Sex and Gender Differences in Neurodegenerative Diseases: Challenges for Therapeutic Opportunities.

The term "neurodegenerative diseases" (NDs) identifies a group of heterogeneous diseases characterized by progressive loss of selectively vulnerable populations of neurons, which progressively deteriorates over time, leading to neuronal dysfunction. Protein aggregation and neuronal loss have been considered the most characteristic hallmarks of NDs, but growing evidence confirms that significant dysregulation of innate immune pathways plays a crucial role as well. NDs vary from multiple sclerosis, in which the autoimmune inflammatory component is predominant, to more "classical" NDs, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy. Of interest, many of the clinical differences reported in NDs seem to be closely linked to sex, which may be justified by the significant changes in immune mechanisms between affected females and males. In this review, we examined some of the most studied NDs by looking at their pathogenic and phenotypical features to highlight sex-related discrepancies, if any, with particular interest in the individuals' responses to treatment. We believe that pointing out these differences in clinical practice may help achieve more successful precision and personalized care.

The Extent of Intracellular Accumulation of Bilirubin Determines Its Anti- or Pro-Oxidant Effect.

BACKGROUND: Severe hyperbilirubinemia can cause permanent neurological damage in particular in neonates, whereas mildly elevated serum bilirubin protects from various oxidative stress-mediated diseases. The present work aimed to establish the intracellular unconjugated bilirubin concentrations (iUCB) thresholds differentiating between anti- and pro-oxidant effects. METHODS: Hepatic (HepG2), heart endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to increasing concentration of bilirubin. iUCB, cytotoxicity, intracellular reactive oxygen species (ROS) concentrations, and antioxidant capacity (50% efficacy concentration (EC50)) were determined. RESULTS: Exposure of SH-SY5Y to UCB concentration > 3.6 µM (iUCB of 25 ng/mg) and >15 µM in H5V and HK2 cells (iUCB of 40 ng/mg) increased intracellular ROS production (p < 0.05). EC50 of the antioxidant activity was 21 µM (iUCB between 5.4 and 21 ng/mg) in HepG2 cells, 0.68 µM (iUCB between 3.3 and 7.5 ng/mg) in SH-SY5Y cells, 2.4 µM (iUCB between 3 and 6.7 ng/mg) in HK2 cells, and 4 µM (iUCB between 4.7 and 7.5 ng/mg) in H5V cells. CONCLUSIONS: In all the cell lines studied, iUCB of around 7 ng/mg protein had antioxidant activities, while iUCB > 25 ng/mg protein resulted in a prooxidant and cytotoxic effects. UCB metabolism was found to be cell-specific resulting in different iUCB.

Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses.

Dendritic cells (DC) have the unique capacities to induce primary T-cell responses. In mice, CD8α(+)DC are specialized to cross-prime CD8(+) T cells and produce interleukin-12 (IL-12) that promotes cytotoxicity. Human BDCA-3(+)DC share several relevant characteristics with CD8α(+)DC, but the capacities of human DC subsets to induce CD8(+) T-cell responses are incompletely understood. Here we compared CD1c(+) myeloid DC (mDC)1, BDCA-3(+)mDC2, and plasmacytoid DC (pDC) in peripheral blood and lymphoid tissues for phenotype, cytokine production, and their capacities to prime cytotoxic T cells. mDC1 were surprisingly the only human DC that secreted high amounts of IL-12p70, but they required combinational Toll-like receptor (TLR) stimulation. mDC2 and pDC produced interferon-λ and interferon-α, respectively. Importantly, mDC1 and mDC2 required different combinations of TLR ligands to cross-present protein antigens to CD8(+) T cells. pDC were inefficient and also expressed lower levels of major histocompatibility complex and co-stimulatory molecules. Nevertheless, all DC induced CD8(+) memory T-cell expansions upon licensing by CD4(+) T cells, and primed naive CD8(+) T cells following appropriate TLR stimulation. However, because mDC1 produced IL-12, they induced the highest levels of cytotoxic molecules. In conclusion, CD1c(+)mDC1 are the relevant source of IL-12 for naive T cells and are fully equipped to cross-prime cytotoxic T-cell responses.

Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIß. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIß isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV--by recruiting PI4KIIIα in the RNA replication complex--hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.

Translational Approach to the Protective Effect of Bilirubin in Diabetic Kidney Disease.

Bilirubin has been regarded as a powerful endogenous antioxidant and anti-inflammatory molecule, able to act on cellular pathways as a hormone. Diabetic kidney disease (DKD) is a common chronic complication of diabetes, and it is the leading cause of end-stage renal disease. Here, we will review the clinical and molecular features of mild hyperbilirubinemia in DKD. The pathogenesis of DKD involves oxidative stress, inflammation, fibrosis, and apoptosis. Serum bilirubin levels are positively correlated with the levels of the antioxidative enzymes as superoxide dismutase, catalase, and glutathione peroxidase, while it is inversely correlated with C-reactive protein, TNF-α, interleukin (IL)-2, IL-6, and IL-10 release in diabetic kidney disease. Bilirubin downregulates NADPH oxidase, reduces the induction of pro-fibrotic factor HIF-1α expression, cleaved caspase-3, and cleaved PARP induction showing lower DNA fragmentation. Recent experimental and clinical studies have demonstrated its effects in the development and progression of renal diseases, pointing out that only very mild elevations of bilirubin concentrations result in real clinical benefits. Future controlled studies are needed to explore the precise role of bilirubin in the pathogenesis of DKD and to understand if the use of serum bilirubin levels as a marker of progression or therapeutic target in DKD is feasible and realistic.

Life-Long Hyperbilirubinemia Exposure and Bilirubin Priming Prevent In Vitro Metabolic Damage.

Background: Unconjugated bilirubin (UCB) is more than the final product of heme catabolism. Mildly elevated systemic bilirubin concentrations, such as in Gilbert syndrome (GS), protect against various oxidative stress-mediated and metabolic diseases, including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, cancer, and age-related disease. The Gunn rat is an animal model of hereditary hyperbilirubinemia widely used in assessing the effect of high serum bilirubin concentration in various organs. The present work aims to understand if life-long hyperbilirubinemia and bilirubin-priming might contribute to protection against atherosclerosis and diabetic nephropathy (DN) at the cellular level. Methods: Primary aortic endothelial cells and podocytes obtained from hyperbilirubinemic homozygous jj and normobilirubinemic heterozygous Nj Gunn rats were exposed to Palmitic Acid (PA) and Angiotensin II (Ang II), respectively, and the effects on cell viability and the activation of damage-related metabolic pathways evaluated. Results were validated on immortalized H5V and HK2 cells exposed to damage after UCB pretreatment. Results: In both primary cell models, cells obtained from jj Gunn rats showed as significantly higher than Nj Gunn rats at any dose of the toxic agent. Reduction in CHOP expression and IL-6 release was observed in jj primary aortic endothelial cells exposed to PA compared to Nj cells. The same occurred on H5V pretreated with Unconjugated bilirubin. Upon Ang II treatment, primary podocytes from jj Gunn rats showed lower DNA fragmentation, cleaved caspase-3, and cleaved PARP induction than primary podocytes from Nj Gunn rats. In HK2 cells, the induction by Ang II of HIF-1α and LOXl2 was significantly reduced by UCB pretreatment. Conclusion: Our data suggest that in models of atherosclerosis and DN life-long hyperbilirubinemia exposure or bilirubin-priming significantly contribute to decrease the injury by enhancing thecellular defensive response.

Association of Serum Bilirubin Level with Metabolic Syndrome and Non-Alcoholic Fatty Liver Disease: A Cross-Sectional Study of 1672 Obese Children.

As in adults, obesity also plays a central role in the development of metabolic syndrome (MS) in children. Non-alcoholic fatty liver disease (NAFLD) is considered a manifestation of MS. Not only MS but also NAFLD seem to be inversely associated with serum bilirubin concentrations, an important endogenous tissue protector when only mild elevated. The aim of the study was to evaluate the association between serum bilirubin levels and the prevalence of MS and NAFLD in Italian obese children and adolescents. A retrospective cross-sectional study was performed in 1672 patients aged from 5 to 18 years. Clinical and laboratory parameters were assessed. NAFLD was measured by liver ultrasonography. The study was approved by the Ethical Committee of the Istituto Auxologico Italiano (research project code 1C021_2020, acronym BILOB). MS was present in 24% and fatty liver (FL) in 38% of this population. Bilirubin was not associated with FL and MS as a whole, but it was inversely associated only with selected components of MS, i.e., large WC, high blood pressure and high triglycerides. Our data suggest that bilirubin is not protective against MS and NAFLD in the presence of severe obesity.

Hepatitis C Virus Deletion Mutants Are Found in Individuals Chronically Infected with Genotype 1 Hepatitis C Virus in Association with Age, High Viral Load and Liver Inflammatory Activity.

Hepatitis C virus (HCV) variants characterized by genomic deletions in the structural protein region have been sporadically detected in liver and serum of hepatitis C patients. These defective genomes are capable of autonomous RNA replication and are packaged into infectious viral particles in cells co-infected with the wild-type virus. The prevalence of such forms in the chronically HCV-infected population and the impact on the severity of liver disease or treatment outcome are currently unknown. In order to determine the prevalence of HCV defective variants and to study their association with clinical characteristics, a screening campaign was performed on pre-therapy serum samples from a well-characterized cohort of previously untreated genotype 1 HCV-infected patients who received treatment with PEG-IFNα and RBV. 132 subjects were successfully analyzed for the presence of defective species exploiting a long-distance nested PCR assay. HCV forms with deletions predominantly affecting E1, E2 and p7 proteins were found in a surprising high fraction of the subjects (25/132, 19%). Their presence was associated with patient older age, higher viral load and increased necroinflammatory activity in the liver. While the presence of circulating HCV carrying deletions in the E1-p7 region did not appear to significantly influence sustained virological response rates to PEG-IFNα/RBV, our study indicates that the presence of these subgenomic HCV mutants could be associated with virological relapse in patients who did not have detectable viremia at the end of the treatment.