Real-time observation of pathophysiological processes during murine experimental Schistosoma japonicum infection using high-resolution ultrasound imaging.

BACKGROUND: Hepatosplenic lesion formation is one of the typical clinical symptoms of schistosomiasis japonica. Although it is established that circum-oval granuloma formation mediated by T lymphocytes is the key event triggering the formation of hepatic lesions, the time-course kinetics of disease progression remains to be fully elucidated. METHODS: The real-time process of the pathophysiology of schistosomiasis japonica from the early to late clinical phase was non-invasively observed in a murine experimental infection model using high-resolution ultrasonography. Together with clinical parameters, including body weight and the levels of serum markers of hepatic damage or fibrosis, ultrasonography was used to assess changes in the liver parenchyma and diameter of the portal vein and portal blood flow velocity. In parallel, parasitological parameters were observed, including egg number in the feces and maturation of parasites. RESULTS: Abnormal high-echo spot patterns in the liver parenchyma, reflecting hepatic fibrosis in ultrasonography, appeared in the liver at 4 weeks post-infection and the pattern became more enlarged and severe over time. This finding was concordant with parasite maturation and initial egg excretion. The serum M2BPGi level markedly increased from 8 weeks post-infection, suggesting sharp deterioration of hepatic fibrosis. At the same time, the diameter of the portal vein, reflecting portal hypertension, became enlarged and reached the peak level at 8 weeks post-infection. Ascites were apparent around the spleen at 9 weeks post-infection, and dilatation of the splenic vein was noted at 10 weeks post-infection. Live adult worms seemed to be detected in the portal vein at 4 weeks post-infection by ultrasonography. CONCLUSIONS: We obtained real-time imaging of the development of hepatosplenic lesions of schistosomiasis japonica in mice. The time-course kinetics of the onset, development, and modulation of each symptom was uncovered. These results are expected to provide new clues for understanding the pathophysiology of human schistosomiasis japonica.

Immunogenicity and anti-fecundity effect of nanoparticle coated glutathione S-transferase (SjGST) DNA vaccine against murine Schistosoma japonicum infection.

There is still urgent need for a vaccine against schistosomiasis, especially in Schistosoma japonicum endemic areas where even a vaccine that will interrupt zoonotic transmission will be potentially effective as an intervention tool. We had developed a novel nanoparticle gene delivery system, which has proven efficacious in gene transfection to target immune cells with complementary adjuvant effect and high protective efficacy in several diseases. Here, we applied this nanoparticle system in combination with S. japonicum glutathione S-transferase (SjGST) DNA vaccine to show the immunogenicity and anti-fecundity effect of the nanoparticle coated vaccine formulation against murine schistosomiasis. The nanoparticle-coated DNA vaccine formulation induced desired immune responses. In comparison with the nanoparticle coated empty vector, it produced significantly increased antigen-specific humoral response, T-helper 1 polarized cytokine environment, higher proportion of IFN-γ producing CD4(+) T-cells and the concomitant decrease in IL-4 producing CD4(+) T-cells. Although there was no effect on worm burden, we recorded a marked reduction in tissue egg burden. There was up to 71.3% decrease in tissue egg burden and 55% reduction in the fecundity of female adult worms. Our data showed that SjGST DNA vaccine, delivered using the nanoparticle gene delivery system, produced anti-fecundity effect on female adult schistosomes as previously described by using conventional subunit vaccine with adjuvant, proving this DNA vaccine formulation as a promising candidate for anti-pathology and transmission blocking application.

A new surveillance and response tool: risk map of infected Oncomelania hupensis detected by Loop-mediated isothermal amplification (LAMP) from pooled samples.

Although schistosomiasis remains a serious health problem worldwide, significant achievements in schistosomiasis control has been made in the People's Republic of China. The disease has been eliminated in five out of 12 endemic provinces, and the prevalence in remaining endemic areas is very low and is heading toward elimination. A rapid and sensitive method for monitoring the distribution of infected Oncomelania hupensis is urgently required. We applied a loop-mediated isothermal amplification (LAMP) assay targeting 28S rDNA for the rapid and effective detection of Schistosoma japonicum DNA in infected and prepatent infected O. hupensis snails. The detection limit of the LAMP method was 100 fg of S. japonicum genomic DNA. To promote the application of the approach in the field, the LAMP assay was used to detect infection in pooled samples of field-collected snails. In the pooled sample detection, snails were collected from 28 endemic areas, and 50 snails from each area were pooled based on the maximum pool size estimation, crushed together and DNA was extracted from each pooled sample as template for the LAMP assay. Based on the formula for detection from pooled samples, the proportion of positive pooled samples and the positive proportion of O. hupensis detected by LAMP of Xima village reached 66.67% and 1.33%, while those of Heini, Hongjia, Yangjiang and Huangshan villages were 33.33% and 0.67%, and those of Tuanzhou and Suliao villages were 16.67% and 0.33%, respectively. The remaining 21 monitoring field sites gave negative results. A risk map for the transmission of schistosomiasis was constructed using ArcMap, based on the positive proportion of O. hupensis infected with S. japonicum, as detected by the LAMP assay, which will form a guide for surveillance and response strategies in high risk areas.

Basophil depletion downregulates Schistosoma mansoni egg-induced granuloma formation.

Granuloma formation around parasite eggs during schistosomal infection is considered to be controlled by Th2 cytokines. However, it is still controversial which cell populations are responsible for the host Th2 cytokine-dependent granuloma formation. Basophils have recently attracted attention because of their ability to produce large amounts of IL-4. Therefore, we investigated whether basophils play an essential role in the induction of granuloma formation induced by Schistosoma mansoni eggs. Together with our previous observation that basophil numbers increased markedly in the spleen at 7 weeks postinfection, immunohistochemical staining using anti-mMCP8 monoclonal antibody (mAb) showed basophil infiltration in the granulomatous lesions formed around parasite eggs. To examine the roles of basophils more directly, we treated mice with anti-CD200R3 mAb to deplete basophils. Depletion of basophils resulted in a reduction of basophil number with concomitant downregulation of egg granuloma formation at 7 weeks postinfection. Moreover, we observed a significant reduction in the size of egg granulomas formed in basophil-depleted mice in the pulmonary granuloma model. Taken together, these findings indicated that basophils are essential for S. mansoni egg-induced granuloma formation, and this may serve as a novel therapeutic target in ameliorating the pathology of schistosomiasis.

Interleukin-4 (IL-4) and IL-13 suppress excessive neutrophil infiltration and hepatocyte damage during acute murine schistosomiasis japonica.

Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4-/- IL-13-/- (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4-/- IL-13-/- IL-17A-/- (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.

Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay.

Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places.

Centromere-specific acetylation of histone H4 in barley detected through three-dimensional microscopy.

Histone acetylation affects chromatin conformation and transcriptional activity. However, the structural role of histone acetylation at specific chromosomal regions, such as the centromere, is poorly understood. In this study, histone H4 acetylation and its localization in barley interphase nuclei are revealed by three-dimensional microscopy. The centromeres form a ring-like allocation near the nuclear membrane in barley. Immunofluorescence studies on non-fixed, interphase nuclei treatment revealed ring-like distribution of the highly acetylated histone H4, located near the nuclear membrane at one pole of the nucleus. This fluorescent structure was similar to the centromere cluster and referred to as hyperacetylated region (HAR). The distribution pattern of the acetylated histone H4 was similar to each of the K5, K8, K12 and K16 lysine residues, although H4 acetylated at K5, K8 and K12 residues was found in almost all nuclei, whereas H4 acetylated at K16 was weakly observed in only half of the nuclei. Each HAR consists of two strongly acetylated cores and a halo-like, less acetylated surrounding area. Fluorescence signals from centromere-specific repetitive sequences of barley, detected through three-dimensional fluorescence in situ hybridization (3D-FISH), co-localized with the HAR corresponding to the K5 residue acetylation, but the signals did not completely overlap each other. These findings indicate that histone acetylation specifically occurring at the centromeres likely have certain structural roles for the centromere.

Cell cycle-dependent and lysine residue-specific dynamic changes of histone H4 acetylation in barley.

Histone acetylation affects chromatin conformation and regulates various cellular functions, such as transcription and cell cycle progression. Although mitosis dependent transcriptional silencing and large-scale chromatin structural changes are well established, acetylation of histone H4 during the mitosis is poorly understood in plants. Here, the dynamics of acetylation of histone H4 in defined genome regions has been examined in the fixed barley cells throughout the mitosis by three-dimensional microscopy. Patterns of strong acetylation of the two lysine residues K5 and K16 of histone H4 in the barley genomes were found to be different. In interphase nuclei, H4 acetylated at K 16 was associated with the gene-rich, telomere-associated hemispheres, whereas K5 acetylation was detected in centromeric regions where the heterochromatin is distributed. Regions of strong K5 acetylation changed dynamically as the cell cycle proceeded. At prometaphase, centromeric acetylation at K5 decreased suddenly, with accompanying rapid increases of acetylation in the nucleolar organizing regions (NORs). Reverse changes occurred at telophase. On the other hand, the strongly acetylated regions of the K16 showed changes compatible with transcriptional activities and chromosome condensation throughout the cell cycle. Telomeric acetylation at K16 was detected throughout the cell cycle, although it was reduced at metaphase which corresponds to the most condensed stage of the chromosomes. It is concluded that dynamic changes in H4 acetylation occur in a lysine residue-, stage-, and region-specific manner and that they correlate with changes in the chromosome structure through the cell cycle.