Placenta: an old organ with new functions.

The transition from oviparity to viviparity and the establishment of feto-maternal communications introduced the placenta as the major anatomical site to provide nutrients, gases, and hormones to the developing fetus. The placenta has endocrine functions, orchestrates maternal adaptations to pregnancy at different periods of pregnancy, and acts as a selective barrier to minimize exposure of developing fetus to xenobiotics, pathogens, and parasites. Despite the fact that this ancient organ is central for establishment of a normal pregnancy in eutherians, the placenta remains one of the least studied organs. The first step of pregnancy, embryo implantation, is finely regulated by the trophoectoderm, the precursor of all trophoblast cells. There is a bidirectional communication between placenta and endometrium leading to decidualization, a critical step for maintenance of pregnancy. There are three-direction interactions between the placenta, maternal immune cells, and the endometrium for adaptation of endometrial immune system to the allogeneic fetus. While 65% of all systemically expressed human proteins have been found in the placenta tissues, it expresses numerous placenta-specific proteins, whose expression are dramatically changed in gestational diseases and could serve as biomarkers for early detection of gestational diseases. Surprisingly, placentation and carcinogenesis exhibit numerous shared features in metabolism and cell behavior, proteins and molecular signatures, signaling pathways, and tissue microenvironment, which proposes the concept of "cancer as ectopic trophoblastic cells". By extensive researches in this novel field, a handful of cancer biomarkers has been discovered. This review paper, which has been inspired in part by our extensive experiences during the past couple of years, highlights new aspects of placental functions with emphasis on its immunomodulatory role in establishment of a successful pregnancy and on a potential link between placentation and carcinogenesis.

Clinico-Pathological and Prognostic Significance of a Combination of Tumor Biomarkers in Iranian Patients With Breast Cancer.

PURPOSE: Breast cancer is one of the most common cancers in the world. It is a multifaceted malignancy with different histopathological and biological features. Molecular biomarkers play an essential role in accurate diagnosis, classification, prognosis, prediction of treatment response, and cancer surveillance. This study investigated the clinico-pathological and prognostic significance of HER3 and ROR1 in breast cancer samples. METHODS: Tissue microarrays (TMA) were constructed using tissue blocks of 444 Iranian breast cancer patients diagnosed with breast cancer. Overall survival (OS) and disease-free survival (DFS) were assessed after 5 years follow-up. TMA slides were stained with monoclonal antibodies against ROR1, HER3, ER, PR, Ki67, P53, HER2 and CK5/6 using IHC and correlation between the investigated tumor markers and the clinico-pathological parameters of patients were analyzed. RESULTS: Our results showed a significant correlation between ROR1 and ER, PR, HER3, and CK5/6 expression. Additionally, there was a significant correlation between HER3 and ER, PR, HER2, and Ki67 expression. Ki67 was also correlated with HER2 and P53 expression. HER3 expression was significantly correlated with tumor stage, lymph node metastasis, perineural invasion, and multifocal tumors. Furthermore, ROR1 expression was significantly associated with tumor metastasis, lympho-vascular invasion, and perineural invasion. While HER2-HER3 coexpression was significantly associated with poor OS, HER3-ROR1 coexpression was associated with lymph node invasion, lymph node metastasis, and distant metastasis. CONCLUSION: ROR1 and HER3 displayed significant association with different clinic-pathological features and in addition to the other tumor biomarkers could be considered as diagnostic and prognostic biomarkers in breast cancer patients.

Discovery of a novel marker for human granulocytes and tissue macrophages: RTL1 revisited.

Here, retrotransposon-like 1 (RTL1) is introduced as a marker for circulating and tissue neutrophils, tissue macrophages, and tumor-associated macrophages (TAM) and neutrophils (TAN). Anti-RTL1 polyclonal and monoclonal antibodies were produced, and their reactivity was examined by Western blotting (WB), ELISA, and immunostaining of human normal and cancer tissues. The reactivity of the anti-RTL1 antibodies with peripheral blood leukocytes and a panel of hematopoietic cell lines was examined. The generated antibodies specifically detected RTL1 in the WB of the placenta and U937 cells. The polyclonal antibody showed excellent reactivity with tissue-resident macrophages, Hofbauer cells, alveolar and splenic macrophages, Kupffer cells, and inflammatory cells in the tonsil, appendix, and gallbladder. In vitro GM-CSF-differentiated macrophages also showed a high level of intracellular RTL1 expression. TAM and TAN also showed excellent reactivity with this antibody. Almost all circulating granulocytes but not lymphocytes or monocytes expressed RTL1 at their surface. Serial sections of the appendix stained with CD15 and RTL1 and placenta stained with CD68 and RTL1 showed a considerable overlap in RTL1 expression in CD15+ granulocytes and CD68+ macrophages. A small percentage of myelomonocytic cell lines was positive for surface RTL1, while promyelocytic, monocytic, megaloblastic, and lymphoblastic cell lines were negative. Endothelial cells of normal and cancer tissues highly expressed RTL1. RTL1 could be considered a new marker for different normal tissue macrophages, TAM, circulating and tissue neutrophils, and TAN.

Investigation of Expression Profile of Placenta-specific 1 (PLAC1) in Acute Myeloid and Lymphoid Leukemias.

Background: Placenta-specific 1 (PLAC1) is one of the cancer-testis-placenta antigens that has no expression in normal tissue except placenta trophoblast and testicular germ cells, but is overexpressed in a variety of solid tumors. There is a lack of studies on the expression of PLAC1 in leukemia. We investigated expression of PLAC1 in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL). Methods: In this study, we investigated expression pattern of PLAC1 gene in peripheral blood and bone marrow mononuclear cells of newly-diagnosed patients with AML (n=31) and ALL (n=31) using quantitative real-time PCR. Normal subjects (n=17) were considered as control. The PLAC1 protein expression in the samples were also detected using western blotting. Results: Our data demonstrated that PLAC1 transcripts had 2.7 and 2.9 fold-change increase in AML and ALL, respectively, compared to normal samples. PLAC1 transcript expression was totally negative in all studied normal subjects. Level of PLAC1 mRNA expression in ALL statistically increased compared to normal samples (p=0.038). However, relative mRNA expression of PLAC1 in AML was not significant in comparison to normal subjects (p=0.848). Furthermore, relative mRNA expression of PLAC1 in AML subtypes was not statistically significant (p=0.756). PLAC1 gene expression showed no difference in demographical clinical and para-clinical parameters. Western blotting confirmed expression of PLAC1 in the ALL and AML samples. Conclusion: Considering PLAC1 expression profile in acute leukemia, PLAC1 could be a potential marker in leukemia which needs complementary studies in the future.

Novel Monoclonal Antibodies Specific for Human Ki67 and P53 Tumor Markers in Breast Cancer Tissue Samples.

Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test. Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs. Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer. Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.

Cancer Is Associated with the Emergence of Placenta-Reactive Autoantibodies.

Placenta-specific antigens are minimally expressed or unexpressed in normal adult tissues, while they are widely expressed in cancer. In the course of carcinogenesis, a vast array of autoantibodies (AAbs) is produced. Here, we used a quantitative approach to determine the reactivity of AAbs in the sera of patients with breast (BrC: N = 100, 100% female, median age: 51 years), gastric (GC: N = 30, 46.6% female, median age: 57 years), bladder (BC: N = 29, 34.4% female, median age: 57 years), and colorectal (CRC: N = 34, 41.1% female, median age: 51 years) cancers against first-trimester (FTP) and full-term placental proteome (TP) in comparison with age- and sex-matched non-cancer individuals. Human-on-human immunohistochemistry was used to determine reactive target cells in FTP. The effect of pregnancy on the emergence of placenta-reactive autoantibodies was tested using sera from pregnant women at different trimesters of pregnancy. Except for BC, patients with BrC (p < 0.0284), GC (p < 0.0002), and CRC (p < 0.0007) had significantly higher levels of placenta-reactive AAbs. BrC (p < 0.0001) and BC (p < 0.0409) in the early stages triggered higher autoantibody reactivity against FTP. The reactivities of BrC sera with FTP did not show an association with ER, PR, or HER2 expression. Pregnancy in the third trimester was associated with the induction of TP- and not FTP-reactive autoantibodies (=0.018). The reactivity of BrC sera with placental proteins was found to be independent of gravidity or abortion. BrC sera showed a very strong and specific pattern of reactivity with scattered cells beneath the syncytiotrophoblast layer. Our results reinforce the concept of the coevolution of placentation and cancer and shed light on the future clinical application of the placental proteome for the non-invasive early detection and treatment of cancer.

Human amniotic epithelial cells exert anti-cancer effects through secretion of immunomodulatory small extracellular vesicles (sEV).

We identified here mechanism by which hAECs exert their anti-cancer effects. We showed that vaccination with live hAEC conferred effective protection against murine colon cancer and melanoma but not against breast cancer in an orthotopic cancer cell inoculation model. hAEC induced strong cross-reactive antibody response to CT26 cells, but not against B16F10 and 4T1 cells. Neither heterotopic injection of tumor cells in AEC-vaccinated mice nor vaccination with hAEC lysate conferred protection against melanoma or colon cancer. Nano-sized AEC-derived small-extracellular vesicles (sEV) (AD-sEV) induced apoptosis in CT26 cells and inhibited their proliferation. Co-administration of AD-sEV with tumor cells substantially inhibited tumor development and increased CTL responses in vaccinated mice. AD-sEV triggered the Warburg's effect leading to Arginine consumption and cancer cell apoptosis. Our results clearly showed that it is AD-sEV but not the cross-reactive immune responses against tumor cells that mediate inhibitory effects of hAEC on cancer development. Our results highlight the potential anti-cancer effects of extracellular vesicles derived from hAEC.

Immunization with placenta-specific 1 (plac1) induces potent anti-tumor responses and prolongs survival in a mouse model of melanoma.

PURPOSE: Melanoma is a malignant and metastatic form of skin cancer, which is not diagnosed in early stages of the disease. Nowadays, immunotherapy is changing the treatment landscape for metastatic melanoma. Placenta-specific1 (PLAC1) is a cancer-testis-placenta (CTP) antigen with differential expression in melanoma tissues. Here, we evaluated the potential of plac1 to induce anti-cancer immune responses as well as to prevent cancer development in a mouse model of melanoma. METHODS: Two proteins containing full extracellular domain (ED) of mouse plac1+KDEL3 and full ED of mouse plac1+ tetanus toxin P2 and P30+ pan DR epitope (PADRE) â€‹+ â€‹KDEL3 were produced and injected in mice to evaluate their capacity to induce anti-cancer immune responses as well as their potential to prevent melanoma development. Induction of plac1-specific humoral and cellular responses as well as tumor-associated parameters were tested in a series of 36 mice. RESULTS: Sera of mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 contained antibodies able to react with surface plac1 in B16F10 â€‹cells. Both proteins induced proliferative cellular immune responses against B16F10 â€‹cells and plac1-specific cytotoxic T cells (CTL) and CD107a â€‹+ â€‹CTL responses, which was higher in mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3. Splenocytes of mice vaccinated with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 exerted a significant cytotoxicity against B16F10 â€‹cells. Vaccination with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 significantly delayed B16F10-induced tumor onset, reduced tumor growth, and increased survival. Tumors induced by B16F10 expressed plac1 in vivo. CONCLUSION: Our results pave the way for development of effective melanoma preventive vaccine in humans, although further studies are needed.

Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor.

BACKGROUND: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. METHODS: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. RESULTS: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. CONCLUSION: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.

Optimization of Expression and Purification of Recombinant Mouse plac1.

Background: Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated. Methods: A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA. Results: The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1 antibody titer was significantly higher in sera of mice immunized with main compared to control plac1. Conclusion: In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses.

Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients.

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor-binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (P < 0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing amino acids (aa) 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and western blotting. Altogether, our data suggest involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of dominant immunogenic epitopes in NP and RBD of SARS-CoV-2 could provide important information for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

Endometrial mesenchymal stem/stromal cells: The Enigma to code messages for generation of functionally active regulatory T cells.

BACKGROUND: Regulatory T cells (Tregs) play an important role in fine-tuning of immune responses and are pivotal for a successful pregnancy. Recently, the importance of mesenchymal stem cells in regulation of immune responses in general and Tregs in particular has been highlighted. Here, we hypothesized that menstrual stromal/stem cells (MenSCs) contribute to uterine immune system regulation through induction of functionally active Tregs. METHODS: MenSCs were collected from 18 apparently healthy women and characterized. Bone marrow mesenchymal stem cells (BMSCs) served as a control. The effect of MenSCs on proliferation of anti-CD3/CD28-stimulated T CD4 + cells and generation of Tregs with or without pre-treatment with mitomycin C, IFN-γ and IL-1ß was evaluated by flow cytometry. The potential role of IDO, PGE2, IL-6, IL-10, and TGF-ß on proliferation of T CD4 + cells and generation of Tregs was assessed using blocking antibodies or agents. IDO activity was evaluated in MenSCs and BMSCs culture supernatants by a colorimetric assay. IL-10 and IFN-γ production in MenSCs-primed T CD4 + was measured using intracellular staining. To investigate the functional properties of Tregs induced by MenSCs, Treg cells were isolated and their functional property to inhibit proliferation of anti-CD3/CD28-stimulated PBMCs was assessed by flow cytometry. RESULTS: According to the results, proliferation of T CD4 + lymphocytes was enhanced in the presence of MenSCs, while pre-treatment of MenSCs with pro-inflammatory cytokines reversed this effect. PGE2 and IDO were the major players in MenSCs-induced T cell proliferation. Non-treated MenSCs decreased the frequency of Tregs, whereas after pre-treatment with IFN-γ and IL-1ß, they induced functional Tregs with ability to inhibit the proliferation of anti-CD3/CD28-stimulated PBMCs. This effect was mediated through IL-6, IL-10, TGF-ß and IDO. IFN-γ/IL-1ß-treated MenSCs induced IL-10 and IFN-γ production in CD4 + T cells. CONCLUSION: Collectively, these findings indicate that immunomodulatory impact of menstrual blood stem cells (MenSCs) on generation of Tregs and inhibition of T cells proliferation is largely dependent on pre-treatment with IFN-γ and IL-1ß. This is the first report on immunomodulatory impact of MenSCs on Tregs and highlights the pivotal role of endometrial stem cells in regulation of local endometrial immune responses.

Expression profiling of RTL1 in human breast cancer tissues and cell lines.

Breast cancer (BC) is the most common cancer in females. In this regard, the identification of molecular alterations driving BC is an immediate need for developing effective immunotherapeutic tools. Here we investigated the expression of a placenta-specific protein, Retrotransposon-like 1 (RTL1) in a series of BC tissues and cell lines. RTL1-specific polyclonal antibody was generated and characterized. Using tissue microarray immunohistochemistry, expression of RTL1 in a total of 147 BC and 36 non-malignant breast tissues was investigated and the association of patient's clinicopathological parameters with RTL1 expression was then examined. Expression of RTL1 in four BC cells was assessed by flow cytometry, immunofluorescent staining and Western blotting. We observed a mixture pattern of nuclear and cytoplasmic RTL1 expression in most tissues examined, however nuclear expression was found to be dominant pattern of expression. The level of nuclear RTL1 expression was significantly higher in BC tissues (P < 0.001). A statistically significant association between nuclear RTL1 expression and histological grade and vascular invasion was found (P < 0.001 and P < 0.05). All cell lines expressed RTL1 with varying degrees at their surface. The most invasive BC cell line MDA-MB-231, compared to T-47D, SKBR3 and MCF7 expressed higher levels of RTL1 at their surface. Cells with a low level of surface expression, expressed high levels of intracellular RTL1 expression. Our antibody reacted with a specific band of about 125 KD in normal human placenta and all cell lines examined. In contrast to placenta, two additional bands were also observed in cancer cell lines. Our results showed for the first time that RTL1 is differentially expressed in BC compared to non-malignant breast tissues and is associated with a higher grade and vascular invasion. In BC cells with high metastatic and invasive potential, this antigen is mostly confined to cell surface compartment indicating the possibility of using antibody-based immunotherapy for advanced metastatic BC patients.

Methylation of TGM-3 Promoter and Its Association with Oral Squamous Cell Carcinoma (OSCC).

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) is among the ten most common cancers worldwide. Hypermethylation of CpG sites in the promoter region and subsequent down-regulation of a tumor suppressor gene, TGM-3 has been proposed to be linked to different types of human cancers including OSCC. In this study, methylation status of CpG sites in the promoter region of TGM-3 has been evaluated in a cohort of patients with OSCC compared to normal controls. METHODS: Forty fresh tissue samples were obtained from newly diagnosed OSCC patients and normal individuals referred to dentistry clinic for tooth extraction. DNA was extracted, bisulfite conversion was performed and it was subjected to PCR using bisulfite-sequencing PCR (BSP) primers. Prepared samples were sequenced on a DNA analyzer with both forward and reverse primers of the region of interest. The peak height values of cytosine and thymine were calculated and methylation levels for each CpG site within the DNA sequence was quantified. RESULTS: Quantitative DNA methylation analyses in CpG islands revealed that it was significantly higher in OSCC patients compared to controls. DNA methylation at CpG1/CpG3/CpG5 (p=0.004-0.01) and CpG1/CpG3 (p=0.001-0.019) sites was associated with tumor stage and grade, respectively. Male OSCC patients had higher methylation rate at CpG3 (p=0.032), while smoker patients showed higher methylation rate at CpG6 (p=0.045). CONCLUSION: These results manifested the contribution of DNA methylation of TGM-3 in OSCC and its potential association with clinico-pathologic parameters in OSCC.

Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells.

OBJECTIVES: Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. DESIGN: This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. Participants/Materials, Setting, and Methods: Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. RESULTS: 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. LIMITATIONS: Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. CONCLUSIONS: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

Mesenchymal stem cells induce expansion of regulatory T cells in abortion-prone mice.

Recurrent pregnancy loss (RPL) is one of the most common complications of early pregnancy associated in most cases with local or systemic immune abnormalities such as the diminished proportion of regulatory T cells (Tregs). Mesenchymal stem cells (MSCs) have been shown to modulate the immune responses by de novo induction and expansion of Tregs. In this study, we analyzed the molecular and cellular mechanisms involved in Treg-associated pregnancy protection following MSCs administration in an abortion-prone mouse mating. In a case-control study, syngeneic abdominal fat-derived MSCs were administered intraperitoneally (i.p) to the DBA/2-mated CBA/J female mice on day 4.5 of pregnancy. Abortion rate, Tregs proportion in spleen and inguinal lymph nodes, Ho1, Foxp3, Pd1 and Ctla4 genes expression at the feto-maternal interface were then measured on day 13.5 of pregnancy using flow cytometry and quantitative RT-PCR, respectively. The abortion rate in MSCs-treated mice reduced significantly and normalized to the level observed in normal pregnant animals. We demonstrated a significant induction of Tregs in inguinal lymph nodes but not in the spleen following MSCs administration. Administration of MSCs remarkably upregulated the expression of Ho1, Foxp3, Pd1 and Ctla4 genes in both placenta and decidua. Here, we show that MSCs therapy could protect the fetus in the abortion-prone mice through Tregs expansion and upregulation of Treg-related genes. These events could establish an immune-privileged microenvironment, which participates in the regulation of detrimental maternal immune responses against the semi-allogeneic fetus.

MenSCs exert a supportive role in establishing a pregnancy-friendly microenvironment by inhibiting TH17 polarization.

OBJECTIVES: Uncontrolled TH17 differentiation has been suggested to play a role in the pathogenesis of pregnancy loss. We recently showed that menstrual blood stromal/stem cells (MenSCs) alter functional features of natural killer cells. Here, we hypothesized that MenSCs could modulate differentiation of TH17 cells. METHOD: MenSCs were collected from 18 apparently healthy women and characterized. Bone marrow mesenchymal stem cells (BMSCs) served as a control. TH17 polarization and proliferation of purified T CD4+ cells were assessed by flow cytometry in a well-defined co-culture system containing T CD4+ cells and MenSCs or BMSCs. Indoleamine 2,3-Dioxygenase (IDO) activity was evaluated in MenSC and BMSC culture supernatants by a colorimetric assay. The impact of MenSCs on expression of transcription factors, RORC, T-bet, Gata3, NRP-1 and Helios were studied by qPCR. RESULTS: MenSCs significantly inhibited TH17 differentiation (p = 0.0383) and percentage of the cells co-expressing IL-17 and IFN-γ (p = 0.0023). PGE2 blockade significantly reduced percentage and proliferation of T CD4+IL-17+ (p = 0.003, p = 0.0018), T CD4+ IFN-γ+ (p = 0.002, p = 0.0022) and T CD4+IL-17+ IFN-γ+ (p = 0.004, p = 0.02) cells. MenSCs produced a considerable activity of IDO (p = 0.0002), induced a significant rise in the Treg frequency (p = 0.0091) and a sharp increase in TH17/Tregs ratio (p = 0.0022). MenSCs increased expression of NRP1 (p = 0.001), while downregulated expression of RORC in T cells (p = 0.001). CONCLUSION: Our results suggest a supportive role for MenSCs in establishing a pregnancy-friendly microenvironment in the uterus and put forth the idea that inherent abnormalities of MenSCs may be a basis for dysregulated endometrial immune network leading to pregnancy loss.

HPV16-E7 Protein T Cell Epitope Prediction and Global Therapeutic Peptide Vaccine Design Based on Human Leukocyte Antigen Frequency: An In-Silico Study.

Cervical cancer is the second most common leading cause of women's death due to cancer worldwide, about 528,000 patients' cases and 266,000 deaths per year, related to human papillomavirus (HPV). Peptide-based vaccines being safe, stable, and easy to produce have demonstrated great potential to develop therapeutic HPV vaccine. In this study, the major histocompatibility complex (MHC) class I, class II T cell epitopes of HPV16-E7 were predicted. Therefore, we designed a plan to find the most effective peptides to prompt appropriate immune responses. For this purpose, retrieving protein sequences, conserved region identification, phylogenic tree construction, T cell epitope prediction, epitope-predicted population coverage calculation, and molecular docking were performed consecutively and most effective immune response prompting peptides were selected. Based on different tools index, six CD8+ T cells and six CD4+ epitopes were chosen. This combination of 12 epitopes created a putative global vaccine with a 95.06% population coverage. These identified peptides can be employed further for peptide analysis and can be used as a peptide or poly-epitope candidates for therapeutic vaccine studies to treat HPV-associated cancers.

Thyroid peroxidase in human endometrium and placenta: a potential target for anti-TPO antibodies.

Autoimmune thyroid disease is the most common endocrine disorder during pregnancy. Thyroid autoantibodies (TAs) have been suggested to serve a role in implantation failure and spontaneous abortion. Until now, there are no data on the potential interaction of TAs with human reproductive organs. Here, we set out for the first time to test this hypothesis by studying the expression of thyroid peroxidase (TPO) at gene and protein level in human reproductive organs. Endometrial samples were taken from normal women, and placenta tissues were collected after full-term caesarian section. Expression of TPO messenger RNA (mRNA) was investigated by qRT-PCR. In addition, polyclonal anti-TPO antibodies were produced and the expression of TPO protein in mentioned tissues was evaluated by immunohistochemistry and Western blot analysis. The reactivity of anti-TPO antibody in human embryos was evaluated by immunofluorescent staining. For the first time, our study showed that TPO is expressed at gene and protein levels in endometrium and placenta. TPO expression was mainly localized to glandular and luminal epithelial cells in the endometrium. In placenta, the syncytiotrophoblasts and invasive trophoblast cells were the main cell types that expressed TPO protein. Specific band of approximately 110 kDa was observed in all endometrial and placental tissues by Western blot analysis. However, no expression of TPO protein was observed in human embryo. TPO expression in endometrium and placenta may explain higher frequency of abortion and infertility in patients with thyroid autoimmunity.

Pyrvinium pamoate induces in-vitro suppression of IL-6 and IL-8 produced by human endometriotic stromal cells.

Endometriosis, a chronic inflammatory disease, is identified by the presence of endometrial tissue outside the uterus. The prevalence of this disease among reproductive-age women is almost 10-15%. High levels of IL-6 and IL-8 have been found in the peritoneal fluid (PF) of women with endometriosis and are involved in its pathogenesis. Isolated stromal cells from 12 ectopic and eutopic endometrial biopsies of women with ovarian endometrioma and also 12 endometrial biopsies of nonendometriotic controls were treated with 1.1 µM pyrvinium pamoate, a Wnt/ß-catenin signaling pathway inhibitor, for 72 hrs. Before treatment, mRNA gene expression and secretion of IL-6 and IL-8 were significantly higher in ectopic (EESCs) than eutopic (EuESCs) and control (CESCs) endometrial stromal cells. After treatment, mRNA gene expression and also secretion of IL-6 and IL-8 were significantly reduced. Our Findings showed that pyrvinium pamoate suppresses the mRNA gene expression and secretion of IL-6 and IL-8 in human endometriotic stromal cells. Additional investigations on this compound are required before clinical application.