Sodium diethyldithiocarbamate trihydrate: an effective and selective compound for hematological malignancies.

Myeloid leukemias and lymphomas are among the most common and well-studied hematological malignancies. However, due to the aggressiveness and rapid progression of certain subtypes, treating these diseases remains a challenge. Considering the promising results of diethyldithiocarbamates in preclinical and clinical oncology trials, this study aimed to investigate the potential of sodium diethyldithiocarbamate trihydrate (DETC) as a prototype for developing new drugs to treat hematological malignancies. In silico analysis using SwissADME was conducted to evaluate the physicochemical characteristics and pharmacokinetic properties of DETC. An in vitro investigation utilizing the MTT assay assessed the cytotoxic effects of DETC on neoplastic and non-neoplastic cell lines. Selectivity was determined using a selectivity index and a hemolysis assay, while the mechanism of cell death in neoplastic cell lines was examined through flow cytometry analysis of pro-apoptotic and anti-apoptotic protein levels. The results demonstrated that the physicochemical characteristics of DETC are suitable for oral administration. Furthermore, the compound showed promising cytotoxic activity against human myeloid leukemia (K562) and Burkitt's lymphoma (Daudi) cell lines, with high selectivity for neoplastic cells over non-neoplastic cells of the bone marrow microenvironment (HS-5 cell line). Moreover, hemolysis was observed only at very high concentrations. The cytotoxicity mechanism of DETC against both neoplastic cell lines involved cell cycle arrest and the production of reactive oxygen species. In K562 cells, cell death was induced via apoptosis. Additional experiments are needed to confirm the exact mechanism of cell death in Daudi Burkitt's lymphoma cells.

Lymphocyte B Subtypes in Peripheral Blood: A Prognostic Biomarker for COVID-19 Patients.

BACKGROUND: In view of the scientific gap in knowledge of the involvement of the B-cell compartment and clinical prognostic in SARS-CoV-2 infection, this work aims to evaluate the B-cell subsets and the presence of specific IgM and IgG, as well as neutralizing antibodies against SARS-CoV-2, in unvaccinated patients diagnosed with COVID-19. METHODS: This study included 133 patients with COVID-19. Cellular components were assessed by flow cytometry, and immunoglobulin levels and reactivity were measured by indirect enzyme-linked immunosorbent assay. RESULTS: Our results showed no changes in less differentiated B cells. However, non-switched memory B cells (NS-MBCs) and class-switched memory B cells (CS-MBCs) were reduced in the patients with moderate disease. Also, plasmablasts and double-negative (DN) or "atypical" memory B cells were increased in groups of patients with moderate to critical conditions. In addition, the production of IgM, IgG, and neutralizing antibodies against SARS-CoV-2 demonstrated a positive correlation between the positivity of antibodies against SARS-CoV-2 and disease severity. Besides being related to the development of a more severe course of the disease, the increase in DN B-cell count also contributed to a poorer disease outcome in patients with a higher percentage of these cells. On the other hand, we observed an increase in the absolute number of CS-MBCs in patients with greater chances of survival. CONCLUSIONS: This study demonstrates that the B-cell compartment may contribute to the development of clinical symptoms of COVID-19, with changes in B-cell subset counts linked to disease course and patient prognosis.

T cell maturation is significantly affected by SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An adequate T cell response is essential not only for fighting disease but also for the creation of immune memory. Thus, the present study aims to evaluate the T cells of patients with moderate, severe and critical COVID-19 not only at the time of illness but also 2 months after diagnosis to observe whether changes in this compartment persist. In this study, 166 COVID-19 patients were stratified into moderate/severe and critical disease categories. The maturation and activation of T cells were evaluated through flow cytometry. In addition, Treg cells were analysed. Until 15 days after diagnosis, patients presented a reduction in absolute and relative T lymphocyte counts. After 2 months, in moderate/severe patients, the counts returned to a similar level as that of the control group. In convalescent patients who had a critical illness, absolute T lymphocyte values increased considerably. Patients with active disease did not show differentiation of T cells. Nonetheless, after 2 months, patients with critical COVID-19 showed a significant increase in CD4+ EMRA (CD45RA+ effector memory) T lymphocytes. Furthermore, COVID-19 patients showed delayed T cell activation and reduced CD8+ suppressor T cells even 2 months after diagnosis. A reduction in CD4+ Treg cells was also observed, and their numbers returned to a similar level as that of healthy controls in convalescent patients. The results demonstrate that COVID-19 patients have a delayed activation and differentiation of T cells. In addition, these patients have a great reduction of T cells with a suppressor phenotype.

Omics-based identification of an NRF2-related auranofin resistance signature in cancer: Insights into drug repurposing.

Auranofin is a thioredoxin reductase-1 inhibitor originally approved for the treatment of rheumatoid arthritis. Recently, auranofin has been repurposed as an anticancer drug, with pharmacological activity reported in multiple cancer types. In this study, we characterized transcriptional and genetic alterations associated with auranofin response in cancer. By integrating data from an auranofin cytotoxicity screen with transcriptome profiling of lung cancer cell lines, we identified an auranofin resistance signature comprising 29 genes, most of which are classical targets of the transcription factor NRF2, such as genes involved in glutathione metabolism (GCLC, GSR, SLC7A11) and thioredoxin system (TXN, TXNRD1). Pan-cancer analysis revealed that mutations in NRF2 pathway genes, namely KEAP1 and NFE2L2, are strongly associated with overexpression of the auranofin resistance gene set. By clustering cancer types based on auranofin resistance signature expression, hepatocellular carcinoma, and a subset of non-small cell lung cancer, head-neck squamous cell carcinoma, and esophageal cancer carrying NFE2L2/KEAP1 mutations were predicted resistant, whereas leukemia, lymphoma, and multiple myeloma were predicted sensitive to auranofin. Cell viability assays in a panel of 20 cancer cell lines confirmed the augmented sensitivity of hematological cancers to auranofin; an effect associated with dependence upon glutathione and decreased expression of NRF2 target genes involved in GSH synthesis and recycling (GCLC, GCLM and GSR) in these cancer types. In summary, the omics-based identification of sensitive/resistant cancers and genetic alterations associated with these phenotypes may guide an appropriate repurposing of auranofin in cancer therapy.

Perinatal exposure to a glyphosate pesticide formulation induces offspring liver damage.

The present study investigated the effects of perinatal exposure to glyphosate-based herbicide (GBH) in offspring's liver. Pregnant Wistar rats were exposed to GBH (70 mg glyphosate/Kg body weight/day) in drinking water from gestation day 5 to postnatal day 15. The perinatal exposure to GBH increased 45Ca2+ influx in offspring's liver. Pharmacological tools indicated a role played by oxidative stress, phospholipase C (PLC) and Akt pathways, as well as voltage-dependent Ca2+ channel modulation on GBH-induced Ca2+ influx in offspring's liver. In addition, changes in the enzymatic antioxidant defense system, decreased GSH content, lipid peroxidation and protein carbonylation suggest a connection between GBH-induced hepatotoxic mechanism and redox imbalance. The perinatal exposure to GBH also increased the enzymatic activities of transaminases and gamma-glutamyl transferase in offspring's liver and blood, suggesting a pesticide-induced liver injury. Moreover, we detected increased iron levels in liver, blood and bone marrow of GBH-exposed rats, which were accompanied by increased transferrin saturation and decreased transferrin levels in blood. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in the liver of rats perinatally exposed to GBH, which were associated with. Increased phospho-p65NFκB immunocontent. Therefore, we propose that excessive amounts of iron in offspring's liver, blood and bone marrow induced by perinatal exposure to GBH may account for iron-driven hepatotoxicity, which was associated with Ca2+ influx, oxidative damage and inflammation. Further studies will clarify whether these events can ultimately impact on liver function.

Involvement of the NF-κB and PI3K/Akt/mTOR pathways in cell death triggered by stypoldione, an o-quinone isolated from the brown algae Stypopodium zonale.

Multiple myeloma (MM) is a clonal plasma cell malignancy that remains incurable to date. Thus, the aims of this study were to evaluate the involvement of the NF-κB and PI3K/Akt/mTOR pathways in the cytotoxicity of stypoldione, an o-quinone isolated from the brown algae Stypopodium zonale, in MM cells (MM1.S). The cytotoxic effect was evaluated in MM1.S cells and peripheral blood mononuclear cells (PBMCs) by MTT assay. The stypoldione reduced the cell viability of MM1.S cells in a concentration and time-dependent manner (IC50 in MM.1S from 2.55 to 5.38 µM). However, it was also cytotoxic to PBMCs, but at a lower range. Additionally, no significant hemolysis was observed even at concentration up to 10 times the IC50 . Apoptotic cell death was confirmed by cell morphology and Annexin V-FITC assay. Stypoldione induced intrinsic and extrinsic apoptosis by increasing FasR expression and reactive oxygen species (ROS) production, inverting the Bax/Bcl-2 ratio, and inducing ΔΨm loss, which resulted in AIF release and caspase-3 activation. It also increased Ki-67 and survivin expression and inhibited the NF-κB and PI3K/Akt/mTOR pathways. These results suggest that stypoldione is a good candidate for the development of new drugs for MM treatment.

The relationship between peripheral immune response and disease severity in SARS-CoV-2-infected subjects: A cross-sectional study.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and marked by an intense inflammatory response and immune dysregulation in the most severe cases. In order to better clarify the relationship between peripheral immune system changes and the severity of COVID-19, this study aimed to evaluate the frequencies and absolute numbers of peripheral subsets of neutrophils, monocytes, and dendritic cells (DCs), in addition to quantifying the levels of inflammatory mediators. One hundred fifty-seven COVID-19 patients were stratified into mild, moderate, severe, and critical disease categories. The cellular components and circulating cytokines were assessed by flow cytometry. Nitric oxide (NOx) and myeloperoxidase (MPO) levels were measured by colourimetric tests. COVID-19 patients presented neutrophilia, with signs of emergency myelopoiesis. Alterations in the monocytic component were observed in patients with moderate to critical illness, with an increase in classical monocytes and a reduction in nonclassical monocytes, in addition to a reduction in the expression of HLA-DR in all subtypes of monocytes, indicating immunosuppression. DCs, especially plasmacytoid DCs, also showed a large reduction in moderate to critical patients. COVID-19 patients showed an increase in MPO, interleukin (IL)-12, IL-6, IL-10, and IL-8, accompanied by a reduction in IL-17A and NOx. IL-10 levels ≥14 pg/ml were strongly related to the worst outcome, with a sensitivity of 78·3% and a specificity of 79·1%. The results of this study indicate the presence of systemic effects induced by COVID-19, which appear to be related to the pathophysiology of the disease, highlighting the potential of IL-10 as a possible prognostic biomarker for COVID-19.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels

ABSTRACT Introduction: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eightto ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. Objective and Method: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. Results: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. Conclusion: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

Synthesis of chalcones derived from 1-naphthylacetophenone and evaluation of their cytotoxic and apoptotic effects in acute leukemia cell lines.

Chalcones and their derivatives have been described as promising compounds with antiproliferative activity against leukemic cells. This study aimed to investigate the cytotoxic effect of three synthetic chalcones derived from 1-naphthylacetophenone (F07, F09, and F10) in acute leukemia cell lines (K562 and Jurkat) and examine the mechanisms of cell death induced by these compounds. The three compounds were cytotoxic to K562 and Jurkat cells, with IC50 values ranging from 1.03 to 31.66 µM. Chalcones induced intrinsic and extrinsic apoptosis, resulting in activation of caspase-3 and DNA fragmentation. F07, F09, and F10 were not cytotoxic to human peripheral blood mononuclear cells, did not produce any significant hemolytic activity, and did not affect platelet aggregation after ADP stimulation. These results, combined with calculations of molecular properties, suggest that chalcones F07, F09, and F10 are promising molecules for the development of novel antileukemic drugs.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels.

INTRODUCTION: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eight- to ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. OBJECTIVE AND METHOD: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. RESULTS: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. CONCLUSION: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

Patterns of dendritic cell and monocyte subsets are associated with disease severity and mortality in liver cirrhosis patients.

Liver cirrhosis is often complicated by an immunological imbalance known as cirrhosis-associated immune dysfunction. This study aimed to investigate disturbances in circulating monocytes and dendritic cells in patients with acute decompensation (AD) of cirrhosis. The sample included 39 adult cirrhotic patients hospitalized for AD, 29 patients with stable cirrhosis (SC), and 30 healthy controls (CTR). Flow cytometry was used to analyze monocyte and dendritic cell subsets in whole blood and quantify cytokines in plasma samples. Cirrhotic groups showed higher frequencies of intermediate monocytes (iMo) than CTR. AD patients had lower percentages of nonclassical monocytes than CTR and SC. Cirrhotic patients had a profound reduction in absolute and relative dendritic cell numbers compared with CTR and showed higher plasmacytoid/classical dendritic cell ratios. Increased plasma levels of IL-6, IL-10, and IL-17A, elevated percentages of CD62L+ monocytes, and reduced HLA-DR expression on classical monocytes (cMo) were also observed in cirrhotic patients. Patients with more advanced liver disease showed increased cMo and reduced tissue macrophages (TiMas) frequencies. It was found that cMo percentages greater than 90.0% within the monocyte compartment and iMo and TiMas percentages lower than 5.7% and 8.6%, respectively, were associated with increased 90-day mortality. Monocytes and dendritic cells are deeply altered in cirrhotic patients, and subset profiles differ between stable and advanced liver disease. High cMo and low TiMas frequencies may be useful biomarkers of disease severity and mortality in liver cirrhosis.

Follow-up and outcome of the twelve-year experience in adult patients with acute promyelocytic leukemia

ABSTRACT Acute promyelocytic leukemia is a subtype of acute myeloid leukemia, characterized by the presence of neoplastic promyelocytes, due to the reciprocal balanced translocation between chromosomes 15 and 17. Currently, with the use of agents that act directly on this molecular change, such as all-trans retinoic acid and arsenic trioxide, APL has shifted from a highly mortal to a curable disease. However, some cases are still at high risk of death, especially early death, and acquiring a better understanding of the clinical and biological factors involving APL is needed to correctly identify and treat such cases. The early suspected diagnosis and prompt initiation of the target therapy are important for better response rates. The follow-up and outcomes, using real-life data from 44 consecutive APL patients, were studied between 2001 and 2013. The overall survival rate was 82.7% and early death was 16%. Almost all patient deaths were due to severe bleeding, which was confirmed by multivariate analysis, as the most important prognostic factor leading to death. A better understanding the pathogenesis of the hemorrhagic complications in APL is needed, as well as the risk factors associated with early death in APL patients, as this has become synonymous with overall mortality.

Follow-up and outcome of the twelve-year experience in adult patients with acute promyelocytic leukemia.

Acute promyelocytic leukemia is a subtype of acute myeloid leukemia, characterized by the presence of neoplastic promyelocytes, due to the reciprocal balanced translocation between chromosomes 15 and 17. Currently, with the use of agents that act directly on this molecular change, such as all-trans retinoic acid and arsenic trioxide, APL has shifted from a highly mortal to a curable disease. However, some cases are still at high risk of death, especially early death, and acquiring a better understanding of the clinical and biological factors involving APL is needed to correctly identify and treat such cases. The early suspected diagnosis and prompt initiation of the target therapy are important for better response rates. The follow-up and outcomes, using real-life data from 44 consecutive APL patients, were studied between 2001 and 2013. The overall survival rate was 82.7% and early death was 16%. Almost all patient deaths were due to severe bleeding, which was confirmed by multivariate analysis, as the most important prognostic factor leading to death. A better understanding the pathogenesis of the hemorrhagic complications in APL is needed, as well as the risk factors associated with early death in APL patients, as this has become synonymous with overall mortality.

Anti-leukemic activity of semisynthetic derivatives of lupeol.

In our previous work, lupeol was isolated from aerial parts of V. scorpioides and modified by semisynthetic approach. The purpose of this study was to investigate the cytotoxicity of lupeol and its derivatives previously prepared on the human K562 acute myeloid leukemia cell and human Jurkat acute lymphoid leukemia cell in vitro. Compounds 3ß-hydroxylup-20(29)-en-30-al (2), lup-20(30)-en-3ß,29-diol (3), 3ß-acetoxylup-20(29)-en-30-al (5) and 3ß-acetoxy-30-hydroxylup-20(29)-ene (6) presented cytotoxicity with IC50 ranging from 11.72 to 56.15 µM at 24 h of incubation for both cell lines. Most of the active compounds (3, 5 and 6) were selective to leukemia cells, in compare with healthy cells. The hemolysis assay showed high blood compatibility of the cytotoxic lupeol derivatives which makes possible an intravenous administration of these compounds aiming to the potential to development of anti-leukemic drugs.

Molecular events and cytotoxic effects of a novel thiosemicarbazone derivative in human leukemia and lymphoma cell lines.

The present study aimed to investigate the cytotoxic effect of 38 new thiosemicarbazone derivatives on hematological neoplastic cells lines and to select the most effective compounds to investigate the main molecular mechanisms involved in cell death. Cytotoxicity screening on Daudi and Jurkat cells revealed that only compound 1b met the selection criteria; therefore, it was chosen for further investigation. Cell viability of Daudi, Jurkat, Molt-4, Namalwa, K562, and MM.1S cell lines decreased in a concentration- and time-dependent manner after compound1b incubation; nevertheless the compound neither caused significant hemolysis nor reduction in peripheral blood mononuclear cell viability. Although no changes were observed on cell cycle or Ki-67 expression, compound1b induced apoptotic-like cell death with mitochondrial involvement, Bax/Bcl-2 inversion, AIF release, survivin inhibition, and caspase-3 activation in both Daudi and Jurkat cells. Furthermore, the compound reduced NFκB expression in Jurkat cells. In Daudi cells, compound1b also decreased CHOP, Akt, pAkt, and MAPK/ERK2 expression, thereby suggesting modulation of UPR, PI3K/Akt/mTOR, and MAPK/ERK signaling pathways. Finally, the compound was able to reduce the cell viability of samples collected from patients with different lymphoid neoplasms subtypes, showing that thiosemicarbazones derivatives could be used in the development of new drugs with anticancer activity.

Immune response of Brazilian farmers exposed to multiple pesticides.

Occupational exposure to pesticides has been identified as a factor that predisposes to disorders of the immune system. Immunosuppression, autoimmunity, cancer of various organs and other diseases in people who apply these products have been reported by the studies. This study aimed to investigate the relationship between occupational exposure to pesticides and the immunological profile in 43 farmers exposed to mixtures of pesticides for at least 15 years. A control group composed of 30 individuals without a history of occupational exposure to pesticides was also evaluated. Peripheral blood samples were processed by flow cytometry and cells were labelled with an 8-color monoclonal antibody panel. Plasma cytokines were also measured. Significant increase in classical monocytes (p < 0.001) and dendritic cells (p < 0.001) in the exposed group was observed as well in total T cells (p = 0.04), central memory CD8 T cells (p = 0.02) and effector memory CD8 T cells (p = 0.01). On the other hand, the activation markers of T cells as the expression of CD57, HLA-DR, CD25 and CD28 were evaluated and no difference was found between groups. When the B cells were analyzed, a significant decrease in total B cells (p = 0.01), regulatory B cells (p < 0.001) and plasmablasts (p < 0.001) in the exposed group, compared to healthy controls, was observed. Pro-inflammatory IL-6 was significantly elevated (p = 0.04) in the plasma of farmers compared to that of controls. The constant antigenic stimulus that occurs during exposure to pesticides can favor the recruitment of dendritic cells and macrophages (APCs) presents in the skin and respiratory tract. In the secondary lymphoid organs, the CD4 T and B cells that process such antigens are possibly undergoing proliferative exhaustion, with the consequent depletion of all mature B subpopulations. The resulting drop in humoral immunity may be offset by an increase in the number of circulating CD8 T lymphocytes due to their cytotoxic action.

Apoptotic events induced by a natural plastoquinone from the marine alga Desmarestia menziesii in lymphoid neoplasms.

There exists an urgent need for the development of new drugs for the treatment of lymphoid neoplasms. The aim of this study was to evaluate the cytotoxic effect of the marine plastoquinone 9'-hydroxysargaquinone (9'-HSQ), focusing on investigation of the mechanism by which it causes death in lymphoid neoplastic cells. This particular plastoquinone reduced the cell viability of different hematological tumor cell lines in a time-dependent and concentration-dependent manner. Intrinsic apoptosis occurred with time-dependent reduction of mitochondrial membrane potential (42.3 ± 1.1% of Daudi cells and 18.6 ± 5.6% of Jurkat cells maintained mitochondrial membrane integrity) and apoptosis-inducing factor release (Daudi: 133.3 ± 8.1%, Jurkat: 125.7 ± 6.9%). Extrinsic apoptosis also occurred, as reflected by increased FasR expression (Daudi: 139.5 ± 7.1%, Jurkat: 126.0 ± 1.0%). Decreases were observed in the expression of Ki-67 proliferation marker (Daudi: 67.5 ± 2.5%, Jurkat: 84.3 ± 3.8%), survivin (Daudi: 66.0 ± 9.9%, Jurkat: 63.1 ± 6.0%), and NF-κB (0.7 ± 0.04% in Jurkat cells). Finally, 9'-HSQ was cytotoxic to neoplastic cells from patients with different lymphoid neoplasms (IC50: 4.9 ± 0.6 to 34.2 ± 0.4 µmol/L). These results provide new information on the apoptotic mechanisms of 9'-HSQ and suggest that it might be a promising alternative for the treatment of lymphoid neoplasms.

Eight-color panel for immune phenotype monitoring by flow cytometry.

Flow cytometry (FC) is a fast and highly informative technology that has gained prominence in immune phenotype monitoring. FC standardization is crucial to obtain reliable results that are comparable among laboratories and immune monitoring studies, as this method is influenced by several variables, including equipment, reagents, staining procedures, and pre-analytical and analytical factors. Recent studies have standardized antibody panels and analytical procedures to analyze circulating immune cells in peripheral blood (PB). However, these panels cannot be adapted for laboratories that perform eight-color FC with liquid reagents. The aim of this study was to design and test an eight-color panel, intended to phenotype the main immune cell subsets in PB using liquid reagents and fresh whole blood samples. Samples were collected from healthy individuals recruited from staff and students and from six chemotherapy patients with leukopenia. The antibody panel was designed on the basis of previous studies. Quality controls comprised antibody titration, fluorescence minus one controls, internal controls, and compensation controls. Samples were analyzed by two operators using an eight-color three-laser FACSCanto II flow cytometer (BD Biosciences, USA) and Infinicyt software (Cytognos, Spain). The proposed eight-color panel is composed of six tubes. Analysis of these tubes allowed evaluation of frequencies and classification of various immune cells, such as naïve T, central memory T, effector memory T, CDRA+ effector memory T, activated T, and regulatory T cells; class-switched B, non-switched B, memory B, regulatory B cells, and plasmablasts; myeloid and plasmacytoid dendritic cells, classical and non-classical monocytes; and immature neutrophils. Immunophenotyping of leukocytes using the proposed panel was efficient to correctly differentiate the majority of immune cell subtypes. It is a promising tool to determine the immunological profile of patients in clinical trials and establish associations with disease prognosis, complications, and outcomes.

MICONIDINE acetate, a new selective and cytotoxic compound with synergic potential, induces cell cycle arrest and apoptosis in leukemia cells.

Plants are important sources of biologically active compounds and they provide unlimited opportunities for the discovery and development of new drug leads, including new chemotherapeutics. Miconidin acetate (MA) is a hydroquinone derivative isolated from E. hiemalis. In this study we demonstrated that MA was cytotoxic against acute leukemia (AL), solid tumor cells and cancer stem cells, with the strongest effect exhibited against AL. Furthermore, it was non-cytotoxic against non-tumor cells and did not cause significant hemolysis. MA blocks the G2/M phase and causes cytostatic effects, acting in a similar way to dexamethasone by increasing PML expression. The compound also triggered intrinsic and extrinsic apoptosis by modulating Bax, FasR and survivin expression. This led to an extensive mitochondrial damage that resulted in AIF, cytochrome c and endonuclease G release, caspase-3 and PARP cleavage and DNA fragmentation. We have further demonstrated that MA was strongly cytotoxic against neoplastic cells collected from patients with different AL subtypes. Interestingly, MA increased the cytotoxic effect of chemotherapeutics cytarabine and vincristine. This study indicates that MA may be a new agent for AL and highlights its potential as a new source of anticancer drugs. Graphical abstract MA blocks G2/M phase with PML expression and KI67 inhibition, ROS generation and intrinsic and extrinsic apoptosis, leading to mitochondrial damage, caspase 3 and PARP cleavage and DNA fragmentation.

Synthesis of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity.

Malignant neoplasms are one of the leading causes of death worldwide and hematologic malignancies, including acute leukemia (AL) is one of the most relevant cancer types. Current available chemotherapeutics are associated with high morbidity and mortality rates, therefore, the search for new molecules with antitumor activity, specific and selective for neoplastic cells, became a great challenge for researchers in the oncology field. As pyrazolines stand out in the literature for their great variety of biological activities, the aim of this study was to synthesize and evaluate the antileukemic activity of five new pyrazoline derivatives. All pyrazolines showed adequate physicochemical properties for a good oral bioavailability. The two unpublished and most effective pyrazoline derivatives have been selected for further experiments. These compounds are highly selective for leukemic cells when compared to non-neoplastic cells and did not cause lysis on human red blood cells. Additionally, selected pyrazolines induced cell cycle arrest at G0/G1 phase and decreased cell proliferation marker KI67. Apoptotic cell death induced by selected pyrazolines was confirmed by morphological analysis, assessment of phosphatidylserine residue exposure and DNA fragmentation. Several factors indicate that both intrinsic and extrinsic apoptosis occurred. These were: increased FasR expression; the predominance of Bax in relation to Bcl-2; the loss of mitochondrial membrane potential; AIF release; decreased expression of survivin (an antiapoptotic protein); and the activation of caspase-3. The selected pyrazolines were also found to be cytotoxic against neoplastic cells collected from the peripheral blood and bone marrow of patients with different subtypes of acute leukemia.