Schistosoma japonicum Infection in Treg-Specific USP21 Knockout Mice.

The E3 deubiquitinating enzyme ubiquitin-specific proteolytic enzyme 21 (USP21) plays vital roles in physiological activities and is required for Treg-cell-mediated immune tolerance. Using a murine model infected with Schistosoma japonicum, we observed that there were more cercariae developed into adults and more eggs deposited in the livers of the USP21fl/flFOXP3Cre (KO) mice. However, immunohistochemistry showed that the degree of egg granuloma formation and liver fibrosis was reduced. In USP21fl/flFOXP3Cre mice, levels of IFN-gamma, IL-4, anti-soluble egg antigen (SEA) IgG and anti-soluble worm antigen preparation (SWAP) IgG increased in blood, as determined using ELISAs and multiplex fluorescent microsphere immunoassays, while the levels of IL-10, lL-17A, IL-23, IL-9, and anti-SEA IgM decreased. In addition, the levels of the USP21 protein and mRNA in the liver and spleen of KO mice decreased. We further observed increased Th1 responses amplified by Tregs (regulatory T cells) and compromised Th17 responses, which alleviated the liver immunopathology. We speculated that these changes were related to polarization of Th1-like Tregs. Our results revealed the roles of USP21 in Treg-cell-mediated regulation of immune interactions between Schistosoma and its host. USP21 may have potential for regulating hepatic fibrosis in patients with schistosomiasis.

C1orf35 contributes to tumorigenesis by activating c-MYC transcription in multiple myeloma.

Multiple myeloma (MM) is a clinically and biologically heterogenous event that accounts for approximately 10% of all hematological malignancies. Chromosome 1 open reading frame 35 (C1orf35) is a gene cloned and identified in our laboratory from a MM cell line (GenBank: AY137773), but little is known about its function. In the current study, we have confirmed that C1orf35 is a candidate oncogene, and it can promote cell cycle progression from G1 to S. Later, we found that C1orf35 is able to affect the cell proliferation by modulating the expression of c-MYC (v-myc myelocytomatosis viral oncogene homolog), and the oncogenic property of C1orf35 can be rescued by c-MYC inhibition. Herein, we found positive association between C1orf35 and c-MYC in MM patients and in MM cell lines. The correlation analysis of the genes coamplified in MM patients from GEO datasets showed a correlation between C1orf35 and c-MYC, and the expression data of different stages of plasma cell neoplasm acquired from GEO datasets showed that the expression of C1orf35 increase with the progression of the disease. This indicates that C1orf35 may play a role in the disease progression. Moreover, C1orf35 can modulate c-MYC expression and rescue c-MYC transcription inhibited by Act D. Finally, we have shown that C1orf35 activates c-MYC transcription by binding to the i-motif of Nuclease hypersensitivity element III1 (NHE III1) in the c-MYC promoter. Not only does our current study advance our knowledge of the pathogenesis and therapeutic landscape of MM, but also of other cancer types and diseases that are initiated with deregulated c-MYC transcription.

Promoter methylation induced epigenetic silencing of DAZAP2, a downstream effector of p38/MAPK pathway, in multiple myeloma cells.

Multiple myeloma (MM) is hematological malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow environment. Previously, we identified DAZAP2 as a candidate cancer suppressor gene, the downregulation of which is regulated by its own promoter methylation status. In the current study, we analyzed the DAZAP2 promoter in MM cell lines KM3, MM.1S, OPM-2, and ARH77 by bisulfite genomic sequencing assay. We identified the binding site for transcription factor cyclic adenosine monophosphate response element binding (CREB) in the DAZAP2 promoter CpG2, and we found that hypermethylation of the CREB binding motif in the DAZAP2 promoter is responsible for the reduced DAZAP2 expression in MM cells. Later we checked the p38/MAPK signaling cascade, which is reported to regulate expression and function of CREB. Our results showed that the p38/MAPK signaling pathway drives the expression of DAZAP2 by phosphorylation of CREB, and hypermethylation of CREB binding motif in DAZAP2 promoter can inhibit binding of CREB to the latter, thus downregulating DAZAP2 expression. Moreover, treating the MM cells with 5-aza-2' deoxycytidine to demethylate DAZAP2 promoter restored the binding of CREB to its binding motif, and thus upregulated DAZAP2 expression. Our results not only identified DAZAP2 as a new downstream target of p38/MAPK/CREB signaling cascade, but we also clarified that the downregulation of DAZAP2 in MM cells is caused by hypermethylation of CREB binding motif in its own promoter region, which implies that demethylation of DAZAP2 promoter can be a novel therapeutic strategy for MM treatment.

[Resistance of rAAV2-MfE77.43-Transferred Mice to Schistosoma japonicum Infection].

Objective: To investigate the resistance of E77.43 gene of Microtus fortis（MfE77.43） to Schistosoma japonicum infection. Methods: MfE77.43 was constructed into the recombinant Adeno-associated virus AAV2. The AAV2-MfE77.43 was transfected into HEK293 cells by the calcium phosphate DNA coprecipitation method. The recombinant rAAV2-MfE77.43 was purified and total RNA was extracted from the transfected cells. The expression of E77.43 was examined by RT-PCR and the purity of rAAV2-MfE77.43 was analyzed by SDS-PAGE. Eighteen KM mice were divided into three groups （n=6 in each group）. Mice in the experiment group were intramuscularly injected on days 0, 3 and 7 with 400 µl recombinant AAV2-MfE77.43 virus which was 5-fold diluted in normal saline. Mice in negative control and blank control groups received same volume of pAAV or normal saline. Venous blood was collected through the tail before each injection, and E77.43 expression in plasma was detected by dot-ELISA method. After the last injection, each mouse was infected with 40 S. japonicum cercariae and sacrificed on day 41 after infection. Adult worms and liver eggs per gram（LEPG） were counted. Worm and egg reduction rate was calculated respectively. Egg granulomas were observed by HE staining. Results: RT-PCR resulted in a 330 bp specific band. SDS-PAGE of virus shell protein revealed three protein bands with M(r) of 87 000, 72 000 and 62 000, respectively. Dot-ELISA showed that E77.43 protein began to be expressed on day 3 after rAAV2-MfE77.43 injection, remaining stable till day 41. The adult worm number and LEPG were 20.16±3.93 and 19 800±2 715, respectively, with a worm and egg reduction rate of 27.3% and 26.2% in the experiment group. While the worm number and LEPG in the negative control group were 29.16±2.44 and 28 000±2 192（P<0.01）, respectively. HE staining and observation revealed fewer eosinophils and inflammatory cells around the liver eggs in the therapy group. Conclusion: The E77.43 gene shows protective effects against S. japonicum infection, indicating that E77.43 may participate in the natural resistance of Microtus fortis to S. japonicum infection.

A Microtus fortis protein, serum albumin, is a novel inhibitor of Schistosoma japonicum schistosomula.

Schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease. Experimental and epidemiological evidence strongly suggests that Microtus fortis ( Mf ) is a naturally resistant vertebrate host of Schistosoma japonicum . In the present study, we found that Mf serum albumin ( Mf -albumin) and the conditioned medium of pcDNA3.1- Mf -albumin caused 46.2% and 38.7% schistosomula death rates in 96 h, respectively, which were significantly higher than that of the negative control (p < 0.05). We also found that mice injected with Mf -albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram (p < 0.05) in comparison to the control animals. To characterise the mechanisms involved in clearance, schistosomula were incubated with fluorescein isothiocyanate-labelled Mf -albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation. Next, digestive tract excretions from schistosomula were collected and the sensitivity of Mf -albumin to digestive tract excretions was evaluated. The results indicated that schistosomula digestive tract excretions showed indigestibility of Mf -albumin. The death of schistosomula could be partially attributed to the lack of digestion of Mf -albumin by digestive tract excretions during the development of the schistosomula stage. Therefore, these data indicate the potential of Mf -albumin as one of the major selective forces for schistosomiasis.

A Microtus fortis protein, serum albumin, is a novel inhibitor of Schistosoma japonicum schistosomula

Schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease. Experimental and epidemiological evidence strongly suggests that Microtus fortis ( Mf ) is a naturally resistant vertebrate host of Schistosoma japonicum . In the present study, we found that Mf serum albumin ( Mf -albumin) and the conditioned medium of pcDNA3.1- Mf -albumin caused 46.2% and 38.7% schistosomula death rates in 96 h, respectively, which were significantly higher than that of the negative control (p < 0.05). We also found that mice injected with Mf -albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram (p < 0.05) in comparison to the control animals. To characterise the mechanisms involved in clearance, schistosomula were incubated with fluorescein isothiocyanate-labelled Mf -albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation. Next, digestive tract excretions from schistosomula were collected and the sensitivity of Mf -albumin to digestive tract excretions was evaluated. The results indicated that schistosomula digestive tract excretions showed indigestibility of Mf -albumin. The death of schistosomula could be partially attributed to the lack of digestion of Mf -albumin by digestive tract excretions during the development of the schistosomula stage. Therefore, these data indicate the potential of Mf -albumin as one of the major selective forces for schistosomiasis.

Karyopherin alpha 2 (KPNA2) is associated with the natural resistance to Schistosoma japanicum infection in Microtus fortis.

Microtus fortis is a naturally vertebrate host resistant to Schistosoma japonicum infection. In order to understand the molecular mechanism and identify the molecules related to the natural resistance to S. japanicum infection of M. fortis, we screened a gene pool named gE76 by expression cloning and proved it to have high anti-schistosomula effects in our previous work. In this study we identified a clone named gE76.44. We found that the conditioned medium of pcDNA1.1-gE76.44 caused 14.0% schistosomula death rate in 96 h, which was significantly higher than that of negative control (P<0.05). The gE76.44 was sequenced and the full-length cDNA was 2008 bp with ORF of 1590bp encoding a polypeptide of 529 amino acid residues. Bioinformatics analysis indicated it was the homologue of karyopherin alpha 2 (KPNA2). To further confirm its anti-schistosome activity, we inserted full length of Mf-KPNA2 (KPNA2 of M. fortis) gene into a retroviral expression vector pLXSN and packaged the recombinant virus with PA317 cells. Mice infected with S. japanicum cercariae were administrated by intravenous injection through tail vein and treated with pLXSN-KPNA2. Adult worms and egg reduction were counted after heart perfusion of mice 42 d after infection. We found that compared with the control, mice injected with Mf-KPNA2 had 39.42% worm burden reduction and 76.50% reduction in LEPG (liver eggs per gram) (P<0.01), indicating its anti-schistosome effect of Mf-KPNA2 in vivo. Taken together, the results suggested Mf-KPNA2 as a novel anti-schistosome molecule in vitro and in vivo.

Identification of the resistance of a novel molecule heat shock protein 90alpha (HSP90alpha) in Microtus fortis to Schistosoma japonicum infection.

Microtus fortis is a naturally resistant vertebrate host of Schistosoma japonicum by preventing completion of parasite's life cycle. Sera of M. fortis were found to have anti-schistosome effect in vitro and in vivo. In order to identify genes associated with the anti-schistosome effect of M. fortis, we screened a M. fortis marrow cDNA expression library by expression cloning and identified a 331-bp clone gC14.75. It was the homologue of heat shock protein 90alpha (HSP90alpha). Full-length of M. fortis HSP90alpha gene, Mf-HSP90alpha, was amplified according to gC14.75 and Cricetulus griseus HSP90alpha. To test the potential anti-schistosome function of Mf-HSP90alpha, we prepared conditioned medium of Mf-HSP90alpha and added it to schistosomula cultured in vitro. It caused 27.0% schistosomula death rate in 96h, which was considerably higher than that of negative control. We transferred Mf-HSP90alpha by retroviral expression vector pLXSN into mice to investigate its anti-schistosome effect in vivo. Compared with those of DMEM injection control, mice injected with Mf-HSP90alpha recombinant retrovirus had 40.8% worm burden reduction and 57.9% reduction in liver eggs per gram (LEPG) indicating its anti-schistosome effect in vivo. Taken together, our results suggested Mf-HSP90alpha as a novel anti-schistosome molecule in vitro and in vivo.