DISSECTING THE ROLE OF THE CYTOPLASMIC MUTANT NUCLEOPHOSMIN IN ACUTE MYELOID LEUKAEMIA DEVELOPMENT

Acute myeloid leukaemia (AML) is a genetic heterogeneous group of diseases, with the largest subgroup showing a mutation in the Nucleophosmin gene (NPM1). Normally the NPM protein localizes mainly in the nucleolus, but in AML blasts it is aberrant localized to the cytoplasm (NPMc+AML). Notably, NPMc+AML patients show peculiar gene expression profiles, treatment response and prognosis. Hence, it has been proposed as an independent category for leukaemia classification according to WHO in 2008. In view of the relevance of NPMc+ mutation to AML pathogenesis and prognosis, understanding its role in leukaemia development represents a major issue in the field. The aim of this PhD project is to get further insight into the relevance of NPMc+ mutations to AML development. To this scope, here it is reported a characterization of a novel mouse model expressing the mutated protein. The hematopoietic restricted expression of the protein induces leukaemia in mice. This data definitively clarify that NPMc+ is an initiating mutation for leukaemia development. However, the long latency and low penetrance of disease onset strongly support the need of cooperating mutations. Since, the high frequency of FLT3-ITD mutations in NPMc+AML, we genetically tested the synergisms between the two abnormalities. To this scope, NPMc+ mice were crossed with FLT3-ITD mice (Lee, 2007). Double mutated mice developed leukaemia with sort latency and full penetrance indicating effective cooperation. Moreover, our data support the two hits model of tumourigenesis, where functional complementary mutations contribute to disease onset. Another major challenge of this project is to understand how NPMc+ affect the biology of normal HSPC and imposes the transition from normal to cancer stem cells. We found that NPMc+ expression perturbs the homeostasis of HSCP and expand the number of LT-HSC by increasing the proliferation rate. However, this enhanced proliferation is not associated to loss of quiescent and functional HSC, which may represent a reservoir of persistent pre-malignant cells available for the accumulation of additional genetic alteration. Further investigation into the biology of per-leukaemic stem cells may give insights into the molecular mechanisms imposed by the oncogene for malignancy transformation and finally may contribute for the development of new therapeutic strategies

GDM-complicated pregnancies: focus on adipokines

Gestational diabetes mellitus (GDM) is a serious complication of pregnancy and is defined as a state of glucose intolerance that is first diagnosed and arises during gestation. Although the pathophysiology of GDM has not yet been thoroughly clarified, insulin resistance and pancreatic β-cell dysfunction are considered critical components of its etiopathogenesis. To sustain fetus growth and guarantee mother health, many significant changes in maternal metabolism are required in normal and high-risk pregnancy accompanied by potential complications. Adipokines, adipose tissue-derived hormones, are proteins with pleiotropic functions including a strong metabolic influence in physiological conditions and during pregnancy too. A growing number of studies suggest that various adipokines including adiponectin, leptin, visfatin, resistin and tumor necrosis factor α (TNF-α) are dysregulated in GDM and might have pathological significance and a prognostic value in this pregnancy disorder. In this review, we will focus on the current knowledge on the role that the aforementioned adipokines play in the development and progression of GDM.Gestational diabetes mellitus (GDM) is a serious complication of pregnancy and is defined as a state of glucose intolerance that is first diagnosed and arises during gestation. Although the pathophysiology of GDM has not yet been thoroughly clarified, insulin resistance and pancreatic β-cell dysfunction are considered critical components of its etiopathogenesis. To sustain fetus growth and guarantee mother health, many significant changes in maternal metabolism are required in normal and high-risk pregnancy accompanied by potential complications. Adipokines, adipose tissue-derived hormones, are proteins with pleiotropic functions including a strong metabolic influence in physiological conditions and during pregnancy too. A growing number of studies suggest that various adipokines including adiponectin, leptin, visfatin, resistin and tumor necrosis factor α (TNF-α) are dysregulated in GDM and might have pathological significance and a prognostic value in this pregnancy disorder. In this review, we will focus on the current knowledge on the role that the aforementioned adipokines play in the development and progression of GDM

Regulation of NFKB through the nuclear processing of p105 (NFKB1) in Epstein-Barr Virus immortalized B cell lines.

Transcription factors of the NF-κB/Rel family are retained in the cytoplasm as inactive complexes through association with IκB inhibitory proteins. Several NF-κB activators induce the proteolysis of IκB proteins, which results in the nuclear translocation and DNA binding of NF-κB complexes. Here, we report a novel mechanism of NF-κB regulation mediated by p105 (NF-κB1) precursor of p50 directly at the nuclear level. In Epstein- Barr virus-immortalized B cells, p105 was found in the nucleus, where it was complexed with p65. In concomitance with NF-κB activation, mitomycin C induced the processing of p105 to p50 in the nucleus, while it did not affect the steady-state protein levels of IκBα and p105 in the cytoplasm. Differently, phorbol 12-myristate 13-acetate induced a significant proteolysis of both IκBα and p105 in the cytoplasm, while it did not affect the protein level of p105 in the nucleus. These results suggest that in Epstein-Barr virus-positive B cell lines the nuclear processing of p105 can contribute to NF-κB activation in response to specific signaling molecules, such as DNA-damaging agents

An NF-kB site in the 5'-untraslated leader region of the Human Immunodeficiency virus type 1 enhances the viral expression in response to NF-kB-activating stimuli.

The 5'-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-κB-responsive element in the trans- activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-κB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-α. The TAR NF-κB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-κB enhancer upstream of the TATA box in the NF-κB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-κB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-κB inducers; (ii) deletion or base pair substitutions of the TAR NF-κB site significantly reduce the HIV-1 LTR activation by NF-κB inducers; (iii) deletions of both the NF-κB sites upstream of the TATA box and the TAR NF- κB site abolish the activation of HIV-1 LTR in response to NF-κB inducers. Moreover, the p50·p65 NF-κB complex binds to the TAR NF-κB sequence and trans-activates the TAR NF-κB-directed expression. The identification of an additional NF-κB site in the HIV-1 LTR points to the relevance of NF-κB factors in the HIV-1 life cycle

Evaluation of low doses BPA-induced perturbation of glycemia by toxicogen-omics points to a primary role of pancreatic islets and to the mechanism of toxicity

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 ? 10-9 M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-[[ampi]]kappa;B pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-[[ampi]]kappa;B activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions

Molecular targets of developmental exposure to bisphenol A in diabesity: a focus on endoderm-derived organs

Several studies associate foetal human exposure to bisphenol A (BPA) to metabolic/endocrine diseases, mainly diabesity. They describe the role of BPA in the disruption of pancreatic beta cell, adipocyte and hepatocyte functions. Indeed, the complexity of the diabesity phenotype is due to the involvement of different endoderm-derived organs, all targets of BPA. Here, we analyse this point delineating a picture of different mechanisms of BPA toxicity in endoderm-derived organs leading to diabesity. Moving from epidemiological data, we summarize the in vivo experimental data of the BPA effects on endoderm-derived organs (thyroid, pancreas, liver, gut, prostate and lung) after prenatal exposure. Mainly, we gather molecular data evidencing harmful effects at low-dose exposure, pointing to the risk to human health. Although the fragmentation of molecular data does not allow a clear conclusion to be drawn, the present work indicates that the developmental exposure to BPA represents a risk for endoderm-derived organs development as it deregulates the gene expression from the earliest developmental stages. A more systematic analysis of BPA impact on the transcriptomes of endoderm-derived organs is still missing. Here, we suggest in vitro toxicogenomics approaches as a tool for the identification of common mechanisms of BPA toxicity leading to the diabesity in organs having the same developmental origin

Improvement of adiponectin in relation to physical performance and body composition in young obese males subjected to twenty-four weeks of training programs

Obesity and related metabolic diseases represent a worldwide health problem. The main factor predisposing to obesity is an unhealthy lifestyle including the lack of physical activity. A pivotal role in the etio-pathogenesis of obesity is carried out by adipose tissue, an endocrine organ secreting several adipokines involved in numerous metabolic and inflammatory processes. Among these, of particular importance is adiponectin, an adipokine involved in the regulation of insulin sensibility and in anti-inflammatory processes. The aim of the study was to determine the effects of 24 weeks of two different training programs polarized (POL) and threshold training (THR) on body composition, physical capacities and adiponectin expression. Thirteen male obese subjects (BMI: 32.0 ± 3.0 kg m-2) followed 24 weeks of two different training programs, POL and THR, consisting of walking or running (or a combination of the two methods) in their normal living conditions. Before (T0) and after the end of the program (T1), the assessment of body composition was assessed by bioelectrical impedance and the concentration of salivary and serum adiponectin was analyzed by enzyme-linked immunosorbent assay and western blotting. Although the results obtained did not show significant differences between the two training programs, body mass and body mass index decreased by a mean of −4.46 ± 2.90 kg and 1.43 ± 0.92 kg m−2 (P < 0.05). Fat mass decreased by −4.47 ± 2.78 kg (P < 0.05). V′O2max increased by a mean of 0.20 ± 0.26 L min−1 (P < 0.05) Also, we observed an increase in saliva and in serum of adiponectin concentrations at T1 compared to T0 by 4.72 ± 3.52 μg mL−1 and 5.22 ± 4.74 ng mL−1 (P < 0.05) respectively. Finally, we found significant correlations between Δ serum adiponectin and Δ Hip (R = −0.686, P = 0.001) and between Δ salivary adiponectin and ΔWaist (R = −0.678, P = 0.011). Our results suggest that a 24 weeks training program, independently from intensity and volume, induces an amelioration of body composition and fitness performance. These improvements are associated with an increase in total and HMW adiponectin expression in both saliva and in serum

Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

Cannabidiolic acid (CBDA) is the main precannabinoid in industrial hemp. It represents a common constituent of hemp seed oil, but mainly abundant in the aerial parts of the plant (including their processing waste). Thus, the optimization of fast and low-cost purification strategies is mandatory, as well as a deep investigation on its nutraceutical and cosmeceutical properties. To this purpose, CBDA content in hemp seed oil is evaluated, and its recovery from wasted leaves is favorably achieved. The cytotoxicity screening towards HaCaT cells, by means of MTT, SRB and LDH release assays, suggested it was not able to decrease cell viability or perturb cell integrity up to 10 μM concentration. Thus, the ability of CBDA to differentially modulate the release of proinflammatory cytokines and chemokines mediators has been evaluated, finding that CBDA decreased IFN-γ, CXCL8, CXCL10, CCL2, CCL4 and CCL5, mostly in a dose-dependent manner, with 10 μM tested concentration exerting the highest activity. These data, together with those from assessing antimicrobial activity against Gram(+) and Gram(-) bacteria and the antibiofilm formation, suggest that CBDA is able to counteract the inflammatory response, also preventing bacteria colonization

The role of diabetes in metastatic melanoma patients treated with nivolumab plus relatlimab

Background The combination of nivolumab + relatlimab is superior to nivolumab alone in the treatment of naive patients and has activity in PD-1 refractory melanoma. We had previously observed a reduced expression of LAG3 in melanoma tissue from patients with type 2 diabetes. Method To evaluate the impact of diabetes on oncological outcomes of patients with advanced melanoma treated with nivolumab plus the LAG3 inhibitor relatlimab we performed a retrospective multicenter study. Results Overall, 129 patients were included: 88 without diabetes before the treatment, 37 who were diagnosed with type 2 diabetes before the start of treatment, and 4 without diabetes before treatment who developed immune checkpoint inhibitor-induced diabetes (ICI-DM). PFS was 21.71 months (95% CI: 15.61–27.81) in patients without diabetes, 10.23 months (95% CI: 5.81–14.66) in patients with type 2 diabetes, and 50.85 months (95% CI: 23.04–78.65) in patients who developed ICI-DM. OS was 37.94 months (95% CI: 31.02–44.85) in patients without diabetes, 22.12 months (95% CI: 14.41–29.85) in those with type 2 diabetes and 57.64 months (95% CI: 42.29–72.99) in those who developed ICI-DM. Multivariate analysis showed that the presence of diabetes and LDH was correlated with OS and PFS. The mean OS was 64.63 months in subjects with low levels of glucose ( 1.5) had a worse prognosis than those whose glucose level had not increased. This result was observed also in subgroups treated either in first line or further lines. Patients who developed ICI-DM during the study period had better outcomes than the overall population and patients without diabetes. Conclusions LAG3 inhibition for treating metastatic or unresectable melanoma has a reduced efficacy in patients with type 2 diabetes, possibly due to a low expression of LAG3 in tumor tissue. Higher level evidence should be obtained