Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis

Generalized pustular psoriasis (GPP) is a rare and yet potentially lethal clinical variant of psoriasis, characterized by the formation of sterile cutaneous pustules, neutrophilia, fever and features of systemic inflammation. We sequenced the exomes of five unrelated individuals diagnosed with GPP. Nonsynonymous, splice-site, insertion, and deletion variants with an estimated population frequency of <0.01 were considered as candidate pathogenic mutations. A homozygous c.338C>T (p.Ser113Leu) missense substitution of IL36RN was identified in two individuals, with a third subject found to be a compound heterozygote for c.338C>T (p.Ser113Leu) and a c.142C>T (p.Arg48Trp) missense substitution. IL36RN (previously known as IL1F5) encodes an IL-1 family receptor antagonist, which opposes the activity of the IL-36A and IL-36G innate cytokines. Homology searches revealed that GPP mutations alter evolutionarily conserved residues. Homozygosity for the c.338C>T (p.Ser113Leu) variant is associated with an elevated proinflammatory response following ex vivo stimulation with IL36A. These findings suggest loss of function of IL36RN as the genetic basis of GPP and implicate innate immune dysregulation in this severe episodic inflammatory disease, thereby highlighting IL-1 signaling as a potential target for therapeutic intervention.     

The nerve growth factor administrated as eye drops activates mature and precursor cells in subventricular zone of adult rats

The possibility to take advantage from the nerve growth factor (NGF) ability to induce recovery of damaged tissue has been largely explored in animal models and humans. Recently, the successful use of the ocular administration of NGF in ophthalmology, and the evidences that from the eyes NGF can access to the brain have stimulated new fields of research and open further perspectives to the clinical application of this neurotrophin. In our previous studies we have demonstrated the efficacy of NGF eye drop treatment to improved behavioural deficits and recover structural and biochemical alterations occurring follow brain lesion in animals. Since NGF exerts neuroreparative effects in brain by acting on mature neurons and neuronal precursors localised in germinal subventricular zone (SVZ), the present study has been aimed to evaluate the effects of NGF eye drop administration on the expression of the mitotic marker Ki67 in brain of adult rats. We found that a single ocular administration (10 μl) of 200 μg/mL NGF solution is sufficient to enhance the distribution of Ki67 positive cells also expressing p75 neurotrophin receptors in the proliferating layer of the SVZ. In addition, NGF treatment induces an increase of levels of brain derived neurotrophic factor (BDNF) in forebrain. This data further supports the efficacy of ocular applied NGF to affect brain activities and suggests that NGF also by inducing local factors, including BDNF, can activate the machinery regulating the proliferation and maturation of neuronal precursor in brain.     

Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K_d = 1.39 ± 0.33 μM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K_d = 1.42 ± 0.15 μM, 2.00 ± 0.2 μM and 2.4 ± 0.4 μM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD.As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na₃VO₄ and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.     

Involvement of the mitochondrial compartment in human NCL fibroblasts

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.     

Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea

The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac mesodermal induction in undifferentiated cells independently of cell proliferation. This analysis suggests that IGF-1Ea may be a good candidate to improve both in vitro production of cardiomyocytes from pluripotent stem cells and in vivo activation of the differentiation program of cardiac progenitor cells.     

Novel large-range mitochondrial DNA deletions and fatal multisystemic disorder with prominent hepatopathy

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.     

Cell death pathology: perspective for human diseases

Apoptosis, a genetically regulated form of cell death with distinct biochemical and morphological features, plays a relevant physiological and pathological role in the organism, being pivotal in the maintenance of tissue development and homeostasis in the adult as well as in the regulation of immune responses. Deregulation of this process causes several human disorders including cancer, autoimmune and neurodegenerative diseases. Thus, modulation of the apoptotic process and of cell death in general, is a potential therapeutic approach for the treatment of several human pathologies.     

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).     

The extended catalysis of glutathione transferase

Glutathione transferase reaches 0.5–0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S] ? [E]. As glutathione transferase lowers the pK_a of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate–enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual “inverted” kinetics whereby the classical trends of v versus E and v versus S are reversed.