Functional Characterization of Erythropoiesis in Fyn-/- mice: a Novel Role of Fyn in Stress Erythropoiesis

Erythropoiesis is a dynamic complex multistep process going from committed erythroid progenitors to erythroid precursors and circulating mature red cells. Erythroid maturation is strictly dependent on EPO signaling cascade. EPO physically interacts with its receptor (EPO-R), which expression is downregulated after the basophilic erythroblasts stage.7 Binding of EPO to EPO-R results in EPO-R conformational change and it requires the activation of Jak2, as primary kinase. STAT5 is a master of erythropoiesis and it resides in cytoplasm. In response to EPO signaling, it binds phospho-tyrosine (Tyr) residues in the nucleus, initiating the transcription of several genes important in terminal erythroid differentiation. Giving the importance of Jak2 kinase as initiator of the EPO signaling cascade, additional kinases, such as Lyn, a Src family kinase, has been described to participate to EPO pathway. Lyn is able to phosphorylate EPO-R, Jak2 itself and STAT5. The activation of EPO/Jak2 signaling pathway is associated with production of reactive oxidative species (ROS), which are also generated by a large amount of iron imported into the cells during heme biosynthesis. During erythropoiesis, ROS might function as second messenger by modulating intracellular signaling pathways. Fyn, a Src kinase, has been previously reported to participate in signaling pathways in response to ROS in various cell types. Here, we explore the potential contribution of Fyn to normal and stress erythropoiesis by studying 2-4 months-old Fyn knockout mouse strain (Fyn-/-) and C57BL/6J as wild-type controls. Fyn-/- mice showed a mild compensated microcytic anemia associated with signs of dyserythropoiesis. Increased ROS levels and Annexin-V+ cells were presented in all Fyn-/- erythroblast subpopulations compared to wild-type, suggesting a possible reduction in the efficiency of erythropoietin (EPO) signaling pathway in the absence of Fyn. Indeed, in Fyn-/- erythroblasts we observed a reduction in Tyr-phosphorylation state of EPO-R associated with a compensatory activation of Jak2 without major change in Lyn activity. A reduction in STAT5 activation resulting in down-regulation of Cish, a known direct STAT5 target gene, was noted in Fyn-/- erythroblasts. This was paralleled by a reduction in GATA1 and increased HSP70 nuclear translocation compared to wild type, supporting a higher cellular prooxidant environment in the absence of Fyn. Using the vitro cell forming colony unit assay, we found a lower CFU-E and BFU-E cells production, which once again was associated with decreased activation of EPO mediated cascade in the absence of Fyn. To explore the possible role of Fyn in stress erythropoiesis, mice were treated with recombinant EPO, phenylhydrazine (PHZ) or doxorubicin (Doxo). Fyn-/- mice showed a low response to EPO compared to wild-type animals and prolonged anemia after either PHZ or Doxo treatment with a delayed hematologic recovery compared to wildtype mice. When we analyzed the expression of a battery of ARE-genes related to oxidative response such as catalase, Gpx, heme-oxygenase 1 and peroxiredoxin-2, we noted up-regulated expression of these genes in sorted Fyn-/- erythroblasts compared to wild-type cells. In agreement, we observed increased activation of the redox-sensitive transcriptional factor Nrf2 targeting ARE-genes, whose regulation has been previously linked to Fyn. In fact, Nrf2 is switched-off by Fyn, ubiquitylated and delivered to the autophagosome by the p62 cargo protein. In Fyn-/- sorted erythroblasts, we observed (i) accumulation of p62 in large clusters; and (ii) reduction of Nrf2-p62 complex compared to wildtype cells. To address the question whether the perturbation of Nrf2-p62 system results in impairment of autophagy in the absence of Fyn, we used LysoTracker to explore late phases of autophagy. Lysosomal progression was defective in Fyn-/- reticulocytes and it was associated with accumulation of p62 during in vitro reticulocyte maturation. These data indicate that the absence of Fyn blocks the Nrf2 post-induction response to oxidation, resulting in impaired autophagy. To validate our working hypothesis, we treated Fyn-/- mice with Rapamycin, an inducer of autophagy. In Fyn-/- mice, Rapamycin treatment resulted in decrease dyserythropoiesis, ROS levels and Annexin V+ cells, associated with reduction in accumulation of p62 in Fyn-/- erythroblasts. Collectively, our data 4 enabled us to document a novel role for Fyn in erythropoiesis, contributing to EPO-R activation and harmonizing the Nrf2-p62 adaptive cellular response against oxidation. Future studies will be designed to further characterize the signaling pathways intersects by Fyn in normal and diseased erythropoiesis

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of \u3b2-thalassemia

Anemia of \u3b2-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of \u3b2-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free \u3b1-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2\u3b1 phosphorylation. The improvement of \u3b2-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of \u3b2-thalassemia

Off-label long acting injectable antipsychotics in real-world clinical practice: a cross-sectional analysis of prescriptive patterns from the STAR Network DEPOT study

Introduction Information on the off-label use of Long-Acting Injectable (LAI) antipsychotics in the real world is lacking. In this study, we aimed to identify the sociodemographic and clinical features of patients treated with on- vs off-label LAIs and predictors of off-label First- or Second-Generation Antipsychotic (FGA vs. SGA) LAI choice in everyday clinical practice. Method In a naturalistic national cohort of 449 patients who initiated LAI treatment in the STAR Network Depot Study, two groups were identified based on off- or on-label prescriptions. A multivariate logistic regression analysis was used to test several clinically relevant variables and identify those associated with the choice of FGA vs SGA prescription in the off-label group. Results SGA LAIs were more commonly prescribed in everyday practice, without significant differences in their on- and off-label use. Approximately 1 in 4 patients received an off-label prescription. In the off-label group, the most frequent diagnoses were bipolar disorder (67.5%) or any personality disorder (23.7%). FGA vs SGA LAI choice was significantly associated with BPRS thought disorder (OR = 1.22, CI95% 1.04 to 1.43, p = 0.015) and hostility/suspiciousness (OR = 0.83, CI95% 0.71 to 0.97, p = 0.017) dimensions. The likelihood of receiving an SGA LAI grew steadily with the increase of the BPRS thought disturbance score. Conversely, a preference towards prescribing an FGA was observed with higher scores at the BPRS hostility/suspiciousness subscale. Conclusion Our study is the first to identify predictors of FGA vs SGA choice in patients treated with off-label LAI antipsychotics. Demographic characteristics, i.e. age, sex, and substance/alcohol use co-morbidities did not appear to influence the choice towards FGAs or SGAs. Despite a lack of evidence, clinicians tend to favour FGA over SGA LAIs in bipolar or personality disorder patients with relevant hostility. Further research is needed to evaluate treatment adherence and clinical effectiveness of these prescriptive patterns

The Role of Attitudes Toward Medication and Treatment Adherence in the Clinical Response to LAIs: Findings From the STAR Network Depot Study

Background: Long-acting injectable (LAI) antipsychotics are efficacious in managing psychotic symptoms in people affected by severe mental disorders, such as schizophrenia and bipolar disorder. The present study aimed to investigate whether attitude toward treatment and treatment adherence represent predictors of symptoms changes over time. Methods: The STAR Network \u201cDepot Study\u201d was a naturalistic, multicenter, observational, prospective study that enrolled people initiating a LAI without restrictions on diagnosis, clinical severity or setting. Participants from 32 Italian centers were assessed at three time points: baseline, 6-month, and 12-month follow-up. Psychopathological symptoms, attitude toward medication and treatment adherence were measured using the Brief Psychiatric Rating Scale (BPRS), the Drug Attitude Inventory (DAI-10) and the Kemp's 7-point scale, respectively. Linear mixed-effects models were used to evaluate whether attitude toward medication and treatment adherence independently predicted symptoms changes over time. Analyses were conducted on the overall sample and then stratified according to the baseline severity (BPRS < 41 or BPRS 65 41). Results: We included 461 participants of which 276 were males. The majority of participants had received a primary diagnosis of a schizophrenia spectrum disorder (71.80%) and initiated a treatment with a second-generation LAI (69.63%). BPRS, DAI-10, and Kemp's scale scores improved over time. Six linear regressions\u2014conducted considering the outcome and predictors at baseline, 6-month, and 12-month follow-up independently\u2014showed that both DAI-10 and Kemp's scale negatively associated with BPRS scores at the three considered time points. Linear mixed-effects models conducted on the overall sample did not show any significant association between attitude toward medication or treatment adherence and changes in psychiatric symptoms over time. However, after stratification according to baseline severity, we found that both DAI-10 and Kemp's scale negatively predicted changes in BPRS scores at 12-month follow-up regardless of baseline severity. The association at 6-month follow-up was confirmed only in the group with moderate or severe symptoms at baseline. Conclusion: Our findings corroborate the importance of improving the quality of relationship between clinicians and patients. Shared decision making and thorough discussions about benefits and side effects may improve the outcome in patients with severe mental disorders

Prescription appropriateness of anti-diabetes drugs in elderly patients hospitalized in a clinical setting: evidence from the REPOSI Register

Diabetes is an increasing global health burden with the highest prevalence (24.0%) observed in elderly people. Older diabetic adults have a greater risk of hospitalization and several geriatric syndromes than older nondiabetic adults. For these conditions, special care is required in prescribing therapies including anti- diabetes drugs. Aim of this study was to evaluate the appropriateness and the adherence to safety recommendations in the prescriptions of glucose-lowering drugs in hospitalized elderly patients with diabetes. Data for this cross-sectional study were obtained from the REgistro POliterapie-Società Italiana Medicina Interna (REPOSI) that collected clinical information on patients aged ≥ 65 years acutely admitted to Italian internal medicine and geriatric non-intensive care units (ICU) from 2010 up to 2019. Prescription appropriateness was assessed according to the 2019 AGS Beers Criteria and anti-diabetes drug data sheets.Among 5349 patients, 1624 (30.3%) had diagnosis of type 2 diabetes. At admission, 37.7% of diabetic patients received treatment with metformin, 37.3% insulin therapy, 16.4% sulfonylureas, and 11.4% glinides. Surprisingly, only 3.1% of diabetic patients were treated with new classes of anti- diabetes drugs. According to prescription criteria, at admission 15.4% of patients treated with metformin and 2.6% with sulfonylureas received inappropriately these treatments. At discharge, the inappropriateness of metformin therapy decreased (10.2%, P < 0.0001). According to Beers criteria, the inappropriate prescriptions of sulfonylureas raised to 29% both at admission and at discharge. This study shows a poor adherence to current guidelines on diabetes management in hospitalized elderly people with a high prevalence of inappropriate use of sulfonylureas according to the Beers criteria

Antidiabetic Drug Prescription Pattern in Hospitalized Older Patients with Diabetes

Objective: To describe the prescription pattern of antidiabetic and cardiovascular drugs in a cohort of hospitalized older patients with diabetes. Methods: Patients with diabetes aged 65 years or older hospitalized in internal medicine and/or geriatric wards throughout Italy and enrolled in the REPOSI (REgistro POliterapuie SIMI—Società Italiana di Medicina Interna) registry from 2010 to 2019 and discharged alive were included. Results: Among 1703 patients with diabetes, 1433 (84.2%) were on treatment with at least one antidiabetic drug at hospital admission, mainly prescribed as monotherapy with insulin (28.3%) or metformin (19.2%). The proportion of treated patients decreased at discharge (N = 1309, 76.9%), with a significant reduction over time. Among those prescribed, the proportion of those with insulin alone increased over time (p = 0.0066), while the proportion of those prescribed sulfonylureas decreased (p < 0.0001). Among patients receiving antidiabetic therapy at discharge, 1063 (81.2%) were also prescribed cardiovascular drugs, mainly with an antihypertensive drug alone or in combination (N = 777, 73.1%). Conclusion: The management of older patients with diabetes in a hospital setting is often sub-optimal, as shown by the increasing trend in insulin at discharge, even if an overall improvement has been highlighted by the prevalent decrease in sulfonylureas prescription

The “Diabetes Comorbidome”: A Different Way for Health Professionals to Approach the Comorbidity Burden of Diabetes

(1) Background: The disease burden related to diabetes is increasing greatly, particularly in older subjects. A more comprehensive approach towards the assessment and management of diabetes’ comorbidities is necessary. The aim of this study was to implement our previous data identifying and representing the prevalence of the comorbidities, their association with mortality, and the strength of their relationship in hospitalized elderly patients with diabetes, developing, at the same time, a new graphic representation model of the comorbidome called “Diabetes Comorbidome”. (2) Methods: Data were collected from the RePoSi register. Comorbidities, socio-demographic data, severity and comorbidity indexes (Cumulative Illness rating Scale CIRS-SI and CIRS-CI), and functional status (Barthel Index), were recorded. Mortality rates were assessed in hospital and 3 and 12 months after discharge. (3) Results: Of the 4714 hospitalized elderly patients, 1378 had diabetes. The comorbidities distribution showed that arterial hypertension (57.1%), ischemic heart disease (31.4%), chronic renal failure (28.8%), atrial fibrillation (25.6%), and COPD (22.7%), were the more frequent in subjects with diabetes. The graphic comorbidome showed that the strongest predictors of death at in hospital and at the 3-month follow-up were dementia and cancer. At the 1-year follow-up, cancer was the first comorbidity independently associated with mortality. (4) Conclusions: The “Diabetes Comorbidome” represents the perfect instrument for determining the prevalence of comorbidities and the strength of their relationship with risk of death, as well as the need for an effective treatment for improving clinical outcomes

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma

About 3-4% of cirrhotic patients develop primary liver cancer every year. Specific serologic markers have not yet been identified for screening of high risk patients. The serpin squamous cell carcinoma antigen (SCCA) is overexpressed in liver cancer and circulating SCCA-IgM complexes have been described in patients with hepatocellular carcinoma (HCC). The aim of the present study was to assess the behavior of SCCA-IgM in relation to HCC development in patients with cirrhosis. A retrospective, longitudinal study was conducted in a cohort of prospectively followed cirrhotic patients. Two groups with similar clinical profile at presentation were studied : group A included 16 patients who developed HCC during a median follow up of 4 years; group B included 17 patients who did not develop HCC during the same time interval. Circulating SCCA-IgM immune complexes were determined using a recently standardized ELISA assay. At presentation similar levels of SCCA-IgM complexes [mean +/- SD: 267.40 +/- 382.25 U/ml vs. 249.10 +/- 446.90 U/ml, p = 0.9006] and of alpha-fetoprotein [AFP; 24.11 +/- 59.04 IU/ml vs. 10.91 +/- 23.34 IU/ml, p = 0.3995] were detected in group A and in group B. The increase over time (phi) of SCCA-IgM, assessed within at least one year before clinical diagnosis of HCC, was remarkably higher in group A than in group B (mean +/- SD = 280.05 +/- 606.71 (U/ml)/year vs. -37.92 +/- 95.94 (U/ml)/year, p = 0.0408), while AFP increase was not significantly different (11.89 +/- 23.27 (IU/ml)/year vs. 3.67 +/- 11.46 (IU/ml)/year, p = 0.2179). Receiver operating characteristic (ROC) curves were plotted for the rate of change in the levels of both markers and the diagnostic accuracy measured as AUROC was higher for SCCA-IgM phi (0.821) than for AFP phi (0.654). In conclusion, the progressive increase of SCCA-IgM over time was associated with liver tumor development, suggesting that monitoring the behavior of SCCA-IgM might become useful to identify cirrhotic patients at higher risk of HCC development