparable time post-infection, mRNAs encoding TNF-α, IL-1β and IL-6 were significantly elevated in group AV as a function of infection. In group AVA as compared to AV, expression of TNF-α and IL-1β mRNAs was lower at 12-24 hours post-infection and expression of IL-6 mRNA was lower at 24 hours post-infection. Compared with control groups, IL-10 mRNA expression in group AV was markedly higher at 12-24 hours of sepsis. Expression of IL-10 mRNA was lower in group AVA as compared to AV at 24 hours of sepsis. Conclusions Antimicrobial therapy reduces expression of toll-like receptors and cytokines in rats with alcohol-induced liver disease complicated by VV sepsis. Monitoring hepatic toll-like receptor and cytokine expression during antibiotic therapy may be valuable for determining the course of VV sepsis in subjects with liver disease.     

Changes of sulfur dioxide, nuclear factor-κB, and interleukin-8 levels in pediatric acute lymphoblastic leukemia with bacterial inflammation

Background Bacterial inflammation is a common complication in patients with leukemia,and sulfur dioxide （SO2） is a bioactive molecule in modulating Gram-negative bacilli infection.This study aimed to examine the changes in SO2,nuclear factor-κB （NF-κB）,and interleukin-8 （IL-8） levels in pediatric acute lymphoblastic leukemia （ALL） with Gram-negative bacterial inflammation.Methods Fifty-five ALL children were enrolled in this study,including 30 males and 25 females,aged 3-13 years,and the median age was 7.8 years.All these children who accepted chemotherapy for ALL were divided into the control group （before chemotherapy）,the infection group （after chemotherapy with infection）,and the recovery group （the infection was controlled after 1 week）.The serum level of SO2 was detected using high performance liquid chromatography with fluorescence assay,and NF-κB and IL-8 levels were measured by enzyme-linked immunosorbent assay （ELISA）.Human THP-1 cells were cultured,induced,and differentiated into macrophages,which were divided into five groups and each group was cultured with different stimulators：lipopolysaccharide （LPS） group,LPS＋L-aspartate-β-hydroxamate （HDX） group,LPS＋SO2 group,SO2,and control groups.NF-κB level and IL-8 protein contents by ELISA were examined in each group.Results In comparison with those of the control group,levels of serum SO2,NF-κB,and IL-8 of the infection group were significantly increased （P ＜0.05）,while those of the recovery group were significantly decreased （P ＜0.05）.A positive correlation was found between levels of serum SO2 and intracellular NF-κB/IL-8,and the correlation coefficients were 0.671 and 0.798 （P ＜0.05）,respectively.According to the results found in human THP-1 cells,levels of NF-κB and IL-8 in LPS group were significantly increased compared with those of the control group （P ＜0.05）; when compared with those in LPS group,levels of NF-κB in LPS＋HDX group further increased significantly （P ＜     

Involvement of Shp-2 phosphatase in IL-1α mediated mouse embryo fi-broblast MMP-1 secretion and migration

Objective: To investigate the re1αtion between Shp-2 (a cytop1αsmic tyrosine phosphotase) and matrix metalloproteinase-1 (MMP-1) in the migration of mouse embryo fibrob1αst cell. Methods: Shp-2 -/- embryo fibrob1αst was separated from Shp-2 knockout mouse on El0. 5, and Shp-2 cDNA was transfected in-to Shp-2 -/- cell. The cell migration was observed with the wound healing, the MMP-1 expression and secre-tion was analyzed by immunoblotting and immunoprecipitation. The activation of MMP-1 in the supernatant was detected with type I col1αgenase activity assay after the stimu1αtion of IL-1α compared with El0. 5 mouse embryo fibrob1αst cell(Shp-2 / ). Results: There was obvious migration in Shp-2 / cells and Shp-2 -/-R cells, but not in Shp-2 -/- cells. The shape of Shp-2 -/- cell was epithelial-like. The expression and se-cretion was increased in Shp-2 / cells and Shp-2 -/-R cells, and it had not changed in Shp-2 -/- cell. The activation of MMP-1 was lower in Shp-2 -/- cells compared to the other cells. Conclusion: IL-1 induces the expression and secretion of MMP-1α at the physical dose, and the cell migration is involved in MMP-1 by way of Shp-2 signal transduction.