Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica

To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5α. The amplified library contained more than 400 positive bacterial clones, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully, the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.

The Role of TGF-β_1 in Mice Hepatic Fibrosis by Schistosomiasis Japonica

To investigate the role of transforming growth factor-β1 (TGF-β1) in mice with hepatic fi- brosis caused by Schistosomiasis Japonica, ELISA,VG staining and multimedia color hieroglyph quan- titative analysis were used to study the change of the serum TGF-β1, liver collagen fiber and reticular fiber in mice. The level of serum TGF-β1 in experimental group was significantly higher than that in control group (P<0.01 or P<0. 05) 8, 10, 12 weeks after infected by schistosomiasis. After infec- tion, the level of liver collagen fiber and reticular fiber, and that of TGF -β1 increased over time (P< 0.01 or P<0. 05). In mice infected by Schistosomiasis Japonica, the level of TGF-β1 increased with prolongation of infection time, and with the increase of liver collagen fiber and reticular fiber. TGFβ1 plays an important role of immunomodulation in hepatic fibrosis formation caused by Schistosomiasis Japonica.

Dynamic changes in the collagen metabolism of liver fibrosis at the transcription level in rabbits with Schistosomiasis japonica

OBJECTIVE: To study the role of the synthesis and degradation of collagen at the transcription level during liver fibrogenesis due to schistosomiasis japonica in rabbits. METHODS: New Zealand rabbits challenged by cercariae of Schistosoma japonicum (S. japonicum) were served as animal models for liver fibrosis. Liver specimens were collected through operations at 4, 6, 8, 10, 12, 16, 20, 24 and 28 wks after challenge. Type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels of liver tissue were detected by RT-PCR + Dot blot. The size of egg granulomas and the degree of liver fibrosis were measured by histopathological examinations. RESULTS: Type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels increased simultaneously in the early stage after challenge. Most of them reached their peak at 10 weeks, and compared with normal controls, type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels increased by 12.0-, 11.0-, 6.6-, 10.0- and 11.0-fold, respectively, coinciding with the change of egg granulomas, i.e., the change in the inflammatory process. Then both collagen and collagenase mRNA levels decreased. Type I, III and IV collagen mRNA levels declined to 2-fold to 3-fold as compared with normal controls (P 0.05) at 28 wks. This study shows that the synthesis and degradation of collagen keep a dynamic balance at the early stage of schistosomiasis japonica challenge, while at the later stages the quantity of collagen synthesis was higher than that of collagen degradation. CONCLUSIONS: It was confirmed at transcription level that when the quantity of collagen synthesis was higher than that of collagen degradation liver fibrogenesis may be resulted in.

Estimating the prevalence of schistosomiasis japonica in China:a serological approach

Background:The prevalence of schistosomiasis japonica has decreased significantly,and the responses changing from control to elimination in Jiangsu Province,P.R.China.How to estimate the change in prevalence of schistosomiasis using only serological data will be important and useful.Methods:We collected serum samples from 2011 to 2015 to build a serum bank from Dantu County of Jiangsu,China.Serum samples were detected by enzyme-linked immunosorbent assay(ELISA),the positive rate and optical density(OD)value were obtained.The Bayesian model including the prior information of sensitivity and specificity of ELISA was established,and the estimated infection rates were obtained for different years,genders and age groups.Results:There was no significant difference in the mean OD between different years and genders,but there was a significant difference between the different age groups.There were statistically significant differences in the positive rate for different years and age groups,but no significant difference at different genders.The estimated infection rate for the five years was 1.288,1.456,1.032,1.485 and 1.358%,respectively.There was no significant difference between different years and between genders,but a significant difference between different age groups.Conclusions:The risk of schistosomiasis transmission in this area still exists,and risk monitoring of schistosomiasis should be strengthened.

Modulation of in vivo granuloma formation related to regulation of in vitro IFN γ and IL 4 expressions in experimental Schistosomiasis japonica

Objective To find out relationship between the in vitro schistosome egg antigen (SEA) stimulated IFN γ and IL 4 mRNA expressions and the in vivo SEA elicited granulomatous responses, the transcriptions of IFN γ and IL 4 gene were investigated in the spleen of BALB/c mice infected with Schistosoma japonicum (S japonicum) Methods Spleens were removed at 0, 3, 5, 8, and 10 and 12 weeks after infection and the spleen cells were incubated in the presence of SEA The extracted RNA was analyzed for IFN γ and IL 4 mRNA by reverse transcription PCR (RT PCR) Newly formed liver granulomas were measured Results The study revealed that no detectable IFN γ and IL 4 mRNA RT PCR products were found in SEA treated spleen cells from uninfected, or 3 week infected mice, whereas IL 4 mRNA was found to be expressed in 5 and 8 week infected mice, and an appreciable enhanced expression of IL 4 mRNA was observed in SEA stimulated spleen cells at 8 week infection than at 5 week infections However, SEA could not induce IFN γ and IL 4 mRNA transcription in 10 and 12 week infected mice, indicating the modulated expression of IFN γ and IL 4 mRNA Conclusion The significant changes of IL 4 mRNA expression in SEA stimulated spleen cells during S japonicum infection were coincident with SEA elicited granuloma formation and modulation in vivo

A novel and effective hyperthermia method for Schistosomiasis japonica prevention and treatment

The emerging resistance to schistosoma has been paid much attention and is in urgent need for a novel strategy to control the prevalent parasitic zoonosis. In this study, the efficiency of hyperthermia therapy was investigated by the skin hyperthermia device. The survival rate of cercariae decreased from 68.15 % （37 ℃, 5 min） to 0 （49℃, 10 min） with the thermal dosages increased, which proved the preventing effect of hyperthermia therapy （P 〈 0.05）. Therapeutic effects were assessed in Schistosoma japonicum-infected BALB/c mice. When the cercarial contact region of the skin was treated at 45-49℃ for 5 min within 8 h of infection, worm reduction rate （WRR） reached 74 %-83 % （P 〈 0.01）. The sensitivity of adult schistosoma to heat was also investigated using microwave intraperitoneal hyperthermia （thermal dosages 42-43℃, 20 min）. The WRR, hepatic shift rates and egg reduction rates were 23.7 %, 40 % and 30 %, respectively, comparing with 80.2 %, 59.6 % and 53.9 % of praziquantel （PZQ）-treated group. Encouraging results have been obtained that hyperthermia can effectively kill schistosomula, especially with the appearance of cercarial dermatitis, while PZQ lacks efficacy against the cercariae. Thus, hyperthermia therapy would show significant benefit in preventing and treatment of schistosoma, especially in the early stage.

Paeoniflorin prevents hepatic fibrosis of Schistosomiasis japonica by inhibiting TGF-β1 production from macrophages in mice

In order to investigate the effect of paeoniflorin(PAE)on hepatic fibrosis of mice with Schistosomiasis japonica in vivo and in vitro,a model of hepatic fibrosis caused by schistosomiasis was established in mice infected with cercariae of Schistosoma japonicum.Then,PAE was orally administered before and after praziquantel treat-ment and both therapeutics were given simultaneously at different time points after the infection.The concentra-tion of serum hyaluronic acid(HA)was determined by radioimmunoassay(RIA).Hepatic granuloma and fib-rosis were evaluated via HE and Masson staining.The expression of α-smooth muscle actin(α-SMA),transform-ing growth factor β1(TGF-β1)and collagen I(Col I)protein was detected by immunohistochemistry.The effect of soluble egg antigen(SEA)and PAE on the pro-duction of TGF-β1 from mouse peritoneal macrophages(PMQs)was investigated by RT-PCR,Western blotting and ELISA.The effect of TGF-β1 in optimum macro-phage-conditioned medium(OPMCM)on the prolifera-tion of hepatic stellate cells(HSCs)and collagen secretion from HSCs with anti-TGF-β1 antibody was explored by MTT assay and ELISA.The results show that PAE could significantly reduce the concentration of serum HA,the size of egg granuloma,the severity of hepatic fibrosis and the expression of a-SMA,TGF-β1 and Col I protein in the pre-treatment group.However,in sim-or post-treatment group,PAE did not have any significant therapeutic effect.TGF-β1 could be secreted from PMQs stimulated by SEA.Meanwhile,the production of TGF-β1 from PMQs could be depressed significantly by PAE in a con-centration-dependent manner.TGF-β1 could promote the proliferation of HSCs and the secretion of collagens.In a word,PAE can prevent hepatic granuloma and fib-rosis caused by schistosomiasis japonica through the inhibition of the secretion of TGF-β1 from PMQs,the proliferation and activation of HSCs and the secretion of collagens from HSCs.

Study on IL-4, IL-5 and IL-10 in Liver and Bone Marrow of Mice Infected with Schistosomiasis Japonica

Summary: To compare Th2 cytokines levels in livers and bone marrow of mice infected with Schistosomiasis Japonica and understand their implication, ABC immunohistochemical staining technique and multimedia pathologic pictures analysis were used to detect the IL 4, IL 5 and IL 10 derived from livers and bone marrow of infected mice. Results showed that in the livers of infected mice, IL 4, IL 5 and IL 10 were obviously increased over time. The level of IL 4 was increased most among the 3 indicators. In bone marrow of infected mice, however, even though IL 4 was slowly increased over time, it was lower than that of livers in 10th and 12th week. The level of IL 5 was higher than that of livers during 12th weeks. Before 10th weeks IL 10 was elevated. After 12th week IL 10 was decreased and obviously lower than that of livers. It is concluded that in mice infected with Schistosomiasis Japonica , the immune response occurred mainly in the liver. Therefore, in bone marrow, IL 5 showed obvious auto secretion. It might be related to its biological ability to induce B cell division and eosinophil differentiation.

Sigmoid colonic carcinoma associated with deposited ova of Schistosoma japonicum:A case report

We report a case of sigmoid colonic carcinoma associated with deposited ova of Schistosoma japonicum. A 57-year old woman presented with a 10-mo history of left lower quadrant abdominal pain and a 2-mo history of bloody stools. She had a significant past medical history of asymptomatic schistosomiasis japonica and constipation. A colonoscopy showed an exophytic fragile neoplasm with an ulcerating surface in the sigmoid colon. During the radical operative procedure, we noted the partially encircling tumor was located in the distal sigmoid colon, and extended into the serosa. Succedent pathological analysis demonstrated the diagnosis of sigmoid colonic ulcerative tubular adenocarcinoma, and showed deposited ova of Schistosoma japonicum in both tumor lesions and mesenteric lymph nodes. Three days after surgery the patient returned to the normal bowel function with one defecation per day. These findings reveal that deposited schistosome ova play a possible role in the carcinogenesis of colorectal cancer.

Effect of Anluohuaxian Tablet Combined with γ-IFN on Schistosomal Liver Fibrosis

The therapeutic effects of anluohuaxian tablet combined with γ-IFN on schistosomal liver fibrosis and its mechanism were studied in a murine model and clinical cases of schistosomal liver fibrosis, Fifty Kunming mice were randomly divided into 5 groups： normal control group, infection control group, anluohuaxian tablet-treated group, γ-IFN-treated group and combined treatment （anluohuaian tablet＋γ-IFN） group. Pathologic changes in liver, including hepatic pigmentation and the size of schistosomal egg granuloma, were observed by HE staining after treatment for 8 weeks. The expression of the type Ⅰ and Ⅲ collagen, and TIMP-1 was detected by immunohistochemistry. TGF-β1 mRNA expression was examined by real-time fluorescent quantitative PCR. Sixty patients with schistosomal liver fibrosis were divided into treatment group and control group. The patients in treatment group were treated with anluohuaxian tablet in combination with γ-IFN for 6 months. Before and after treatment, the changes of symptoms and signs, liver function, serum liver fibrosis indexes and imaging indexes were observed. The results showed that as compared with infection control group, all forms of treatments relieved the hepatic pathological injury with apparently diminished size of schistosomal egg nodules and decreased percentage of pigmentation （P〈0.05）. Furthermore, the expression of collagen Ⅰ and Ⅲ, TIMP-1, and TGF-β1 mRNA in combined treatment group was significantly decreased as compared with anluohuaxian tablet-treated and γ-IFN-treated groups （P〈0.05）. In the clinical observation, the serum liver fibrosis indexes, the portal vein width as well as the spleen thickness was significantly reduced in treatment group as compared with control group （P〈0.05）. It was concluded that the combined use of anluohuaxian tablet with γ-IFN in schistosomal liver fibrosis could protect liver function, alleviate liver fibrosis, and could be used as a choice in treating patients with schiatosomal liver fibrosis.

Pentoxifylline Inhibits Liver Fibrosis via Hedgehog Signaling Pathway

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog（HH） signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline（PTX） on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate（PMA） was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen（SEA）, and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8（CCK-8） was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and（or） PTX to detect the transforming growth factor-β gene expression. The m RNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The m RNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells（HSCs）. The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.

Approaches being used in the national schistosomiasis elimination programme in China:a review

Schistosomiasis japonica,caused by the human blood fluke Schistosoma japonicum,remains a major public health problem in China,although great success has been achieved.The control efforts during the past half-decade,notably the wide implementation of the new integrated strategy with emphasis on control of the source of S.japonicum infection across the country since 2004,has greatly reduced S.japonicum in humans,livestock,and intermediate host Oncomelania hupensis snails,and transmission control of schistosomiasis was achieved in China in 2015.A two-stage roadmap was therefore proposed for schistosomiasis elimination in 2015,with aims to achieve transmission interruption by 2020 and achieve disease elimination by 2025 in the country.During the last two decades,a variety of approaches,which target the epidemiological factors of schistosomiasis japonica have been developed,in order to block the transmission cycle of the parasite.These approaches have been employed in the national or local schistosomiasis control activities,and facilitated,at least in part,the progress of the schistosomiasis elimination programs.Here,we present an approach to control the source of S.japonicum infection,three new tools for snail control,three approaches for detecting and monitoring S.japonicum infection,and a novel model for health education.These approaches are considered to play a great role in the stage moving towards transmission interruption and elimination of schistosomiasis in China.

Interruption of schistosomiasis transmission in mountainous and hilly regions with an integrated strategy:a longitudinal case study in Sichuan,China

Background:Schistosomiasis remains a major public health concern in China.Since 2004,an integrated strategy was developed to control the transmission of Schistosoma japonicum in China.However,the long-term effectiveness of this integrated strategy for the interruption of schistosomiasis transmission remains unknown in the mountainous and hilly regions of China until now.This longitudinal study aims to evaluate the effectiveness of the integrated strategy on transmission interruption of schistosomiasis in Sichuan Province from 2005 through 2014.Methods:The data regarding replacement of bovines with machines,improved sanitation,access to clean water,construction of public toilets and household latrines,snail control,chemotherapy,and health education were captured from the annual report of the schistosomiasis control programmes in Sichuan Province from 2005 to 2014,and S.japonicum infection in humans,bovines and snails were estimated to evaluate the effectiveness of the integrated strategy.Results:During the 10-year period from 2005 through 2014,a total of 536568 machines were used to replace bovines,and 3284333 household lavatories and 15523 public latrines were built.Tap water was supplied to 19116344 residents living in the endemic villages.A total of 230098 hm2 snail habitats were given molluscicide treatment,and 357233 hm2 snail habitats received environmental improvements.There were 7268138 humans and 840845 bovines given praziquantel chemotherapy.During the 10-year study period,information,education and communication(IEC)materials were provided to village officers,teachers and schoolchildren.The 10-year implementation of the integrated strategy resulted in a great reduction in S.japonicum infection in humans,bovines and snails.Since 2007,no acute infection was detected,and no schistosomiasis cases or infected bovines were identified since 2012.In addition,the snail habitats reduced by 62.39%in 2014 as compared to that in 2005,and no S.japonicum infection was identified in snails since 2007.By 2014,88.9%of the endemic counties achieved the transmission interruption of schistosomiasis and transmission control of schistosmiasis was achieved in the whole province in 2008.Conclusion:The government-directed and multi-department integrated strategy is effective for interrupting the transmission of schistosomiasis in the mountainous and hilly regions of China.

Three Gorges Dam:the changing trend of snail density in the Yangtze River basin between 1990 and 2019

Background The area of Oncomelania hupensis snail remains around 3.6 billion m^(2),with newly emerging and reemergent habitats continuing to appear in recent years.This study aimed to explore the long-term dynamics of snail density before and after the operation of Three Gorges Dam(TGD).Methods Data of snail survey between 1990 and 2019 were collected from electronic databases and national schistosomiasis surveillance.Meta-analysis was conducted to estimate the snail density.Joinpoint model was used to identify the changing trend and inflection point.Inverse distance weighted interpolation(IDW)was used to determine the spatial distribution of recent snail density.Results A total of 3777 snail survey sites with a precise location of village or beach were identified.For the downstream area,snail density peaked in 1998(1.635/0.11 m^(2),95%CI:1.220,2.189)and fluctuated at a relatively high level before 2003,then declined steadily from 2003 to 2012.Snail density maintained lower than 0.150/0.11 m^(2) between 2012 and 2019.Joinpoint model identified the inflection of 2003,and a significant decreasing trend from 2003 to 2012 with an annual percentage change(APC)being−20.56%(95%CI:−24.15,−16.80).For the upstream area,snail density peaked in 2005(0.760/0.11 m^(2),95%CI:0.479,1.207)and was generally greater than 0.300/0.11 m2 before 2005.Snail density was generally lower than 0.150/0.11 m^(2) after 2011.Snail density showed a significant decreasing trend from 1990 to 2019 with an APC being−6.05%(95%CI:−7.97,−7.09),and no inflection was identified.IDW showed the areas with a high snail density existed in Poyang Lake,Dongting Lake,Jianghan Plain,and the Anhui branch of the Yangtze River between 2015 and 2019.Conclusions Snail density exhibited a fluctuating downward trend in the Yangtze River basin.In the downstream area,the operation of TGD accelerated the decline of snail density during the first decade period,then snail density fluctuated at a relatively low level.There still exists local areas with a high snail density.Long-term control and monitoring of snails need to be insisted on and strengthened.

Co-parasitism of intestinal protozoa and Schistosoma japonicum in a rural community in the Philippines

Background:Co-parasitism is a frequent occurrence in impoverished communities in the tropics resulting in a considerable disease burden.While there are extensive reports of intestinal helminthiases,including schistosomiasis japonica,the occurrence and extent of diseases caused by intestinal protozoa(IP)have yet to be investigated in depth in the Philippines.We present a detailed analysis of polyparasitism in a rural community of Northern Samar,focusing on co-infections of IP with Schistosoma japonicum.Methods:A descriptive cross sectional study was carried out in 2015 across 18 barangays(villages)endemic for S.japonicum in Northern Samar,the Philippines to assess the burden of human schistosomiasis and IP infections.Faecal samples collected from 412 participants from the 18 barangays were included in the final molecular analysis.A multiplex quantitative PCR assay was developed and used for the detection of Blastocystis spp.,Entamoeba histolytica,Cryptosporidium spp.and Giardia duodenalis in stool samples.The findings were combined with previous results of droplet digital PCR diagnosis of individuals from the same 18 barangays infected with S.japonicum determined using the same stool samples for analysis.Results:Mean age of the study participants was 40.3 years(95%CI:38.8-41.8)with 53%(n=218)being males.Prevalence of S.japonicum(74.5%)and Blastocystis spp.(58.7%)was significantly higher compared to other infections,with E.histolytica having the lowest prevalence(12.1%).A majority of individuals were infected with more than one parasite with two infections being most common(n=175,42.5%).The prevalence of individuals with two parasites was significantly higher than all others with 27.9%(n=115)subjects harbouring a single parasite species.Of individuals with two infections,S.japonicum and Blastocystis spp.were the most common combination(n=110,62.9%).Examining age within the population,58.5%(n=38)of school-aged children and 60.1%(n=14)of women of child bearing age harboured at least two parasite species.Conclusions:The study revealed that polyparasitism with IP infections and schistosomiasis japonica is highly prevalent in individuals in Northern Samar which likely contributes to the significant public health and socio-economic burden suffered by this population.More generally,the findings are of relevance when considering implementation of integrated control strategies for intestinal parasites.