Diagnostic leukapheresis reveals distinct phenotypes of NSCLC circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs. METHODS: DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations. RESULTS: Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes. CONCLUSIONS: In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

A Drosophila model of cigarette smoke induced COPD identifies Nrf2 signaling as an expedient target for intervention.

Chronic obstructive pulmonary disease (COPD) is among the most important causes of death. Signaling systems that are relevant for tissue repair and detoxification of reactive oxygen species or xenobiotics are thought to be impaired in lungs of patients suffering from this disease. Here, we developed a simple cigarette smoke induced Drosophila model of COPD based on chronic cigarette smoke exposure that recapitulates major pathological hallmarks of the disease and thus can be used to investigate new therapeutic strategies. Chronic cigarette smoke exposure led to premature death of the animals and induced a set of phenotypes reminiscent of those seen in COPD patients, including reduced physical activity, reduced body fat, increased metabolic rate and a substantial reduction of the respiratory surface. A detailed transcriptomic analysis revealed that especially the TGF-ß, Nrf2 and the JAK/STAT signaling pathways are altered by chronic cigarette smoke exposure. Based on these results, we focused on Nrf2 signaling. A pharmacological intervention study performed with oltipraz, an activator of Nrf2 signaling, increased survival of cigarette smoke exposed animals significantly. Thus, the Drosophila COPD model recapitulates many major hallmarks of COPD and it is highly useful to evaluate the potential of alternative therapeutic strategies.

Safety and efficacy of fingolimod in patients with relapsing-remitting multiple sclerosis (FREEDOMS II): a double-blind, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: Fingolimod has shown reductions in clinical and MRI disease activity in patients with relapsing-remitting multiple sclerosis. We further assessed the efficacy and safety of fingolimod in such patients. METHODS: We did this placebo-controlled, double-blind phase 3 study predominantly in the USA (101 of 117 centres). Using a computer-generated sequence, we randomly allocated eligible patients-those aged 18-55 years with relapsing-remitting multiple sclerosis-to receive fingolimod 0·5 mg, fingolimod 1·25 mg, or placebo orally once daily (1:1:1; stratified by study centre). On Nov 12, 2009, all patients assigned to fingolimod 1·25 mg were switched to the 0·5 mg dose in a blinded manner after a review of data from other phase 3 trials and recommendation from the data and safety monitoring board, but were analysed as being in the 1·25 mg group in the primary outcome analysis. Our primary endpoint was annualised relapse rate at month 24, analysed by intention to treat. Secondary endpoints included percentage brain volume change (PBVC) from baseline and time-to-disability-progression confirmed at 3 months. This trial is registered with ClinicalTrilals.gov, number NCT00355134. FINDINGS: Between June 30, 2006, and March 4, 2009, we enrolled and randomly allocated 1083 patients: 370 to fingolimod 1·25 mg, 358 to fingolimod 0·5 mg, and 355 to placebo. Mean annualised relapse rate was 0·40 (95% CI 0·34-0·48) in patients given placebo and 0·21 (0·17-0·25) in patients given fingolimod 0·5 mg: rate ratio 0·52 (95% CI 0·40-0·66; p<0·0001), corresponding to a reduction of 48% with fingolimod 0·5 mg versus placebo. Mean PBVC was -0·86 (SD 1·22) for fingolimod 0·5 mg versus -1·28 (1·50) for placebo (treatment difference -0·41, 95% CI -0·62 to -0·20; p=0·0002). We recorded no statistically significant between-group difference in confirmed disability progression (hazard rate 0·83 with fingolimod 0·5 mg vs placebo; 95% CI 0·61-1·12; p=0·227). Fingolimod 0·5 mg caused more of the following adverse events versus placebo: lymphopenia (27 [8%] patients vs 0 patients), increased alanine aminotransferase (29 [8%] vs six [2%]), herpes zoster infection (nine [3%] vs three [1%]), hypertension (32 [9%] vs 11 [3%]), first-dose bradycardia (five [1%] vs one [<0·5%]), and first-degree atrioventricular block (17 [5%] vs seven [2%]). 53 (15%) of 358 patients given fingolimod 0·5 mg and 45 (13%) of 355 patients given placebo had serious adverse events over 24 months, which included basal-cell carcinoma (ten [3%] patients vs two [1%] patients), macular oedema (three [1%] vs two [1%]), infections (11 [3%] vs four [1%]), and neoplasms (13 [4%] vs eight [2%]). INTERPRETATION: Our findings expand knowledge of the safety profile of fingolimod and strengthen evidence for its beneficial effects on relapse rates in patients with relapsing-remitting multiple sclerosis. We saw no effect of fingolimod on disability progression. Our findings substantiate the beneficial profile of fingolimod as a disease-modifying agent in the management of patients with relapsing-remitting multiple sclerosis. FUNDING: Novartis Pharma AG.

Fingolimod in relapsing multiple sclerosis: An integrated analysis of safety findings.

BACKGROUND: Fingolimod 0.5mg once daily is the first approved oral therapy for relapsing multiple sclerosis (MS). OBJECTIVE: To report integrated long-term safety data from phase 2/3 fingolimod studies. METHODS: Descriptive safety data are reported from the FTY720 Research Evaluating Effects of Daily Oral Therapy in Multiple Sclerosis (FREEDOMS) study, a 24-month, randomized, double-blind study comparing fingolimod 0.5mg and 1.25mg with placebo, and an All Studies group (patients who received fingolimod 0.5mg (n=1640) or 1.25-0.5mg (n=1776) in phase 2/3 studies and associated extensions). Relevant post-marketing experience, up to December 2011, is included. RESULTS: The incidence of adverse events (AEs) and serious AEs (SAEs) was similar with fingolimod and placebo in FREEDOMS. In the All Studies group, fingolimod 0.5mg was associated with transient, rarely symptomatic (0.5%), bradycardia and second-degree atrioventricular block on treatment initiation, minor blood pressure increases, frequent (9%) but generally asymptomatic liver enzyme elevations, and macular oedema (0.4%). The incidences of infections (including serious and herpes infections), malignancies, SAEs and treatment discontinuations due to AEs were similar with fingolimod 0.5mg and placebo. CONCLUSION: The safety profile of fingolimod has been well characterized in this large combined trial population. Although infrequent SAEs can occur, there is no increased risk of infections, malignancies or serious cardiovascular events versus placebo.

Induction of periimplantitis in dental implants.

Development, progression, and therapy of periimplantitis are nonresolved emerging problems. The aim of this pilot study was to establish a model for periimplantitis in mice to have a base for tests with immune-deficient knockout organisms to improve the knowledge about development and progression of periimplantitis and to develop further therapeutic options.In 8 mice, titanium implants were inserted in the median of the palate. Four of these implants had ligatures (periimplantitis group). After 2 weeks, the animals received a special diet enriched with sugar and flavor. After 9 weeks, micro-computed tomography (micro-CT) examinations to evaluate the periimplant tissue and histologies were performed.Dental implant insertions within the oral cavity are possible in living mice. Implants with ligatures showed significantly larger periimplant bone defects than controls. The radiologic findings were confirmed by histology. At the end of the observation period, the portion of implants lost was higher in the ligature group.This is the first publication to describe the insertion of dental implants in living mice. In addition, it is the first time that periimplant infection could be induced in that species. This model will pave the way to study knockout mice with reduced or even enhanced resistance to periimplantitis.

Deep sequencing reveals differential expression of microRNAs in favorable versus unfavorable neuroblastoma.

Small non-coding RNAs, in particular microRNAs(miRNAs), regulate fine-tuning of gene expression and can act as oncogenes or tumor suppressor genes. Differential miRNA expression has been reported to be of functional relevance for tumor biology. Using next-generation sequencing, the unbiased and absolute quantification of the small RNA transcriptome is now feasible. Neuroblastoma(NB) is an embryonal tumor with highly variable clinical course. We analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads. MiRNA expression profiles obtained by deep sequencing correlated well with real-time PCR data. Cluster analysis differentiated between favorable and unfavorable NBs, and the miRNA transcriptomes of these two groups were significantly different. Oncogenic miRNAs of the miR17-92 cluster and the miR-181 family were overexpressed in unfavorable NBs. In contrast, the putative tumor suppressive microRNAs, miR-542-5p and miR-628, were expressed in favorable NBs and virtually absent in unfavorable NBs. In-depth sequence analysis revealed extensive post-transcriptional miRNA editing. Of 13 identified novel miRNAs, three were further analyzed, and expression could be confirmed in a cohort of 70 NBs.

Brivaracetam (UCB 34714).

Brivaracetam (UCB 34714) is chemically related to levetiracetam (LEV, Keppra). It possesses a binding affinity for the synaptic vesicle protein 2A (SV2A) ten-fold above that of LEV and also shows an ability to inhibit Na+ channels. This correlates with a higher potency in suppressing epileptiform responses in vitro and a more potent and complete suppression of different seizure types in animals with an acquired or genetic epilepsy. Brivaracetam has been tested in a comprehensive safety pharmacology, toxicology, developmental toxicology, and genotoxicity program. It is of low acute toxicity, target organ for toxic effects is the hepatobiliary tract. Carcinogenicity studies are ongoing. Human pharmacology studies have shown that brivaracetam has a half-life of 8 h and nearly complete bioavailability. Brivaracetam is primarily metabolized via hydrolysis of the acetamide group and CYP2C8-mediated hydroxylation. Its metabolites are not pharmacologically active. Excretion of over 95% of the dose, including metabolites, occurs renally within 72 h. Healthy volunteer studies demonstrated a favorable tolerability profile. Treatment emergent adverse events were mild to moderate, mostly of CNS origin, and resolved within 24 hrs, with decreasing incidence after repeated intake. Drug-drug interaction studies with high dose of brivaracetam (400 mg/d) showed a dose-dependent increase of carbamazepine-epoxide levels. No significant interaction with low doses of phenytoin was observed at the same high dose levels of brivaracetam, and only a moderate pharmacokinetic interaction with an oral contraceptive, without impact on hormonal levels or ovulation, was observed. The pharmacokinetic profile of brivaracetam is unaltered in elderly subjects or those with impaired renal function. Clearance of brivaracetam is reduced in patients with hepatic insufficiency. In the photoparoxysmal response model in patients with photosensitive epilepsy brivaracetam was effective at all tested doses (10 - 80 mg) in reducing or abolishing EEG discharges evoked by a photic stimulus. Phase 2 studies in patients with refractory partial onset seizures have recently been completed.