Evaluation of CD39, CD73, HIF-1α, and their related miRNAs expression in decidua of preeclampsia cases compared to healthy pregnant women.

BACKGROUND: The Preeclampsia (PE) molecular mechanisms are not fully revealed and different biological processes are involved in the pathogenesis of PE. We aimed to evaluate adenosine and hypoxia-related signaling molecules in PE patients in the current study. METHODS: Decidua tissue and peripheral blood samples were taken from 25 healthy pregnant and 25 PE women at delivery time. CD39, CD73, and Hypoxia-inducible factor-alpha (HIF-α) were evaluated in mRNA and protein level using real-time PCR and western blotting techniques, respectively. Also, miR-30a, miR-206, and miR-18a expression were evaluated by real-time PCR. At last, secretion levels of IGF and TGF-ß in the taken serum of blood samples were measured by ELISA. RESULTS: Our results revealed that Expression of CD39 is decreased in PE cases versus healthy controls at mRNA and protein levels (p = 0.0003 for both). CD73 and HIF-α showed an increased level of expression in PE patients at RNA and protein status (p = 0.0157 and p < 0.0001 for protein evaluation of CD73 and HIF-α, respectively). The miRNA-30a (p = 0.0037) and miR-206 (p = 0.0113) showed elevated expression in the decidua of the PE group. The concentration of secreted IGF-1 (p = 0.0002) and TGF-ß (p = 0.0101) in serum samples of PE cases compared to the healthy group were decreased. CONCLUSION: In conclusion, our results showed that aberrant expression of molecules that are involved in ATP catabolism and the hypoxic conditions is observed in PE cases and involved in their hypertension and inflammation could be served as PE prognosis by more confirming in comprehensive future studies. miR-206 and miR-30a play a role by regulating CD39 and CD73 as molecules that are involved in ATP catabolism as well as regulating the production of IGF-1 in the process of hypertension, which is the main feature in patients with preeclampsia. On the other hand, decreased level of miR-18a lead to upregulation of HIF-1a, and the consequence condition of hypoxia increases hypertension and inflammation in these patients.

Simultaneous silencing of the A2aR and PD-1 immune checkpoints by siRNA-loaded nanoparticles enhances the immunotherapeutic potential of dendritic cell vaccine in tumor experimental models.

Following various immunotherapies, lack of proper anti-tumor immune responses is considered a significant problem in novel cancer therapeutic approaches. The expression of inhibitory checkpoint molecules on tumor-infiltrating T cells is one of the main reasons for the ineffectiveness of various immunotherapies. Therefore, we decided to inhibit two of the most important immune checkpoints expressed on tumor-associated T cells, PD-1 and A2aR. Ligation of PD-1 with PD-L1 and A2aR with adenosine significantly suppress T cell responses against tumor cells. Whitin tumors, specific inhibition of these molecules on T cells is of particular importance for successful immunotherapy as well as the elimination of treatment-associated side-effects. Thus, in this study, superparamagnetic iron oxide (SPION) nanoparticles (NPs) were covered by chitosan lactate (CL), functionalized with TAT peptide, and loaded with siRNA molecules against PD-1 and A2aR. Appropriate physicochemical properties of the prepared NPs resulted in efficient delivery of siRNA to tumor-derived T cells and suppressed the expression of A2aR and PD-1, ex vivo. T cell functions such as cytokine secretion and proliferation were considerably enhanced by the downregulation of these molecules which led to an increase in their survival time. Interestingly, treatment of CT26 and 4T1 mouse tumors with siRNA-loaded NPs not only inhibited tumor growth but also markedly increased anti-tumor immune responses and survival time. The results strongly support the efficacy of SPION-CL-TAT NPs loaded with anti-PD-1/A2aR siRNAs in cancer therapy and their further development for cancer patients in the near future.

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies.

Siglec-F (SF) is a surface glycoprotein expressed by mouse eosinophils and induces caspase- and mitochondria-dependent apoptosis after engagement with its cognate ligand or specific antibodies. This targeting eosinophils by monoclonal antibodies may help diverse diseases associated with increased frequency of eosinophils including allergy and asthma. In this paper, production of murine and rat monoclonal antibodies (mAbs) against Siglec-F has been addressed. Balb/c mice were immunized with siglec-F1 (SF1) and siglec-F2 (SF2) synthetic peptides conjugated to a carrier protein. Rats were immunized with Chinese hamster ovary CHO cells overexpressing Siglec-F (CHO-SF) or with Siglec-F-human immunoglobulin FC fusion protein (CHO-SF-Ig). Hybridomas were produced by standard protocol and screened for their reactivity by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and flow cytometry. In parallel, polyclonal antibodies were generated in New Zealand White rabbits immunized with SF1 and SF2 peptides. Three mouse and three rat mAbs were generated against synthetic peptides and SF-Ig, respectively. All mouse monoclonal and rabbit polyclonal antibodies reacted well with immunizing molecules in ELISA and detected specific band of Siglec-F in WB. However, they failed to detect native molecule in flow cytometry analysis. Quite the contrary, rat mAbs did not reacted with the denatured protein in WB, instead exhibited significant reactivity with CHO-SF cells in flow cytometry. Based on the heavily glycosylated nature of Siglec-F, it seems that generation of anti-SF antibodies able to detect native protein needs a properly folded molecule for immunization. Monoclonal antibodies reported here are invaluable tools for studying linear and conformation epitopes of SF and tracing mouse eosinophils.