Publications by authors named "Katia Fettucciari"

35 Publications

Proinflammatory Cytokines: Possible Accomplices for the Systemic Effects of Toxin B.

J Inflamm Res 2021 11;14:57-62. Epub 2021 Jan 11.

Gastroenterology, Hepatology & Digestive Endoscopy Section, Department of Medicine, University of Perugia Medical School, Perugia, Italy.

infection (CDI) has a serious impact on the healthcare system, and most of its pathogenic effects are mainly due to the activity of toxins A and B (TcdA and TcdB, respectively). The molecular mechanisms of their cytotoxic activity are well known, especially in the colon, where the infection occurs and normally remains localized. However, the mechanisms causing toxic effects on various systemic organs (extraintestinal manifestations) with frequent lethal outcomes in some patients affected by CDI are still poorly understood. Few studies are available that demonstrate low serum levels of Tcds in both experimental animal models and patients with CDI. Until now, it has remained unclear how low levels of circulating Tcds could lead to serious toxic effects. On the basis of our previous in vitro studies, in which the proinflammatory cytokines TNF-alpha and IFN-gamma strongly potentiated the toxic activity of low doses of TcdB, we hypothesize that the presence of both TcdB in the circulation and a systemic proinflammatory cytokine storm may be responsible for the selective severe effects of TcdB in some patients. This may occur in patients with severe CDI and systemic Tcds, in whom proinflammatory cytokines such as TNF-alpha and IFN-gamma reach a significant concentration in the circulation. This hypothesis could identify therapeutic interventions based on the reduction or neutralization of the indirect toxic action of these cytokines.
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http://dx.doi.org/10.2147/JIR.S287096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810702PMC
January 2021

Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

Antioxidants (Basel) 2020 Dec 29;10(1). Epub 2020 Dec 29.

Department of Medicine and Surgery, Section of Physiology and Biochemistry, University of Perugia, Piazza dell'Università, 1, 06123 Perugia PG, Italy.

Age-related retinal degenerations, including age-related macular degeneration (AMD), are caused by the loss of retinal pigmented epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD, deeply linked to the aging process, also involves oxidative stress and inflammatory responses. However, the molecular mechanisms contributing to the shift from healthy aging to AMD are still poorly understood. Since RPE cells in the retina are chronically exposed to a pro-oxidant microenvironment throughout life, we simulated in vivo conditions by growing ARPE-19 cells in the presence of 10 μM HO for several passages. This long-term oxidative insult induced senescence in ARPE-19 cells without affecting cell proliferation. Global proteomic analysis revealed a dysregulated expression in proteins involved in antioxidant response, mitochondrial homeostasis, and extracellular matrix organization. The analyses of mitochondrial functionality showed increased mitochondrial biogenesis and ATP generation and improved response to oxidative stress. The latter, however, was linked to nuclear factor-κB (NF-κB) rather than nuclear factor erythroid 2-related factor 2 (Nrf2) activation. NF-κB hyperactivation also resulted in increased pro-inflammatory cytokines expression and inflammasome activation. Moreover, in response to additional pro-inflammatory insults, senescent ARPE-19 cells underwent an exaggerated inflammatory reaction. Our results indicate senescence as an important link between chronic oxidative insult and detrimental chronic inflammation, with possible future repercussions for therapeutic interventions.
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http://dx.doi.org/10.3390/antiox10010025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823845PMC
December 2020

The efficacy of the anticancer 3-bromopyruvate is potentiated by antimycin and menadione by unbalancing mitochondrial ROS production and disposal in U118 glioblastoma cells.

Heliyon 2020 Dec 19;6(12):e05741. Epub 2020 Dec 19.

Department of Experimental Medicine, University of Perugia, 06132, Perugia, Italy.

Metabolic reprogramming of tumour cells sustains cancer progression. Similar to other cancer cells, glioblastoma cells exhibit an increased glycolytic flow, which encourages the use of antiglycolytics as an effective complementary therapy. We used the antiglycolytic 3-bromopyruvate (3BP) as a metabolic modifier to treat U118 glioblastoma cells and investigated the toxic effects and the conditions to increase drug effectiveness at the lowest concentration. Cellular vitality was not affected by 3BP concentrations lower than 40 μM, although p-Akt dephosphorylation, p53 degradation, and ATP reduction occurred already at 30 μM 3BP. ROS generated in mitochondria were enhanced at 30 μM 3BP, possibly by unbalancing their generation and their disposal because of glutathione peroxidase inhibition. ROS triggered JNK and ERK phosphorylation, and cyt c release outside mitochondria, not accompanied by caspases-9 and -3 activation, probably due to 3BP-dependent alkylation of cysteine residues at caspase-9 catalytic site. To explore the possibility of sensitizing cells to 3BP treatment, we exploited 3BP effects on mitochondria by using 30 μM 3BP in association with antimycin A or menadione concentrations that in themselves exhibit poor toxicity. 3BP effect on cyt c release and cell vitality loss was potentiated due the greater oxidative stress induced by antimycin or menadione association with 3BP, supporting a preeminent role of mitochondrial ROS in 3BP toxicity. Indeed, the scavenger of mitochondrial superoxide MitoTEMPO counteracted 3BP-induced cyt c release and weakened the potentiating effect of 3BP/antimycin association. In conclusion, the biochemical mechanisms leading U118 glioblastoma cells to viability loss following 3BP treatment rely on mitochondrial ROS-dependent pathways. Their potentiation at low 3BP concentrations is consistent with the goal to minimize the toxic effect of the drug towards non-cancer cells.
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http://dx.doi.org/10.1016/j.heliyon.2020.e05741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753915PMC
December 2020

The cytotoxic synergy between Clostridioides difficile toxin B and proinflammatory cytokines: an unholy alliance favoring the onset of Clostridioides difficile infection and relapses.

Microbiologyopen 2020 08 12;9(8):e1061. Epub 2020 Jul 12.

Department of Experimental Medicine, University of Perugia Medical School, Perugia, Italy.

Clostridioides difficile infection (CDI) represents an important health problem worldwide, with significant morbidity and mortality. This infection has also high recurrence rates, whose pathophysiological grounds are still poorly understood. Based on our experiments in vitro with Clostridioides difficile toxin B and existing experimental and clinical evidence, we propose that primary CDI and relapses might be favored by a mechanism that involves the enhancement of the toxicity of toxin B by proinflammatory cytokines, tumor necrosis factor alpha, and interferon gamma on the enteric glial cells and their network in an environment characterized by a strong dysmicrobism.
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http://dx.doi.org/10.1002/mbo3.1061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424247PMC
August 2020

Acetamidine-Based iNOS Inhibitors as Molecular Tools to Counteract Inflammation in BV2 Microglial Cells.

Molecules 2020 Jun 6;25(11). Epub 2020 Jun 6.

Department of Pharmacy, University G. d'Annunzio, Via dei Vestini 31, 66100 Chieti, Italy.

Neurodegenerative diseases are associated with increased levels of nitric oxide (NO) mainly produced by microglial cells through inducible nitric oxide synthase (iNOS) whose expression is induced by inflammatory stimuli. NO can both exert cytotoxic functions and induce a metabolic switch by inhibiting oxidative phosphorylation and upregulating glycolytic flux. Here, we investigated whether two newly synthesized acetamidine based iNOS inhibitors, namely CM292 and CM544, could inhibit lipopolysaccharide (LPS)-induced BV2 microglial cell activation, focusing on both inflammatory and metabolic profiles. We found that CM292 and CM544, without affecting iNOS protein expression, reduced NO production and reverted LPS-induced inflammatory and cytotoxic response. Furthermore, in the presence of the inflammatory stimulus, both the inhibitors increased the expression of glycolytic enzymes. In particular, CM292 significantly reduced nuclear accumulation of pyruvate kinase M2, increased mitochondrial membrane potential and oxygen consumption rate, and augmented the expression of pyruvate dehydrogenase, pointing to a metabolic switch toward oxidative phosphorylation. These data confirm the role played by NO in the connection between cell bioenergetics profile and inflammation, and suggest the potential usefulness of iNOS inhibitors in redirecting microglia from detrimental to pro-regenerative phenotype.
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http://dx.doi.org/10.3390/molecules25112646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321217PMC
June 2020

Guanylin, Uroguanylin and Guanylate Cyclase-C Are Expressed in the Gastrointestinal Tract of Horses.

Front Physiol 2019 27;10:1237. Epub 2019 Sep 27.

School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy.

Guanylate cyclase-C (GC-C) is a multifunctional receptor encoded by the gene, representing an attractive target for therapy in several gastrointestinal diseases in humans. Little is known about this system in horses. We investigated for the first time the gene expression of guanylin, uroguanylin and GC-C receptors in different horse's gastrointestinal tracts. Tissue samples from stomach, duodenum, jejunum, ileum, head and body of cecum, left and right dorsal colon, left and right ventral colon, pelvic flexure, transverse colon, descending colon and rectum were collected from adult horses within 1 h . For each sample, total RNA was extracted from 100 mg of ground tissue, and qRT-PCR performed on , and transcripts on a CFX96 Touch instrument. Data analysis was carried out with Bio-Rad CFX Manager software, and genes of interest normalized relative to the abundance of the two reference genes (). Additionally, the protein expression levels of GC-C receptor were analyzed through western blotting. A common pattern of expression throughout the gastrointestinal lumen for all three investigated transcripts was found. The expression of , and genes was higher in jejunum, ileum, descending colon and rectum. The levels of expression of GC-C protein confirmed these data. The findings of this study might open new scenarios for the therapeutic approach to enteric diseases of horse using selective agonists of GC-C.
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http://dx.doi.org/10.3389/fphys.2019.01237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776823PMC
September 2019

Gentamicin Targets Acid Sphingomyelinase in Cancer: The Case of the Human Gastric Cancer NCI-N87 Cells.

Int J Mol Sci 2019 Sep 6;20(18). Epub 2019 Sep 6.

Department of Pharmaceutical Sciences, University of Perugia, 06126 Perugia, Italy.

Emerging literature implicates acid sphingomyelinase in tumor sensitivity/resistance to anticancer treatments. Gentamicin is a drug commonly used as an antimicrobial but its serendipity effects have been shown. Even though many evidences on the role of gentamicin in cancer have been reported, its mechanism of action is poorly understood. Here, we explored acid sphingomyelinase as a possible new target of gentamicin in cancer. Since gastric cancer is one of the most common cancers and represents the second cause of death in the world, we performed the study in NCI-N87 gastric cancer cell line. The effect of the drug resulted in the inhibition of cell proliferation, including a reduction of cell number and viability, in the decrease of MIB-1 proliferative index as well as in the upregulation of cyclin-dependent kinase inhibitor 1A and 1B ( and ), and growth arrest and DNA-damage 45A () genes. The cytotoxicity was apoptotic as shown by FACS analysis. Additionally, gentamicin reduced HER2 protein, indicating a minor tumor aggressiveness. To further define the involvement of sphingomyelin metabolism in the response to the drug, gene and protein expression of acid and neutral sphingomeylinase was analyzed in comparison with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and vitamin D receptor (VDR), molecules involved in cancer. Gentamicin induced a downregulation of , , and neutral sphingomyelinase and a strong upregulation of acid sphingomyelinase. Of note, we identified the same upregulation of acid sphingomyelinase upon gentamicin treatment in other cancer cells and not in normal cells. These findings provide new insights into acid sphingomyelinase as therapeutic target, reinforcing studies on the potential role of gentamicin in anticancer therapy.
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http://dx.doi.org/10.3390/ijms20184375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770866PMC
September 2019

Clostridium difficile toxin B induces senescence in enteric glial cells: A potential new mechanism of Clostridium difficile pathogenesis.

Biochim Biophys Acta Mol Cell Res 2018 12 6;1865(12):1945-1958. Epub 2018 Oct 6.

Department of Medicine, University of Perugia Medical School, Perugia, Italy; Gastroenterology and Hepatology Section, Santa Maria della Misericordia Hospital, Perugia, Italy.

Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs. Rat-transformed EGCs were treated with 10 ng/ml TcdB for 6 h-48 h, or for 48 h, followed by incubation for additional 4 or 11 days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays. TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-β‑galactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, c‑Myc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin‑2 and Sirtuin‑3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways. In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.
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http://dx.doi.org/10.1016/j.bbamcr.2018.10.007DOI Listing
December 2018

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 08 2;1863(8):895-908. Epub 2018 May 2.

Department of Experimental Medicine, University of Perugia, 06132 Perugia, Italy. Electronic address:

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.
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http://dx.doi.org/10.1016/j.bbalip.2018.04.021DOI Listing
August 2018

Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by HO and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-kB desensitization: Implication for smokers-related osteoporosis.

Free Radic Biol Med 2018 03 31;117:6-17. Epub 2018 Jan 31.

Department of Experimental Medicine, University of Perugia, Italy. Electronic address:

Nicotine contained in cigarette smoke contributes to the onset of several diseases, including osteoporosis, whose emerging pathogenic mechanism is associated with osteoblasts apoptosis. Scanty information is available on the molecular mechanisms of nicotine on osteoblasts apoptosis and, consequently, on an important aspect of the pathogenesis of smokers-related osteoporosis. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a major precursor of advanced glycation end products (AGEs), potent pro-apoptotic agents. Hydroimidazolone (MG-H1) is the major AGE derived from the spontaneous MG adduction of arginine residues. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 was involved in the apoptosis induced by 0.1 and 1µM nicotine in human primary osteoblasts chronically exposed for 11 and 21 days. By using gene overexpression/silencing and scavenging/inhibitory agents, we demonstrated that nicotine induces a significant intracellular accumulation of hydrogen peroxide (HO) that, by inhibiting Glo1, drives MG-H1 accumulation/release. MG-H1, in turn, triggers HO overproduction via receptor for AGEs (RAGE) and, in parallel, an apoptotic mitochondrial pathway by inducing Transglutaminase 2 (TG2) downregulation-dependent NF-kB desensitization. Measurements of HO, Glo1 and MG-H1 circulating levels in smokers compared with non-smokers or in smokers with osteoporosis compared with those without this bone-related disease supported the results obtained in vitro. Our findings newly pose the antiglycation enzymatic defense Glo1 and MG-H1 among the molecular events involved in nicotine-induced reactive oxygen species-mediated osteoblasts apoptosis, a crucial event in smoker-related osteoporosis, and suggest novel exposure markers in health surveillance programmes related to smokers-associated osteoporosis.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.01.017DOI Listing
March 2018

Clostridium difficile-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

Cell Mol Life Sci 2018 04 28;75(7):1145-1149. Epub 2017 Dec 28.

Department of Experimental Medicine, University of Perugia Medical School, Perugia, Italy.

Post-infectious irritable bowel syndrome is a well-defined pathological entity that develops in about one-third of subjects after an acute infection (bacterial, viral) or parasitic infestation. Only recently it has been documented that an high incidence of post-infectious irritable bowel syndrome occurs after Clostridium difficile infection. However, until now it is not known why in some patients recovered from this infection the gastrointestinal disturbances persist for months or years. Based on our in vitro studies on enteric glial cells exposed to the effects of C. difficile toxin B, we hypothesize that persistence of symptoms up to the development of irritable bowel syndrome might be due to a disturbance/impairment of the correct functions of the enteroglial intestinal network.
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http://dx.doi.org/10.1007/s00018-017-2736-1DOI Listing
April 2018

VDR independent induction of acid-sphingomyelinase by 1,23(OH) D in gastric cancer cells: Impact on apoptosis and cell morphology.

Biochimie 2018 Mar 20;146:35-42. Epub 2017 Nov 20.

Department of Pharmaceutical Science, University of Perugia, Italy. Electronic address:

1 alpha,25-dihydroxyvitamin D (1,23(OH) D) is known to play a dual role in cancer, by promoting or inhibiting carcinogenesis via 1,23(OH) D receptor (VDR) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Fok I polymorphism of VDR may indirectly influence the receptor levels through autoregulation. The involvement of neutral sphingomyelinase in the non-classic VDR-mediated genomic pathway response to 1,23(OH) D treatment has been reported. Until now no information were reported about Fok I polymorphism of VDR in NCI-N87 human gastric cancer cells and the relation between acid sphingomyelinase and 1,23(OH) D. Herein, we showed that NCI-N87 human gastric cancer cells are homozygous for the Fok I 'C' allele; resulting in a three amino acid-truncated protein form of the VDR. Surprisingly 1,23(OH) D treatments strongly down-regulated the expression of VDR whereas acid sphingomyelinase and PTEN expression were upregulated. No changes of neutral sphingomyelinase expression were observed after 1,23(OH) D treatment, whereas acid sphingomyelinase activity increased. Furthermore 1,23(OH) D induced over-expression of caspase 8, CDKN2B, MAP3K5, cytochrome C apoptotic genes. Morphological analysis highlighted some very large round or oval cells and small cells with angular or fusiform extensions, confirmed by MIB-1 immunodetection and Hercep test. Taken together our results indicated that the action of 1,23(OH) D in gastric cancer cells was independent on 1,23(OH) D receptor and suggested the acid sphingomyelinase as a possible target to induce molecular events.
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http://dx.doi.org/10.1016/j.biochi.2017.11.011DOI Listing
March 2018

Enteric glial cells counteract Clostridium difficile Toxin B through a NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways.

Sci Rep 2017 03 28;7:45569. Epub 2017 Mar 28.

Department of Experimental Medicine, University of Perugia, Perugia, Italy.

Enteric glial cells (EGCs) are components of the intestinal epithelial barrier essential for regulating the enteric nervous system. Clostridium difficile is the most common cause of antibiotic-associated colitis, toxin B (TcdB) being the major virulence factor, due to its ability to breach the intestinal epithelial barrier and to act on other cell types. Here we investigated TcdB effects on EGCs and the activated molecular mechanisms. Already at 2 hours, TcdB triggered ROS formation originating from NADPH-oxidase, as demonstrated by their reduction in the presence of the NADPH-oxidase inhibitor ML171. Although EGCs mitochondria support almost completely the cellular ATP need, TcdB exerted weak effects on EGCs in terms of ATP and mitochondrial functionality, mitochondrial ROS production occurring as a late event. ROS activated the JNK signalling and overexpression of the proapoptotic Bim not followed by cytochrome c or AIF release to activate the downstream apoptotic cascade. EGCs underwent DNA fragmentation through activation of the ROS/JNK/caspase-3 axis, evidenced by the ability of ML171, N-acetylcysteine, and the JNK inhibitor SP600125 to inhibit caspase-3 or to contrast apoptosis. Therefore, TcdB aggressiveness towards EGCs is mainly restricted to the cytosolic compartment, which represents a peculiar feature, since TcdB primarily influences mitochondria in other cellular types.
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http://dx.doi.org/10.1038/srep45569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368562PMC
March 2017

Effects of Single-Dose Prucalopride on Intestinal Hypomotility in Horses: Preliminary Observations.

Sci Rep 2017 01 27;7:41526. Epub 2017 Jan 27.

Department of Medicine, Perugia University School of Medicine, Piazzale Lucio Severi, 1, 06159 San Sisto (Perugia), Italy.

Abnormalities of gastrointestinal motility are often a challenge in horses; however, the use of prokinetic drugs in such conditions must be firmly established yet. For this reason we carried out a preliminary study on the effects of prucalopride on intestinal motor activity of horses with gut hypomotility. The effect of prucalopride per os by oral dose syringe (2 mg/100 kg body weight) was assessed by abdominal ultrasound (evaluating duodenal, cecal, and colonic motor activity) in six horses with gut hypomotility. After administration of prucalopride, a significant increase of contractile activity was found in the duodenum at 30 minutes (p = 0.0005), 60 minutes (p = 0.01) and 90 minutes (p = 0.01), whereas in the cecum and in the left colon the increase was only present at 60 minutes (p = 0.03, and p = 0.02, respectively). No changes from baseline heart and respiratory rate or behavior side effects were observed after administration of the drug and throughout the observation period. Prucalopride may be a useful adjunct to the therapeutic armamentary for treating hypomotile upper gut conditions of horses. Dosing information is however needed to establish its actual clinical efficacy and its proper effects on the large bowel in these animals.
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http://dx.doi.org/10.1038/srep41526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269739PMC
January 2017

Enteric glial cells are susceptible to Clostridium difficile toxin B.

Cell Mol Life Sci 2017 04 28;74(8):1527-1551. Epub 2016 Nov 28.

Department of Medicine, Gastroenterology, Hepatology and Digestive Endoscopy Section, Perugia University, Perugia, Italy.

Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1-10 ng/ml for 1.5-48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.
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http://dx.doi.org/10.1007/s00018-016-2426-4DOI Listing
April 2017

Macrophage induced gelsolin in response to Group B Streptococcus (GBS) infection.

Cell Microbiol 2015 Jan 9;17(1):79-104. Epub 2014 Sep 9.

Department of Experimental Medicine, Perugia University, Perugia, Italy.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences. We have shown that interaction of macrophages with GBS causes macrophage calpain activation, cytoskeletal disruption and apoptosis, consequences of intracellular calcium increase induced by membrane permeability alterations provoked by GBS-β-haemolysin. Open question remains about what effect calcium influx has on other calcium-sensing proteins such as gelsolin, involved in cytoskeleton modulation and apoptosis. Therefore we analysed the effect of GBS-III-COH31:macrophage interaction on gelsolin expression. Here we demonstrate that an early macrophage response to GBS-III-COH31 is a very strong gelsolin increase, which occurs in a time- and infection-ratio-dependent manner. This is not due to transcriptional events, translation events, protein turnover alterations, or protein-kinase activation, but to calcium influx, calpain activation and caspase-3 degradation. In fact, EGTA and PD150606 (calpain inhibitor) prevented gelsolin increase while BAF (caspase inhibitor) enhanced it. Since gelsolin increase is induced by highly β-haemolytic GBS-III-NEM316 and GBS-V-10/84, but not by weakly β-haemolytic GBS, or GBS-III-COH31 in conditions suppressing β-haemolysin expression/activity and the presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor), GBS-β-haemolysin is solely responsible for gelsolin increase causing, through membrane permeability defects, calcium influx and calpain activation. Early gelsolin increase could represent a macrophage response to antagonize apoptosis since gelsolin knockdown increases macrophage susceptibility to GBS-induced apoptosis. This response seems to be GBS specific because macrophage apoptosis by Staurosporine or Cycloeximide does not induce gelsolin.
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http://dx.doi.org/10.1111/cmi.12338DOI Listing
January 2015

Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids.

BMC Pharmacol Toxicol 2014 Jul 3;15:35. Epub 2014 Jul 3.

Department of Medicine, University of Perugia, Perugia, Italy.

Background: Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR.

Methods: Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student's t-test was employed for statistical evaluation.

Results: We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk and Lck genes. To investigate the function associated with Itk up-regulation, dexamethasone-induced apoptosis of thymocytes from Itk-deficient mice was evaluated. Our results demonstrated that Itk deficiency causes increased sensitivity to dexamethasone but not to other pro-apoptotic stimuli.

Conclusions: Modulation of Itk, Txk, and Lck in thymocytes and mature lymphocytes is another mechanism by which glucocorticoids modulate T-cell activation and differentiation. Itk up-regulation plays a protective role in dexamethasone-treated thymocytes.
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http://dx.doi.org/10.1186/2050-6511-15-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105561PMC
July 2014

Impairment of brain mitochondrial functions by β-hemolytic Group B Streptococcus. Effect of cardiolipin and phosphatidylcholine.

J Bioenerg Biomembr 2013 Dec 25;45(6):519-29. Epub 2013 Aug 25.

Department of Internal Medicine, Section of Biochemistry, University of Perugia, Via Gambuli, 1, 06156, Perugia, Italy.

Group B Streptococcus (GBS) causes severe infection in the central nervous system. In this study, brain mitochondrial function was investigated by simulating infection of isolated mitochondria with GBS, which resulted in loss of mitochondrial activity. The β-hemolysin expressing strains GBS-III-NEM316 and GBS-III-COH31, but not the gGBS-III-COH31 that does not express β-hemolysin, caused dissipation of preformed mitochondrial membrane potential (Δψm). This indicates that β-hemolysin is responsible for decreasing of the reducing power of mitochondria. GBS-III-COH31 interacted with mitochondria causing increase of oxygen consumption, due to uncoupling of respiration, blocking of ATP synthesis, and cytochrome c release outside mitochondria. Moreover, the mitochondrial systems contributing to the control of cellular Ca(2+) uptake were lost. In spite of these alterations, mitochondrial phospholipid content and composition did not change significantly, as evaluated by MALDI-TOF mass spectrometry. However, exogenous cardiolipin (CL) and dipalmitoylphosphatidylcholine (DPPC) attenuated the uncoupling effect of GBS-III-COH31, although with different mechanisms. CL was effective only when fused to the inner mitochondrial membrane, probably reducing the extent of GBS-induced proton leakage. DPPC, which is not able to fuse with mitochondrial membranes, exerted its effect outside mitochondria, likely by shielding mitochondria against GBS β-hemolysin attack.
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http://dx.doi.org/10.1007/s10863-013-9525-9DOI Listing
December 2013

A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells.

Int J Biochem Cell Biol 2013 Apr 16;45(4):836-44. Epub 2013 Jan 16.

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, via del Giochetto 06122, Perugia, Italy.

Methylglyoxal is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Methylglyoxal cytotoxicity appears to occur through cell-cycle arrest but, more often, through induction of apoptosis. In this study we examined whether, and through which molecular mechanism, methylglyoxal affects the growth of poorly aggressive LNCaP and invasive PC3 human prostate cancer cells, where its role has not been exhaustively investigated yet. We demonstrated that methylglyoxal is cytotoxic on LNCaP and PC3 and that such cytotoxicity occurs not via cell proliferation but apoptosis control. Moreover, we demonstrated that methylglyoxal cytotoxicity, potentiated by the silencing of its major scavenging enzyme Glyoxalase I, occurred via different apoptotic responses in LNCaP and PC3 cells that also showed a different susceptibility to this metabolite. Finally, we showed that the observed methylglyoxal apoptogenic role involved different molecular pathways, specifically mediated by methylglyoxal or methylglyoxal-derived argpyrimidine intracellular accumulation and NF-kB signaling-pathway. In particular, in LNCaP cells, methylglyoxal, through the accumulation of argpyrimidine, desensitized the key cell survival NF-kB signaling pathway, which was consistent with the modulation of NF-kB-regulated genes, triggering a mitochondrial apoptotic pathway. The results suggest that this physiological compound merits investigation as a potential chemo-preventive/-therapeutic agent, in differently aggressive prostate cancers.
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http://dx.doi.org/10.1016/j.biocel.2013.01.003DOI Listing
April 2013

Notch1 modulates mesenchymal stem cells mediated regulatory T-cell induction.

Eur J Immunol 2013 Jan 5;43(1):182-7. Epub 2012 Dec 5.

Hematology and Clinical Immunology Section, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy.

Notch1 signaling is involved in regulatory T (Treg)-cell differentiation. We previously demonstrated that, when cocultured with CD3(+) cells, mesenchymal stem cells (MSCs) induced a T-cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4(+) T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI-I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC-induced CD4(+) CD25(high) FOXP3(+) cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF-β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg-cell induction mediated by MSCs.
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http://dx.doi.org/10.1002/eji.201242643DOI Listing
January 2013

γ-Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and notch down-regulation.

Int J Cancer 2013 Apr 17;132(8):1940-53. Epub 2012 Oct 17.

Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, University of Perugia, Perugia, Italy.

γ-Secretase inhibitors (GSIs) have been proposed for combined therapies of malignancies with a dysregulated Notch signaling. GSI I (Z-Leu-Leu-Nle-CHO) induces apoptosis of some tumor cells by inhibiting proteasome and Notch activity. Alterations in these two cell survival regulators contribute to apoptosis resistance of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the mechanisms whereby GSI I increases apoptosis of primary CLL cells. Time-course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress apoptotic signaling, and a consistent Noxa protein up-regulation. These events precede, and some of them contribute to, mitochondrial alterations, which occur notwithstanding Mcl-1 accumulation induced by GSI I. In CLL cells, GSI I inhibits Notch1 and Notch2 activation only in the late apoptotic phases, suggesting that this event does not initiate CLL cell apoptosis. However, Notch inhibition may contribute to amplify GSI I-induced CLL cell apoptosis, given that Notch activation sustains the survival of these cells, as demonstrated by the evidence that both Notch1 and Notch2 down-regulation by small-interfering RNA accelerates spontaneous CLL cell apoptosis. Overall, our results show that GSI I triggers CLL cell apoptosis by inhibiting proteasome activity and enhancing ER stress, and amplifies it by blocking Notch activation. These findings suggest the potential relevance of simultaneously targeting these three important apoptosis regulators as a novel therapeutic strategy for CLL.
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http://dx.doi.org/10.1002/ijc.27863DOI Listing
April 2013

Role of glyoxalase I in the proliferation and apoptosis control of human LNCaP and PC3 prostate cancer cells.

Prostate 2013 Jan 1;73(2):121-32. Epub 2012 Jun 1.

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.

Background: Glyoxalase I (GLOI) detoxifies reactive dicarbonyls, as methylglyoxal (MG) that, directly or through the formation of MG-derived adducts, is a growth inhibitor and apoptosis inducer. GLOI has been considered a general marker of cell proliferation, but a direct link between the two has yet to be demonstrated. The aim of the present work was to clarify whether GLOI was involved in the proliferation control of LNCaP and PC3 human prostate cancer cells or might play a different role in the growth regulation of these cells.

Methods: RNA interference was used to study the role of GLOI in cell proliferation or apoptosis. Cell proliferation was evaluated by [3H]thymidine incorporation assay and flow cytometry, that was also used to analyze apoptosis. Real-time TaqMan polymerase chain reaction and spectrophotometric analyses were used to study transcript levels or specific activity, respectively. Proteins levels were analyzed by Western blot. MG was measured by high-performance liquid chromatography.

Results: We found that GLOI is not implicated in the proliferation control of LNCaP and PC3 cells but plays a role in the apoptosis of invasive prostate cancer PC3 cells, through a mechanism involving a specific MG-adduct and NF-kB signaling pathway.

Conclusions: Our data represent the first systematic demonstration that GLOI cannot be considered a general marker of cell proliferation and that acts as a pro-survival factor in invasive PC3 cells by elusing apoptosis. GLOI may be involved in prostate cancer progression, via the control of key molecules in the mitochondrial apoptotic mechanism, through NF-kB signaling pathway.
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http://dx.doi.org/10.1002/pros.22547DOI Listing
January 2013

Eicosapentaenoic acid demethylates a single CpG that mediates expression of tumor suppressor CCAAT/enhancer-binding protein delta in U937 leukemia cells.

J Biol Chem 2011 Aug 9;286(31):27092-102. Epub 2011 Jun 9.

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia 06126, Italy.

Polyunsaturated fatty acids (PUFAs) inhibit proliferation and induce differentiation in leukemia cells. To investigate the molecular mechanisms whereby fatty acids affect these processes, U937 leukemia cells were conditioned with stearic, oleic, linolenic, α-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids. PUFAs affected proliferation; eicosapentaenoic acid (EPA) was the most potent on cell cycle progression. EPA enhanced the expression of the myeloid lineage-specific transcription factors CCAAT/enhancer-binding proteins (C/EBPβ and C/EBPδ), PU.1, and c-Jun, resulting in increased expression of the monocyte lineage-specific target gene, the macrophage colony-stimulating factor receptor. Indeed, it is known that PU.1 and C/EBPs interact with their consensus sequences on a small DNA fragment of macrophage colony-stimulating factor receptor promoter, which is a determinant for expression. We demonstrated that C/EBPβ and C/EBPδ bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBPδ, which has been reported to be a tumor suppressor gene silenced by promoter hypermethylation in U937 cells. After U937 conditioning with EPA and bisulfite sequencing of the -370/-20 CpG island on the C/EBPδ promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBPδ gene basal expression. Our results provide evidence for a new role of PUFAs in the regulation of gene expression. Moreover, we demonstrated for the first time that re-expression of the tumor suppressor C/EBPδ is controlled by the methylation state of a site-specific CpG dinucleotide.
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http://dx.doi.org/10.1074/jbc.M111.253609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149302PMC
August 2011

Group B Streptococcus (GBS) disrupts by calpain activation the actin and microtubule cytoskeleton of macrophages.

Cell Microbiol 2011 Jun 18;13(6):859-84. Epub 2011 Mar 18.

General Pathology and Immunology Section, Perugia University, Perugia, Italy.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress β-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through β-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01584.xDOI Listing
June 2011

Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL.

Blood 2010 Oct 13;116(15):2713-23. Epub 2010 Jul 13.

Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, University of Perugia, Perugia, Italy.

A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8-mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.
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http://dx.doi.org/10.1182/blood-2010-03-275628DOI Listing
October 2010

Protein expression changes induced in murine peritoneal macrophages by Group B Streptococcus.

Proteomics 2010 Jun;10(11):2099-112

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.

Protein expression changes induced in thioglycolate-elicited peritoneal murine macrophages (M Phi) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control M Phi and M Phi incubated 2 h with live or heat-inactivated GBS were separated by 2-DE. Proteins whose expression was significantly different in infected M Phi, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in M Phi by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS-infected M Phi. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose-cycle enzymes being down-regulated in M Phi infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.
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http://dx.doi.org/10.1002/pmic.200900642DOI Listing
June 2010

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells.

Blood 2009 Jan 16;113(4):856-65. Epub 2008 Sep 16.

Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, University of Perugia, Perugia.

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.
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http://dx.doi.org/10.1182/blood-2008-02-139725DOI Listing
January 2009

GITR-GITRL system, a novel player in shock and inflammation.

ScientificWorldJournal 2007 May 1;7:533-66. Epub 2007 May 1.

Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università di Perugia, Perugia, Italy.

Glucocorticoid-induced TNFR-Related (GITR) protein is a member of the tumor necrosis factor receptor superfamily that modulates acquired and natural immune response. It is expressed in several cells and tissues, including T cells, natural killer cells, and, at lower levels, in cells of innate immunity. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting and endothelial cells. Recent evidence suggests that the GITR/GITRL system participates in the development of inflammatory responses, including shock, either due to early response of neutrophils and macrophages, or together with autoimmune/allergic pathogenesis. The pro-inflammatory role of the GITR/GITRL system is due to: 1) modulation of the extravasation process, 2) activation of innate immunity cells, 3) activation of effector T cells also favored by partial inhibition of suppressor T cells and modulation of dendritic function. This review summarizes the in vivo role of the GITR/GITRL system in inflammation and shock, explaining the mechanisms responsible for their effects, considering the interplay among the different cells of the immune system and transduction pathways activated by GITR and GITRL triggering. The hidden aspects about GITR/GITRL function, crucial for treatment planning of inflammatory diseases and shock by modulation of this system is stressed.
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http://dx.doi.org/10.1100/tsw.2007.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901298PMC
May 2007

Interleukin-7-engineered mesenchymal cells: in vitro effects on naive T-cell population.

Biol Blood Marrow Transplant 2006 Dec;12(12):1250-60

Hematology and Clinical Immunology Section, Department of Clinical and Experimental Medicine, Perugia University, Perugia, Italy.

T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7.
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http://dx.doi.org/10.1016/j.bbmt.2006.09.001DOI Listing
December 2006

Modulation of pro- and antiapoptotic molecules in double-positive (CD4+CD8+) thymocytes following dexamethasone treatment.

J Pharmacol Exp Ther 2006 Nov 16;319(2):887-97. Epub 2006 Aug 16.

Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università di Perugia, Istituto di Biotecnologie Trapiantologiche and Polo Scientifico e Didattico di Terni, Perugia, Italy.

Glucocorticoids play a role in regulation of T lymphocytes homeostasis and development. In particular, glucocorticoid treatment induces massive apoptosis of CD4(+)CD8(+) double-positive (DP) thymocytes. This effect is due to many mechanisms, mainly driven by modulation of gene transcription. To find out which genes are modulated, we analyzed DP thymocytes treated for 3 h with dexamethasone (a synthetic glucocorticoid) by global gene expression profiling. Results indicate modulation of 163 genes, also confirmed by either RNase protection assay or real-time polymerase chain reaction. In particular, dexamethasone caused down-regulation of genes promoting DP thymocyte survival (e.g., Notch1, suppressor of cytokine signaling 1, and inhibitor of DNA binding 3) or modulation of genes activating cell death through the ceramide pathway (UDP-glucose ceramide glucosyltransferase, sphingosine 1-phosphate phosphatase, dihydroceramide desaturase, isoform 1, and G protein-coupled receptor 65) or through the mitochondrial machinery. Among the latter, there are Bcl-2 family members (Bim, Bfl-1, Bcl-xL, and Bcl-xbeta), genes involved in the control of redox status (thioredoxin reductase, thioredoxin reductase inhibitor, and NADP(+)-dependent isocitrate dehydrogenase) and genes belonging to Tis11 family that are involved in mRNA stability. Our study suggests that dexamethasone treatment of DP thymocytes modulates several genes belonging to apoptosis-related systems that can contribute to their apoptosis.
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http://dx.doi.org/10.1124/jpet.106.108480DOI Listing
November 2006