In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency.

BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

Predictors of lung entrapment in malignant pleural effusion.

INTRODUCTION: Malignant pleural effusion (MPE) is a common complication in advanced stages of malignancy and is associated with poor prognosis. Non-expandable lung (NEL) often occurs and its presence influences the MPE approach. Our main objective was to assess risk factors for malignant NEL. METHODS: Patients diagnosed with pathologically confirmed MPE between January 2012 and December 2018 in our institution were retrospectively analyzed. Demographic and clinical data of patients were reviewed and compared according to the presence or absence of NEL. A univariate and multivariate binary logistic regression analysis were used to determine predictors of the development of NEL. RESULTS: Of 365 patients included, 68 (18.6%) had NEL. After multivariate analysis, we found that loculated MPE (OR 8.63, 95%CI 4.30-17.33, p<0.001), complete hemithorax opacification (OR 2.81, 95%CI 1.17-6.76, p<0.021), lung cancer (OR 2.09, 95%CI 1.01-4.31, p=0.047) and higher effusion-serum LDH ratio (OR 1.09, 95%CI 1.00-1.17, p=0.039) were independent predictors of malignant NEL. There were no significant differences compared with expandable lung group regarding time from primary malignancy diagnosis to MPE diagnosis (3.0, IQR 0.0-75.8 vs 2.0, IQR 0.0-75.5 weeks, p=0.942) or MPE symptoms onset to MPE diagnosis (4.0, IQR 1.0-9.0 vs 3.0, IQR 1.0-9.0 weeks, p=0.497). Patients with NEL had a higher number of therapeutic pleural drainages (3.0, IQR 2.0-6.0 vs 2.0, IQR 1.0-3.0; p<0.001) and longer hospital stay (32.5, IQR 15.5-46.3 vs 21.0, IQR 11.0-36.0, p=0.007), measured in hospitalization days until the end of life, than patients with expandable lung. The rate of recurrence of pleural effusion was not significantly different between groups (p=0.291). Overall survival (OS) was 3.0 (95%CI, 2.3-3.7) months, regardless of lung expandability (p=0.923). CONCLUSION: Loculated MPE, complete hemithorax opacification, lung cancer and a higher effusion-serum LDH ratio were found to be independent predictors for NEL. These patients underwent thoracocenteses more frequently and had longer hospitalization days, although without significant impact in the OS.

Predictors of immune-related adverse events and outcomes in patients with NSCLC treated with immune-checkpoint inhibitors.

OBJECTIVE: To identify predictors of immune-related adverse events (IRAEs) in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). Assess associations between outcomes and the development of IRAEs. METHODS: Retrospective analysis of patients with NSCLC treated with ICIs between 2016 and 2020 in the Pulmonology Department of our hospital. Patients with and without IRAEs were compared. A logistic regression analysis was performed to determine predictors of IRAEs. Progression-free survival (PFS) and overall survival (OS) curves were calculated using the Kaplan-Meier method, and the long-rank test was used to assess survival differences between groups. Univariate and multivariate Cox proportional-hazards regression models were used to identify factors associated with PFS and OS. The value considered statistically significant was p≤0.05. RESULTS: A total of 184 patients (77.7% men, mean age 66.9±9.5 years) treated with ICIs were analyzed. During follow-up, 49 (26.6%) patients developed IRAEs and 149 (81.0%) died. According to the multivariate logistic regression analysis, treatment with statins (OR:3.15; p = 0.007), previous systemic corticosteroid therapy (OR:3.99; p = 0.001), disease controlled as response to ICI (OR:5.93; p < 0.001) and higher hemoglobin values (OR:1.28; p = 0.040) were independent predictors for the development of IRAEs. Patients who developed IRAEs had significantly longer medians of PFS (41.0 vs 9.0 weeks, p < 0.001) and OS (89.0 vs 28.0 weeks; p < 0.001). CONCLUSIONS: Patients treated with statins, pre-ICI systemic corticosteroids, higher baseline hemoglobin value and controlled disease as initial response to ICI had a higher risk of developing IRAEs. The development of IRAEs was associated with better outcomes.

OSA patients not treated with PAP - Evolution over 5 years according to the Baveno classification and cardiovascular outcomes.

INTRODUCTION: The evolution of patients with obstructive sleep apnea (OSA) non-eligible for PAP-therapy at diagnosis is unknown. Currently, the severity of OSA is based on the apnea-hypopnea index (AHI), but its prognostic relevance has raised concerns. The Baveno classification may allow a better stratification of severity and therapeutic guidance in OSA. METHODS: Patients with AHI≥5/h in 2015, classified into Baveno groups A and B and non-eligible for PAP therapy at diagnosis and over 5 years, were analyzed. Patients were reclassified into Baveno groups (A-D) and changes in groups over 5 years were explored. Patients in Baveno groups C and D, who developed major cardiovascular comorbidities (CVC) or end-organ damage (EOD group), were compared with patients in Baveno groups A and B (non-EOD group). To identify predictors of the development of major CVC or EOD, a logistic regression analysis was performed. RESULTS: There were 76 patients, 58% male, mean age 51.9 ± 10.1 years, mean body mass index (BMI) of 30.3 ± 5.0 kg/m2 and median AHI of 8.9 (5.9-12.0) events/h. At diagnosis, 46% and 54% of patients were classified into Baveno group A and group B, respectively. In total, 21% of patients developed major CVC or EOD (Baveno group C or D); higher age (p = 0.011) and BMI (p = 0.004) and a higher percentage of central apneas (p = 0.012) at diagnosis significantly predicted it, while sex, sleepiness, insomnia, AHI, ODI and T90 were not. CONCLUSIONS: A significant percentage of patients non-eligible for PAP-therapy at diagnosis of OSA developed CVC or EOD; higher age and BMI and a higher percentage of central apneas were significant predictors.

TMD splitting functions in [Formula: see text] factorization: the real contribution to the gluon-to-gluon splitting.

We calculate the transverse momentum dependent gluon-to-gluon splitting function within [Formula: see text]-factorization, generalizing the framework employed in the calculation of the quark splitting functions in Hautmann et al. (Nucl Phys B 865:54-66, arXiv:1205.1759, 2012), Gituliar et al. (JHEP 01:181, arXiv:1511.08439, 2016), Hentschinski et al. (Phys Rev D 94(11):114013, arXiv:1607.01507, 2016) and demonstrate at the same time the consistency of the extended formalism with previous results. While existing versions of [Formula: see text] factorized evolution equations contain already a gluon-to-gluon splitting function i.e. the leading order Balitsky-Fadin-Kuraev-Lipatov (BFKL) kernel or the Ciafaloni-Catani-Fiorani-Marchesini (CCFM) kernel, the obtained splitting function has the important property that it reduces both to the leading order BFKL kernel in the high energy limit, to the Dokshitzer-Gribov-Lipatov-Altarelli-Parisi (DGLAP) gluon-to-gluon splitting function in the collinear limit as well as to the CCFM kernel in the soft limit. At the same time we demonstrate that this splitting kernel can be obtained from a direct calculation of the QCD Feynman diagrams, based on a combined implementation of the Curci-Furmanski-Petronzio formalism for the calculation of the collinear splitting functions and the framework of high energy factorization.

Young microbes for adult obesity.

Gut microbes are active participants of host metabolism. At birth, child physiology is committed towards healthiness or sickness depending, in part, on maternal condition (i.e. lean vs obesity) and delivery. Finally, changes from breastfeeding to solid food also account to define gut microbiota ecology in adulthood. Nowadays, alterations of gut microbiota, named dysbiosis, are acquired risk factors for multiple diseases, especially type 2 diabetes and obesity. Despite important evidence linking nutrition to dysbiosis to energetic dysmetabolism, molecular mechanisms for causality are still missing. That the status of gut microbiota of mother and child is crucial for future diseases is witnessed by adulthood overweight and obesity observed in children with dysbiosis. In this short review we highlight the importance of early life events related to the microbiota and their impact on future adult disease risk. Therefore, our effort to treat or prevent metabolic diseases should be addressed towards early or previous life steps, when microbial decisions are going to affect our metabolic fate.

Factor XIII-A dynamics in acute myocardial infarction: a novel prognostic biomarker?

After acute myocardial infarction (MI) the damaged heart has to be repaired. Factor XIII (FXIII) is considered a key molecule in promoting heart healing. FXIII deficiency was associated to cardiac rupture and anomalous remodelling in MI. During MI, FXIII contributes firstly to the intracoronary thrombus formation and shortly after to heal the myocardial lesion. To quantify the real contribution of FXIII in this process, and to explore its possible prognostic role, we monitored the FXIII-A subunit levels in 350 acute MI patients during the first six days (d0-d5) plus a control at 30-60 days (d30). A one-year follow-up was performed for all the patients. A transient drop in the FXIII-A mean level was noted in the whole cohort of patients (FXIII-Ad0 99.48 ± 30.5 vs FXIII-Ad5 76.51 ± 27.02; p< 0.0001). Interestingly, those who developed post-MI heart failure showed the highest drop (FXIII-Ad5 52.1 ± 25.2) and they already presented with low levels at recruitment. Similarly, those who died showed the same FXIII-A dynamic (FXIII-Ad5 54.0 ± 22.5). Conversely, patients who remained free of major adverse cardiac events, had lower consuming (FXIII-Ad0 103.6 ± 29.1 vs FXIII-Ad5 84.4 ± 24.5; p< 0.0001). Interestingly, the FXIII-A drop was independent from the amount of injury assessed by TnT and CKMB levels. The survival analysis ascribed an increased probability of early death or heart failure inversely related to FXIII-A quartiles (FXIII-A25th< 59.5 %; hazard ratio 4.25; 2.2-5.1; p< 0.0001). Different FXIII-A dynamics and levels could be utilised as early prognostic indicators during acute MI, revealing the individual potential to heal and suggesting tailored treatments to avoid heart failure or its extreme consequence.

Managing the manager: gut microbes, stem cells and metabolism.

One major discovery of the last decade in the field of metabolic diseases is that the microorganisms comprising the gut microbiota are now considered a metabolic "organ", modulating multiple functions of the host, such as intestinal immune system maturation, adiposity, cardiac metabolism, liver triglyceride storage, and brain development and behaviour. The corresponding mechanisms involve increased energy harvesting through the production by microbiota of short-chain fatty acids for use by the host, and the release of pro-inflammatory compounds, such as lipopolysaccharide (LPS), flagellin and peptidoglycan. In particular, a high-fat diet (HFD) modifies gut microbiota, resulting in an increase of plasma LPS levels known as "metabolic endotoxaemia", a major driver of the onset of metabolic diseases through a CD14-dependent mechanism. The LPS-sensitive cell types can be seen within bone marrow-derived cells (BMC), which are involved in the development of inflammation in the adipose tissue of obese and type 2 diabetic mice. Furthermore, the expression of LPS receptor/cofactor CD14 cells from the stromal vascular fraction of adipose depots can also be directly targeted by LPS to initiate precursor cell development and adiposity. Moreover, data from the literature also indicate an impact of gut microbiota on intestinal stem cells. Thus, this mini review presents the experimental evidence supporting a relationship between gut microbiota and stem cells as a new axis of metabolic homoeostasis control.

GRB 130427A: a nearby ordinary monster.

Long-duration gamma-ray bursts (GRBs) are an extremely rare outcome of the collapse of massive stars and are typically found in the distant universe. Because of its intrinsic luminosity (L ~ 3 × 10(53) ergs per second) and its relative proximity (z = 0.34), GRB 130427A reached the highest fluence observed in the γ-ray band. Here, we present a comprehensive multiwavelength view of GRB 130427A with Swift, the 2-meter Liverpool and Faulkes telescopes, and by other ground-based facilities, highlighting the evolution of the burst emission from the prompt to the afterglow phase. The properties of GRB 130427A are similar to those of the most luminous, high-redshift GRBs, suggesting that a common central engine is responsible for producing GRBs in both the contemporary and the early universe and over the full range of GRB isotropic energies.

Inflammation and insulin resistance exert dual effects on adipose tissue tumor protein 53 expression.

OBJECTIVE: The purpose of this study was to investigate the expression of human adipose tissue protein 53 (p53) in subjects who varied widely in terms of obesity and insulin resistance. We also analyzed different in vivo and in vitro models to try to comprehend the associations found in humans. METHODS: p53 was analyzed in human adipose and isolated adipocytes, in high fat-fed and GLP-1R KO mice, during in vitro adipogenesis, and in adipocytes after high glucose, rosiglitazone and inflammatory conditions. The effects of surgery-induced weight loss and ex vivo metformin were also evaluated. RESULTS: Omental (OM) p53 gene expression (+27%, P=0.001) and protein (+11%, P=0.04) were increased in obese subjects and high fat diet-induced obese mice (+86%, P=0.018). Although the obesity-associated inflammatory milieu was associated with increased OM p53, this was negatively related to insulin resistance and glycated hemoglobin, and positively with biomarkers for insulin sensitivity. Multiple linear regression analyses revealed that glycated hemoglobin (P<0.0001) and body mass index (P=0.048) contributed independently to explain 13.7% (P<0.0001) of the OM p53 variance. Accordingly, the improvement of insulin sensitivity with surgery-induced weight loss (+51%, P=0.01) and metformin (+42%, P=0.02) led to increased adipose p53. While the glucose-intolerant GLP-1R KO mice showed decreased mesenteric p53 (-45.4%, P=0.017), high glucose led to decreased p53 in pre-adipocytes (-27%, P<0.0001). Inflammatory treatments led to increased p53 (+35%, P<0.0001), while Rs downregulated this expression (-40%, P=0.005) in mature adipocytes. CONCLUSION: Inflammation and insulin resistance exert dual effects on adipose p53, which seems to be the final result of these opposing forces.

[Metabolic therapy at the edge between human hosts and gut microbes]. / La thérapie métabolique à l'interface entre l'homme et le microbiote intestinal.

Personalized medicine is becoming day-after-day more urgent taking into account the great diversity characterizing patients affected by a given pathology, especially metabolic diseases. In fact, antidiabetic/obesity treatments have shown a reduced or no effect at all in some patients, representing a major challenge physicians have to face worldwide. Therefore, efforts have to be put to identify individual factors affecting our susceptibility towards a given medication. In that regard, gut microbiota may stand for the missing piece of the metabolic puzzle regulating host response, since its role in the induction of metabolic diseases has now been achieved. In fact, we firstly provided a bacterial explanation for the low-grade chronic inflammation featuring metabolic diseases, by showing the lipopolysaccharide as a trigger and risk factor of such pathologies. However, despite similar lineages of microbes characterize the gut of people, important differences still remain, which may be responsible for opposite effect of treatments such as pre- or probiotics, whose efficacy seems to be governed by the own gut microbiota of subjects. We have recently shown that gut microbiota is associated to the inclination to resist or not high-fat diet-induced type 2 diabetes in mice. In addition, the direct targeting of gut microbes by dietary fibers reversed the observed metabolic phenotype. These results, together with the literature, strongly suggest gut microbiota as a new target for the development of personalized metabolic therapy.

[Impact of periodontal disease on arterial pressure in diabetic mice]. / Impact de la maladie parodontale sur la pression artérielle des souris diabétiques.

Diabetes-driven cardiovascular diseases represent a high challenge for developed countries. Periodontal disease is strictly linked to the aforementioned diseases, due to its Gram negative-driven inflammation. Thus, we investigated the effects of periodontal disease on arterial pressure during the development of diabetes in mice. To this aim, C57BL/6 female mice were colonized with pathogens of periodontal tissue (Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum) for 1month, whereas another group of mice did not undergo the colonization. Subsequently, all mice were fed a high-fat carbohydrate-free diet for 3months. Then, arterial pressure was measured in vivo and a tomodensitometric analysis of mandibles was realized as well. Our results show increased mandibular bone-loss induced by colonization with periopathogens. In addition, periodontal infection augmented glucose-intolerance and systolic and diastolic arterial pressure, parameters already known to be affected by a fat-diet. In conclusion, we show here that periodontal disease amplifies metabolic troubles and deregulates arterial pressure, emerging as a new axis of metabolic investigation.

Circulating lipopolysaccharide-binding protein (LBP) as a marker of obesity-related insulin resistance.

OBJECTIVE: Lipopolysaccharide-binding protein (LBP) is a 65-kDa acute-phase protein present in blood at high concentrations, known to be derived from the liver. We aimed to gain insights into the association of circulating LBP with insulin resistance in humans and mice. METHODS, DESIGN AND MEASUREMENTS: We studied the cross-sectional (n=222) and weight loss-induced (n=34) associations of LBP (enzyme-linked immunosorbent assay) with inflammatory and metabolic parameters (including minimal model-measured insulin sensitivity), and the effects of high-fat diet (HFD), metformin and genetic insulin sensitization (glucagon-like peptide 1 receptor knockout model) in mice. RESULTS: Circulating LBP concentration was significantly increased in subjects with type 2 diabetes and dramatically increased in subjects with morbid obesity. LBP was significantly associated with insulin sensitivity and different inflammatory markers and decreased after weight loss (22.2 ± 5.8 vs 16.2 ± 9.3 µg ml(-1), P<0.0001) in association with changes in body mass index and insulin sensitivity. Circulating LBP concentration was increased in HFD mice, whereas decreased in glucagon-like peptide 1 receptor knockout mice (significantly more insulin sensitive than wild-type mice) and after metformin administration. CONCLUSION: LBP is an inflammatory marker associated with obesity-related insulin resistance.

Involvement of tissue bacteria in the onset of diabetes in humans: evidence for a concept.

AIMS/HYPOTHESIS: Evidence suggests that bacterial components in blood could play an early role in events leading to diabetes. To test this hypothesis, we studied the capacity of a broadly specific bacterial marker (16S rDNA) to predict the onset of diabetes and obesity in a general population. METHODS: Data from an Epidemiological Study on the Insulin Resistance Syndrome (D.E.S.I.R.) is a longitudinal study with the primary aim of describing the history of the metabolic syndrome. The 16S rDNA concentration was measured in blood at baseline and its relationship with incident diabetes and obesity over 9 years of follow-up was assessed. In addition, in a nested case-control study in which participants later developed diabetes, bacterial phylotypes present in blood were identified by pyrosequencing of the overall 16S rDNA gene content. RESULTS: We analysed 3,280 participants without diabetes or obesity at baseline. The 16S rDNA concentration was higher in those destined to have diabetes. No difference was observed regarding obesity. However, the 16S rDNA concentration was higher in those who had abdominal adiposity at the end of follow-up. The adjusted OR (95% CIs) for incident diabetes and for abdominal adiposity were 1.35 (1.11, 1.60), p = 0.002 and 1.18 (1.03, 1.34), p = 0.01, respectively. Moreover, pyrosequencing analyses showed that participants destined to have diabetes and the controls shared a core blood microbiota, mostly composed of the Proteobacteria phylum (85-90%). CONCLUSIONS/INTERPRETATION: 16S rDNA was shown to be an independent marker of the risk of diabetes. These findings are evidence for the concept that tissue bacteria are involved in the onset of diabetes in humans.