Phytochemical characterization and biological activities of Plectranthus barbatus Andrews

Plectranthus barbatus Andrews (Lamiaceae) is widely distributed in the world and has a range of popular therapeutic indications. This work aimed to evaluate the phytochemical characterization of two leaf extracts of P. barbatus, and their antimicrobial, antineoplastic and immunomodulatory potential. After collection, herborization and obtainment of the P. barbatus aqueous extract (PBA) and acetone:water 7:3 P. barbatus organic extract (PBO), the phytochemical characterization was performed by high-performance liquid chromatography (HPLC). The antimicrobial activity was performed to determine the minimum inhibitory concentration (MIC) against eight bacterial strains using the microdilution test and the fungus Trichophyton rubrum by disc diffusion assay and microdilution test. Cytotoxicity was assessed by MTT and trypan blue methods in normal peripheral blood mononuclear cells (PBMCs) at concentrations ranged between 0.1 to 100 µg.mL-1 and in neoplastic cell lines Toledo, K562, DU-145 and PANC-1 at 1, 10 and 100 µg.mL-1 . Immunomodulatory activity, was evaluated by sandwich ELISA of proinflammatory cytokines at BALB/c mice splenocytes cultures supernatant. Both extracts presented flavonoids, cinnamic derivatives, steroids and ellagic acid. PBO showed bacteriostatic activity against Acinetobacter baumannii (MIC = 250 µg.mL-1) clinical isolate and PBA fungistatic activity against Trichophyton rubrum (MIC = 800 µg.mL-1). The extracts did not exhibit toxicity to PBMCs and neoplastic cells (IC50 > 100 µg.mL-1). Additionally, PBO at 100 µg.mL-1 significantly inhibited IFN-γ and IL-17A cytokines (p = 0.03). Plectranthus barbatus is a potential candidate for therapeutic use due to its low toxicity in healthy human cells and exhibits biological activities of medical interest as bacteriostatic, fungistatic and immunomodulatory.

Emoxipine Modulates Concentration-Dependent Effects of Cytarabine and Cyclocytidine on Activation of Human T Cells

Title: Emoxipine modulates concentration-dependent effects of cytarabine and cyclocytidine on activation of human T cells. Introduction: Both cytarabine and cyclocytidine are used in the treatment of acute myeloid leukemia. Well known that cytarabine and other related cytosine-based nucleoside analogues are being toxic to tumor cells by increasing levels of cellular oxidative stress as it could be abrogated by antioxidants. However, very little is known both about both the effects of combinations of antimetabolites with antioxidants on the cytotoxic innate and adaptive immune cells and whether lymphocytes toxicity affects its anticancer efficiency. Aim: To estimate effects of cytarabine and cyclocytidine with emoxipine on in vitro activated human T cells at concentrations reached during in vivo treatment with high doses, conventional doses and low doses. Materials and Methods: T cells derived from blood donors were activated in vitro in cell culture medium alone or supplemented with cytarabine 0.1-10.0 μM or cyclocytidine 0.1-10.0 μM. Cell characteristics were assessed by flow cytometry. Results: Only cytarabine 1.0-10.0 μM had both antiproliferative and proapoptotic effects. Additionally, these cytarabine concentrations increased the γIFN-producing by CD3+CD4+ T cells and did not affect the release of this cytokine by CD3+CD8+ T cells. In contrast, the lowest concentration (0.1 μM) did not have or showed minor antiproliferative or cytotoxic effects, did not alter the release of γIFN. Cyclocytidine did not affect viability of normal peripheral blood mononuclear cells but decreased the proliferative capacity of activated normal T cells in dose-dependent manner. Additionally, cyclocytidine altered the percentage of γIFN-producing proliferative CD3+CD8+ cytotoxic T cells for any concentration tested (0.1, 1.0, 1 and 10.0 μM) meanwhile highly suppressed the number of the whole amount of CD3+CD8+ cells and did not affect the release of cytokines by CD3+CD4+ T cells. The study of the expression of the CD107a marker showed a significant stimulating effect of 10 µm of citarabine on the activation of subpopulations of T-lymphocytes (CD3+) and cytotoxic T-lymphocytes (CD3+CD8+).

Carbon-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics.

The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood and rely on fluorescent reporters. Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Abs-CD conjugates developed using this strategy were used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) to validate the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.

10.02 Genomic HLA homozygosity is frequent in esophageal adenocarcinoma and related to low immunogenicity

BackgroundClassical human leukocyte antigen (HLA) class I molecules are expressed by most somatic cells and present peptides to cytotoxic T cells. The HLA-genotype of an individual contains up to six different HLA-I molecules and defines the repertoire of peptides that can be presented to cytotoxic T cells. Homozygosity for one or more HLA-loci could translate in a smaller repertoire of tumour neoantigens possibly presented to cytotoxic T cells in an individual and potentially predispose such individuals with a disadvantage to fight a nascent tumour.Material and MethodsHigh-resolution HLA-genotyping from germline normal DNA of 80 esophago-gastric adenocarcinoma (EGA) patients was performed with the NGS method by Illumina. Whole exome sequencing (WES) was performed on tumor tissue and normal peripheral blood cells (n=39). The data were processed, and non-synonymous mutations were called. The amount of potential high-affinity binders derived from 10 cancer testis antigens (CTAs) frequently expressed in EGA and non-synonymous mutations obtained from WES data were determined using an in-silico approach for MHC-binding (IEDB.org). RNA-extraction and gene expression profiling were performed using the NanoString technology.ResultsWe compared the frequency of HLA homozygosity in EGA patients to an HLA-matched reference population derived from a large cohort of bone marrow donors (n=7.615 out of 615.017 donors). We demonstrate that EGA patients are more likely to be homozygous for at least one HLA-I gene than the control population. In EGA patients, 35% of HLA-A, -B, and -C alleles were homozygous in comparison with 19% of HLA alleles among the HLA-matched general population. This difference corresponded to an odds ratio (OR) for homozygosity of 2.282 (95% confidence interval (CI) 1.442-3.615, p<0.001). The odds ratios for homozygosity at HLA-A (OR=1.885, CI=1.111-3.236, p<0.05), HLA-B (OR=3.045, CI=1.346-6.499, p<0.05) and HLA-C (OR=2.170, CI=1.445-3.579, p<0.05) were significantly different. We then aimed to estimate the influence of HLA-homozygosity in the context of tumour immune surveillance. Predictions by IEDB analysis resource tool indeed showed a reduced repertoire of high and moderate-affinity MHC-binders (both CTA-derived and mutation-derived peptides) in the homozygous cohort. Our findings demonstrate a reduced amount of potentially immunogenic peptides in EGA patients with HLA-homozygosity for at least one locus, which may result in impaired cancer immunosurveillance. In line with this observation, we also found increased levels of CTA expression in homozygous compared to heterozygous patients. After artificial modification of the genotype of homozygous patients to a heterozygous genotype, the set of predicted good-binding peptides was comparable to the heterozygous cohort.ConclusionOur results highlight the effect of HLA-I homozygosity on the immunopeptidome as important prerequisite of anti-tumor immunity. The high frequency of genomic HLA-I homozygosity observed in the EGA cohort may reflect an increased cancer risk for these patients. Together with previous reports demonstrating reduced survival after checkpoint therapy, our study suggests consideration of germ-line HLA-homozygosity for the design and interpretation of immunotherapeutic trials.Disclosure InformationM.A. Garcia-Marquez: None. M. Thelen: None. E. Bauer: None. K. Wennhold: None. J. Lehmann: None. D. Keller: None. B. Gathof: None. L. Maas: None. J. George: None. C. Bruns: None. A. Quaas: None. M. von Bergwelt-Baildon: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; Astellas, Roche, MSD. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS. M. Peifer: None. H.A. Schlößer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Astra Zeneca. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS.

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

Molecular Mechanisms Responsible for In Vitro Cytotoxic Attributes of Conyza bonariensis Extract against Lymphoblastic Leukaemia Jurkat Cells

Background: Conyza bonariensis is known to have anti-cancer properties. Objective: The study investigated the in vitro pro-apoptotic properties of Conyza bonariensis (C. bonariensis) towards human lymphoblastic leukemia Jurkat cells. Methods: C. bonariensis are extracted with non-polar solvent by maceration. MTS cell viability assay was employed to determine the cytotoxic activity of the extract towards human leukemia Jurket cells and normal Peripheral Blood Mononuclear Cells (PBMCs) cells. The phytochemical composition of the extract was chemically characterized using HPLC. Flow cytometric studies (FACS) were conducted to explore the pro-apoptotic potential of the extract. Western blot studies were employed to identify the molecular targets involved in the induction of apoptosis. Results: The n-hexane extract showed selective cytotoxic activity towards Jurkat cells. FACS analysis indicated that the extract induced early and late apoptosis in Jurkat cells. Western blot studies revealed that the extract down-regulated the expression of DNMT1, SIRT1, and UHRF1 with a simultaneous up-regulation of the expression of p73 and caspases-3 proteins. HPLC characterization of the extract revealed the presence of phenolic compounds. Conclusion: Overall these findings demonstrate that the anticancer effects of a Conyza bonariensis extract towards human lymphoblastic leukemiais due to the modulation of the activity of multiple oncogenic and tumor suppressor proteins and that its phenolic content is involved are proposed to be responsible for these activities.

BCL11B Upregulates the Expression of NF-κB in T Cells Stimulated with SEA

BackgroundNF-κB is one of the most important inflammatory mediators in the tumour microenvironment promotes inflammation-induced cancer. Many studies report that NF-κB is activated in many kinds of leukaemia, so that some researchers by inhibiting NF-κB to treat leukaemia. The overexpression of BCL11B has been primarily reported in T cell malignancies. Some studies have reported that BCL11B is a potential transcriptional repressor which has several splice isoforms. MiR-21-5p promotes cell proliferation by targeting BCL11B in Thp-1 human monocytes. Both NF-κB and BCL11B transcription factors are related to inflammation and leukaemia. Whether there is correlation between NF-κB and BCL11B transcription factors? If BCL11B is a potential important transcription factor in treatment of T lymphocyte leukaemia? In this study, we stimulated Jurkat cells and normal peripheral blood T cells with staphylococcal enterotoxin A(SEA), and we detected the expression of both the BCL11B and NF-κB genes and their respective proteins at different stimulation times. Then, the BCL11B gene was suppressed by BCL11B-siRNA and detected at another stimulation time. ResultsWe found that the mRNA expression of BCL11B and NF-κB increased in two kinds of T cell lines over time which stimulated by SEA or PMA+IO. A similar result was confirmed for BCL11B and NF-κB protein expression. While the expression of the NF-κB protein did not increase on equal conditions in the BCL11B-knockdown group. ConclusionOur result suggested that the gene and protein expression levels of both BCL11B and NF-κB were related, and BCL11B regulates NF-κB expression in Jukat cells and healthy human peripheral blood through the TCR signalling pathway. This study reveals that BCL11B can be used as a new therapeutic target for chronic inflammation and T cell leukaemia pathogenesis.

Raabin-WBC: a large free access dataset of white blood cells from normal peripheral blood

Accurate and early detection of peripheral white blood cell anomalies plays a crucial role in the evaluation of an individual's well-being. The emergence of new technologies such as artificial intelligence can be very effective in achieving this. In this regard, most of the state-of-the-art methods use deep neural networks. Data can significantly influence the performance and generalization power of machine learning approaches, especially deep neural networks. To that end, we collected a large free available dataset of white blood cells from normal peripheral blood samples called Raabin-WBC. Our dataset contains about 40000 white blood cells and artifacts (color spots). To reassure correct data, a significant number of cells were labeled by two experts, and the ground truth of nucleus and cytoplasm were extracted by experts for some cells (about 1145), as well. To provide the necessary diversity, various smears have been imaged. Hence, two different cameras and two different microscopes were used. The Raabin-WBC dataset can be used for different machine learning tasks such as classification, detection, segmentation, and localization. We also did some primary deep learning experiments on Raabin-WBC, and we showed how the generalization power of machine learning methods, especially deep neural networks, was affected by the mentioned diversity.

Unexplained Fatigue in an Otherwise Healthy Man Linked to Kikuchi-Fujimoto Disease, A Case Report

Kikuchi histiocytic necrotizing lymphadenitis is a benign and self-limited illness usually characterized by cervical lymphadenopathy and fever. We present a case of a 42-year male who complained of extreme fatigue for 2 weeks. On laboratory workup, he had leucopenia and thrombocytopenia with normal peripheral blood and bone marrow examination. The radiological investigation revealed multiple enlarged lymph nodes in the left axilla and left supraclavicular region. The subsequent excisional biopsy of the axillary node clinched the diagnosis of Kikuchi- Fujimoto disease. The patient was completely recovered and laboratory parameters were normal with supportive treatment. Kikuchi- Fujimoto disease should be considered in patients with unexplained fatigue with lymphadenopathy and early biopsy prevents unnecessary investigations as well as potentially harmful treatments.

Raabin-WBC: a large free access dataset of white blood cells from normal peripheral blood

Accurate and early detection of peripheral white blood cell anomalies plays a crucial role in the evaluation of an individual's well-being. The emergence of new technologies such as artificial intelligence can be very effective in achieving this. In this regard, most of the state-of-the-art methods use deep neural networks. Data can significantly influence the performance and generalization power of machine learning approaches, especially deep neural networks. To that end, we collected a large free available dataset of white blood cells from normal peripheral blood samples called Raabin-WBC. Our dataset contains about 40000 white blood cells and artifacts (color spots). To reassure correct data, a significant number of cells were labeled by two experts, and the ground truth of nucleus and cytoplasm were extracted by experts for some cells (about 1145), as well. To provide the necessary diversity, various smears have been imaged. Hence, two different cameras and two different microscopes were used. The Raabin-WBC dataset can be used for different machine learning tasks such as classification, detection, segmentation, and localization. We also did some primary deep learning experiments on Raabin-WBC, and we showed how the generalization power of machine learning methods, especially deep neural networks, was affected by the mentioned diversity.