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.

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.

[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.

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.

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.

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.

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.

Spectrum of F8 gene mutations in haemophilia A patients from a region of Italy: identification of 23 new mutations.

SUMMARY: Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by a lack or decrease of coagulation factor VIII activity. The molecular diagnosis of HA is challenging and a variety of different mutations have been identified throughout the F8 gene. Our aim was to detect the causative mutation in 266 HA patients from Emilia-Romagna region (Italy) and in all suspected carriers. Molecular analysis of F8 in 201 HA patients (152 index cases) was performed with a combination of several indirect and direct molecular approaches, such as long distance polymerase chain reaction, multiplex ligation-dependent probe amplification, denaturing high performance liquid chromatography and direct sequencing. The analysis revealed 78 different mutations, 23 of which were novel, not having been reported in national or international databases. The detection rate was 100%, 86% and 89% in patients with severe, moderate and mild HA, respectively. The information provided by this registry will be helpful for monitoring the treatment of HA patients in Emilia-Romagna and also for reliable genetic counselling of affected families in the future.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

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.

Neglect-associated constructional disorders: a paradoxical phenomenon?

Five neglect patients without diffuse cognitive impairment or overt constructional disabilities were asked to bisect lines and rectangles and to copy rectangles bisected in their midplane. As a group, patients showed the usual rightward bias in bisecting lines and a milder deviation in bisecting horizontally-aligned rectangles, but showed a leftward deviation of the subjective midline in the copying task. This was due to drawing the left half shorter with respect to normal controls but three patients also drew the right half longer (the total length was the same as that of controls). A possible interpretation of rectangle copying results in these three patients is that they could create a representation of the stimulus to be copied accurately enough to reproduce its total length correctly but the subjective distribution of right and left space within that representation was unbalanced. However, specific experimental work is needed to verify why our patients with mild to moderate unilateral spatial neglect overrepresented the left side in a line bisection task and underrepresented it in a copying task.

A modified functional global test to measure protein C, protein S activities and the activated protein C-resistance phenotype.

Identifying a defect affecting the protein C/protein S (PC/PS) anticoagulant system, using a single global test, has recently become possible thanks to a new methodological approach based on the activation of endogenous plasma PC by Protac, derived from Agkistrodon Contortix snake venom (ACV). The introduction of a commercial test (ProC Global), ACV-based, provides a useful tool for the screening of thrombotic patients since the most frequent causes of inherited thrombophilia are found in the PC/PS system. The test provides information only on the global activity of the anticoagulant pathway but not on PC and PS activity or on the factor V related conditions (e.g., FV Leiden). The present study shows that by carrying out the test alternating the presence of PC-, PS-, or FV-deficient plasma and using appropriate amounts of ACV, it is possible to increase the specificity of the test to correctly evaluate respectively the PC or PS activities or the activated protein C resistance condition (APC-R). These simple modifications applied to the original commercial test allow to detect exactly, using a single, basic methodology, the principal defects affecting the PC/PS anticoagulant pathway. Furthermore, carrying out the tests on an automated coagulometer, in combination or not with the classic ProC Global assay, it is possible to use a unique reagent profile to simultaneously investigate in the same or different samples, the PC, PS, and APC-R defect.

A photometric method for the dosage of factor XIII applied to the study of chronic hepatopathies.

The photometric method of Fickenscher et al. for the determination of factor XIII (FXIII) activity has been used in the study of 35 patients with severe chronic hepatopathy, in comparison with 25 normal subjects. The FXIII proteic fractions a and b were determined by quantitative immuno-electrophoresis after Laurell. The plasmatic FXIII activity, as well as the proteic fractions a and b, were significantly reduced in hepatopatic patients, in comparison to controls, and proportional to the prolongation of prothrombin times. Ratios between functional and immunological levels of FXIII in hepatopatics were similar to those observed in controls. These results confirm the involvement of fibrin stabilization deficiency in the coagulation defect of severe chronic hepatopathies. The correlations between functional and antigenic values are in agreement with the hepatic origin of FXIII. The method of Fickenscher has been proved to be rapid and simple, and it may be useful in the routine study of hepatopathies, for a better knowledge of the role of FXIII deficiency in the complex coagulopathy of liver diseases, as well as of other acquired FXIII deficiencies.