mTOR Inhibition Is Effective against Growth, Survival and Migration, but Not against Microglia Activation in Preclinical Glioma Models.

Initially introduced in therapy as immunosuppressants, the selective inhibitors of mTORC1 have been approved for the treatment of solid tumors. Novel non-selective inhibitors of mTOR are currently under preclinical and clinical developments in oncology, attempting to overcome some limitations associated with selective inhibitors, such as the development of tumor resistance. Looking at the possible clinical exploitation in the treatment of glioblastoma multiforme, in this study we used the human glioblastoma cell lines U87MG, T98G and microglia (CHME-5) to compare the effects of a non-selective mTOR inhibitor, sapanisertib, with those of rapamycin in a large array of experimental paradigms, including (i) the expression of factors involved in the mTOR signaling cascade, (ii) cell viability and mortality, (iii) cell migration and autophagy, and (iv) the profile of activation in tumor-associated microglia. We could distinguish between effects of the two compounds that were overlapping or similar, although with differences in potency and or/time-course, and effects that were diverging or even opposite. Among the latter, especially relevant is the difference in the profile of microglia activation, with rapamycin being an overall inhibitor of microglia activation, whereas sapanisertib was found to induce an M2-profile, which is usually associated with poor clinical outcomes.

Use of High-Dose Nebulized Colistimethate in Patients with Colistin-Only Susceptible Acinetobacter baumannii VAP: Clinical, Pharmacokinetic and Microbiome Features.

(1) Background: Colistin-only susceptible (COS) Acinetobacter baumannii (AB) ventilator-associated pneumonia (VAP) represents a clinical challenge in the Intensive Care Unit (ICU) due to the negligible lung diffusion of this molecule and the low-grade evidence on efficacy of its nebulization. (2) Methods: We conducted a prospective observational study on 134 ICU patients with COS-AB VAP to describe the 'real life' clinical use of high-dose (5 MIU q8) aerosolized colistin, using a vibrating mesh nebulizer. Lung pharmacokinetics and microbiome features were investigated. (3) Results: Patients were enrolled during the COVID-19 pandemic with the ICU presenting a SAPS II of 42 [32-57]. At VAP diagnosis, the median PaO2/FiO2 was 120 [100-164], 40.3% were in septic shock, and 24.6% had secondary bacteremia. The twenty-eight day mortality was 50.7% with 60.4% and 40.3% rates of clinical cure and microbiological eradication, respectively. We did not observe any drug-related adverse events. Epithelial lining fluid colistin concentrations were far above the CRAB minimal-inhibitory concentration and the duration of nebulized therapy was an independent predictor of microbiological eradication (12 [9.75-14] vs. 7 [4-13] days, OR (95% CI): 1.069 (1.003-1.138), p = 0.039). (4) Conclusions: High-dose and prolonged colistin nebulization, using a vibrating mesh, was a safe adjunctive therapeutic strategy for COS-AB VAP. Its right place and efficacy in this setting warrant investigation in interventional studies.

The effects of painless nerve growth factor on human microglia polarization.

Previous studies in the rat suggest that microglial cells represent a potential druggable target for nerve growth factor (NGF) in the brain. The painless human Nerve Growth Factor (hNGFp) is a recombinant mutated form of human nerve growth factor (hNGF) that shows identical neurotrophic and neuroprotective properties of wild-type NGF but displays at least 10-fold lower algogenic activity. From the pharmacological point of view, hNGFp is a biased tropomyosin receptor kinase A (TrkA) agonist and displays a significantly lower affinity for the p75 neurotrophin receptor (p75NTR). This study aimed to evaluate the expression of TrkA and p75NTR NGF receptors in two different human microglia cell lines, and to investigate the effects of hNGFp and wild-type NGF (NGF) on L-arginine metabolism, taken as a marker of microglia polarization. Both NGF receptors are expressed in human microglia cell lines and are effective in transducing signals triggered by NGF and hNGFp. The latter and, to a lesser extent, NGF inhibit cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these cells. Conversely NGF but not hNGFp stimulates arginase-mediated urea production.

The effects of CHF6467, a new mutated form of NGF, on cell models of human glioblastoma. A comparison with wild-type NGF.

CHF6467 is a mutated form of human recombinant nerve growth factor (NGF). The mutation selectively disrupts the binding of NGF to its p75NTR receptor while maintaining the affinity toward TrkA receptor. Because of such different profile of receptor interaction, CHF6467 maintains unaltered the neurotrophic and neuroprotective properties of wild-type NGF but shows reduced algogenic activity.In this study, we investigated the effects of CHF6467 on mortality, proliferation, cell-damage and migration in three human glioblastoma cell lines (U87MG, T98G, LN18), and in the rat astrocytoma C6 cells. Both CHF6467 and wild-type NGF, given in the range 1-50 ng/ml, did not modify cell proliferation, metabolism and migration, as well as the number of live/dead cells.The present in vitro data are predictive of a lack of tumorigenic activity by both wild-type NGF and CHF6467 on these cell types in vivo, and warrant for CHF6467 further clinical development.

PDIA3 Expression in Glioblastoma Modulates Macrophage/Microglia Pro-Tumor Activation.

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia-glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia.

Pharmacokinetics of high-dose tigecycline in critically ill patients with severe infections.

BACKGROUND: In critically ill patients, the use of high tigecycline dosages (HD TGC) (200 mg/day) has been recently increasing but few pharmacokinetic/pharmacodynamic (PK/PD) data are available. We designed a prospective observational study to describe the pharmacokinetic/pharmacodynamic (PK/PD) profile of HD TGC in a cohort of critically ill patients with severe infections. RESULTS: This was a single centre, prospective, observational study that was conducted in the 20-bed mixed ICU of a 1500-bed teaching hospital in Rome, Italy. In all patients admitted to the ICU between 2015 and 2018, who received TGC (200 mg loading dose, then 100 mg q12) for the treatment of documented infections, serial blood samples were collected to measure steady-state TGC concentrations. Moreover, epithelial lining fluid (ELF) concentrations were determined in patients with nosocomial pneumonia. Amongst the 32 non-obese patients included, 11 had a treatment failure, whilst the other 21 subjects successfully eradicated the infection. There were no between-group differences in terms of demographic aspects and main comorbidities. In nosocomial pneumonia, for a target AUC0-24/MIC of 4.5, 75% of the patients would be successfully treated in presence of 0.5 mcg/mL MIC value and all the patients obtained the PK target with MIC ≤ 0.12 mcg/mL. In intra-abdominal infections (IAI), for a target AUC0-24/MIC of 6.96, at least 50% of the patients would be adequately treated against bacteria with MIC ≤ 0.5 mcg/mL. Finally, in skin and soft-tissue infections (SSTI), for a target AUC0-24/MIC of 17.9 only 25% of the patients obtained the PK target at MIC values of 0.5 mcg/mL and less than 10% were adequately treated against germs with MIC value ≥ 1 mcg/mL. HD TGC showed a relevant pulmonary penetration with a median and IQR ELF/plasma ratio (%) of 152.9 [73.5-386.8]. CONCLUSIONS: The use of HD TGC is associated with satisfactory plasmatic and pulmonary concentrations for the treatment of severe infections due to fully susceptible bacteria (MIC < 0.5 mcg/mL). Even higher dosages and combination strategies may be suggested in presence of difficult to treat pathogens, especially in case of SSTI and IAI.

DNA inhibitors for the treatment of brain tumors.

Introduction: The worldwide incidence of central nervous system (CNS) primary tumors is increasing. Most of the chemotherapeutic agents used for treating these cancer types induce DNA damage, and their activity is affected by the functional status of repair systems involved in the detection or correction of DNA lesions. Unfortunately, treatment of malignant high-grade tumors is still an unmet medical need.Areas covered: We summarize the action mechanisms of the main DNA inhibitors used for the treatment of brain tumors. In addition, studies on new agents or drug combinations investigated for this indication are reviewed, focusing our attention on clinical trials that in the last 3 years have been completed, terminated or are still recruiting patients.Expert opinion: Much still needs to be done to render aggressive CNS tumors curable or at least to transform them from lethal to chronic diseases, as it is possible for other cancer types. Drugs with improved penetration in the CNS, toxicity profile, and activity against primary and recurrent tumors are eagerly needed. Targeted agents with innovative mechanisms of action and ability to harness the cells of the tumor microenvironment against cancer cells represent a promising approach for improving the clinical outcome of CNS tumors.

Phospho-mTOR expression in human glioblastoma microglia-macrophage cells.

The glioblastoma (GBM) immune microenvironment is highly heterogeneous, and microglia may represent 30-70% of the entire tumor. However, the role of microglia and other specific immune populations is poorly characterized. Activation of mTOR signaling occurs in numerous human cancers and has roles in microglia-glioma cell interactions. We now show in human tumor specimens (42 patients), that 39% of tumor-associated microglial (TAM) cells express mTOR phosphorylated at Ser-2448; and similar mTOR activation is observed using a human microglia-glioma interaction paradigm. In addition, we confirm previous studies that microglia express urea and ARG1 (taken as M2 marker) in the presence of glioma cells, and this phenotype is down-regulated in the presence of a mTOR inhibitor. These results suggest that mTOR suppression in GBM patients might induce a reduction of the M2 phenotype expression in up to 40% of all TAMs. Since the M2 profile of microglial activation is believed to be associated with tumor progression, reductions in that phenotype may represent an additional anti-tumor mechanism of action of mTOR inhibitors, along with direct anti-proliferative activities.

Pro-Inflammatory Activation of A New Immortalized Human Microglia Cell Line.

: The characterization of human microglia has been hampered by poor availability of human cell sources. However, microglia is involved in the physiopathology of multiple sclerosis, Alzheimer's disease, Parkinson's disease, HIV dementia, retinal degenerative diseases, cancer, and many other conditions. Therefore, there is an important need to have experimental paradigms of human microglia characterized and usable to study the role of microglia in the different pathologies in which it is involved. In the present work, we carried out an extensive characterization of Immortalized Human Microglia-SV40 cell line (IMhu), marketed by Applied Biological Material. The functional response of IMhu to a large variety of stimuli was studied. In particular, we investigated morphology, mortality, and changes in the production of different cytokines and chemokines, both under basal conditions and after stimulation. Moreover, western blotting analysis was conducted on phospho-mTOR (Ser 2448) and downstream parameters, p-P70S6K and 4EBP1, in order to understand if IMhu can be used for evaluations of mTOR pathway. In conclusion, IMhu cells proved to be a useful experimental model to investigate the physiopathology of inflammatory disease that involved microglia cells, including pathological conditions that involved the mTOR pathway.