Flow cytometric analysis of innate lymphoid cells: challenges and solutions.

Introduction: The three groups of helper innate lymphoid cells (ILCs), namely ILC1, ILC2 and ILC3, have been identified by flow cytometry by combinations of cell surface markers. Here, we review various ways ILCs are currently identified, focusing on potential problems and their solutions. The first step to identify all ILCs is to exclude other lymphocytes and myeloid cells by their lineage-specific markers (Lin). However, the Lin cocktail varies in various studies, and the definition of Lin- population containing ILCs is often ambiguous, resulting in contamination of Lin+ cells, particularly T cells. Method: We have designed combinations of cell surface markers to identify ILC populations in various tissues of B6 mice by flow cytometry. To minimize T cell contamination, TCR/CD3ϵ antibodies were used separately from the Lin cocktail. ILCs identified by surface markers are confirmed by the expression of the transcription factors GATA3, RORγt, T-bet and Eomes. Result: ILC1s in the B6 mouse liver are identified by Lin-NKp46+NK1.1+TCR/CD3ϵ-CD49a+CD49b-. However, defining ILC1s in other tissues remains a challenge. ILC2s in the lung are identified by Lin-TCR/CD3ϵ- Thy1+CD127+ST2+ whereas ILC2s in the small intestine and liver are identified by Lin-TCR/CD3ϵ-Thy1+GATA3+RORγt-. ILC3s in B6 mouse spleen, liver, lung and small intestine are identified by Lin-TCR/CD3ϵ- Thy1+CD127+RORγt+. Discussion: The ILC population is heterogeneous and the strategies to identify ILCs have to be designed for each ILC population and tissue. Excluding T cells in all cases is crucial, and a combination of transcription factors GATA3, RORγt, T-bet, and Eomes should be used to identify ILCs. Using CD3ϵ/TCRs in a different fluorochrome not in Lin cocktail minimizes contamination of T cells specifically identify individual ILC populations in various tissues.

Migration of Lung Resident Group 2 Innate Lymphoid Cells Link Allergic Lung Inflammation and Liver Immunity.

Group 2 innate lymphoid cells (ILC2s) are tissue resident in the lung and activated by inhaled allergens via epithelial-derived alarmins including IL-33. Activated ILC2s proliferate, produce IL-5 and IL-13, and induce eosinophilic inflammation. Here, we report that intranasal IL-33 or the protease allergen papain administration resulted in increased numbers of ILC2s not only in the lung but also in peripheral blood and liver. Analyses of IL-33 treated parabiosis mice showed that the increase in lung ILC2s was due to proliferation of lung resident ILC2s, whereas the increase in liver ILC2s was due to the migration of activated lung ILC2s. Lung-derived ILC2s induced eosinophilic hepatitis and expression of fibrosis-related genes. Intranasal IL-33 pre-treatment also attenuated concanavalin A-induced acute hepatitis and cirrhosis. These results suggest that activated lung resident ILC2s emigrate from the lung, circulate, settle in the liver and promote type 2 inflammation and attenuate type 1 inflammation.

Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets.

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor-related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2- population different from conventional IL-18Rα-ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

Myeloid-derived suppressor cells: Important contributors to tumor progression and metastasis.

Myeloid-derived suppressor cells (MDSCs) are traditionally considered among the major components of the immunosuppressive tumor microenvironment (TME). However, there is currently increasing evidence indicating that MDSCs in addition to suppression of immune surveillance is also involved in an array of non-immunological functions like augmenting metastatic potential of tumor cells. Indeed, MDSCs can promote metastasis in animal models and cancer patients through promoting premetastatic niche formation, tumor angiogenesis and invasion. Moreover, MDSC frequency and function have been associated with progressive disease and correlated with clinical outcome. This review will summarize and discusses the data demonstrating the role for MDSCs in tumor metastasis.

The Cytotoxic and Apoptotic Effects of Scrophularia Atropatana Extracts on Human Breast Cancer Cells.

Purpose: Breast cancer is the most frequent malignancy diagnosed in women both in developed and developing countries. Natural products especially those from herbal origin have high potential in producing drug components with a source of novel structures. The present study was designed to explore the cytotoxic effects and the cell death mechanism of Scrophularia atropatana extracts. Methods: MTT assay was employed to evaluate the cytotoxic activity of the extracts of S. atropatana on the MCF-7 as well as non-malignant cells. Furthermore, induction of apoptosis was evaluated by TUNEL assay, cell death detection ELISA, DNA fragmentation test, western blotting and Real Time PCR. Results: In vitro exposures of the MCF-7 cells with different concentration of S. atropatana extract significantly inhibited their growth and viability and induced apoptosis in the MCF-7 cells. Cleavage PARP protein, decrease in the mRNA expression levels of bcl-2 and increase expression of Caspase-3 and Caspase-9 mRNA, highlights that the induction of apoptosis was the main mechanism of cell death. Moreover the expression study of Caspase-9 mRNA showed that, the extracts have induced apoptosis via intrinsic mitochondrial pathway. Conclusion: Our results demonstrated that dichloromethane extract of Scrophularia atropatana has an apoptotic effects and it can be developed as anticancer agents.

An in vitro ethnopharmacological study on Prangos ferulacea: a wound healing agent.

Introduction: Traditionally Prangos ferulacea root is being used as an effective wound healing agent especially for pus-filled wounds both in human and stocks in the western north of Iran. Regarding the subject we decided to study P. ferulacea roots essential oil (PFE) for its antimicrobial and wound healing activities. Methods: The in vitro wound healing activity of PFE was evaluated in the mouse fibroblast cell line L929 using MTT assay of cell viability and cytotoxicity indices. Scratch assay as an in vitro model of wound healing assay was also conducted in this study. Moreover, the type I collagen content was used as an indicator of progress in wound healing process using Sircol collagen assay. Besides, PFE was subjected to GC/MS to identify the chemical constituents, and antimicrobical property was also evaluated against S. aureus, S. epidermidis, E. coli, P. aeruginosa,S. paratyphi and C. albicans using agar dilution method. Results: GC/MS analysis showed that the monoterpene hydrocarbones dominated in PFE, amounting to a total percentage of 95.1% with the major constituents: ß-Phellandrene (32.1%), m-Tolualdehyde (26.2%), and δ-3-carene (25.8%). PFE inhibited the growth of S. aureus and P. aeruginusa with the MIC value of 20 µg/mL. In addition, at the second day of treatment, PFE at concentrations of 4 and 16 µg/mL significantly (P<0.001) enhanced the migration rate of L929 cells by 87.05±2.4 and 63.5±0.08 %, respectively. Moreover, the collagen production by L929 cells was increased greatly (P<0.001). Conclusion: It is proposed that the excellent antimicrobial activity along with the significant increase of migration rate and collagen production by fibroblast cells might be associated with the high content and synergistic effect of the monoterpens, corroborating the traditional usage of this plant as a wound healing agent.

Cytotoxic and Apoptotic Activities of Methanolic Subfractions of Scrophularia oxysepala against Human Breast Cancer Cell Line.

Herbs have played a positive role in medicine for thousands of years. In the current study, we investigated the cytotoxicity effects of Scrophularia oxysepala methanolic subfractions and the underlying mechanism responsible for cell death in human breast carcinoma (MCF-7 cells) and mouse fibrosarcoma (WEHI-164 cells). From 60% and 80% methanolic fractions, four subfractions (Fa, Fb, Fc, and Fd), yielded from size exclusion by Sephadex-LH20 column chromatography, were chosen. MTT assay revealed that all subfractions significantly reduced cell viability after 24 h and 36 h in a dose-dependent manner; it is worth noting that Fa and Fb subfractions had the highest cytotoxicity, with IC50 values of 52.9 and 61.2 µg/mL in MCF-7 at 24 h, respectively. ELISA, TUNEL, and DNA fragmentation assay revealed that antiproliferative effects of all subfractions were associated with apoptosis on cancer cells, without any significant effect on L929 normal cells. qRT-PCR data showed that, after 24 h treatment with IC50 concentrations of the subfractions, caspase-3 expression was increased in cancer cells while the expression of Bcl-2 was decreased. S. oxysepala methanolic subfractions induce apoptosis in MCF-7 and WEHI-164 cells and could be considered as a source of natural anticancer agents.

Dichloromethane fractions of Scrophularia oxysepala extract induce apoptosis in MCF-7 human breast cancer cells.

Breast cancer is a prevalent malignancy among women, especially in developing countries. A large number of anticancer agents with herbal origins have been reported. Hence, herbals may play an essential role in prevention and treatment of cancers. We investigated cytotoxic effects of dichloromethane fractions of Scrophularia oxysepala extract on the MCF-7 breast cancer cell line. The cytotoxic activity of Scrophularia oxysepala fractions on the MCF-7 cells was assessed using Trypan Blue dye exclusion and MTT (3-(4, 5-dimetylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assays. In addition, apoptosis induction was determined using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis. Quantitative Real-Time PCR was also used for analyzing the changes in Caspase-3, Caspase-9, and Bcl-2 genes' expression. Results revealed an effective inhibition of growth and viability in MCF-7 cells treated with dichloromethane fractions. Cell death assay and DNA fragmentation analysis using the TUNEL test confirmed the induction of apoptosis in the MCF-7 cell line. Further, the fractions have resulted in an increased expression of Caspase-3 and Caspase-9 mRNA, which highlights the possibility of apoptosis in the treatments. The expression study of Caspase-9 mRNA confirmed that, the fractions have triggered apoptosis via intrinsic mitochondrial pathway. In summary, fractions of Scrophularia oxysepala extract were found to be promising in growth inhibition and induction of apoptosis in MCF-7 breast cancer cells.

Methanolic Fractions of Ornithogalum cuspidatum Induce Apoptosis in PC-3 Prostate Cancer Cell Line and WEHI-164 Fibrosarcoma Cancer Cell Line.

PURPOSE: The present study, was aimed to assess the cytotoxic effects of Ornithogalum cuspidatum methanolic fractions on PC-3, prostate cancer cells and WEHI-164, Fibrosarcoma cells. METHODS: Methanolic fractions of O. cuspidatum were prepared using solid phase extraction and the cells were treated with different concentrations for 12 and 24 hours. Cytotoxicity and cell viability were measured by MTT assay. ELISA was also employed to assess the histone-associated DNA fragments and the involvement of apoptotic mechanisms. RESULTS: 10 and 20% fractions had not significant cytotoxic effects (p>0.05) but other fractions exerted growth inhibition on both cancer cell lines (p<0.05). After 24h of incubation with 40, 60, 80 and 100% fractions, the IC50 values were: 165, 85, 65 and 45µg/ml on PC-3 cells and 200, 96, 76 and 73µg/ml against WEHI-164 cell line, respectively. ELISA results also revealed that, both cell lines had undergone apoptosis. CONCLUSION: It is deduced that, 80% and 100% methanolic fractions had significant anti-proliferative and apoptotic impacts on PC-3 and WEHI-164 cells in vitro and could be considered for developing chemo-preventive substances.

In vitro Cytotoxic Activity of Four Plants Used in Persian Traditional Medicine.

PURPOSE: The aim of this study was to investigate in vitro cytotoxic activity of four methanolic crude plant extracts against panel cell lines. METHODS: Methanolic extracts were tested for their possible antitumor activity and cytotoxicity using the 3-(4,5-dimetylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay on six cancer cell lines; non-Hodgkin's B-cell lymphoma (Raji), human leukemic monocyte lymphoma (U937), human acute myelocytic leukemia (KG-1A), human breast carcinoma (MCF-7 cells), human Prostate Cancer (PC3) and mouse fibrosarcoma (WEHI-164) cell lines and one normal cell line; Human Umbilical Vein Endothelial Cells (HUVEC). RESULTS: All species showed dose dependent inhibition of cell proliferation. IC50 values ranging from 25.66±1.2 to 205.11±1.3 µg/ml. The highest cytotoxic activity Chelidonium majus L> Ferulago Angulata DC> Echinophora platyloba DC> Salvia officinalis L, respectively. CONCLUSION: all extracts demonstrate promising cytotoxicity activity as a natural resource for future bio-guided fractionation and isolation of potential antitumor agents.

Induction of apoptosis by grape seed extract (Vitis vinifera) in oral squamous cell carcinoma.

Development of novel therapeutic modalities is crucial for the treatment of oral squamous cell carcinoma (OSCC). Recent scientific studies have been focused on herbal medicines as potent anti-cancer drug candidates. This study is the first to investigate the cytotoxic effects and the mechanism of cell death induced by grape seed extract (GSE) in oral squamous cell carcinoma (KB cells). MTT (3-(4,5-dimetylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) and trypan blue assays were performed in KB cells as well as human umbilical vein endothelial cells (HUVEC) were used to analyze the cytotoxic activity of GSE. Furthermore, the apoptosis-inducing action of the extract was determined by TUNEL, DNA fragmentation and cell death analysis. Statistical significance was determined by analysis of variance (ANOVA), followed by Duncan's test at a significance level of P≤0.05. The results showed apoptotic potential of GSE, confirmed by significant inhibition of cell growth and viability in a dose- and time- dependent manner without inducing damage to non-cancerous cell line HUVEC. The results of this study suggest that this plant contains potential bioactive compound(s) for the treatment of oral squamous cell carcinoma.

Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

BACKGROUND: CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. OBJECTIVE: The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. METHODS: To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. RESULTS: Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. CONCLUSIONS: The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.