miR-146a and miR-146b regulate the expression of ICAM-1 in giant cell arteritis.

Giant cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries. MiRNAs are small, non-coding RNAs that inhibit gene expression at post-transcriptional level. Several miRNAs have been shown to be dysregulated in temporal artery biopsies (TABs) from GCA patients, but their role is unknown. The aims of the present work were: to gain insight into the link between inflammation and miRNA up-regulation in GCA; to identify the role of miR-146a and miR-146b. Primary cultures from TABs were treated with IL-1ß, IL-6, soluble IL-6R (sIL6R), IL-17, IL-22, IFNγ, LPS and PolyIC. Correlations between cytokine mRNA and miRNA levels were determined in inflamed TABs. Primary cultures from TABs, human aortic endothelial and smooth muscle cells and ex-vivo TAB sections were transfected with synthetic miR-146a and miR-146b to mimic miRNA activities. Cell viability, target gene expression, cytokine levels in culture supernatants were assayed. Treatment of primary cultures from TABs with IL-1ß and IL-17 increased miR-146a expression while IL-1ß, IL-6+sIL6R and IFNγ increased miR-146b expression. IFNγ and IL-1ß mRNA levels correlated with miR-146a/b levels. Following transfection, cell viability decreased only in primary cultures from TABs. Moreover, transfection of miR-146a/b mimics increased ICAM-1 gene expression and production of the soluble form of ICAM-1 by primary cultures from TABs and by ex-vivo TABs. ICAM-1 expression was higher in inflamed than normal TABs and ICAM-1 levels correlated with miR-146a/b levels. Expression of miR-146a and miR-146b in GCA appeared to be driven by inflammatory cytokines (e.g. IL-1ß, IFNγ). miR-146a and miR-146b seem responsible for the increase of soluble ICAM-1.

CCL18, CHI3L1, ANG2, IL-6 systemic levels are associated with the extent of lung damage and radiomic features in SARS-CoV-2 infection.

OBJECTIVE AND DESIGN: We aimed to identify cytokines whose concentrations are related to lung damage, radiomic features, and clinical outcomes in COVID-19 patients. MATERIAL OR SUBJECTS: Two hundred twenty-six patients with SARS-CoV-2 infection and chest computed tomography (CT) images were enrolled. METHODS: CCL18, CHI3L1/YKL-40, GAL3, ANG2, IP-10, IL-10, TNFα, IL-6, soluble gp130, soluble IL-6R were quantified in plasma samples using Luminex assays. The Mann-Whitney U test, the Kruskal-Wallis test, correlation and regression analyses were performed. Mediation analyses were used to investigate the possible causal relationships between cytokines, lung damage, and outcomes. AVIEW lung cancer screening software, pyradiomics, and XGBoost classifier were used for radiomic feature analyses. RESULTS: CCL18, CHI3L1, and ANG2 systemic levels mainly reflected the extent of lung injury. Increased levels of every cytokine, but particularly of IL-6, were associated with the three outcomes: hospitalization, mechanical ventilation, and death. Soluble IL-6R showed a slight protective effect on death. The effect of age on COVID-19 outcomes was partially mediated by cytokine levels, while CT scores considerably mediated the effect of cytokine levels on outcomes. Radiomic-feature-based models confirmed the association between lung imaging characteristics and CCL18 and CHI3L1. CONCLUSION: Data suggest a causal link between cytokines (risk factor), lung damage (mediator), and COVID-19 outcomes.

Study of gene polymorphisms as predictors of treatment efficacy and toxicity in patients with indolent non-hodgkin lymphomas and mantle cell lymphoma receiving bendamustine and rituximab.

Bendamustine is used in combination with rituximab (BR) to treat indolent non-Hodgkin lymphomas (iNHL) and mantle cell lymphoma (MCL). The variability in treatment efficacy and toxicity could be related to single nucleotide polymorphisms (SNPs) in immune response genes. We would like to show a correlation between SNPs and treatment outcome in iNHL and MCL patients receiving BR. We investigated some SNPs that had already been associated with NHL outcome. Samples were genotyped for the IL2 (rs2069762), IL10 (rs1800890, rs10494879), VEGFA (rs3025039), IL8 (rs4073), CFH (rs1065489) and MTHFR (rs1801131) SNPs by allelic discrimination assays. We enrolled 70 patients that received rituximab 375 mg/m2 and bendamustine 90 mg/m2 every 28 days, both as first-line treatment and ≥ second-line regimens. Overall response rate was 97·1% (complete response [CR] rate 73·9%). Treatment toxicity included grade 3-4 neutropenia (24/70 patients), infections (21/70 patients; 1/70 grade 3), skin rash (26/70 patients; 2/70 grade 3). After a median follow-up of 24 months we did find any correlation between the analysed SNPs, CR rate and PFS. However, we demonstrated an association between the SNP in IL2 (rs2069762) and the onset of skin rash (P = 0·0001). Our study suggests a role for cytokine SNPs in bendamustine-related toxicity, which could represent a promising research field.

GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LßT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, ß-catenin activation and mouse luteinizing-hormone ß-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LßT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LßT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced ß-catenin activation, Lhb gene expression increase occurring upon LßT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

Validation of a double-color ELISpot assay of IFN-γ and IL-4 production in human peripheral blood mononuclear cells.

The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest.

Comparable cytokine release ex-vivo by whole blood from COVID-19 patients with and without non-invasive ventilation.

T cells are key players in the resolution of the infection by SARS-CoV-2. A delay in their activation can lead to severe COVID-19. The present work aimed to identify differences in cytokine release by T cells ex-vivo between COVID-19 patients in the acute phase, showing diverse disease severity. Concentrations of IFNγ, Granzyme B, IL-6, IL-10, IL-17A, IL-18, IP-10, MCP-1, and TNFα were evaluated after stimulation ex-vivo of whole blood samples with peptides from SARS-CoV-2 spike protein and a mitogen as well as without stimulation. Samples derived from hospitalized COVID-19 patients and SARS-CoV-2 vaccinated controls (CTR). Patients were classified on disease severity considering the necessity of non-invasive ventilation (NIV). Samples from patients requiring NIV revealed a similar release of cytokines compared with patients without NIV. COVID-19 patients showed higher spontaneous production of IFNγ and IP-10, lower production of MCP-1 after SARS-CoV-2 peptide stimulation and lower production of IFNγ, IL-10, IL-17A, Granzyme B, IP-10 after mitogenic stimulus compared with CTR. In conclusion, differences in T cell responses evaluated ex-vivo by a whole blood-based cytokine release assay do not appear to explain the need for non-invasive ventilation in COVID-19 patients.

Complement activation predicts negative outcomes in COVID-19: The experience from Northen Italian patients.

Coronavirus disease 19 (COVID-19) may present as a multi-organ disease with a hyperinflammatory and prothrombotic response (immunothrombosis) in addition to upper and lower airway involvement. Previous data showed that complement activation plays a role in immunothrombosis mainly in severe forms. The study aimed to investigate whether complement involvement is present in the early phases of the disease and can be predictive of a negative outcome. We enrolled 97 symptomatic patients with a positive RT-PCR for SARS-CoV-2 presenting to the emergency room. The patients with mild symptoms/lung involvement at CT-scan were discharged and the remaining were hospitalized. All the patients were evaluated after a 4-week follow-up and classified as mild (n. 54), moderate (n. 17) or severe COVID-19 (n. 26). Blood samples collected before starting any anti-inflammatory/immunosuppressive therapy were assessed for soluble C5b-9 (sC5b-9) and C5a plasma levels by ELISA, and for the following serum mediators by ELLA: IL-1ß, IL-6, IL-8, TNFα, IL-4, IL-10, IL-12p70, IFNγ, IFNα, VEGF-A, VEGF-B, GM-CSF, IL-2, IL-17A, VEGFR2, BLyS. Additional routine laboratory parameters were measured (fibrin fragment D-dimer, C-reactive protein, ferritin, white blood cells, neutrophils, lymphocytes, monocytes, platelets, prothrombin time, activated partial thromboplastin time, and fibrinogen). Fifty age and sex-matched healthy controls were also evaluated. SC5b-9 and C5a plasma levels were significantly increased in the hospitalized patients (moderate and severe) in comparison with the non-hospitalized mild group. SC5b9 and C5a plasma levels were predictive of the disease severity evaluated one month later. IL-6, IL-8, TNFα, IL-10 and complement split products were higher in moderate/severe versus non-hospitalized mild COVID-19 patients and healthy controls but with a huge heterogeneity. SC5b-9 and C5a plasma levels correlated positively with CRP, ferritin values and the neutrophil/lymphocyte ratio. Complement can be activated in the very early phases of the disease, even in mild non-hospitalized patients. Complement activation can be observed even when pro-inflammatory cytokines are not increased, and predicts a negative outcome.

Pro-Inflammatory and Pro-Oxidative Changes During Nilotinib Treatment in CML Patients: Results of a Prospective Multicenter Front-Line TKIs Study (KIARO Study).

Tyrosine kinase inhibitors (TKI) may offer a normal life expectancy to Chronic Myeloid Leukemia (CML) patients. However, a higher than expected incidence of arterial occlusive events (AOEs) was observed during treatment with nilotinib. We previously showed an "inflammatory status" during nilotinib that may explain the increased incidence of AOEs. Thus, we conducted this prospective KIARO study involving 186 CML patients (89 imatinib, 59 nilotinib, 38 dasatinib). Interleukin 6 (IL6), interleukin 10 (IL10), Tumor Necrosis Factor-α (TNFα), oxLDL, and high-sensitivity C-reactive protein (hs-CRP) plasma levels were measured at diagnosis and during treatment, with the aim to investigate changes in the inflammatory status favoring AOEs of each patient. Clinical and biochemical pro-atherothrombotic profiles and the 10-year SCORE chart were also evaluated. We showed a pro-inflammatory/pro-oxidative milieu increasing along treatment with nilotinib compared with imatinib or dasatinib, as demonstrated by higher hs-CRP and oxLDL levels and increased IL6/IL10 and TNFα/IL10 ratios only in nilotinib cohort. After median follow-up of 23.3 months starting from TKI, 10/186 patients (5.4%) suffered an AOE. Approximately 5/10 (50%) AOEs occurred during nilotinib treatment despite a lower 10-year SCORE and a lower median age in this subgroup. A longer follow-up is needed to further confirm the active role of nilotinib in AOEs pathogenesis.

Minimal Residual Disease in Multiple Myeloma: State of the Art and Applications in Clinical Practice.

Novel drugs have revolutionized multiple myeloma therapy in the last 20 years, with median survival that has doubled to up to 8-10 years. The introduction of therapeutic strategies, such as consolidation and maintenance after autologous stem cell transplants, has also ameliorated clinical results. The goal of modern therapies is becoming not only complete remission, but also the deepest possible remission. In this context, the evaluation of minimal residual disease by techniques such as next-generation sequencing (NGS) and next-generation flow (NGF) is becoming part of all new clinical trials that test drug efficacy. This review focuses on minimal residual disease approaches in clinical trials, with particular attention to real-world practices.

Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission.

Chronic myeloid leukemia (CML) patients in sustained "deep molecular response" may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38- LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38- stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/µL, range 0.27-698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/µL; range 0.0012-0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/µL; range 0.006-0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that "circulating" CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a "stem cell response" threshold to achieve and maintain TFR are ongoing.