Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases

Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models

Isoscaling in neck fragmentation

Production of intermediate mass fragments (IMF) has been studied in semi-peripheral 124Sn (35AMeV) + 64Ni and 112Sn (35AMeV) + 58Ni reactions. Our recently proposed new method of an analysis of the neck- like fragmentation processes that provides information on the IMFs time equence and time scale is reviewed. Isotopic analysis of so characterized IMFs gives evidence for neutron enrichment of mid-velocity fragments. A clear isoscaling behavior is found despite the short emission time scale. Evolution of the isoscaling parameters from semi-peripheral to central collisions is discussed

Platelet gel supplementation in long bone nonunions treated by external fixation.

The aim of the present study was to evaluate the use of previously frozen, thawed platelet gel supplementation to accelerate the healing of long bone nonunions treated by external fixation. DESIGN: Prospective case series with historical controls. SETTING: University Hospital. PATIENTS: Twenty patients affected by tibial, humeral, or forearm atrophic nonunions were treated by percutaneous stabilization with unilateral external fixators and injection of autologous platelet gel. The healing time was compared to the result obtained in a historical control group treated without platelet gel supplementation. MAIN OUTCOME MEASUREMENTS: Consolidation rate and radiographic healing time of nonunions in the 2 groups were assessed by independent blinded observers. The nonunion was judged to be healed when bridging callus formation on both radiographic views was observed on at least 3 of 4 cortices. RESULTS: The healing rate of nonunion was 90\% (18/20) in platelet gel cases and 85\% (17/20) in controls, respectively (P = 0.633). The mean time until radiographic consolidation in nonunions supplemented with platelet gel (147 +/- 63 days) was not different to the result in the control group (153 +/- 61 days; P = 0.784). Analyzing the mean healing time for separate segments, no differences were noted between study and control group-that is, tibia: 112 +/- 43 and 130 +/- 5 days, respectively (P = 0.382); humerus, 225 +/- 36 and 202 +/- 70 days, respectively (P = 0.530). CONCLUSION: The present study failed to show the clinical usefulness of isolated percutaneous platelet gel supplementation in long bone nonunions treated by external fixation; however, caution should be exercised in interpreting this result because the actual numbers are small and the statistical power is limited

Use of autologous platelet-rich plasma (PRP) in periodontal defect treatment after extraction of impacted mandibular third molars.

PURPOSE: The extraction of mesioangular impacted third molars may cause multiple periodontal defects at the distal root of the second molar. Platelet-rich plasma (PRP) is a material containing many autologous growth factors that may be used in repairing and preventing periodontal complications at the distal root of the second molar adjacent to the extracted third molar. PATIENTS AND METHODS: We analyzed the effects of autologous PRP on periodontal tissues after extraction of the third molar in 18 young patients (age, 21-26 years). Inclusion criteria were the presence of a pocket distal to the mandibular second molar with a probing depth>or=7.5 mm and a probing attachment level>or=6 mm. RESULTS: We observed, at 12 weeks after surgery, a notable reduction in the probing depth and an improvement in the probing attachment level in those cases treated with PRP compared with the controls, as well as formation of new bone tissue in the bone defect. CONCLUSION: We showed that PRP is effective in inducing and accelerating bone regeneration for the treatment of periodontal defects at the distal root of the mandibular second molar after surgical extraction of a mesioangular, deeply impacted mandibular third molar

The usefulness of 111indium-oxine autologous platelet gel graft imaging to evaluate osteoinduction in patients undergoing surgery of jaw bone defects.

Autologous platelet gel (AGP) is a source of concentrated growth factors contained in the platelet granules used to enhance bone quality and, especially, quicken bone formation in regeneration techniques, and also ameliorate the haemostasis in anti-coagulated patient management. The purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111In -Oxine and to evaluate its usefulness, as a marker of bone osteoinduction by means of scintigraphy, after in vivo application in patients with jaw bone defects following cystic lesion enucleation and the extraction of deeply impacted lower third molar. All patients included in the study presented mandible bone defects following cyst enucleation or deeply impacted lower third molar extraction. In sterile conditions, 111In-Oxine AGP was added during the bone-milling phase of the graft preparation and then applied to the bone defects. The scintigraphy was performed 2 hours after the application of labelled AGP (early scan) and at 24, 48, 72, 384 hours (delayed scan). At early scan all the patients presented a high concentration of 111In-Oxine AGP, which was easily recognized at the level of jaw defect. Limited diffusion of AGP was seen in the tissue surrounding the bone defect; this activity was attributed to the presence, in the PRP, of a quote of autologous granulocytes, as marker of inflammatory process, which was labelled with 111In-Oxine. In order to demonstrate the persistence and stability of labelling AGP, abdominal scintigraphies were performed to assess the presence of activity in the liver, spleen and bone marrow. None of the patients presented appreciable activity in these organs. The labelled AGP topically applied showed high uptake values, without statistically significant activity in the surrounding tissues or in critical organs during the early phase, as well as in delayed controls, and confirmed a very low grade of loss of 111In-Oxine from the bone defect. The scintigraphy represents a useful method of assessing the success of surgical procedure for jaw bone defects performed with autogenous grafts. It is well accepted by the patients, offering at the same time a sensitive method of studying uptake of topically applied AGP and to follow up kinetics of AGP in order to correlate quantitative data of the platelet gel life span with evolution of the bone remodelling process. Finally, the labelled granulocytes around the bone defect allow to assess the inflammatory process evolution derived from the surgical technique

Iron depletion before HCV antiviral therapy: A pilot, randomized, controlled trial.

It is not known whether iron depletion before pegylated IFN or combination treatment improves sustained virological response (SVR) rate in patients with chronic hepatitis C, despite its use in clinical practice in this setting. We aimed to investigate whether blood letting improves the efficacy (SVR) and tolerability of PEG-IFNalpha2b + Ribavirin in chronic hepatitis C patients. Patients with chronic hepatitis C and ferritin >100 ng/mL were randomized to: (1) repeated phlebotomies to obtain a ferritin level <50 ng/mL followed by pegylated-Interferon alpha2b + ribavirin (active arm); or (2) pegylated-Interferon alpha2b + ribavirin (control arm). Primary endpoint was SVR rate, secondary endpoint was frequency of clinical and laboratory grade 3-4 adverse events. Thirty-three patients were enrolled in the study (19 in active arm, 14 in control arm). The 19 patients in the active arm underwent a median of 5 phlebotomies (range: 1-9) to achieve the targeted ferritin (<50 ng/mL). Phlebotomies significantly reduced ferritin, iron, transferrin saturation, aspartate aminotransferase, alanine aminotransferase, and hemoglobin levels. Platelet count significantly increased, whereas HCV-RNA levels remained unchanged. After antiviral therapy overall SVR was 31.6% in active arm and 21.4% in control arm (P = 0.698). Considering only the 18 patients who were naive to antiviral therapy, SVR was 60% in active arm versus 25% in control arm (P = 0.188). Tolerability, drug dose reduction or withdrawal were similar in the two arms. In conclusion phlebotomies do not increase the overall efficacy of antiviral therapy. However, the strong trend to higher SVR in naive patients undergoing phlebotomies warrants further investigation. J. Clin. Apheresis 2009. (c) 2009 Wiley-Liss, Inc

Peculiar Subcellular Localization of Fas Antigen in Human and Mouse Spermatozoa

The highly polarized structure and function of mammalian spermatozoa dictate that these cells compartmentalize specific metabolic and signaling pathways to regions where they are needed. Fas was initially identified as membrane receptor for pro-apoptotic signals, has been recently recognized as a molecule with pleiotropic functions. In this article, we provide evidence of a peculiar Fas localization: it is closely associated to the perinucleus, mainly at the level of the inner acrosomal membrane, as well as in the inner compartment of mitochondria. Immunoelectron microscopy and Western blot analysis indicated that intracellular Fas was associated with mitochondria in mouse epididymal spermatozoa. Accordingly, also in human ejaculated sperm, immunofluorescence analysis showed Fas localized in the middle piece of sperm flagellum where mitochondria are grouped. The potential functional implications of these findings are discussed. Microsc. Res. Tech. 72:573-579, 2009. (C) 2009 Wiley-Liss, Inc

Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1

Platelet components have found successful clinical utilization to initiate or to accelerate tissue-repair mechanisms. However, the molecular pathways by which platelet factors contribute to tissue regeneration have not been fully elucidated. We have studied the effect of thrombin-activated platelets (TAPs) on cell growth in vivo and in cultured cell systems. Application of TAPs to ulcerative skin lesions of diabetic patients induced local activation of ERK1/2 and Akt/PKB. Moreover, when applied to cultured human skin fibroblasts, TAPs promoted cell growth and DNA synthesis and activated platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 receptor tyrosine kinases. PDGF was released by TAPs and rapidly achieved a plateau. At variance, the release of IGF-1 was mainly provided by the TAPs-stimulated fibroblasts and progressively increased up to 48 h. The PDGF-R blocker Ag1296 reduced the activation of Akt/PKB and, at a lesser extent, of ERK1/2. Conversely, inhibition of IGF-1 signaling by Ag1024 and expression of a dominant-negative IGF-1R mutant selectively reduced the stimulation of ERK1/2 by TAPs and fibroblast-released factors, with minor changes of Akt/PKB activity. Thus, platelet factors promote fibroblast growth by acutely activating Akt/PKB and ERK1/2. Sustained activation of ERK1/2, however, requires autocrine production of IGF-1 by TAPs-stimulated fibroblasts

Association between ccttt pentanucleotide repeat in the inducible nitric oxide synthase promoter polymorphism and achalasia

Background and aim: It is suggested that achalasia represents an autoimmune disorder in which a triggering factor (probably a virus) is the starter of an uncontrolled destruction of inhibitory neurons of the LES (myenteric plexus), mainly those expressing nitric oxide synthase (nNOS). Nitric oxide may represent an ideal candidate to explain the spread of inflammation and inhibitory nerve degeneration occurring in achalasia patients because: a) depending upon its concentration, it is involved in both defence against infections and inhibitory neurotransmission; b) excessive concentrations of NO have been demonstrated In Vitro to be neurotoxic, particularly for NOS expressing neurons; c) its production by different isoforms of NOS is genetically regulated. The aim of the present study was to assess whether the functional polymorphism in the pentanucleotide repeat (CCTTT) of iNOS gene promoter is involved in susceptibility to achalasia. Methods: Genomic DNA was isolated from peripheral leukocytes of 181 unrelated Italian Caucasian patients with sporadic achalasia and 220 healthy subjects matched for age (+/-5 yr) and gender. Genotyping of the pentanucle- otide repeat (CCTTT)n, in the iNOS promoter was performed by PCR and by capillary electrophoresis. PCR fragments were sized by Genescan software. Data were analysed by Fisher's exact test : a) considering allele frequencies (i.e. n of repeats) ; b) dichomotizing the subjects in those having long and short repeats form (> 11 and < 12, respectively). Results: The distribution of alleles having various repeat numbers ranged from 8 to 15, with a peak frequency at 12 repeats for both controls and patients. Analysis of the allele frequencies revealed that individuals carrying 10 and 12 CCTTT repeats were respectively less and more frequent in achalasia (16 vs 28%, OR 0.5, 95% CI 0.3-0.5, p= 0.008 and 34 vs. 25% OR 1.6, 95% CI 1-2.4, p= 0.04). Nosignificant differences were observed for the other (CCTTT)n. Moreover achalasia individuals having more than 11 repeats were more frequent (47 vs. 34%, OR 1.7, 95% CI 1.1-2.6, p= 0.009). Conclusion: We showed that higher pentanucleo- tide repeats in the iNOS gene promoter are involved in the susceptibility to sporadic achalasia. Since In Vitro data showed that the iNOS promoter activity increases in parallel with the repeat number of (CCTTT)n, we conclude that individuals carrying longer forms have an increased risk of achalasia by higher nitric oxide productio