Immunomodulatory effects of antiangiogenic tyrosine kinase inhibitors in renal cell carcinoma models: Impact on following anti-PD-1 treatments.

The approval of immune checkpoint inhibitors (ICIs) has revolutionized the management of metastatic renal cell carcinoma (RCC), introducing several ICI-based combinations as the new standard of care for affected patients. Nonetheless, monotherapy with antiangiogenic tyrosine kinase inhibitors (TKIs), such as pazopanib or sunitinib, still represents a first-line treatment option for selected patients belonging to the favorable risk group according to the International mRCC Database Consortium (IMDC) model. After TKI monotherapy, the main second-line option is represented by ICI monotherapy with the anti-Programmed Death Receptor 1(PD-1) nivolumab. To date, the expected clinical outcomes are similar with pazopanib or sunitinib and there is no clear indication for selecting one TKI over the other. Moreover, their impact on subsequent ICI treatment outcomes is not well defined, yet. Based on these premises, we investigated the immunomodulatory activity of these drugs in vitro and in vivo.Both TKIs induced Programmed Cell Death Ligand-1 (PD-L1) expression and soluble PD-L1 release in RCC cells, and hampered T cell activation, reducing cytokine production and the proportion of activated T cells. Nevertheless, in a syngeneic co-culture system with peripheral blood mononuclear cells (PBMCs) and tumor cells, incubation with anti-PD-1 antibody following TKIs treatment significantly restored T cell function, potentiating the cytotoxic effects against tumor cells. Pazopanib and sunitinib followed by anti-PD-1 antibody produced a comparable inhibition of tumor growth in a RCC syngeneic mouse model. Our findings suggest that pazopanib and sunitinib, showing similar immunomodulatory effects, may have a comparable impact on the subsequent effectiveness of PD-1/PD-L1 blockade.

Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Functionally Relevant Cytokine/Receptor Axes in Myelofibrosis.

Dysregulated inflammatory signaling is a key feature of myeloproliferative neoplasms (MPNs), most notably of myelofibrosis (MF). Indeed, MF is considered the prototype of onco-inflammatory hematologic cancers. While increased levels of circulatory and bone marrow cytokines are a well-established feature of all MPNs, a very recent body of literature is intriguingly pinpointing the selective overexpression of cytokine receptors by MF hematopoietic stem and progenitor cells (HSPCs), which, by contrast, are nearly absent or scarcely expressed in essential thrombocythemia (ET) or polycythemia vera (PV) cells. This new evidence suggests that MF CD34+ cells are uniquely capable of sensing inflammation, and that activation of specific cytokine signaling axes may contribute to the peculiar aggressive phenotype and biological behavior of this disorder. In this review, we will cover the main cytokine systems peculiarly activated in MF and how cytokine receptor targeting is shaping a novel therapeutic avenue in this disease.

Interplay between Protein Kinase C Epsilon and Reactive Oxygen Species during Myogenic Differentiation.

Reactive oxygen species (ROS) are currently recognized as a key driver of several physiological processes. Increasing evidence indicates that ROS levels can affect myogenic differentiation, but the molecular mechanisms still need to be elucidated. Protein kinase C (PKC) epsilon (PKCe) promotes muscle stem cell differentiation and regeneration of skeletal muscle after injury. PKCs play a tissue-specific role in redox biology, with specific isoforms being both a target of ROS and an up-stream regulator of ROS production. Therefore, we hypothesized that PKCe represents a molecular link between redox homeostasis and myogenic differentiation. We used an in vitro model of a mouse myoblast cell line (C2C12) to study the PKC-redox axis. We demonstrated that the transition from a myoblast to myotube is typified by increased PKCe protein content and decreased ROS. Intriguingly, the expression of the antioxidant enzyme superoxide dismutase 2 (SOD2) is significantly higher in the late phases of myogenic differentiation, mimicking PKCe protein content. Furthermore, we demonstrated that PKCe inhibition increases ROS and reduces SOD2 protein content while SOD2 silencing did not affect PKCe protein content, suggesting that the kinase could be an up-stream regulator of SOD2. To support this hypothesis, we found that in C2C12 cells, PKCe interacts with Nrf2, whose activation induces SOD2 transcription. Overall, our results indicate that PKCe is capable of activating the antioxidant signaling preventing ROS accumulation in a myotube, eventually promoting myogenic differentiation.

Tracking fibrosis in myeloproliferative neoplasms by CCR2 expression on CD34+ cells.

In myeloproliferative neoplasm (MPNs), bone marrow fibrosis - mainly driven by the neoplastic megakaryocytic clone - dictates a more severe disease stage with dismal prognosis and higher risk of leukemic evolution. Therefore, accurate patient allocation into different disease categories and timely identification of fibrotic transformation are mandatory for adequate treatment planning. Diagnostic strategy still mainly relies on clinical/laboratory assessment and bone marrow histopathology, which, however, requires an invasive procedure and frequently poses challenges also to expert hemopathologists. Here we tested the diagnostic accuracy of the detection, by flow cytometry, of CCR2+CD34+ cells to discriminate among MPN subtypes with different degrees of bone marrow fibrosis. We found that the detection of CCR2 on MPN CD34+ cells has a very good diagnostic accuracy for the differential diagnosis between "true" ET and prePMF (AUC 0.892, P<0.0001), and a good diagnostic accuracy for the differential diagnosis between prePMF and overtPMF (AUC 0.817, P=0.0089). Remarkably, in MPN population, the percentage of CCR2-expressing cells parallels the degree of bone marrow fibrosis. In ET/PV patients with a clinical picture suggestive for transition into spent phase, we demonstrated that only patients with confirmed secondary MF showed significantly higher levels of CCR2+CD34+ cells. Overall, flow cytometric CCR2+CD34+ cell detection can be envisioned in support of conventional bone marrow histopathology in compelling clinical scenarios, with the great advantage of being extremely rapid. For patients in follow-up, its role can be conceived as an initial patient screening for subsequent bone marrow biopsy when disease evolution is suspected.

Buffering Adaptive Immunity by Hydrogen Sulfide.

T cell-mediated adaptive immunity is designed to respond to non-self antigens and pathogens through the activation and proliferation of various T cell populations. T helper 1 (Th1), Th2, Th17 and Treg cells finely orchestrate cellular responses through a plethora of paracrine and autocrine stimuli that include cytokines, autacoids, and hormones. Hydrogen sulfide (H2S) is one of these mediators able to induce/inhibit immunological responses, playing a role in inflammatory and autoimmune diseases, neurological disorders, asthma, acute pancreatitis, and sepsis. Both endogenous and exogenous H2S modulate numerous important cell signaling pathways. In monocytes, polymorphonuclear, and T cells H2S impacts on activation, survival, proliferation, polarization, adhesion pathways, and modulates cytokine production and sensitivity to chemokines. Here, we offer a comprehensive review on the role of H2S as a natural buffer able to maintain over time a functional balance between Th1, Th2, Th17 and Treg immunological responses.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during l-asparaginase treatment.

The Genetic Makeup of Myeloproliferative Neoplasms: Role of Germline Variants in Defining Disease Risk, Phenotypic Diversity and Outcome.

Myeloproliferative neoplasms are hematologic malignancies typified by a substantial heritable component. Germline variants may affect the risk of developing a MPN, as documented by GWAS studies on large patient cohorts. In addition, once the MPN occurred, inherited host genetic factors can be responsible for tuning the disease phenotypic presentation, outcome, and response to therapy. This review covered the polymorphisms that have been variably associated to MPNs, discussing them in the functional perspective of the biological pathways involved. Finally, we reviewed host genetic determinants of clonal hematopoiesis, a pre-malignant state that may anticipate overt hematologic neoplasms including MPNs.

Hydrogen Sulfide Inhibits TMPRSS2 in Human Airway Epithelial Cells: Implications for SARS-CoV-2 Infection.

The COVID-19 pandemic has now affected around 190 million people worldwide, accounting for more than 4 million confirmed deaths. Besides ongoing global vaccination, finding protective and therapeutic strategies is an urgent clinical need. SARS-CoV-2 mostly infects the host organism via the respiratory system, requiring angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enter target cells. Therefore, these surface proteins are considered potential druggable targets. Hydrogen sulfide (H2S) is a gasotransmitter produced by several cell types and is also part of natural compounds, such as sulfurous waters that are often inhaled as low-intensity therapy and prevention in different respiratory conditions. H2S is a potent biological mediator, with anti-oxidant, anti-inflammatory, and, as more recently shown, also anti-viral activities. Considering that respiratory epithelial cells can be directly exposed to H2S by inhalation, here we tested the in vitro effects of H2S-donors on TMPRSS2 and ACE2 expression in human upper and lower airway epithelial cells. We showed that H2S significantly reduces the expression of TMPRSS2 without modifying ACE2 expression both in respiratory cell lines and primary human upper and lower airway epithelial cells. Results suggest that inhalational exposure of respiratory epithelial cells to natural H2S sources may hinder SARS-CoV-2 entry into airway epithelial cells and, consequently, potentially prevent the virus from spreading into the lower respiratory tract and the lung.

Impact of the rs1024611 Polymorphism of CCL2 on the Pathophysiology and Outcome of Primary Myelofibrosis.

Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2-one of the most potent immunomodulatory chemokines-to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes; ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation; iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications.

Cytokine Profiling in Myeloproliferative Neoplasms: Overview on Phenotype Correlation, Outcome Prediction, and Role of Genetic Variants.

Among hematologic malignancies, the classic Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered a model of inflammation-related cancer development. In this context, the use of immune-modulating agents has recently expanded the MPN therapeutic scenario. Cytokines are key mediators of an auto-amplifying, detrimental cross-talk between the MPN clone and the tumor microenvironment represented by immune, stromal, and endothelial cells. This review focuses on recent advances in cytokine-profiling of MPN patients, analyzing different expression patterns among the three main Philadelphia-negative (Ph-negative) MPNs, as well as correlations with disease molecular profile, phenotype, progression, and outcome. The role of the megakaryocytic clone as the main source of cytokines, particularly in myelofibrosis, is also reviewed. Finally, we report emerging intriguing evidence on the contribution of host genetic variants to the chronic pro-inflammatory state that typifies MPNs.

NK cells: A double edge sword against SARS-CoV-2.

Natural killer (NK) cells are pivotal effectors of the innate immunity protecting an individual from microbes. They are the first line of defense against invading viruses, given their substantial ability to directly target infected cells without the need for specific antigen presentation. By establishing cellular networks with a variety of cell types such as dendritic cells, NK cells can also amplify and modulate antiviral adaptive immune responses. In this review, we will examine the role of NK cells in SARS-COV2 infections causing the ongoing COVID19 pandemic, keeping in mind the controversial role of NK cells specifically in viral respiratory infections and in inflammatory-driven lung damage. We discuss lessons learnt from previous coronavirus outbreaks in humans (caused by SARS-CoV-1 and MERS-COV).

ROS in Platelet Biology: Functional Aspects and Methodological Insights.

Reactive oxygen species (ROS) and mitochondria play a pivotal role in regulating platelet functions. Platelet activation determines a drastic change in redox balance and in platelet metabolism. Indeed, several signaling pathways have been demonstrated to induce ROS production by NAPDH oxidase (NOX) and mitochondria, upon platelet activation. Platelet-derived ROS, in turn, boost further ROS production and consequent platelet activation, adhesion and recruitment in an auto-amplifying loop. This vicious circle results in a platelet procoagulant phenotype and apoptosis, both accounting for the high thrombotic risk in oxidative stress-related diseases. This review sought to elucidate molecular mechanisms underlying ROS production upon platelet activation and the effects of an altered redox balance on platelet function, focusing on the main advances that have been made in platelet redox biology. Furthermore, given the increasing interest in this field, we also describe the up-to-date methods for detecting platelets, ROS and the platelet bioenergetic profile, which have been proposed as potential disease biomarkers.

Sodium-glucose cotransporter 2 inhibitors antagonize lipotoxicity in human myeloid angiogenic cells and ADP-dependent activation in human platelets: potential relevance to prevention of cardiovascular events.

BACKGROUND: The clear evidence of cardiovascular benefits in cardiovascular outcome trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes might suggest an effect on atherosclerotic plaque vulnerability and/or thrombosis, in which myeloid angiogenic cells (MAC) and platelets (PLT) are implicated. We tested the effects of SGLT2i on inflammation and oxidant stress in a model of stearic acid (SA)-induced lipotoxicity in MAC and on PLT activation. The possible involvement of the Na+/H+ exchanger (NHE) was also explored. METHOD: MAC and PLT were isolated from peripheral blood of healthy subjects and incubated with/without SGLT2i [empagliflozin (EMPA) and dapagliflozin (DAPA) 1-100 µM] to assess their effects on SA (100 µM)-induced readouts of inflammation, oxidant stress and apoptosis in MAC and on expression of PLT activation markers by flow-cytometry after ADP-stimulation. Potential NHE involvement was tested with amiloride (aspecific NHE inhibitor) or cariporide (NHE1 inhibitor). Differences among culture conditions were identified using one-way ANOVA or Friedman test. RESULTS: NHE isoforms (1,5-9), but not SGLT2 expression, were expressed in MAC and PLT. EMPA and DAPA (100 µM) significantly reduced SA-induced inflammation (IL1ß, TNFα, MCP1), oxidant stress (SOD2, TXN, HO1), but not apoptosis in MAC. EMPA and DAPA (both 1 µM) reduced PLT activation (CD62p and PAC1 expression). SGLT2i effects were mimicked by amiloride, and only partially by cariporide, in MAC, and by both inhibitors in PLT. CONCLUSIONS: EMPA and DAPA ameliorated lipotoxic damage in stearate-treated MAC, and reduced ADP-stimulated PLT activation, potentially via NHE-inhibition, thereby pointing to plaque stabilization and/or thrombosis inhibition as potential mechanism(s) involved in SGLT2i-mediated cardiovascular protection.

One-shoulder carrying school backpack strongly affects gait swing phase and pelvic tilt: a case study.

The use of backpacks is common to both adults and children and often leads to the onset of musculoskeletal discomforts. Although a large number of studies have focused on the optimal load for children schoolbags, there is no general consensus. Here we report a 13-yr old girl case study, showing the impact of weight and wearing the school backpack on gait parameters. The variation of gait parameters and pelvis angles in different conditions were studied: without backpack (CTRL), or with backpack at 10% Body Weight (10BW), 15% BW (15BW) and 20% BW (20BW), carried "on both shoulders" (2S), "on one shoulder" (1S), or "with one hand" (1H). Swing phase was comparably modified by 2S/20BW and 1S/10BW conditions, suggesting that a lower backpack weight was sufficient to induce gait alterations when carried in asymmetrical conditions. Pelvic tilt, which was preserved by a two-shoulders distributed 10% BW load (2S/10BW), was strongly  reduced in asymmetrical condition (1S/10BW), suggesting that a low weight carried on a single shoulder generates postural modifications including reduction of pelvic tilting, which is known to be associated to low back pain.

Sighting acute myocardial infarction through platelet gene expression.

Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs.

Sulphurous thermal water inhalation impacts respiratory metabolic parameters in heavy smokers.

Sulphurous thermal water inhalations have been traditionally used in the treatment of airway diseases. In vivo and in vitro studies reported that they ameliorate mucus rheology, mucociliary clearance and reduce inflammation. Cigarette smoking induces an inflammatory damage, with consequent remodeling of respiratory airways, which in turn affect pulmonary functions. Despite the anti-inflammatory effects of H2S are clinically documented in several airway inflammatory diseases, data on the effects of sulphurous thermal water treatment on pulmonary function and biomarkers of airways inflammation in smokers are still scant. Therefore, we investigated whether a conventional cycle of sulphurous thermal water inhalation produced changes in markers of respiratory inflammation and function. A cohort of 504 heavy current and former smokers underwent 10-day cycles of sulphurous thermal water inhalation. Pulmonary function and metabolic analyses on exhaled breath condensate were then performed at day 0 and after the 10-day treatment. Spirometric data did not change after spa therapy, while exhaled breath condensate analysis revealed that a single 10-day cycle of sulphurous water inhalation was sufficient to induce a statistically significant increase of citrulline levels along with a decrease in ornithine levels, thus shifting arginine metabolism towards a reduced nitric oxide production, i.e. an anti-inflammatory profile. Overall, sulphurous thermal water inhalation impacts on arginine catatabolic intermediates of airways cells, shifting their metabolic balance towards a reduction of the inflammatory activity, with potential benefits for smokers.

Mechano-biological model of glioblastoma cells in response to osmotic stress.

This work investigates the mechano-biological features of cells cultured in monolayers in response to different osmotic conditions. In-vitro experiments have been performed to quantify the long-term effects of prolonged osmotic stresses on the morphology and proliferation capacity of glioblastoma cells. The experimental results highlight that both hypotonic and hypertonic conditions affect the proliferative rate of glioblastoma cells on different cell cycle phases. Moreover, glioblastoma cells in hypertonic conditions display a flattened and elongated shape. The latter effect is explained using a nonlinear elastic model for the single cell. Due to a crossover between the free energy contributions related to the cytosol and the cytoskeletal fibers, a critical osmotic stress determines a morphological transition from a uniformly compressed to an elongated shape.

Combination of Platelet expression of PKCepsilon and cardiac troponin-I for early diagnosis of chest pain patients in the emergency department.

A rapid differential diagnosis of the clinical conditions underlying chest pain is a relevant clinical issue. Specifically, a fast rule-in or -out of acute myocardial infarction (AMI) is mandatory to improve diagnostic outcome and cost-effectiveness of patient management. We demonstrated that Protein Kinase C (PKC) epsilon is selectively expressed by platelets from AMI patients, accounting for increased platelet activation. Thus, we hypothesized that PKCepsilon-expressing platelets may represent a pathophysiological marker of AMI that could be utilized in combination with troponin-I, the conventional marker of cardiac injury, to add diagnostic information in chest pain workup. In 94 chest pain patients consecutively admitted to Parma University Hospital, we tested the diagnostic performance of flow-cytometric detection of PKCepsilon expressing platelets in discriminating AMI vs. non-AMI conditions. We demonstrated that PKCepsilon-expressing platelets were significantly higher in patients with AMI. Flow cytometry detection of PKCepsilon-expressing platelets showed high sensitivity and specificity (87.5% and 84.4%, respectively) and good diagnostic accuracy (AUC: 0.875). The combination of PKCepsilon expressing platelets and cardiac troponin clearly discriminates patients with 100% and 0% of probability to be affected by AMI. Overall, we highlighted a dual marker strategy potentially useful for a rapid rule-in or -out of myocardial infarction in chest pain patients.