Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4+ clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-γ in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-γ after melanoma stimulation. No CD4+ clones responded to stimulation with recipient haemopoietic cells. The majority of CD8+ clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4+ clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma

Use of an innovative cuff pressure control and subglottic secretions drainage system in COVID-19 ARDS patients undergoing pronation

We conducted a proof of concept study where Anapnoguard endotracheal tubes and its control unit were used in 15 patients with COVID-19 acute respiratory distress syndrome. Anapnoguard system provides suction, venting, rinsing of subglottic space and controls cuff pressure detecting air leakage through the cuff. Alpha-amylase and pepsin levels, as oropharyngeal and gastric microaspiration markers, were assessed from 85 tracheal aspirates in the first 72 h after connection to the system. Oropharyngeal microaspiration occurred in 47 cases (55\%). Episodes of gastric microaspiration were not detected. Patient positioning, either prone or supine, did not affect alpha-amylase and pepsin concentration in tracheal secretions. Ventilator-associated pneumonia (VAP) rate was 40\%. The use of the AG system provided effective cuff pressure control and subglottic secretions drainage. Despite this, no reduction in the incidence of VAP has been demonstrated, compared to data reported in the current COVID-19 literature. The value of this new technology is worth of being evaluated for the prevention of ventilator-associated respiratory tract infections

Physiological Comparison of High-Flow Nasal Cannula and Helmet Noninvasive Ventilation in Acute Hypoxemic Respiratory Failure

Rationale: High-flow nasal cannula (HFNC) and helmet noninvasive ventilation (NIV) are used for the management of acute hypoxemic respiratory failure.Objectives: Physiological comparison of HFNC and helmet NIV in patients with hypoxemia.Methods: Fifteen patients with hypoxemia with PaO2/FiO2\u2009<\u2009200 mm Hg received helmet NIV (positive end-expiratory pressure\u2009 65\u200910 cm H2O, pressure support\u2009=\u200910-15 cm H2O) and HFNC (50 L/min) in randomized crossover order. Arterial blood gases, dyspnea, and comfort were recorded. Inspiratory effort was estimated by esophageal pressure (Pes) swings. Pes-simplified pressure-time product and transpulmonary pressure swings were measured.Measurements and Main Results: As compared with HFNC, helmet NIV increased PaO2/FiO2 (median [interquartile range]: 255 mm Hg [140-299] vs. 138 [101-172]; P\u2009=\u20090.001) and lowered inspiratory effort (7 cm H2O [4-11] vs. 15 [8-19]; P\u2009=\u20090.001) in all patients. Inspiratory effort reduction by NIV was linearly related to inspiratory effort during HFNC (r\u2009=\u20090.84; P\u2009<\u20090.001). Helmet NIV reduced respiratory rate (24 breaths/min [23-31] vs. 29 [26-32]; P\u2009=\u20090.027), Pes-simplified pressure-time product (93 cm H2O\u2009 c5\u2009s\u2009 c5\u2009min-1 [43-138] vs. 200 [168-335]; P\u2009=\u20090.001), and dyspnea (visual analog scale 3 [2-5] vs. 8 [6-9]; P\u2009=\u20090.002), without affecting PaCO2 (P\u2009=\u20090.80) and comfort (P\u2009=\u20090.50). In the overall cohort, transpulmonary pressure swings were not different between treatments (NIV\u2009=\u200918 cm H2O [14-21] vs. HFNC\u2009=\u200915 [8-19]; P\u2009=\u20090.11), but patients exhibiting lower inspiratory effort on HFNC experienced increases in transpulmonary pressure swings with helmet NIV. Higher transpulmonary pressure swings during NIV were associated with subsequent need for intubation.Conclusions: As compared with HFNC in hypoxemic respiratory failure, helmet NIV improves oxygenation, reduces dyspnea, inspiratory effort, and simplified pressure-time product, with similar transpulmonary pressure swings, PaCO2, and comfort

Apoptosis and telomeres shortening related to HIV-1 induced oxidative stress in an astrocytoma cell line

Background: Oxidative stress plays a key role in the neuropathogenesis of Human Immunodeficiency Virus-1 (HIV-1) infection causing apoptosis of astroglia cells and neurons. Recent data have shown that oxidative stress is also responsible for the acceleration of human fibroblast telomere shortening in vitro. In the present study we analyzed the potential relations occurring between free radicals formation and telomere length during HIV-1 mediated astroglial death. Results: To this end, U373 human astrocytoma cells have been directly exposed to X4-using HIV- 1IIIB strain, for 1, 3 or 5 days and treated (where requested) with N-acetylcysteine (NAC), a cysteine donor involved in the synthesis of glutathione (GSH, a cellular antioxidant) and apoptosis has been evaluated by FACS analysis. Quantitative-FISH (Q-FISH) has been employed for studying the telomere length while intracellular reduced/oxidized glutathione (GSH/GSSG) ratio has been determined by High-Performance Liquid Chromatography (HPLC). Incubation of U373 with HIV- 1IIIB led to significant induction of cellular apoptosis that was reduced in the presence of 1 mM NAC. Moreover, NAC improved the GSH/GSSG, a sensitive indicator of oxidative stress, that significantly decreased after HIV-1IIIB exposure in U373. Analysis of telomere length in HIV-1 exposed U373 showed a statistically significant telomere shortening, that was completely reverted in NAC-treated U373. Conclusion: Our results support the role of HIV-1-mediated oxidative stress in astrocytic death and the importance of antioxidant compounds in preventing these cellular damages. Moreover, these data indicate that the telomere structure, target for oxidative damage, could be the key sensor of cell apoptosis induced by oxidative stress after HIV infection