Brazilian Green Propolis: Effects In Vitro and In Vivo on Trypanosoma cruzi

The composition of a Brazilian green propolis ethanolic extract (Et-Bra) and its effect on Trypanosoma cruzi trypomastigotes and other pathogenic microorganisms have already been reported. Here, we further investigated Et-Bra targets in T. cruzi and its effect on experimental infection of mice. The IC50/4 days for inhibition of amastigote proliferation was 8.5 ± 1.8 μg mL−1, with no damage to the host cells. In epimastigotes Et-Bra induced alterations in reservosomes, Golgi complex and mitochondrion. These effects were confirmed by flow cytometry analysis. In trypomastigotes, Et-Bra led to the loss of plasma membrane integrity. The in vitro studies indicate that Et-Bra interferes in the functionality of the plasma membrane in trypomastigotes and of reservosomes and mitochondrion in epimastigotes. Acutely infected mice were treated orally with Et-Bra and the parasitemia, mortality and GPT, GOT, CK and urea levels were monitored. The extract (25–300 mg kg−1 body weight/day for 10 days) reduced the parasitemia, although not at significant levels; increased the survival of the animals and did not induce any hepatic, muscular lesion or renal toxicity. Since Et-Bra was not toxic to the animals, it could be assayed in combination with other drugs. Et-Bra could be a potential metacyclogenesis blocker, considering its effect on reservosomes, which are an important energy source during parasite differentiation

KITD816V mutation in blood for the diagnostic screening of systemic mastocytosis and mast cell activation syndromes

[Background]: Current diagnostic algorithms for systemic mastocytosis (SM) rely on the detection of KITD816V in blood to trigger subsequent bone marrow (BM) investigations. [Methods]: Here, we correlated the KITD816V mutational status of paired blood and BM samples from 368 adults diagnosed with mast cell activation syndrome (MCAS) and mastocytosis and determined the potential utility of investigating KITD816V in genomic DNA from blood-purified myeloid cell populations to increase diagnostic sensitivity. In a subset of 69 patients, we further evaluated the kinetics of the KITD816V cell burden during follow-up and its association with disease outcome. [Results]: Our results showed a high correlation (P < .0001) between the KITD816V mutation burden in blood and BM (74% concordant samples), but with a lower mean of KITD816V-mutated cells in blood (P = .0004) and a high rate of discordant BM+/blood− samples particularly among clonal MCAS (73%) and BM mastocytosis (51%), but also in cutaneous mastocytosis (9%), indolent SM (15%), and well-differentiated variants of indolent SM (7%). Purification of different compartments of blood-derived myeloid cells was done in 28 patients who were BM mast cell (MC)+/blood− for KITD816V, revealing KITD816V-mutated eosinophils (56%), basophils (25%), neutrophils (29%), and/or monocytes (31%) in most (61%) patients. Prognostically, the presence of ≥3.5% KITD816V-mutated cells (P < .0001) and an unstable KITD816V mutation cell burden (P < .0001) in blood and/or BM were both associated with a significantly shortened progression-free survival (PFS). [Conclusions]: These results confirm the high specificity but limited sensitivity of KITD816V analysis in whole blood for the diagnostic screening of SM and other primary MCAS, which might be overcome by assessing the mutation in blood-purified myeloid cell populations.This work was supported by grants from the Fundación Española de Mastocitosis (Madrid, Spain; grant number: FEM2021-SAM) and Blueprint Medicines Corporation (Cambridge, MA). PNN was supported by a grant of Government of Castilla y León (Orden EDU 875 2021), Spain; co-financed with the European Social Fund (BDNS (Identif.): 540787). We also thank the Agencia Estatal de Investigación (AEI) and European Regional Development Fund (FEDER) for the grant (EQC2019-005419-P) within the Subprograma Estatal de Infraestructuras de Investigación y Equipamiento Científico Técnico de 2019

Frequency and prognostic impact of blood-circulating tumor mast cells in mastocytosis

Circulating tumor mast cells (CTMCs) have been identified in the blood of a small number of patients with advanced systemic mastocytosis (SM). However, data are limited about their frequency and prognostic impact in patients with MC activation syndrome (MCAS), cutaneous mastocytosis (CM) and nonadvanced SM. We investigated the presence of CTMCs and MC-committed CD34+ precursors in the blood of 214 patients with MCAS, CM, or SM using highly sensitive next-generation flow cytometry. CTMCs were detected at progressively lower counts in almost all patients with advanced SM (96%) and smoldering SM (SSM; 100%), nearly half of the patients (45%) with indolent SM (ISM), and a few patients (7%) with bone marrow (BM) mastocytosis but were systematically absent in patients with CM and MCAS (P < .0001). In contrast to CTMC counts, the number of MC-committed CD34+ precursors progressively decreased from MCAS, CM, and BM mastocytosis to ISM, SSM, and advanced SM (P < .0001). Clinically, the presence (and number) of CTMCs in blood of patients with SM in general and nonadvanced SM (ISM and BM mastocytosis) in particular was associated with more adverse features of the disease, poorer-risk prognostic subgroups as defined by the International Prognostic Scoring System for advanced SM (P < .0001) and the Global Prognostic Score for mastocytosis (P < .0001), and a significantly shortened progression-free survival (P < .0001) and overall survival (P = .01). On the basis of our results, CTMCs emerge as a novel candidate biomarker of disseminated disease in SM that is strongly associated with advanced SM and poorer prognosis in patients with ISM

Altered innate immune profile in blood of systemic mastocytosis patients

[Background]: Mast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM. [Methods]: Overall, we studied 115 SM patients - 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)- and 32 healthy donors (HD). Spontaneous and in vitro-stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated. [Results]: SM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1β, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ-stimulated blood monocytes to produce IL1β and TGFβ. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non-classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL+ DC was specifically encountered in BMM cases. [Conclusion]: These results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.This study has been funded by Instituto de Salud Carlos III (ISCIII) (grant number PI19/01166; and Centro de Investigación Biomédica en Red de Cáncer [CIBERONC] programme, grant number CB16/12/00400) and co-funded by the European Union (EU). We thank the support of the Spanish National DNA Bank Carlos III (www.bancoadn.org; biobank ID B.0000716; supported by ISCIII and co-founded by EU [grant number PT20/00085]) for providing plasma samples. APP was supported by a grant of the Government of Castilla y León (Orden EDU/556/2019), Spain; co-financed with the “European Regional Development Fund” (BDNS, Identif.:422058). We thank the support of the Spanish Association of Mastocytosis and Related Diseases

Proposed global prognostic score for systemic mastocytosis: a retrospective prognostic modelling study

[Background]: Several risk stratification models have been proposed in recent years for systemic mastocytosis but have not been directly compared. Here we designed and validated a risk stratification model for progression-free survival (PFS) and overall survival (OS) in systemic mastocytosis on the basis of all currently available prognostic factors, and compared its predictive capacity for patient outcome with that of other risk scores.[Methods]: We did a retrospective prognostic modelling study based on patients diagnosed with systemic mastocytosis between March 1, 1983, and Oct 11, 2019. In a discovery cohort of 422 patients from centres of the Spanish Network on Mastocytosis (REMA), we evaluated previously identified, independent prognostic features for prognostic effect on PFS and OS by multivariable analysis, and designed a global prognostic score for mastocytosis (GPSM) aimed at predicting PFS (GPSM-PFS) and OS (GPSM-OS) by including only those variables that showed independent prognostic value (p<0·05). The GPSM scores were validated in an independent cohort of 853 patients from centres in Europe and the USA, and compared with pre-existing risk models in the total patient series (n=1275), with use of Harrells' concordance index (C-index) as a readout of the ability of each model to risk-stratify patients according to survival outcomes.[Findings]: Our GPSM-PFS and GPSM-OS models were based on unique combinations of independent prognostic factors for PFS (platelet count ≤100 × 109 cells per L, serum β2-microglobulin ≥2·5 μg/mL, and serum baseline tryptase ≥125 μg/L) and OS (haemoglobin ≤110 g/L, serum alkaline phosphatase ≥140 IU/L, and at least one mutation in SRSF2, ASXL1, RUNX1, or DNMT3A). The models showed clear discrimination between low-risk and high-risk patients in terms of worse PFS and OS prognoses in the discovery and validation cohorts, and further discrimination of intermediate-risk patients. The GPSM-PFS score was an accurate predictor of PFS in systemic mastocytosis (C-index 0·90 [95% CI 0·87–0·93], vs values ranging from 0·85 to 0·88 for pre-existing models), particularly in non-advanced systemic mastocytosis (C-index 0·85 [0·76–0·92], within the range for pre-existing models of 0·80 to 0·93). Additionally, the GPSM-OS score was able to accurately predict OS in the entire cohort (C-index 0·92 [0·89–0·94], vs 0·67 to 0·90 for pre-existing models), and showed some capacity to predict OS in advanced systemic mastocytosis (C-index 0·72 [0·66–0·78], vs 0·64 to 0·73 for pre-existing models).[Interpretation]: All evaluated risk classifications predicted survival outcomes in systemic mastocytosis. The REMA-PFS and GPSM-PFS models for PFS, and the International Prognostic Scoring System for advanced systemic mastocytosis and GPSM-OS model for OS emerged as the most accurate models, indicating that robust prognostication might be prospectively achieved on the basis of biomarkers that are accessible in diagnostic laboratories worldwide.Carlos III Health Institute, European Regional Development Fund, Spanish Association of Mastocytosis and Related Diseases, Rare Diseases Strategy of the Spanish National Health System, Junta of Castile and León, Charles and Ann Johnson Foundation, Stanford Cancer Institute Innovation Fund, Austrian Science Fund

Induced Pluripotent Stem Cells in Drug Discovery and Neurodegenerative Disease Modelling

Induced pluripotent stem cells (iPSCs) are derived from reprogrammed adult somatic cells. These adult cells are manipulated in vitro to express genes and factors essential for acquiring and maintaining embryonic stem cell (ESC) properties. This technology is widely applied in many fields, and much attention has been given to developing iPSC-based disease models to validate drug discovery platforms and study the pathophysiological molecular processes underlying disease onset. Especially in neurological diseases, there is a great need for iPSC-based technological research, as these cells can be obtained from each patient and carry the individual’s bulk of genetic mutations and unique properties. Moreover, iPSCs can differentiate into multiple cell types. These are essential characteristics, since the study of neurological diseases is affected by the limited access to injury sites, the need for in vitro models composed of various cell types, the complexity of reproducing the brain’s anatomy, the challenges of postmortem cell culture, and ethical issues. Neurodegenerative diseases strongly impact global health due to their high incidence, symptom severity, and lack of effective therapies. Recently, analyses using disease specific, iPSC-based models confirmed the efficacy of these models for testing multiple drugs. This review summarizes the advances in iPSC technology used in disease modelling and drug testing, with a primary focus on neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases

Multiparametric Color Tendency Analysis (MCTA): A Method to Analyze Several Flow Cytometry Labelings Simultaneously.

Despite the remarkable evolution of flow cytometers, fluorescent probes, and flow cytometry analysis software, most users still follow the same ways for data analysis. Conventional flow cytometry analysis relies on the creation of dot plot sequences, based on two fluorescence parameters at a time, to evidence phenotypically distinct populations. Thus, reaching conclusions about the biological characteristics of the samples is a fragmented and challenging process. We present here the MCTA (Multiparametric Color Tendency Analysis), a method for data analysis that considers multiple labelings simultaneously, extending and complementing conventional analysis. The MCTA method executes the background fluorescence exclusion, spillover compensation, and a user-defined gating strategy for subpopulation analysis. The results are then presented in conventional FSC x SSC dot plots with statistical data. For each event, the method converts each of the multiple fluorescence colors under analysis into a vector, with longer vectors being attributed to more intense labelings. Then, the MCTA generates a resultant vector, which is therefore mostly influenced by predominant labelings. The radial position of this resultant vector corresponds to a resultant color, making it easy to visualize phenotypic modulations among cellular subpopulations. Besides, it is a deterministic method that quickly assigns a resulting color to all events that obey the gating strategy, with no polymeric regions defined by the user or downsampling. The MCTA application generates a single dot plot showing all events in the FCS file, but a resultant color is attributed only to those that obey the gating strategy. Therefore, it can also help to evidence rare events or unpredicted subpopulations naturally excluded from the regions defined by the user. We believe that the MCTA method adds a new perspective over multiparametric flow cytometry analysis while evidencing modulations of molecular labeling profiles based on multiple fluorescences. Availability and implementation: The instructions for the MCTA application is freely available at https://github.com/flowcytometry/MCTA