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.

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.

Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

Serological assay for anti-SARS-CoV-2 antibodies improves sensitivity of diagnosis of COVID-19 patients.

The emergence of SARS-CoV-2, responsible for coronavirus disease-2019 (COVID-19), has become a major global health problem. The molecular testing is the accepted assay in SARS-CoV-2 detection. However, there are several reasons for low sensitivity by RNA detection, causing challenges in SARS-CoV-2 diagnosis. In this study, we aimed to investigate serological patterns of SARS-CoV-2 specific IgM, and IgG in 111 hospitalized, and 34 recovered COVID-19 patients and 311 prepandemic normal serum specimens by ELISA. The validity of the ELISA kits was evaluated using samples from normal and recovered cases. This showed that 98.1%, and 98.4% of prepandemic normal samples were negative for anti-SARS-CoV-2 IgM, and IgG, respectively. Assessment of 34 COVID-19 confirmed recovered patients showed a test sensitivity of 76.5%, and 94.1% for IgM, and IgG, respectively. In COVID-19 hospitalized patients, 42.3%, and 51.4% were positive for IgM and IgG, respectively. Viral RNA was not detectable in 43.3% of the hospitalized patients. Interestingly, combined molecular and serological testing improved the sensitivity of COVID-19 diagnosis to 79.6%. Using PCR with combined IgM/IgG results augmented the patient diagnosis sensitivity to 65.3% and 87.2% in ≤ 7 days, and > 7 days intervals, respectively. Overall, serological tests in combination with PCR can improve the sensitivity of COVID-19 diagnosis.

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.

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.

Expression of Human Placenta-specific 1 (PLAC1) in CHO-K1 Cells.

BACKGROUND: Placenta-specific 1 (PLAC1), as a new Cancer/Testis Antigen (CTA), is frequently expressed in a variety of cancers and localized to cytoplasm and plasma membrane. Surface expression of cancer target antigens is of great importance that enables antibody-mediated cancer immunotherapy. The aim of the current study was to express the intact human PLAC1 protein on plasma membrane of a eukaryotic cell as a model for future anti-PLAC1-based cancer immunotherapy. METHODS: In the first approach, entire human PLAC1 gene including its own Signal Peptide (SP) was cloned into pIRES2-EGFP and LeGO-iG2 vectors and expressed in CHO-K1 cells. In the second approach, cytosolic and Signal-Anchor (SA) sequence of Transferrin Receptor Protein 1 (TFR1) were fused to extracellular portion of PLAC1 and expressed as above. Expression of PLAC1 was then assessed using Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western Blot (WB), Immunocytochemistry (ICC), Immunofluorescence (IF) and Flow Cytometry (FC). RESULTS: The first approach resulted in the expression of PLAC1 in submembranous but not in the surface of transfected CHO-K1 cells. Using the chimeric human PLAC1 construct, the same intracellular expression pattern was observed. CONCLUSION: These results indicated that there are some yet unknown PLAC1 localization signals employed by cancer cells for surface expression of PLAC1.

Zinc-Phosphate Nanoparticles as a Novel Anticancer Agent: An In Vitro Evaluation of Their Ability to Induce Apoptosis.

In the current study, zinc-phosphate nanoparticles (ZnPNPs) were investigated for the first time due to their anticancer activity against breast cancer Michigan Cancer Foundation-7 (MCF-7) cell line. The modification of such nanoparticles (NPs) was further examined for physicochemical characterization using various techniques such as powder X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential calculation, field emission scanning electron microscopy (FESEM), energy-dispersed spectroscopy (EDS), and Fourier transform infrared (FTIR) spectroscopy. Then, the newly fabricated ZnPNPs were tested for their in vitro cell cytotoxicity against breast cancer MCF-7 cells and noncancerous human embryonic kidney HEK293 cells, using MTT assay as a colorimetric one to assess cell metabolic activity for 24 h. The apoptotic efficacy of the NPs was subsequently confirmed through data obtained from Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit and cell cycle analysis. Determination of reactive oxygen species (ROS) generation was further performed via flow cytometry. Additionally, the expression of tumor suppressor genes p53 was analyzed using real-time polymerase chain reaction (PCR). Also, the prepared NPs showed a mean particle size of 38 nm. The measurements correspondingly showed that the cytotoxicity of MCF-7 cells depends on the concentration of NPs (IC50 = 80.112 µg/mL). MCF-7 cells were associated with initiation of apoptotic pathway in cells. Additionally, flow cytometry revealed cell cycle arrest in sub-G1 phase. ROS production was also obtained after treatment with IC50 concentration. According to annexin V-FITC/PI staining kit data, the percentage of early and late apoptotic cells was 78.2% in those treated with ZnPNPs. Moreover, the real-time PCR results demonstrated the ability of NPs in upregulating p53 gene expression. In summary, the data demonstrated that fabricated ZnPNPs had prominence to act as antitumor agents in breast cancer therapy.

Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1).

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Placental Kisspeptins Differentially Modulate Vital Parameters of Estrogen Receptor-Positive and -Negative Breast Cancer Cells.

Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2-30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production.

Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells.

BACKGROUND: Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). METHODS: Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. RESULTS: WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p < 0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p < 0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner (p < 0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p < 0.05). CONCLUSION: Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus.

Comparable vitamin D3 metabolism in the endometrium of patients with recurrent spontaneous abortion and fertile controls.

Vitamin D exerts important roles during pregnancy, and its deficiency may be associated with several pregnancy complications, including pregnancy loss, yet no data are available for molecules involved in vitamin D metabolism in patients with unexplained recurrent spontaneous abortion. In this study, we investigated possible difference in endometrial expression of vitamin D3 receptor (VDR), 1α-hydroxylase (CYP27B1), and 24-hydroxylase (CYP24A1) in women with recurrent spontaneous abortion (n = 8) and healthy controls (n = 8). Gene expression of VDR, CYP27B1, and CYP24A1 was determined by real-time PCR, while VDR and CYP27B1 proteins were localized by immunohistochemistry and their abundance was validated by Western blot. We found that both patient and control groups expressed comparable levels of endometrial VDR, CYP27B1, and CYP24A1 transcripts. In line with the gene-expression results, CYP27B1 and different isoforms of VDR protein were present at the same abundance in the endometria of both groups. No significant alteration in VDR and CYP27B1 immunoreactivity pattern was found in the endometrium of patients compared to fertile controls, however. The results of the present study, therefore, do not support the hypothesis of differential expression of key molecules involved in vitamin D3 metabolism in the endometrium of recurrent spontaneous abortion patients and fertile controls.

Synthesis and investigation of new Hesperadin analogues antitumor effects on HeLa cells.

Hesperadin is one of the indolinones that was designed against the ATP-binding site of Aurora kinase. This molecule inhibits Aurora B kinase by phosphorylation of histone H3. In this study, new derivatives of Hesperadin containing an amide group in their structures were synthesized through sequential Ugi/palladium-catalyzed approach and in vitro antitumor activity of new compounds were evaluated by cell proliferation assay. The results show that compounds 6f, 6i, 6l, and 6o were dose-dependently inhibited in different concentrations, and IC50 values were between 35 and 43 nM. It seems that lipophilic substitution on the indolinone core with the ability to form additional hydrogen bond might lead to increased stability of structure and activity of new Hesperadin analogues.

Menstrual blood-derived stromal stem cells from women with and without endometriosis reveal different phenotypic and functional characteristics.

Retrograde flow of menstrual blood cells during menstruation is considered as the dominant theory for the development of endometriosis. Moreover, current evidence suggests that endometrial-derived stem cells are key players in the pathogenesis of endometriosis. In particular, endometrial stromal stem cells have been suggested to be involved in the pathogenesis of this disease. Here, we aimed to use menstrual blood, as a novel source of endometrial stem cells, to investigate whether stromal stem cells from endometriosis (E-MenSCs) and non-endometriosis (NE-MenSCs) women differed regarding their morphology, CD marker expression pattern, proliferation, invasion and adhesion capacities and their ability to express certain immunomodulatory molecules. E-MenSCs were morphologically different from NE-MenSCs and showed higher expression of CD9, CD10 and CD29. Furthermore, E-MenSCs had higher proliferation and invasion potentials compared with NE-MenSCs. The amount of indoleamine 2,3-dioxygenase-1 (IDO1) and cyclooxygenase-2 (COX-2) in E-MenSCs co-cultured with allogenic peripheral blood mononuclear cells (PBMCs) was shown to be higher both at the gene and protein levels, and higher IDO1 activity was detected in the endometriosis group. However, NE-MenSCs revealed increased concentrations of forkhead transcription factor-3 (FOXP3) when compared with E-MenSCs. Nonetheless, interferon (IFN)-γ, Interleukin (IL)-10 and monocyte chemoattractant protein-1 (MCP-1) levels were higher in the supernatant of E-MenSCs-PBMC co-cultures. Here, we showed that there are inherent differences between E-MenSCs and NE-MenSCs. These findings propose the key role MenSCs could play in the pathogenesis of endometriosis and further support the retrograde and stem cell theories of endometriosis. Hence, considering its renewable and easily available nature, menstrual blood could be viewed as a reliable and inexpensive material for studies addressing the cellular and molecular aspects of endometriosis.

Conjugation of Monoclonal Antibodies to Super Paramagnetic Iron Oxide Nanoparticles for Detection of her2/neu Antigen on Breast Cancer Cell Lines.

Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide (SPIO) nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20µg antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques.