ABSTRACT
PURPOSE: Common Variable Immunodeficiency (CVID) is characterized by hypogammaglobulinemia and failure of specific antibody production due to B-cell defects. However, studies have documented various T-cell abnormalities, potentially linked to viral complications. The frequency of Cytomegalovirus (CMV) replication in CVID cohorts is poorly studied. To address this gap in knowledge, we set up an observational study with the objectives of identifying CVID patients with active viraemia (CMV, Epstein-Barr virus (EBV)), evaluating potential correlations with immunophenotypic characteristics, clinical outcome, and the dynamic progression of clinical phenotypes over time. METHODS: 31 CVID patients were retrospectively analysed according to viraemia, clinical and immunologic characteristics. 21 patients with non CVID humoral immunodeficiency were also evaluated as control. RESULTS: Active viral replication of CMV and/or EBV was observed in 25% of all patients. CMV replication was detected only in CVID patients (16%). CVID patients with active viral replication showed reduced HLA-DR+ NK counts when compared with CMV-DNA negative CVID patients. Viraemic patients had lower counts of LIN-DNAMbright and LIN-CD16+ inflammatory lymphoid precursors which correlated with NK-cell subsets. Analysis of the dynamic progression of CVID clinical phenotypes over time, showed that the initial infectious phenotype progressed to complicated phenotypes with time. All CMV viraemic patients had complicated disease. CONCLUSION: Taken together, an impaired production of inflammatory precursors and NK activation is present in CVID patients with active viraemia. Since "Complicated" CVID occurs as a function of disease duration, there is need for an accurate evaluation of this aspect to improve classification and clinical management of CVID patients.
Subject(s)
Common Variable Immunodeficiency , Cytomegalovirus Infections , Cytomegalovirus , Herpesvirus 4, Human , Virus Replication , Humans , Common Variable Immunodeficiency/immunology , Common Variable Immunodeficiency/complications , Male , Female , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Cytomegalovirus/physiology , Adult , Middle Aged , Herpesvirus 4, Human/physiology , Herpesvirus 4, Human/immunology , Retrospective Studies , Killer Cells, Natural/immunology , Young Adult , Viremia/immunology , Epstein-Barr Virus Infections/immunology , Immunophenotyping , Aged , AdolescentABSTRACT
BACKGROUND AND AIMS: While serum osteopontin (OPN)'s established role in cardiometabolic risk is recognized, its potential as a predictor of metabolic syndrome (MetS) improvement through a urine assay has not yet been demonstrated. In this study, we propose its potential predictive role over a 12-month period of standard care, with the ability to complement anthropometric measures. METHODS AND RESULTS: Hierarchical clustering revealed a notable association of urinary OPN (uOPN) with MetS criteria and overcame anthropometric measures in predicting the improvement at 12 months (OR of 2.74 [95% CI 1.32 to 6.29]). uOPN significantly contributed to the homogeneity of the nodes in the random forest and ultimately enhanced the performance of anthropometric measures when assessed for accuracy and area under the curve (AUC). CONCLUSION: Our findings offer insights into potential applications in cardiometabolic medicine for uOPN, which is easily detectable in non-invasive biological samples through an affordable assay.
Subject(s)
Biomarkers , Metabolic Syndrome , Osteopontin , Predictive Value of Tests , Urinalysis , Metabolic Syndrome/diagnosis , Metabolic Syndrome/urine , Metabolic Syndrome/blood , Humans , Osteopontin/urine , Osteopontin/blood , Male , Female , Biomarkers/urine , Biomarkers/blood , Middle Aged , Time Factors , Adult , Treatment Outcome , Aged , Cardiometabolic Risk FactorsABSTRACT
Complex diseases such as Multiple Sclerosis (MS) cover a wide range of biological scales, from genes and proteins to cells and tissues, up to the full organism. In fact, any phenotype for an organism is dictated by the interplay among these scales. We conducted a multilayer network analysis and deep phenotyping with multi-omics data (genomics, phosphoproteomics and cytomics), brain and retinal imaging, and clinical data, obtained from a multicenter prospective cohort of 328 patients and 90 healthy controls. Multilayer networks were constructed using mutual information for topological analysis, and Boolean simulations were constructed using Pearson correlation to identified paths within and among all layers. The path more commonly found from the Boolean simulations connects protein MK03, with total T cells, the thickness of the retinal nerve fiber layer (RNFL), and the walking speed. This path contains nodes involved in protein phosphorylation, glial cell differentiation, and regulation of stress-activated MAPK cascade, among others. Specific paths identified were subsequently analyzed by flow cytometry at the single-cell level. Combinations of several proteins (GSK3AB, HSBP1 or RS6) and immune cells (Th17, Th1 non-classic, CD8, CD8 Treg, CD56 neg, and B memory) were part of the paths explaining the clinical phenotype. The advantage of the path identified from the Boolean simulations is that it connects information about these known biological pathways with the layers at higher scales (retina damage and disability). Overall, the identified paths provide a means to connect the molecular aspects of MS with the overall phenotype.
Subject(s)
Multiple Sclerosis , Humans , Prospective Studies , Tomography, Optical Coherence/methods , Retina , Brain , Heat-Shock ProteinsABSTRACT
Here, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses. Applying a high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses allowed identification of B cell epitopes and relate them to their evolutionary and structural properties. We identify hotspots of pre-existing immunity and identify cross-reactive epitopes that contribute to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves, and SARS-CoV-2 variants. Lastly, we show that mutations in spike and nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts.
ABSTRACT
BACKGROUND AND AIMS: Inflammation due to the excess of nutrient intake plays an important role in the pathophysiology of metabolic syndrome (MetS). Here, the potential influence of neutrophils and their degranulation markers on MetS improvement upon dietary and behavioral counselling, has been investigated. Specifically, we aimed at investigating their role as potential predictors of metabolic syndrome improvements. METHODS AND RESULTS: patients with MetS (n = 127) received behavioral and dietary recommendations before follow-up at 6 months. Serum levels of matrix metalloproteinases (MMP)8, MMP9, myeloperoxidase (MPO), tissue inhibitor of MMP (TIMP)-1, TIMP-2, TIMP-3 and resistin were tested at baseline. In the whole cohort, baseline levels of proinflammatory MMP8, MMP9 and MPO increased together with the number of MetS criteria. Seventy-three (57%) patients experienced a reduction in MetS-defining criteria at follow-up. With respect to those with no improvement, such individuals showed lower weight and waist circumference at enrolment, less frequent smoking habits, higher levels of triglycerides and lower circulating MMP8. At logistic regression analysis, baseline MMP8 showed negative predictive ability (odds ratio (OR) 0.979 [0.961-0.997]; p = 0.025) against MetS improvement. Such findings hold true even when included in the backward stepwise logistic regression model confirming MMP8 as an independent predictor (OR 0.970 [0.949-0.993]; p = 0.009). Receiver operating characteristic (ROC) curve confirmed the predictive ability of MMP8 combined in a model including baseline MetS criteria and waist circumference. Bootstrap resampling analysis internally validated our findings. CONCLUSION: Improvement of MetS is independently associated with baseline low MMP-8 levels, suggesting a pivotal role for inflammation in metabolic alteration.
Subject(s)
Metabolic Syndrome , Humans , Metabolic Syndrome/diagnosis , Matrix Metalloproteinase 8 , Matrix Metalloproteinase 9 , Neutrophils/metabolism , Biomarkers , Inflammation , ROC Curve , Waist CircumferenceABSTRACT
Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcomes were previously correlated with Notch4 expression on Tregs, here, we show that Tregs in MIS-C were destabilized through a Notch1-dependent mechanism. Genetic analysis revealed that patients with MIS-C had enrichment of rare deleterious variants affecting inflammation and autoimmunity pathways, including dominant-negative mutations in the Notch1 regulators NUMB and NUMBL leading to Notch1 upregulation. Notch1 signaling in Tregs induced CD22, leading to their destabilization in a mTORC1-dependent manner and to the promotion of systemic inflammation. These results identify a Notch1/CD22 signaling axis that disrupts Treg function in MIS-C and point to distinct immune checkpoints controlled by individual Treg Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Child , COVID-19/genetics , T-Lymphocytes, Regulatory , Inflammation/genetics , Receptor, Notch1/genetics , Sialic Acid Binding Ig-like Lectin 2ABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained. OBJECTIVES: To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1ß, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes. METHODS: We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line. RESULTS: We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1ß in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation. CONCLUSIONS: These results provide further evidence that IL-1ß targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.
Subject(s)
COVID-19 Drug Treatment , Inflammasomes , Anti-Inflammatory Agents , Cytokine Release Syndrome/drug therapy , Cytokines/metabolism , DNA, Complementary , Humans , Inflammasomes/metabolism , Interleukin 1 Receptor Antagonist Protein/therapeutic use , Interleukin-1beta/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Proteins , Receptors, Interleukin-1 , SARS-CoV-2ABSTRACT
OBJECTIVE: The immune response arises from a fine balance of mechanisms that provide for surveillance, tolerance, and elimination of dangers. Sulfavant A (SULF A) is a sulfolipid with a promising adjuvant activity. Here we studied the mechanism of action of SULF A and addressed the identification of its molecular target in human dendritic cells (hDCs). METHODS: Adjuvant effect and immunological response to SULF A were assessed on DCs derived from human donors. In addition to testing various reporter cells, target identification and downstream signalling was supported by a reverse pharmacology approach based on antibody blocking and gene silencing, crosstalk with TLR pathways, use of human allogeneic mixed lymphocyte reaction. RESULTS: SULF A binds to the Triggering Receptor Expressed on Myeloid cells-2 (TREM2) and initiates an unconventional maturation of hDCs leading to enhanced migration activity and up-regulation of MHC and co-stimulatory molecules without release of conventional cytokines. This response involves the SYK-NFAT axis and is compromised by blockade or gene silencing of TREM2. Activation by SULF A preserved the DC functions to excite the allogeneic T cell response, and increased interleukin-10 release after lipopolysaccharide stimulation. CONCLUSION: SULF A is the first synthetic small molecule that binds to TREM2. The receptor engagement drives differentiation of an unprecedented DC phenotype (homeDCs) that contributes to immune homeostasis without compromising lymphocyte activation and immunogenic response. This mechanism fully supports the adjuvant and immunoregulatory activity of SULF A. We also propose that the biological properties of SULF A can be of interest in various physiopathological mechanisms and therapies involving TREM2.
Subject(s)
Dendritic Cells , Lymphocyte Activation , Cytokines/metabolism , Dendritic Cells/metabolism , Homeostasis , LigandsABSTRACT
Background and Aim: High lipoprotein(a) [Lp(a)] is a well-established cardiovascular (CV) risk factor, but the effect of mildly elevated Lp(a) on CV health is largely unknown. Our aim was to evaluate if Lp(a) is associated with the severity of carotid atherosclerosis (CA) in the specific subset of metabolic syndrome (MetS). Patients and Methods: Subjects with diagnosed MetS and ultrasound-assessed CA were enrolled. Those patients were categorized according to the severity of CA (moderate vs. severe), and the circulating levels of Lp(a) alongside with clinical, anthropometric, and biochemical data were collected. Results: Sixty-five patients were finally included: twenty-five with moderate and forty with severe CA (all with asymptomatic disease). Intergroup comparison showed Lp(a) as the only significantly different variable [6 (2-12) mg/dl vs. 11.5 (6-29.5) mg/dl; p = 0.018]. Circulating levels of Lp(a) were also confirmed as the only variable independently associated with severity of CA at logistic regression analysis [OR 2.9 (95% CI 1.1-7.8); p = 0.040]. ROC curve analysis for Lp(a) confirmed a serum level of 10 mg/dl as the best cut-off value [AUC 0.675 (95% CI 0.548-0.786)]. Although sensitivity and specificity were suboptimal (69.0 and 70.4%, respectively)-likely due to the small sample size-this result is in line with those previously reported in the literature. Conclusion: Lp(a) is independently associated with severity of CA in the subgroup of MetS patients.
ABSTRACT
Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcome were previously correlated with Notch4 expression on regulatory T (Treg) cells, here we show that the Treg cells in MIS-C are destabilized in association with increased Notch1 expression. Genetic analysis revealed that MIS-C patients were enriched in rare deleterious variant impacting inflammation and autoimmunity pathways, including dominant negative mutations in the Notch1 regulators NUMB and NUMBL. Notch1 signaling in Treg cells induced CD22, leading to their destabilization in an mTORC1 dependent manner and to the promotion of systemic inflammation. These results establish a Notch1-CD22 signaling axis that disrupts Treg cell function in MIS-C and point to distinct immune checkpoints controlled by individual Treg cell Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.
ABSTRACT
Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.
Subject(s)
Antineoplastic Agents , Multiple Myeloma , Antineoplastic Agents/therapeutic use , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic , Cell Line, Tumor , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2/metabolism , SulfonamidesABSTRACT
We studied pretransplant minimal residual disease (MRD) in 224 patients (median age 44 years; range 17-65) with acute myeloid leukemia (AML) undergoing allogeneic stem cell transplant (HSCT) in complete remission. MRD was evaluated on marrow samples using multicolor flow cytometry and assessment of WT1 gene expression. Both methods showed a strong prognostic value and their combination allowed the identification of three groups of patients with different risk of relapse. In multivariate analysis, combined MRD was the only predictor of cumulative incidence of relapse, regardless of donor type, conditioning regimen, first or second CR at HSCT, HSCT year, and ELN risk group. Multivariate regression model showed that only negative combined MRD status (P < .001) and myeloablative conditioning (P = .004) were independently associated with better OS. Among MRD-positive patients, a reduced incidence of relapse was observed in patients receiving haplo transplant (P < .05) and in patients who showed grade II-IV aGVHD (P < .03). In patients with negative combined MRD, the intensity of conditioning regimen did not affect the overall favorable outcome. We suggest that pretransplant MRD evaluation combined with transplant-related factors can identify AML patients at higher risk for relapse and might help in defining the overall transplant strategy.
Subject(s)
Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Neoplasm, Residual , Female , Humans , Male , Middle Aged , Transplantation, Homologous , Treatment OutcomeABSTRACT
A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.
Subject(s)
Host-Pathogen Interactions , Immunity, Cellular , Pneumonia, Viral/etiology , Pneumonia, Viral/metabolism , Receptor, Notch4/metabolism , Signal Transduction , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Amphiregulin/pharmacology , Animals , Biomarkers , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility , Host-Pathogen Interactions/immunology , Humans , Immunohistochemistry , Immunomodulation/drug effects , Inflammation Mediators/metabolism , Influenza A virus/physiology , Lung/immunology , Lung/metabolism , Lung/pathology , Lung/virology , Mice , Mice, Transgenic , Pneumonia, Viral/pathology , Receptor, Notch4/antagonists & inhibitors , Receptor, Notch4/genetics , Severity of Illness IndexABSTRACT
Allergy is an inflammatory process determined by a cascade of immune events characterized by T-helper 2 lymphocytes polarization leading to interleukin-4 upregulation, IgE secretion, and mast cell and eosinophil activation. HLA-G molecules, both in membrane-bound and in soluble forms, are known to play a key immunoregulatory role and their involvement in allergic diseases is supported by increasing literature data. HLA-G expression and secretion is specifically induced in peripheral blood mononuclear cells of allergic patients after in vitro incubation with the causal allergen. Elevated levels of soluble HLA-G molecules are detected in serum of patients with allergic rhinitis correlating with allergen-specific IgE levels, clinical severity, drug consumption and response to allergen-specific immunotherapy. HLA-G genetic polymorphisms confer susceptibility to allergic asthma development and high levels of soluble HLA-G molecules are found in plasma and bronchoalveolar lavage fluid of patients with allergic asthma correlating with allergen-specific IgE levels. Interestingly, allergic pregnant women have lower plasma sHLA-G levels than non-allergic women during the 3rd trimester of pregnancy and at delivery. Finally, in allergic patients with atopic dermatitis HLA-G molecules are expressed by T cells, monocytes-macrophages and Langerhans cells infiltrating the dermis. Although at present is difficult to completely define the role of HLA-G molecules in allergic diseases, it may be suggested that they are specifically expressed and secreted by immune cells during the allergic reaction in an attempt to suppress allergic inflammation.
Subject(s)
HLA-G Antigens/immunology , Hypersensitivity/immunology , Animals , HumansABSTRACT
Prediabetes is often observed in patients with Metabolic Syndrome (MetS) and might be associated with metabolic and inflammatory alterations. Here, we investigated whether the inflammatory molecule osteopontin (OPN) might have a prognostic impact in a cohort of MetS patients (n = 85) with baseline normal glycaemia or impaired fasting glucose (IFG) over one year of recommended pharmacological treatments and Mediterranean diet. Patients were then followed up for 12 months with intermediate evaluation after 6 months. At all time points, anthropometric and clinical data were recorded, alongside with haematological and biochemical profiles, including serum concentrations of OPN. As expected, Mediterranean diet improves glycaemic profile in patients with IFG. Baseline serum OPN failed to be associated with baseline anthropometric or biochemical variables. At baseline, higher levels of OPN were shown in patients with IFG as compared to normal glycaemia. Two distinct subgroups of patients in whom OPN decreased or remained stable/increased at follow-up were identified. When higher serum OPN levels were observed at baseline, greater reduction was observed at 1-year follow-up. Reduction in circulating OPN levels was associated with metabolic improvement in terms of blood pressure, LDL-c, HDL-c, and glycaemia. At both univariate and adjusted logistic regression analyses, serum OPN emerged as an independent predictor of glycaemic profile improvement at 1-year follow-up (adjOR 1.05 [1.00-1.10]; P = .041). In conclusion, pharmacological and dietetic interventions improved glycaemic profile in patients with MetS. In particular, glycaemic improvement was demonstrated in patients who also reduce circulating OPN levels. Higher OPN levels at baseline predict normalization of glycaemic profile.
Subject(s)
Blood Glucose/metabolism , Diet, Mediterranean , Glucose Intolerance/diet therapy , Metabolic Syndrome/diet therapy , Osteopontin/blood , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Female , Glucose Intolerance/metabolism , Humans , Logistic Models , Male , Metabolic Syndrome/metabolism , Middle Aged , Pilot Projects , PrognosisABSTRACT
Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.
Subject(s)
Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , Agammaglobulinaemia Tyrosine Kinase , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases , PyrimidinesABSTRACT
Thrombolysis is the gold standard treatment for acute ischemic stroke. Besides its fibrinolytic role, recombinant tissue plasminogen activator (r-tPA) holds several non-fibrinolytic functions. Here, we investigated the potential role of r-tPA on human primary neutrophil migration in vitro. By means of modified Boyden chamber migration assay and checkerboard analysis we showed a dose-dependent chemotactic effect of r-TPA with a maximum effect reached by 0.03 mg/mL (0.003-1 mg/mL). Pre-incubation with MAP kinases inhibitors allowed the identification of PI3K/Akt, but not ERK1/2 as the intracellular pathway mediating the observed effects. Furthermore, by means of real-time PCR, immunocytochemistry and cytofluorimetry we demonstrated that the r-tPA receptor low density lipoprotein receptor-related protein 1 (LRP-1) is synthetized and expressed by neutrophils in response to r-tPA and TNF-α. Inhibition of LRP-1 by receptor-associated protein (RAP), prevented r-tPA-mediated F-actin polymerization, migration and signal through Akt but not ERK1/2. Lastly, also neutrophil degranulation in response to r-tPA seems to be mediated by LRP-1 under adhesion conditions. In conclusion, we show that r-tPA induces neutrophil chemotaxis through LRP-1/Akt pathway. Blunting r-tPA-mediated neutrophil activation might be beneficial as an adjuvant therapy to thrombolysis in this setting.
Subject(s)
Chemotaxis/drug effects , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , MAP Kinase Signaling System/drug effects , Neutrophils/metabolism , Tissue Plasminogen Activator/pharmacology , Adult , Female , Humans , Male , Middle Aged , Neutrophils/cytology , Recombinant Proteins/pharmacologyABSTRACT
Metabolic reprogramming is emerging as a cancer vulnerability that could be therapeutically exploitable using different approaches, including amino acid depletion for those tumors that rely on exogenous amino acids for their maintenance. Ê-Asparaginase (ASNase) has contributed to a significant improvement in acute lymphoblastic leukemia outcomes; however, toxicity and resistance limit its clinical use in other tumors. Here, we report that, in multiple myeloma (MM) cells, the DNA methylation status is significantly associated with reduced expression of ASNase-related gene signatures, thus suggesting ASNase sensitivity for this tumor. Therefore, we tested the effects of ASNase purified from Erwinia chrysanthemi (Erw-ASNase), combined with the next-generation proteasome inhibitor (PI) carfilzomib. We observed an impressive synergistic effect on MM cells, whereas normal peripheral blood mononuclear cells were not affected. Importantly, this effect was associated with increased reactive oxygen species (ROS) generation, compounded mitochondrial damage, and Nrf2 upregulation, regardless of the c-Myc oncogenic-specific program. Furthermore, the cotreatment resulted in genomic instability and DNA repair mechanism impairment via increased mitochondrial oxidative stress, which further enhanced its antitumor activity. Interestingly, carfilzomib-resistant cells were found to be highly dependent on amino acid starvation, as reflected by their higher sensitivity to Erw-ASNase treatment compared with isogenic cells. Overall, by affecting several cellular programs, Erw-ASNase makes MM cells more vulnerable to carfilzomib, providing proof of concept for clinical use of this combination as a novel strategy to enhance PI sensitivity in MM patients.
Subject(s)
Amino Acids , Asparaginase , Asparaginase/pharmacology , Cell Death , Humans , Leukocytes, Mononuclear , Mitochondria , Oligopeptides , Reactive Oxygen SpeciesABSTRACT
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a poor prognosis myeloid malignancy characterized by an atypical phenotype (CD123+, CD56+, and CD4+). We reported that BPDCN-like phenotype (CD123+ and either CD56+ or CD4+ or both) confers poor prognosis to acute myeloblastic leukemia (AML) patients with mutated NPM1. Here, we evaluated the incidence and the prognostic relevance of BPDCN-like phenotype in cytogenetically normal AML (CN-AML) patients. From 2006 to 2016, 83 young (age <60 yrs), consecutive, CN-AML patients underwent intensive treatment. Fifteen patients (18%) showed a BPDCN-like phenotype with no difference between NPM1-mutated (mut) and NPM1-wt patients. It did not significantly affect survival neither in the whole cohort, nor in NPM1-wt patients. However, as reported, it conferred a dismal prognosis in NPM1-mut AML (p < 0.001), irrespectively of the mutational status for FLT3-ITD. In conclusion we show that BPDCN-like phenotype displays a negative prognostic relevance only in NPM1-mutated AML.