Sphingolipids in Childhood Asthma and Obesity (SOAP Study): A Protocol of a Cross-Sectional Study.

Asthma and obesity are two of the most common chronic conditions in children and adolescents. There is increasing evidence that sphingolipid metabolism is altered in childhood asthma and is linked to airway hyperreactivity. Dysregulated sphingolipid metabolism is also reported in obesity. However, the functional link between sphingolipid metabolism, asthma, and obesity is not completely understood. This paper describes the protocol of an ongoing study on sphingolipids that aims to examine the pathophysiology of sphingolipids in childhood asthma and obesity. In addition, this study aims to explore the novel biomarkers through a comprehensive multi-omics approach including genomics, genome-wide DNA methylation, RNA-Seq, microRNA (miRNA) profiling, lipidomics, metabolomics, and cytokine profiling. This is a cross-sectional study aiming to recruit 440 children from different groups: children with asthma and normal weight (n = 100), asthma with overweight or obesity (n = 100), overweight or obesity (n = 100), normal weight (n = 70), and siblings of asthmatic children with normal weight, overweight, or obesity (n = 70). These participants will be recruited from the pediatric pulmonology, pediatric endocrinology, and general pediatric outpatient clinics at Sidra Medicine, Doha, Qatar. Information will be obtained from self-reported questionnaires on asthma, quality of life, food frequency (FFQ), and a 3-day food diary that are completed by the children and their parents. Clinical measurements will include anthropometry, blood pressure, biochemistry, bioelectrical impedance, and pulmonary function tests. Blood samples will be obtained for sphingolipid analysis, serine palmitoyltransferase (SPT) assay, whole-genome sequencing (WGS), genome-wide DNA methylation study, RNA-Seq, miRNA profiling, metabolomics, lipidomics, and cytokine analysis. Group comparisons of continuous outcome variables will be carried out by a one-way analysis of variance or the Kruskal-Wallis test using an appropriate pairwise multiple comparison test. The chi-squared test or a Fisher's exact test will be used to test the associations between categorical variables. Finally, multivariate analysis will be carried out to integrate the clinical data with multi-omics data. This study will help us to understand the role of dysregulated sphingolipid metabolism in obesity and asthma. In addition, the multi-omics data from the study will help to identify novel genetic and epigenetic signatures, inflammatory markers, and mechanistic pathways that link asthma and obesity in children. Furthermore, the integration of clinical and multi-omics data will help us to uncover the potential interactions between these diseases and to offer a new paradigm for the treatment of pediatric obesity-associated asthma.

Chimeric antigen receptor T-cells targeting IL-1RAP: a promising new cellular immunotherapy to treat acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are the basis of refractory/relapsed (R/R) disease in 80% of patients with AML not receiving allogeneic transplantation. METHODS: In this study, we showed that the interleukin-1 receptor accessory protein (IL-1RAP) protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared with the most common potential AML targets, since it is not expressed by the normal hematopoietic stem cell. After establishing the proof of concept for the efficacy of chimeric antigen receptor (CAR) T-cells targeting IL-1RAP in chronic myeloid leukemia, we hypothesized that third-generation IL-1RAP CAR T-cells could eliminate AML LSCs, where the medical need is not covered. RESULTS: We first demonstrated that IL-1RAP CAR T-cells can be produced from AML T-cells at the time of diagnosis and at relapse. In vitro and in vivo, we showed the effectiveness of IL-1RAP CAR T-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous IL-1RAP CAR T-cells against primary cells from patients with AML at diagnosis or at relapse. In patient-derived relapsed AML xenograft models, we confirmed that IL-1RAP CAR T-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen, are cytotoxic against primary AML cells and increased overall survival. CONCLUSION: In conclusion, our preclinical results suggest that IL-1RAP CAR T-based adoptive therapy could be a promising strategy in AML treatment and it warrants the clinical investigation of this CAR T-cell therapy.

Apis mellifera syriaca Venom: Evaluation of Its Anticoagulant Effect, Proteolytic Activity, and Cytotoxicity along with Its Two Main Compounds-MEL and PLA2-On HeLa Cancer Cells.

Bee venom (BV) is one of the most remarkable natural products that has been a subject of studies since ancient times. Recent studies have shown that Apis mellifera syriaca venom possesses antibacterial as well as cytotoxic effects on cancer cell lines. The venom contains a variety of bioactive molecules-mainly melittin (MEL) and phospholipase A2 (PLA2), as well as other compounds that are not well characterized. In this work, we continue the biological characterization of A. mellifera syriaca venom by testing its anticoagulant effect on human plasma using the prothrombin time (PT) test, as well as assessing its proteolytic activity. In addition, the cytotoxicity of the crude venom-and of its two main components, MEL and PLA2-was tested on HeLa cancer cell lines for the first time. The results obtained showed the capacity of A. mellifera syriaca venom to increase clotting time, thereby proving its anticoagulant effect. Moreover, the venom did not demonstrate a significant proteolytic activity unless administrated at concentrations ≥ 5 mg/mL. Finally, we showed that crude A. mellifera syriaca venom, along with MEL, exhibit a strong in vitro cytotoxic effect on HeLa cancer cell lines, even at low concentrations. In summary, our findings could serve as a basis for the development of new natural-based drug candidates in the therapeutic field.

Overcoming target epitope masking resistance that can occur on low-antigen-expresser AML blasts after IL-1RAP chimeric antigen receptor T cell therapy using the inducible caspase 9 suicide gene safety switch.

Although chimeric antigen receptor CAR) T cell immunotherapies are an undeniable and unequivocal success, knowledge obtained from the monitoring of the first clinical trials targeting the CD19 antigen in B malignancies, either refractory/relapsed acute lymphoid leukemia (ALL) or lymphomas, contributed to the identification of tumor cell escape in about 30-50% of B-ALL. Resistance occurred due to loss of surface expression of the antigen (rCD19-) or to the early disappearance or inactivation of CAR T cells (rCD19+). In a recently reported clinical case, rCD19- relapse resulted from masking of the antigen by the CAR at the surface of B-ALL leukemia cells following the unexpected viral transduction of a leukemic cell present in the cytapheresis sample. The objective of this work was to reproduce this epitope-masking resistance model, in the context of acute myeloid leukemia (AML), based on our immunotherapeutic CAR T cell model targeting the accessory protein of the interleukin-1 receptor (IL-1RAP) expressed by leukemic stem cells. As AML primary blasts express different levels of IL-1RAP, we modeled transduction of different AML tumor cell lines screened for density of antigenic sites with our lentiviral vectors carrying a third-generation IL-1RAP CAR, an iCASP9 suicide gene, and a truncated CD19 surface gene. We demonstrated that primary AML blasts can be easily transduced (74.55 ± 21.29%, n = 4) and that CAR T cytotoxicity to IL-1RAP is inversely correlated with epitope masking in relation to the number of antigenic sites expressed on the surface of IL-1RAP+ lines. Importantly, we showed that, in vitro, a 24-h exposure of IL-1RAP+/CAR+ leukemia lines to Rimiducid eliminated >85% of the cells. We confirmed that the expression of IL-1RAP CAR by an IL-1RAP+ leukemic cell, by decreasing the membrane availability of the targeted antigen, can induce resistance while a high epitope density maintains sensitivity to CAR T cells. Moreover, the presence of the iCASP9/Rimiducid suicide system safety switch makes this immunotherapy approach safe for application in a future phase 1 clinical trial.

The Cytotoxic and Apoptotic Effects of the Brown Algae Colpomenia sinuosa are Mediated by the Generation of Reactive Oxygen Species.

Brown algae are a novel resource of biogenic molecules, however few studies have been conducted in the Mediterranean to assess the cytotoxic mechanisms of algal-derived compounds. This study focuses on the antineoplastic activity of extracts from non-investigated algae of the Lebanese coast, Colpomenia sinuosa. Extracts' antineoplastic activities were evaluated by MTT and trypan blue on different tumorigenic cells. Results indicated that the most potent extract was obtained by soxhlet using dichloromethane:methanol solvent (DM soxhlet) against HCT-116. Wound healing assay confirmed that this extract decreased the migration potential of HCT-116 cells with minimal effects on non-tumorigenic cells. It also induced an increase in the subG1 population as determined by flow cytometry. Western blot analysis demonstrated that apoptosis in treated HCT-116 cells was induced via upregulation of p21 protein and downregulation of the anti-apoptotic Bcl 2, which led to caspases activation. The latter, catalyzes the degradation of PARP-1, and thus suppresses cancer proliferation. Morphological alterations, further confirmed apoptosis. A strong pro-oxidant activity evidenced by the enhanced generation of reactive oxygen species (ROS) was observed in HCT-116 treated cells. Interestingly, a strong antioxidant effectively blocked effect induced by the extract. These results indicate that C. sinuosa is a source of bioactive compounds possessing pro-apoptotic and anti-migratory efficacy.

First Characterization of The Venom from Apis mellifera syriaca, A Honeybee from The Middle East Region.

Bee venom is a mixture of several components with proven therapeutic benefits, among which are anti-inflammatory, analgesic, and various cardiovascular conditions. In this work, we analyzed for the first time the proteomic content and biological properties of the crude venom from Apis mellifera syriaca, a honeybee from the Middle East region. Using high-performance liquid chromatography-tandem mass spectrometry, we evidence the venom contains phospholipase A2, hyaluronidase, mast cell-degranulating peptide, adolapin, apamin, and melittin. The latter was purified by solid phase extraction method (SPE) and tested in parallel with crude venom for biological activities. Precisely, crude venom-but not melittin-exhibited antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa strains. Alongside, hemolytic activity was observed in human blood subjected to the venom at high doses. A. mellifera syriaca venom displayed antioxidant activities, and not surprisingly, PLA2 catalytic activity. Eventually, the venom proved to exert antiproliferative effects against MCF-7 and 3T3 cancer cells lines. This first report of a new bee venom opens new avenues for therapeutic uses of bee venoms.

Flexible and enantioselective access to jaspine B and biologically active chain-modified analogues thereof.

Whereas the all-cis tetrahydrofuran framework of the cytotoxic anhydrophytosphingosine jaspine B is considered as a relevant pharmacophore, little is known about the influence of the aliphatic chain of this amphiphilic molecule on its activity. We developed a synthetic strategy allowing flexible introduction of various lipophilic fragments in the jaspine's skeleton. The route was validated with two distinct approaches to jaspine B. Five chain-modified analogues were also prepared. Biological evaluation of these derivatives demonstrated a good correlation between their cytotoxicity and their capacity to inhibit conversion of ceramide into sphingomyelin in melanoma cells. A series of potent and selective inhibitors of sphingomyelin production was thus identified. Furthermore, the good overall potency of an omega-aminated analogue allowed us to dissociate of the pharmacological action of jaspine B from its amphiphilic nature.

The natural marine anhydrophytosphingosine, Jaspine B, induces apoptosis in melanoma cells by interfering with ceramide metabolism.

Marine environment has frequently afforded a variety of biologically active compounds with strong anticancer and cytotoxic properties. In the present study, the mechanism of action of Jaspine B, an anhydrophytosphingosine derivative isolated from the marine sponge Jaspis sp., was investigated. Jaspine B was able to dose- and time-dependently decrease the viability of murine B16 and human SK-Mel28 melanoma cells. On these cells, Jaspine B treatment triggered cell death by typical apoptosis as illustrated by phosphatidylserine externalization, the release of cytochrome c and caspase processing. These effects were associated with increased intracellular ceramide levels owing to perturbed ceramide metabolism. Indeed, Jaspine B exposure strongly inhibited the activity of sphingomyelin synthase (SMS), an enzyme that converts de novo ceramide into the membrane lipid sphingomyelin. Moreover, whereas Jaspine B-induced cell death was enhanced in SMS1-depleted cells, it was strongly inhibited in cells that stably overexpress human SMS1. Finally, the cytotoxic effects of Jaspine B truncated analogs were also shown to be dependent on SMS activity. Altogether, Jaspine B is able to kill melanoma cells by acting on SMS activity and consequently on ceramide formation, and may represent a new class of cytotoxic compounds with potential applications in anticancer melanoma therapy.

Functions of sphingolipid metabolism in mammals--lessons from genetic defects.

Much is known about the pathways that control the biosynthesis, transport and degradation of sphingolipids. During the last two decades, considerable progress has been made regarding the roles this complex group of lipids play in maintaining membrane integrity and modulating responses to numerous signals. Further novel insights have been provided by the analysis of newly discovered genetic diseases in humans as well as in animal models harboring mutations in the genes whose products control sphingolipid metabolism and action. Through the description of the phenotypic consequences of genetic defects resulting in the loss of activity of the many proteins that synthesize, transport, bind, or degrade sphingolipids, this review summarizes the (patho)physiological functions of these lipids.

Synthesis and biological evaluation of conformationally constrained analogues of the antitubercular agent ethambutol.

Three (S)-prolinol-derived conformationally restricted analogues of the antitubercular agent ethambutol were prepared and tested against Mycobacterium tuberculosis.