The sensitivity of acute myeloid leukemia cells to cytarabine is increased by suppressing the expression of Heme oxygenase-1 and hypoxia-inducible factor 1-alpha.

BACKGROUND: Acute myeloid leukemia (AML), a malignancy Often resistant to common chemotherapy regimens (Cytarabine (Ara-c) + Daunorubicin (DNR)), is accompanied by frequent relapses. Many factors are involved in causing chemoresistance. Heme Oxygenase-1 (HO-1) and Hypoxia-Inducible Factor 1-alpha (HIF-1α) are two of the most well-known genes, reported to be overexpressed in AML and promote resistance against chemotherapy according to several studies. The main chemotherapy agent used for AML treatment is Ara-c. We hypothesized that simultaneous targeting of HO-1 and HIF-1α could sensitize AML cells to Ara-c. METHOD: In this study, we used our recently developed, Trans-Activator of Transcription (TAT) - Chitosan-Carboxymethyl Dextran (CCMD) - Poly Ethylene Glycol (PEG) - Nanoparticles (NPs), to deliver Ara-c along with siRNA molecules against the HO-1 and HIF-1α genes to AML primary cells (ex vivo) and cell lines including THP-1, KG-1, and HL-60 (in vitro). Subsequently, the effect of the single or combinational treatment on the growth, proliferation, apoptosis, and Reactive Oxygen Species (ROS) formation was evaluated. RESULTS: The designed NPs had a high potential in transfecting cells with siRNAs and drug. The results demonstrated that treatment of cells with Ara-c elevated the generation of ROS in the cells while decreasing the proliferation potential. Following the silencing of HO-1, the rate of apoptosis and ROS generation in response to Ara-c increased significantly. While proliferation and growth inhibition were considerably evident in HIF-1α-siRNA-transfected-AML cells compared to cells treated with free Ara-c. We found that the co-inhibition of genes could further sensitize AML cells to Ara-c treatment. CONCLUSIONS: As far as we are aware, this study is the first to simultaneously inhibit the HO-1 and HIF-1α genes in AML using NPs. It can be concluded that HO-1 causes chemoresistance by protecting cells from ROS damage. Whereas, HIF-1α mostly exerts prolific and direct anti-apoptotic effects. These findings imply that simultaneous inhibition of HO-1 and HIF-1α can overcome Ara-c resistance and help improve the prognosis of AML patients.

The role of exhausted natural killer cells in the immunopathogenesis and treatment of leukemia.

The immune responses to cancer cells involve both innate and acquired immune cells. In the meantime, the most attention has been drawn to the adaptive immune cells, especially T cells, while, it is now well known that the innate immune cells, especially natural killer (NK) cells, play a vital role in defending against malignancies. While the immune cells are trying to eliminate malignant cells, cancer cells try to prevent the function of these cells and suppress immune responses. The suppression of NK cells in various cancers can lead to the induction of an exhausted phenotype in NK cells, which will impair their function. Recent studies have shown that the occurrence of this phenotype in various types of leukemic malignancies can affect the prognosis of the disease, and targeting these cells may be considered a new immunotherapy method in the treatment of leukemia. Therefore, a detailed study of exhausted NK cells in leukemic diseases can help both to understand the mechanisms of leukemia progression and to design new treatment methods by creating a deeper understanding of these cells. Here, we will comprehensively review the immunobiology of exhausted NK cells and their role in various leukemic malignancies. Video Abstract.

Detection of receptor tyrosine kinase-orphan receptor-2 using an electrochemical immunosensor modified with electrospun nanofibers comprising polyvinylpyrrolidone, soy, and gold nanoparticles.

An electrochemical immunosensing platform was developed for the detection of receptor tyrosine kinase-orphan receptor-2 (ROR2) at a glassy carbon electrode (GCE) modified with the electrospun nanofiber containing polyvinylpyrrolidone (PVP), soy, and Au nanoparticles (AuNPs). The PVP/soy/AuNP nanofiber exhibited good electrochemical behavior due to synergistic effects between PVP, soy, and AuNPs. The PVP/soy in the modified film provided good mechanical strength, high porosity, flexible structures, and high specific surface area. On the other hand, the presence of AuNPs effectively improved conductivity, as well as the immobilization of anti-ROR2 on the modified GCE, leading to enhanced sensitivity. Various characterization approaches such as FE-SEM, FTIR, and EDS were used for investigating the morphological and structural features, and the elemental composition. The designed immunosensor performance was investigated using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). Under optimum conditions with a working potential range from -0.2 to 0.6 V (vs. SCE), sensitivity, linear range (LR), limit of detection (LOD), and correlation coefficient (R2) were acquired at 122.26 µA/cm2 dec, 0.01-1000 pg/mL, 3.39 fg/mL, and 0.9974, respectively. Furthermore, the determination of ROR2 in human plasma samples using the designed immunosensing platform was examined and exhibited satisfactory results including good selectivity against other proteins, reproducibility, and cyclic stability.

Targeted Silencing of NRF2 by rituximab-conjugated nanoparticles increases the sensitivity of chronic lymphoblastic leukemia cells to Cyclophosphamide.

BACKGROUND: Targeting influential factors in resistance to chemotherapy is one way to increase the effectiveness of chemotherapeutics. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway overexpresses in chronic lymphocytic leukemia (CLL) cells and appears to have a significant part in their survival and chemotherapy resistance. Here we produced novel nanoparticles (NPs) specific for CD20-expressing CLL cells with simultaneous anti-Nrf2 and cytotoxic properties. METHODS: Chitosan lactate (CL) was used to produce the primary NPs which were then respectively loaded with rituximab (RTX), anti-Nrf2 Small interfering RNA (siRNAs) and Cyclophosphamide (CP) to prepare the final version of the NPs (NP-Nrf2_siRNA-CP). All interventions were done on both peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMNCs). RESULTS: NP-Nrf2_siRNA-CP had satisfying physicochemical properties, showed controlled anti-Nrf2 siRNA/CP release, and were efficiently transfected into CLL primary cells (both PBMCs and BMNCs). NP-Nrf2_siRNA-CP were significantly capable of cell apoptosis induction and proliferation prevention marked by respectively decreased and increased anti-apoptotic and pro-apoptotic factors. Furthermore, use of anti-Nrf2 siRNA was corresponding to elevated sensitivity of CLL cells to CP. CONCLUSION: Our findings imply that the combination therapy of malignant CLL cells with RTX, CP and anti-Nrf2 siRNA is a novel and efficient therapeutic strategy that was capable of destroying malignant cells. Furthermore, the use of NPs as a multiple drug delivery method showed fulfilling properties; however, the need for further future studies is undeniable. Video Abstract.

Evaluation of the anti-tumor effects of an anti-Human Epidermal growth factor receptor 2 (HER2) monoclonal antibody in combination with CD11b+/Gr-1+ myeloid cells depletion using a recombinant peptibody in 4 T1-HER2 tumor model.

INTRODUCTION: Clinical efficacy of Human Epidermal growth factor Receptor 2 (HER2) targeted strategies is limited due to impaired anti-tumor responses negatively regulated by immunosuppressive cells. We thus, investigated the inhibitory effects of an anti-HER2 monoclonal antibody (1 T0 mAb) in combination with CD11b+/Gr-1+ myeloid cells depletion in 4 T1-HER2 tumor model. METHODS: BALB/c mice were challenged with human HER2-expressing 4 T1 murine breast cancer cell line. A week post tumor challenge, each mouse received 50 µg of a myeloid cells specific peptibody every other day, or 10 mg/kg of 1 T0 mAb two times a week, and their combination for two weeks. The treatments effect on tumor growth was measured by calculating tumor size. Also, the frequencies of CD11b+/Gr-1+ cells and T lymphocytes were measured by flow cytometry. RESULTS: Peptibody treated mice indicated tumor regression and 40 % of the mice eradicated their primary tumors. The peptibody was capable to deplete notably splenic CD11b+/Gr-1+ cells as well as intratumoral CD11b+/Gr-1+ cells (P < 0.0001) and led to an increased number of tumor infiltrating CD8+ T cells (3.3 folds) and also that of resident tumor draining lymph nodes (TDLNs) (3 folds). Combination of peptibody and 1 T0 mAb resulted in enhanced expansion of tumor infiltrating CD4 + and CD8+ T cells which was associated with tumor eradication in 60 % of the mice. CONCLUSIONS: Peptibody is able to deplete CD11b+/Gr-1+ cells and increase anti-tumoral effects of the 1 T0 mAb in tumor eradication. Thus, this myeloid population have critical roles in development of tumors and their depletion is associated with induction of anti-tumoral responses.

A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

The prognostic and therapeutic potential of HO-1 in leukemia and MDS.

BACKGROUND: Heme oxygenase-1 (HO-1), a heme-degrading enzyme, is proven to have anti-apoptotic effects in several malignancies. In addition, HO-1 is reported to cause chemoresistance and increase cell survival. Growing evidence indicates that HO-1 contributes to the course of hematological malignancies as well. Here, the expression pattern, prognostic value, and the effect of HO-1 targeting in HMs are discussed. MAIN BODY: According to the recent literature, it was discovered that HO-1 is overexpressed in myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), acute myeloblastic leukemia (AML), and acute lymphoblastic leukemia (ALL) cells and is associated with high-risk disease. Furthermore, in addition to HO-1 expression by leukemic and MDS cells, CML, AML, and ALL leukemic stem cells express this protein as well, making it a potential target for eliminating minimal residual disease (MRD). Moreover, it was concluded that HO-1 induces tumor progression and prevents apoptosis through various pathways. CONCLUSION: HO-1 has great potential in determining the prognosis of leukemia and MDS patients. HO-1 induces resistance to several chemotherapeutic agents as well as tyrosine kinase inhibitors and following its inhibition, chemo-sensitivity increases. Moreover, the exact role of HO-1 in Chronic Lymphocytic Leukemia (CLL) is yet unknown. While findings illustrate that MDS and other leukemic patients could benefit from HO-1 targeting. Future studies can help broaden our knowledge regarding the role of HO-1 in MDS and leukemia. Video abstract.

The role of SF3B1 and NOTCH1 in the pathogenesis of leukemia.

The discovery of new genes/pathways improves our knowledge of cancer pathogenesis and presents novel potential therapeutic options. For instance, splicing factor 3b subunit 1 (SF3B1) and NOTCH1 genetic alterations have been identified at a high frequency in hematological malignancies, such as leukemia, and may be related to the prognosis of involved patients because they change the nature of malignancies in different ways like mediating therapeutic resistance; therefore, studying these gene/pathways is essential. This review aims to discuss SF3B1 and NOTCH1 roles in the pathogenesis of various types of leukemia and the therapeutic potential of targeting these genes or their mutations to provide a foundation for leukemia treatment.

Preeclampsia-Derived Exosomes Imbalance the Activity of Th17 and Treg in PBMCs from Healthy Pregnant Women.

The disturbance of maternofetal immune tolerance is identified as one of the important issues in the pathology of preeclampsia (PE). PE exosomes are believed to possess significant roles in immune abnormalities. In this study, to assess the possible effects of PE exosomes in the pathophysiology of preeclampsia patients, exosomes were isolated from the serum of PE patients and incubated with peripheral blood mononuclear cells (PBMCs) of healthy pregnant women. Also, exosomes from healthy pregnant women were utilized as the control. Th17/Treg ratio in PE and healthy pregnant women and the effects of PE exosomes on expression level of Th17 and Treg transcription factors, as well as their related cytokines in PBMCs of healthy pregnant women, were evaluated. A significant decrease in Treg cell number and increase in Th17 cells and Th17/Treg ratio were observed in PE patients. Following PE-exosome intervention, a significant increase in mRNA expression level of RORγt, IL-17, IL-23, IL-1ß, and IL-6, and significant decrease in IL-10 and TGFß were evident. On the other hand, no significant difference in FoxP3 level was detected. Additionally, increased IL-6, IL-17, IL-23, and IL-1ß levels and decreased IL-10 level in the supernatant of cultured PBMCs from healthy pregnant women following PE-exosome intervention were exhibited. However, TGF-ß level did not change significantly. Based on our findings, PE exosomes are able to alter the activity of Th17 and Treg cells as well as their related gene expression and cytokine profiles. These findings support the probable role of PE exosomes in PE pathogenesis.

Targeting the CTLA-4/B7 axes in glioblastoma: preclinical evidence and clinical interventions.

INTRODUCTION: Glioblastoma Multiforme (GBM) is one of the fatal cancers of the Central Nervous System (CNS). A variety of reasons exist for why previous immunotherapy strategies, especially Immune Checkpoint Blockers (ICBs), did not work in treating GBM patients. The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a key immune checkpoint receptor. Its overexpression in cancer and immune cells causes tumor cell progression. CTLA-4 suppresses anti-tumor responses inside the GBM tumor-immune microenvironment. AREAS COVERED: It has been attempted to explain the immunobiology of CTLA-4 as well as its interaction with different immune cells and cancer cells that lead to GBM progression. Additionally, CTLA-4 targeting studies have been reviewed and CTLA-4 combination therapy, as a promising therapeutic target and strategy for GBM immunotherapy, is recommended. EXPERT OPINION: CTLA-4 could be a possible supplement for future cancer immunotherapies of GBM. However, many challenges remain such as the high toxicity of CTLA-4 blockers, and the unresponsiveness of most patients to immunotherapy. For the future clinical success of CTLA-4 blocker therapy, combination approaches with other targeted treatments would be a potentially effective strategy. Going forward, predictive biomarkers can be used to reduce trial timelines and increase the chance of success.

Myeloid-derived suppressor cells (MDSCs) depletion by cabozantinib improves the efficacy of anti-HER2 antibody-based immunotherapy in a 4T1-HER2 murine breast cancer model.

BACKGROUND: Clinical trials using Cabozantinib have shown promising results in metastatic breast cancer. This efficacy mainly results from removing and/or polarization of tumor-promoting myeloid cells. Nevertheless, whether such myeloid-derived suppressor cells (MDSCs) depletion can be used to improve the efficacy of anti-HER2 antibodies in early breast cancer has not been defined yet. METHODS: BALB/c mice were inoculated with 4T1 and 4T1-HER2 murine tumor cell lines, and after 7 days, the mice were divided into different groups. Cabozantinib was orally administrated for 15 consecutive days, and anti-HER2 monoclonal antibody (mAb) 1 T0 was intraperitoneally injected twice a week. Tumor size was measured every other day. RESULTS: Our findings indicated that Cabozantinib combined with anti-HER2 mAb dramatically reduced tumor growth and increased tumor rejection (p = 0.0001). Flow cytometry analysis showed MDSC population decreased in TME, lymph nodes, and spleens by roughly 20%, 0.8%, and 35%, respectively. Myeloid suppressive phenotype was altered through inhibition of the expression of immunosuppressive factor Arg-1. Cytokine profiling of different groups indicated that the level of INF-γ was approximately two times higher than that in the control group, and IL-17 increased compared to the control group. However, IL-4 level was significantly reduced in the groups treated with Cabozantinib. These could bring about a 10% increase in CD8+ infiltration into the tumor bed and activation of tumor-draining lymph nodes and splenic T-lymphocytes. CONCLUSION: Collectively, our data provide pre-clinical evidence for using Cabozantinib to reshape the primary TME, which can enhance the effectiveness of anti-HER2 mAb immunotherapy in primary breast cancer.

A ROR1 Small Molecule Inhibitor (KAN0441571C) Induced Significant Apoptosis of Mantle Cell Lymphoma (MCL) Cells.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL.

Combined inhibition of EZH2 and CD73 molecules by folic acid-conjugated SPION-TMC nanocarriers loaded with siRNA molecules prevents TNBC progression and restores anti-tumor responses.

BACKGROUND: Abnormal function or overexpression of CD73 and EZH2 within the tumor microenvironment and tumor cells enhances tumor growth and progression, and in many cases, causes drug resistance. Hence, it seems that silencing the expression of CD73 and EZH2 molecules in breast cancer reduces cancer development and enhances anti-tumor immune responses. METHODS: we used siRNA-loaded superparamagnetic iron oxide (SPIONs) nanoparticles (NPs) coated with trimethyl chitosan (TMC) and functionalized with folic acid for co-delivery of EZH2/CD73 siRNAs to 4 T1 murine cancer cells both in vitro and in vivo. RESULTS: Combination therapy markedly inhibited cancer cells' proliferation, migration, and viability and induced apoptosis in vitro. Moreover, in vivo administration of this combination therapy promoted tumor regression and induced anti-tumor immune responses. DISCUSSION: The findings indicated the CD73/EZH2 factors inhibition by SPION-TMC-FA NPs as a promising therapeutic strategy in breast cancer treatment.

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.

Cloning, expression and characterization of a peptibody to deplete myeloid derived suppressor cells in a murine mammary carcinoma model.

BACKGROUND: Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we expressed and characterized a peptibody which had previously been defined by phage display technique capable of recognizing and depleting murine MDSCs. MATERIALS AND METHODS: Using splicing by overlap extension (SOE) PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. RESULTS: The purified peptibody appeared as a 70 KDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P < 0.05). CONCLUSION: In this study, a peptibody capable of depleting intratumoral MDSCs, was successfully expressed and purified. Our results imply that it could be considered as a potential tool for research on cancer immunotherapy.

Expression level of immune checkpoint inhibitory factors in preeclampsia.

Preeclampsia (PE) is a severe complication in pregnancy, and its symptoms (proteinuria and hypertension) manifest after 20 weeks of gestation, affecting up to 8 % of pregnancies. The pregnant women's immune system uses different tolerance mechanisms to deal with a semi-allogeneic fetus. The T-cell subsets including CD8+, CD4+, and Treg play a critical role in maintaining pregnancies. The expression of immune checkpoint molecules in T-cells can ensure pregnancy at the feto-maternal interface by controlling immune responses. This research aims to evaluate the expression level of immune checkpoint factors, including PD-1, LAG-3, CTLA-4, and TIM-3 in normal pregnant women and PE patients. Decidual tissue was collected from 50 participants (25 PE and 25 control). For evaluating the genes expression, real-time PCR was employed. The western blot was used to assess the proteins level. The results of real-time PCR indicated significantly decreased expression level of these immune checkpoints in PE patients. In parallel to gene expression results, the protein level of PD-1, LAG-3, CTLA-4, and TIM-3 in the PE group was also reduced. We revealed that the profile of proteins and genes expression of immune checkpoints in the decidua of PE mothers are different from normal pregnancy and these results indicate aberrant expression of immune checkpoints such as PD-1, LAG-3, CTLA-4, and TIM-3 may cause maladaptation immune response which results in PE manifestation.