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Francesco Raggi,1 Chiara Rossi,1 Francesco Faita,2 Mariarosaria Distaso,1 Claudia Kusmic,2 Anna Solini1 1Department of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Pisa, Italy; 2Institute of Clinical Physiology, Italian National Research Council, Pisa, ItalyCorrespondence: Anna Solini; Francesco Raggi, Department of Surgical Medical Molecular and Critical Area Pathology, University of Pisa, Via Roma 67, Pisa, I-56126, Italy, Tel +39-50-993482 ; +39-50-992861, Fax +39-50-553235, Email anna.solini@med.unipi.it; francesco.raggi@unipi.itPurpose: Low-grade inflammation contributes to heart failure in obesity or type 2 diabetes mellitus. The P2X7 receptor (P2X7R) is a key regulator of several pro-inflammatory responses in multiple tissues and organs; however, its involvement in the onset of heart dysfunction remains unclear. The study evaluated the role of P2X7R as a cardiac function regulator in C57BL/6J wild-type (WT) and P2X7R knockout (KO) mice by inducing systemic inflammation with high fat diet (HFD).Methods: Specific parameters of systolic and diastolic function and heart morphology were measured in vivo before animal sacrifice by high-frequency ultrasonographic analysis. Gene and protein expression of cardiac biomarkers associated with inflammatory-oxidative pathways were evaluated by real-time PCR and Western Blotting.Results: P2X7R-mediated up-regulation of the NLRP3-caspase-1 complex, increased expression of key oxidative stress (NOS-2, TNFα), and chemotactic (MCP-1) mediators were revealed in WT-HFD animals. In KO-HFD mice, such inflammatory-oxidative pathway was silent. Nevertheless, HFD induced in vivo a clear alteration of diastolic pattern (E/A: p < 0.03 vs WT-HFD) and a cardiac morphologic remodelling (left ventricular mass: p < 0.05 vs WT-HFD) only in P2X7R KO animals. Surprisingly, the transcriptional and protein expression of IL-1β and IL-6, usually regulated through P2X7R activation, were significantly higher in KO-HFD than in WT-HFD mice (both p < 0.05). Furthermore, an up-regulation of miR-214 and a down-regulation of miR-126 in heart of HFD-KO mice were observed, suggesting a link between such epigenetic dysregulation and cytokine overexpression as a potential pathophysiologic mechanism concurring to the progressive cardiac dysfunction.Conclusion: These findings seem to suggest a cardioprotective role of P2X7R toward this tissue-specific inflammatory damage, likely through tissue homeostasis and organ functionality preservation.Keywords: low-grade inflammation, high fat diet, diastolic dysfunction, P2X7 receptor

Edoardo Biancalana,1,* Martina Chiriacò,1,* Paolo Sciarrone,1 Alessandro Mengozzi,1 Sandra Mechelli,2 Stefano Taddei,1 Anna Solini3 1Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy; 2Section of General Medicine IV AUOP, University of Pisa, Pisa, Italy; 3Department of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Pisa, Italy*These authors contributed equally to this workCorrespondence: Anna SoliniDepartment of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Pisa, I-56126, ItalyTel +39-050993482Fax +39-050553235Email anna.solini@med.unipi.itBackground: Remdesivir, an antiviral agent able to reduce inflammatory cascade accompanying severe, life-threatening pneumonia, became the first drug approved by the Food and Drug Administration for the treatment of hospitalized patients with coronavirus 2 related severe acute respiratory syndrome (SARS CoV2). As from its previously known clinical indications, the use of remdesivir in the presence of severe renal impairment is contraindicated; however, the impact of remdesivir on renal function in aging patients has not been elucidated.Subjects and Methods: This retrospective observational study involved 109 individuals consecutively admitted in internal medicine section, Azienda Ospedaliero Universitaria Pisana hospital, in November–December 2020 due to a confirmed diagnosis of SARS CoV2 and receiving remdesivir according to international inclusion criteria. Biochemical variables at admission were evaluated, together with slopes of estimated glomerular filtration rate (eGFR) built during remdesivir treatment. Participants were followed until discharge or exitus.Results: Patients were stratified according to age (80 formed the study cohort and 29 served as controls); CKD stage III was present in 46% of them. No patients showed any sign of deteriorated renal function during remdesivir. Fourteen patients in the elderly cohort deceased; their eGFR at baseline was significantly lower. Recovered patients were characterized by a relevant eGFR gaining during remdesivir treatment.Conclusion: We show here for the first time as remdesivir does not influence eGFR in a cohort of elderly people hospitalized for SARS CoV2, and that eGFR gain during such treatment is coupled with a better prognosis.Keywords: remdesivir, SARS CoV2, glomerular filtration rate, aging, chronic kidney disease

Abstract Background A Chronic Kidney Disease (CKD) Epidemiology Collaboration (EPI) formula not including a Black race coefficient has been recently developed and is now recommended in the US. The new (2021) equation was shown to yield higher estimated glomerular filtration rate (eGFR) values than the old (2009) one in a non-Black general population sample, thus reclassifying a significant number of individuals to a better eGFR category. However, reclassified individuals were previously shown to have a lower risk of progression to end-stage kidney disease, but higher adjusted risks for all-cause death and morbidity and mortality from cardiovascular disease than those not reclassified. This study evaluated the prognostic impact of switching from the 2009 to the 2021 CKD-EPI equation in non-Black individuals with type 2 diabetes. Methods The Renal Insufficiency And Cardiovascular Events (RIACE) was a prospective cohort study enrolling 15,773 Caucasian patients in 19 Italian centers in 2006–2008. Cardiometabolic risk profile, treatments, complications, and comorbidities were assessed at baseline and eGFR was calculated with the two equations. Vital status was retrieved on 31 October 2015 for 15,656 participants (99.3%). Results With the 2021 equation, the eGFR value increased in all patients, except for 293 individuals with a 2009 eGFR ≥ 105 ml·min− 1·1.73 m− 2. The median difference was 4.10 ml·min− 1·1.73 m− 2 and was higher in males, older individuals and those in the G2 category. Reclassification decreased the percentage of patients with reduced eGFR from 17.28 to 13.96% and with any CKD from 36.23 to 34.03%. Reclassified individuals had better cardiometabolic risk profile and lower prevalence of complications and use of medications than non-reclassified individuals. Risk of death versus the 2009 G1 category was lower for reclassified than non-reclassified participants in all eGFR categories and, particularly, in each 2009 eGFR category, though difference was significant only in the G4-G5 category. The Receiver Operator Characteristic curves were statistically, but not clinically different with the two equations. Conclusion Changing from the 2009 to the 2021 CKD-EPI equation results in higher eGFR and lower CKD prevalence, with a lower risk of death in reclassified patients with an eGFR

Abstract Background Metabolic dysfunction-associated Steatotic Liver Disease (MASLD) displays a worse prognosis in subjects with type 2 diabetes (T2D); effective treatments are, so far, scanty. Semaglutide showed efficacy in improving steatohepatitis. We longitudinally observed a MASLD cohort of T2D subjects starting semaglutide, to detect an improvement of non-invasive surrogates of steatosis and fibro-inflammatory liver involvement, evaluating the role of mild alcohol consumption. Patients and methods In 62 overweight/obese T2D subjects with MASLD (36 non-drinker and 26 mild alcohol consumers), anthropometric, bio-humoral and transient elastography (TE) data were collected before (T0) and after an average time of 6.4 month (T1) from injective semaglutide prescription. Circulating levels of hormones (GIP, GLP-1, glucagon, insulin) and inflammatory markers (TNFα, MCP-1, IL-18, IL-10) were measured. Steatotic and necro-inflammatory liver involvement was evaluated with FibroScan controlled attenuation parameter (CAP) and liver stiffness (LS), respectively. Results Significant (p

Abstract Background Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. Results By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host’s regulatory mechanisms to create an environment conducive for virion production. Conclusions The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Abstract Background Foot ulcers and/or infections are common long-term complications of diabetes and are associated with increased mortality, especially from cardiovascular disease, though only a few studies have investigated the independent contribution of these events to risk of death. This study aimed at assessing the association of history of diabetic foot with all-cause mortality in individuals with type 2 diabetes, independent of cardiovascular risk factors, other complications, and comorbidities. Methods This prospective cohort study enrolled 15,773 Caucasian patients in 19 Italian centers in the years 2006–2008. Prior lower extremity, coronary, and cerebrovascular events and major comorbidities were ascertained by medical records, diabetic retinopathy by fundoscopy, diabetic kidney disease by albuminuria and estimated glomerular filtration rate, cardiovascular risk factors by standard methods. All-cause mortality was retrieved for 15,656 patients on 31 October 2015. Results At baseline, 892 patients (5.7%) had a history of diabetic foot, including ulcer/gangrene and/or amputation (n = 565; 3.58%), with (n = 126; 0.80%) or without (n = 439; 2.78%) lower limb revascularization, and revascularization alone (n = 330; 2.09%). History of diabetic foot was associated with all-cause death over a 7.42-year follow-up (adjusted hazard ratio, 1.502 [95% confidence interval, 1.346–1.676], p

Obesity is a heterogeneous condition which results from complex interactions among sex/gender, sociocultural, environmental, and biological factors. Obesity is more prevalent in women in most developed countries, and several clinical and psychological obesity complications show sex-specific patterns. Females differ regarding fat distribution, with males tending to store more visceral fat, which is highly correlated to increased cardiovascular risk. Although women are more likely to be diagnosed with obesity and appear more motivated to lose weight, as confirmed by their greater representation in clinical trials, males show better outcomes in terms of body weight and intra-abdominal fat loss and improvements in the metabolic risk profile. However, only a few relatively recent studies have investigated gender differences in obesity, and sex/gender is rarely considered in the assessment and management of the disease. This review summarizes the evidence of gender differences in obesity prevalence, contributing factors, clinical complications, and psychological challenges. In addition, we explored gender differences in response to obesity treatments in the specific context of new anti-obesity drugs.

Summary: Background: Despite the overall improvement in care, people with type 2 diabetes (T2D) experience an excess risk of end-stage kidney disease. We evaluated the long-term effectiveness of dapagliflozin on kidney function and albuminuria in patients with T2D. Methods: We included patients with T2D who initiated dapagliflozin or comparators from 2015 to 2020. Propensity score matching (PSM) was performed to balance the two groups. The primary endpoint was the change in estimated glomerular filtration rate (eGFR) from baseline to the end of observation. Secondary endpoints included changes in albuminuria and loss of kidney function. Findings: We analysed two matched groups of 6197 patients each. The comparator group included DPP-4 inhibitors (40%), GLP-1RA (22.3%), sulphonylureas (16.1%), pioglitazone (8%), metformin (5.8%), or acarbose (4%). Only 6.4% had baseline eGFR 30 mg/g. During a mean follow-up of 2.5 year, eGFR declined significantly less in the dapagliflozin vs comparator group by 1.81 ml/min/1.73 m2 (95% C.I. from 1.13 to 2.48; p

BackgroundDietary fat consumption, involved in the pathogenesis of insulin resistance and impaired glucose metabolism, is linked with decline in cognitive functions, dementia, and development of Parkinson’s disease and Alzheimer’s disease. Mature IL-1β, requiring the activation of the P2X7 receptor (P2X7R)-inflammasome complex, is an important mediator of neuroinflammation. The aim of the study was to test whether P2X7R activation might interfere with systemic and cerebral metabolic homeostasis.MethodsWe treated WT and P2X7R KO mice with a high-fat diet (HFD) for 16 weeks, evaluating the effects on the Substantia Nigra and Hippocampus, target areas of damage in several forms of cognitive impairment.ResultsHFD-treated WT and P2X7R KO mice showed a different brain mRNA profile of Insulin and Igf-1, with these genes and relative receptors, more expressed in KO mice. Unlike P2X7R KO mice, WT mice treated with HFD displayed a diameter reduction in dopaminergic neurons in the Substantia Nigra, accompanied by an increased IBA1 expression in this area; they also showed poor performances during Y-Maze and Morris Water Maze, tasks involving Hippocampus activity. Conversely, Parkin, whose reduction might promote neuronal cell death, was increased in the brain of P2X7R KO animals.ConclusionWe report for the first time that HFD induces damage in dopaminergic neurons of the Substantia Nigra and a Hippocampus-related worse cognitive performance, both attenuated in the absence of P2X7R. The involved mechanisms might differ in the two brain areas, with a predominant role of inflammation in the Substantia Nigra and a metabolic derangement in the Hippocampus.

Abstract Background Endothelial dysfunction is an independent risk factor for cardiovascular events. Inflammatory mediators released by the adipose tissue can lead to local insulin resistance and endothelial dysfunction. This study addressed the relationship of adipocytokines with endothelial function and blood pressure. Methods In 92 newly diagnosed, drug-naïve essential hypertensive patients (HT, mean age 49 yrs) without organ damage and 66 normotensive subjects (NT, mean age 47 yrs), by an automated system, we measured endothelium-dependent and -independent vasodilation as brachial artery flow-mediated dilation before and after administration of glyceryl-trinitrate. Retinol binding protein-4 (RBP4) and resistin levels were determined by ELISA and RIA, respectively. Oxidative stress was evaluated by measuring serum malondyaldehyde (MDA). Results Flow-mediated dilation was significantly (p = 0.03) lower in HT (5.3 ± 2.6%) than NT (6.1 ± 3.1%), while response to glyceryl-trinitrate (7.5 ± 3.7% vs 7.9 ± 3.4%) was similar. RBP4 (60.6 ± 25.1 vs 61.3 ± 25.9 μg/ml), resistin (18.8 ± 5.3 vs 19.9 ± 6.1 ng/ml) and MDA levels (2.39 ± 1.26 vs 2.08 ± 1.17 nmol/ml) were not different in HT and NT. RBP4 (r = −0.25; p = 0.04) and resistin levels (r = −0.29; p = 0.03) were related to flow-mediated dilation in NT, but not in HT (r = −0.03 and r = −0.10, respectively). In NT, multivariate analysis including RBP4 and confounders showed that only BMI or waist circumference remained related to flow- mediated dilation. In the multivariate model including resistin and confounders, BMI, age and resistin were significantly related to flow-mediated dilation, while only age significant correlated with this parameter when BMI was replaced by waist circumference. Conclusions Adipocytokine levels may be independent predictors of endothelial dysfunction in the peripheral circulation of healthy subjects, providing a pathophysiological link between inflammation from adipose tissue and early vascular alterations.

Sodium glucose cotransporter 2 inhibitors have proven strong efficacy in reducing end-stage renal disease in patients with type 2 diabetes. We are presenting here the case of a 40-year-old woman with acquired partial lipodystrophy, type 2 diabetes and essential hypertension complicated by chronic kidney disease and proteinuria in the nephrotic range. She first came to our attention in 2012; estimated glomerular filtration rate (eGFR) was 41.5 ml/min/1.73 m2 and total proteinuria was 375 mg/24h; she was treated with dual renin angiotensin system blocking. Proteinuria significantly increased during the following years, reaching a nephrotic range (>5 g/day). A kidney biopsy revealed a tubule-interstitial involvement compatible with type 2 diabetes. Leptin replacement therapy, started in 2018, improved glycaemic control and lipid profile, also determining a reduction in insulin total daily dose. In 2019, after the publication of the CREDENCE study, canagliflozin was started on top of losartan and ramipril. After an initial, expected eGFR drop, kidney function stabilized, and albuminuria significantly reduced (from 4120 to 984 mg/24h), while serum potassium showed only minimal increase. At last follow-up (2022) total proteinuria was still reducing (510 mg/24h), while kidney function was substantially unchanged (eGFR 40 ml/min/1.73 m2). This case report suggests that, despite not recommended in international guidelines, the use of SGLT2i in combination with dual renin angiotensin system blockade should be considered in specific conditions and under close clinical monitoring.