Ku protein as a potential human T-cell leukemia virus type 1 (HTLV-1) Tax target in clastogenic chromosomal instability of mammalian cells

The HTLV-1 Tax oncoprotein rapidly induces cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) in cells. Tax is thought to have both aneuploidogenic and clastogenic effects. To examine the cellular target for Tax which might mechanistically explain the clastogenic phenomenon, we tested the ability of Tax to induce MN in rodents cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNAPKcs). We found that cells genetically mutated in Ku80 were refractory to Tax's induction of MN while cells knocked-out for DNAPKcs showed increased number of Tax-induced MN. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation) which measures the number of DNA-breaks in cells that contained unprotected 3'-OH ends, we observed that Tax increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells. Taken together, our findings suggest Ku80 as a cellular factor targeted by Tax in engendering clastogenic DNA damage

The translational value of calcium pyrophosphate deposition disease experimental mouse models

The deposition of calcium pyrophosphate (CPP) crystals in joint tissues causes acute and chronic arthritis that commonly affect the adult and elderly population. Experimental calcium pyrophosphate deposition disease (CPPD) models are divided into genetically modified models and crystal-induced inflammation models. The former do not reproduce phenotypes overlapping with the human disease, while in the latter, the direct injection of crystals into the ankles, dorsal air pouch or peritoneum constitutes a useful and reliable methodology that resembles the CPP induced-inflammatory condition in humans. The translational importance of the induced model is also strengthened by the fact that the key molecular and cellular mediators involved in inflammation are shared between humans and laboratory rodents. Although, in vivo models are indispensable tools for studying the pathogenesis of the CPPD and testing new therapies, their development is still at an early stage and major efforts are needed to address this issue. Here, we analyze the strenghts and limitations of each currently available CPPD in vivo model, and critically discuss their translational value

Oxidative stress by the mitochondrial monoamine oxidase B mediates calcium pyrophosphate crystal-induced arthritis

Objective: Calcium pyrophosphate (CPP) crystal deposition in the joints is associated with a heterogeneous set of debilitating syndromes characterized by inflammation and pain, for which no effective therapies are currently available. As we found that the mitochondrial enzyme monoamine oxidase B (MAO-B) plays a fundamental role in promoting inflammatory pathways, this study aims at assessing the efficacy of two clinical-grade inhibitors (iMAO-Bs) in preclinical models of this disease, to pave the way for a novel treatment. Methods: We tested our hypothesis in two murine models of CPP-induced arthritis, by measuring cytokine and chemokine levels, along with immune cell recruitment. iMAO-Bs (rasagiline and safinamide) were administered either before or after crystal injection. To elucidate the molecular mechanism, we challenged in vitro primed macrophages with CPP crystals and assessed the impact of iMAO-Bs in dampening proinflammatory cytokines and in preserving mitochondrial function. Results: Both in preventive and therapeutic in vivo protocols, iMAO-Bs blunted the release of proinflammatory cytokines (interleukin (IL)-6 and IL1-β) and chemokines (CXCL10, CXCL1, CCL2 and CCL5) (n>6 mice/group). Importantly, they also significantly reduced ankle swelling (50.3% vs 17.1% [P<0.001] and 23.1% [P=0.005] for rasagiline and safinamide, respectively). Mechanistically, iMAO-Bs dampened the burst of reactive oxygen species (ROS) and the mitochondrial dysfunction triggered by CPP crystals in isolated macrophages. Moreover, iMAO-Bs blunted cytokine secretion and NLRP3 inflammasome activation through inhibition of the NF-κB and STAT3 pathways. Conclusion: iMAO-Bs dampen inflammation in murine models of crystal-induced arthropathy, thereby uncovering MAO-B as a promising target to treat these diseases

Effetti dei Microcristalli di Urato Monosodico sull'apoptosi spontanea ed induzione di frammenti di DNA instabile nei sinoviociti

The deposition of Urate Monosodium crystals (MSU) in the articular and periarticular tissues is cause of the onset of inflammatory states associated to acute gout. Despite several and different pathogenetic hypotheses proposed, the molecular mechanism that controls the acute inflammatory attack induced by the microcrystals is still unknown. The aim of this study, through a model of acute inflammation in vitro mediated by MSU crystals , is verify the possible cito- and genotoxic effects induced on primary cultures of synoviocites derived from synovial fluid of 6 patients (3 affected by OA and 3 by AP). The purpose of this research is to determinate if the inflammation can influence the instability of genoma of the involved cells and what are the possible consequences in these cells concern the incidence of the apoptoss, proliferation, DNA integrity, formation of DNA unstable fragments and other aberrant figures as micronuclei. Experimentally the synoviocites primary cultures have been stimulated for 24 hours with different concentrations of MSU crystals (0,01 mg/ml; 0,025 mg/ml; 0,05 mg/ml; 0,075 mg/ml; 0,1 mg/ml, 0,25 mg/ml and 0,5 mg/ml). The same conditions have been repeated on CHO cells cultures, from Chinese Hamster, that represents one of the cellular systems more used and standardized for studies about in vitro mutagenesis.. For every experimental point the followings techniques have been applied: to) Tunel Test in order to analyze and quantify apoptotic cells b)In situ Incorporation of digoxigenin-dUTP for the staining of DNA breaks in the interphase nuclei and within the fragments of unstable DNA. c) cytological analysis for the determination of the mitotic index, of the defective mitoses and of other cytological parameters of genomic instability as presence of micronuclei, nuclear vesicles and anaphasic bridges. The stimulation for 24 hours of the CHO cell lines of Chinese Hamster has underlined as the presence of the MSU crystals induces on this cellular population a clastogenic effect. In fact the incidence of the cells marked by the digoxigenin-dUTP probe, that localizes in correspondence to free 3'-OH inside the DNA, increases in a dose dependent manner . The same results is noticed for other aberrations as the MN , aberrant mitotic figures, NPB and NBUD. Also the Mitotic index shows a meaningful decrease related on the MSU crystals dose used. The stimulation of the OA and AP derived synoviocytes primary cultures from confirm that , under these conditions, the MSU crystals induced a DNA damage in way analogous to how observed in precedence in CHO cell line used as control system. Nevertheless for all the types of DNA damage considered , the synoviocites derived from synovial fluid of patients with AP shows, from basal level as in presence of increasing stimulations of MSU crystals, a meaningful incidence of DNA damage greater in comparison to the synoviocites derived from OA. Such greater sensibility for the potentially genotoxic agents could derive from the fact that the primary cultures from AP originate from a synovial liquid, in general, more inflammatory in comparison to OA derived cells. In presence of stimulation with MSU crystals the frequency of apoptotic cells in CHO cells and OA primary cultures shows a meaningful decrement with values inferior to the 1% of incidence on the total population. Increasing the dose of stimulation the frequency of apoptotic cells increases in fucntion of the dose, in linear way for CHO cell line, while for the OA synoviocities , apoptosis increases. Subsequently for doses of MSU greater than 0,1mg/ml apoptosis slightly decreases. To basal level apoptosia of the AP synoviocites results very lower in comparison to the other cultures; nevertheless this primary culture cells respond in a very analogous way to the CHO cells and AP synoviocites in the presence of increasing doses of MSU crystals. Observing these data can be hypothesized that, relatively to apoptosis, the presence of MSU crystals, also already to concentrations of 0,01mg/ml acts with a " switch on/switch off "way, inhibiting a molecular apoptotic pathwayi for then subsequently activate an alternative for it in sensitive manner to the dose of stimulation. The data proposed in this study, even certainly still preliminary ,indicate that this simple experimental model can result very useful to appraise the DNA damage within joint tissues cell populations involved in the articular inflammatory pathologies and to characterize their behavior and evolution of it under these conditions. In perspective, analogous tests in vitro on cells obtained from the synovial fluid of patients with arthropathies could be predictive, also from the clinical point of view, in to preventively appraise the evolution of a date cellular population under conditions of chronic and acute inflammatory stress and in order to understand the degree of potential citotoxicity that a determined pharmacological treatment could induce to cellular level in a individual patient

Ku protein as a potential human T-cell leukemia virus type 1 (HTLV-1) Tax target in clastogenic chromosomal instability of mammalian cells

The HTLV-1 Tax oncoprotein rapidly induces cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) in cells. Tax is thought to have both aneuploidogenic and clastogenic effects. To examine the cellular target for Tax which might mechanistically explain the clastogenic phenomenon, we tested the ability of Tax to induce MN in rodents cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNAPKcs). We found that cells genetically mutated in Ku80 were refractory to Tax's induction of MN while cells knocked-out for DNAPKcs showed increased number of Tax-induced MN. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation) which measures the number of DNA-breaks in cells that contained unprotected 3'-OH ends, we observed that Tax increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells. Taken together, our findings suggest Ku80 as a cellular factor targeted by Tax in engendering clastogenic DNA damage

Totally stapled gastrojejunal anastomosis using hybrid NOTES: single 12-mm trocar approach in a porcine model.

none7Background The aim of this study was to evaluate the feasibility of a totally stapled gastrojejunal anastomosis performed using one transabdominal 12-mm trocar and a gastroscope in a porcine model. Methods The procedure was carried out on six domestic pigs weighing 45 kg using a hybrid technique with a gastroscope and a 12-mm Hasson trocar, positioned in the left hypochondrium. At the end of the procedure a mechanical circular 21-mm gastrojejunal anastomosis was performed by inserting the stapler through a small gastrotomy after enlarging the trocar incision. Results In all six cases the procedure was completed through a single 3 cm abdominal incision and without complications. The mean operating time was 2 h, and endoscopic investigation showed that the anastomoses were intact, patent, and airtight. Conclusions Totally stapled gastrojejunal anastomosis using a hybrid NOTES—single 12-mm trocar approach is a feasible procedure in the porcine model. Further survival studies are warranted, particularly to evaluate the functional results of this procedure.pubblicato online il 5 aprile 2012noneL. POLESE; S. MERIGLIANO; B. MUNGO; R. RIZZATO; R. LUISETTO; E. ANCONA; L. NORBERTOPolese, Lino; Merigliano, Stefano; B., Mungo; R., Rizzato; Luisetto, Roberto; Ancona, Ermanno; Norberto, Lorenz