I. Giardino | Università degli Studi di Foggia (original) (raw)

Papers by I. Giardino

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Background An unresolved question in coeliac disease is to understand how some toxic gliadin pept... more Background An unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31e43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines. Aim We addressed whether the epithelial uptake of p31e43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response. Methods The time course of intracellular delivery to lysosomes of p31e43, pa-2 or pa-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)g ubiquitination and p42/44emitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis. Results After 24 h of challenge p31e43, but not pa-2 or pa-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARg. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARg levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation. Conclusion p31e43 accumulation in lysosomes leads to epithelial activation via the ROSeTG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARg downregulation.

The Journal of Immunology, 2009

Journal of pediatric gastroenterology and nutrition, 1990

In an ophthalmologic study of 90 children with symptomatic giardiasis, ocular alterations were fo... more In an ophthalmologic study of 90 children with symptomatic giardiasis, ocular alterations were found in 10. Eight of these subjects presented an extensive "salt and pepper" degeneration of the pigmented epithelium involving 360 degrees of the midperiphery of both eyes. In one of the eight children, the pigmented epithelium showed atrophic areas, and in another there was a small hard exudate in the left eye. Of 2 remaining of the 10 children with ocular alterations, 1 presented with slight decoloration of the temporal half of the optic disc, and the other was affected by chorioretinitis. After single-dose antiprotozoic therapy (tinidazole 50 mg/kg), parasitologic tests were negative in all subjects and remained so throughout a 1-year follow-up. However, the characteristic epithelial lesion remained unaltered in all eight children for the entire follow-up period, as well as the optic disc decoloration in the only observed case. The child affected by chorioretinitis recovered...

Neurology, 2002

Acute disseminated encephalomyelitis (ADEM) is an inflammatory demyelinating disease of the CNS. ... more Acute disseminated encephalomyelitis (ADEM) is an inflammatory demyelinating disease of the CNS. Few pediatric series have been published, with retrospective and short-term follow-up studies. To describe a cohort of pediatric patients with ADEM and to determine whether clinical and neuroimaging findings predict outcome. A prospective study was conducted between March 1988 and July 2000 on 84 consecutive children with ADEM at the National Pediatric Hospital &amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Dr. J. P. Garrahan.&amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; Mean age at onset was 5.3 +/- 3.9 years, with a significant male predominance. Sixty-two patients (74%) had a preceding viral illness or vaccination. Acute hemiparesis (76%), unilateral or bilateral long tract signs (85%), and changes in mental state (69%) were the most prominent presenting features. Four MRI groups were identified: ADEM with small lesions (62%), with large lesions (24%), with additional bithalamic involvement (12%), and acute hemorrhagic encephalomyelitis (2%). Of the 54 children whose CSF samples were analyzed, none showed intrathecal oligoclonal bands. The use of high-dose corticosteroid treatment, particularly IV methylprednisolone, was associated with good recovery and resolution of MRI lesions. After a mean follow-up of 6.6 +/- 3.8 years, 90% of children showed a monophasic course, and 10% a biphasic disease. Eighty-nine percent of patients show at present Expanded Disability Status Scale scores of 0 to 2.5. Eleven percent have disability scores of 3 to 6.5. Childhood acute disseminated encephalomyelitis is a benign condition, affecting boys more frequently. No association was found between MRI groups and disability. Disability was related to optic nerve involvement at presentation. Even in relapsing cases, the distinction between acute disseminated encephalomyelitis and MS was possible on the basis of long-term clinical and neuroimaging follow-up and the absence of oligoclonal bands in CSF.

Metabolism, 2003

A time-delayed fluorescence immunoassay was developed for the determination of serum levels of me... more A time-delayed fluorescence immunoassay was developed for the determination of serum levels of methylglyoxal (MG)-derived hydroimidazolone using a monoclonal antiserum raised against Nalpha-acetyl-Ndelta-(5-hydro-5-methyl)-4-imidazolone, Europium-labeled anti-mouse IgG antiserum as indicator, and MG modified bovine serum albumin (BSA) as standard. Serum levels of hydroimidazolone were measured in 45 patients with type 2 diabetes aged 59.4 +/- 6.1 (mean +/- SD) years and with duration of diabetes of 7.3 +/- 3.1 years, and in 19 nondiabetic controls aged 56.3 +/- 4.3 years. The serum levels of hydroimidazolone were significantly higher in patients compared to controls: median, 3.0 (5-95 percentile, 1.6 to 5.4) U/mg protein versus 1.9 (1.2 to 2.8) U/mg protein (P =.0005). Significant positive correlations were observed between the serum levels of hydroimidazolone and serum levels of advanced glycation end products (AGEs), measured with a polyclonal anti-AGE antibody: r = 0.59 for patients (P &amp;lt;.0001), and r = 0.65 for controls (P =.002). Similarly, significant correlations were also found between serum levels of hydroimidazolone and N(epsilon)-(carboxymethyl)-lysine (CML): r = 0.36 in patients and r = 0.55 for controls (both P =.02). Serum hydroimidazolone levels did not correlate with fasting plasma glucose or hemoglobin A(1c) (HbA(1c)) levels. The observed differences between patients with diabetes and nondiabetic controls seem to be comparable to differences measured for other AGE compounds.

Journal of Clinical Investigation, 1994

Intracellular sugars are more reactive glycosylating agents than glucose. In vitro nonenzymatic g... more Intracellular sugars are more reactive glycosylating agents than glucose. In vitro nonenzymatic glycosylation of basic fibroblast growth factor (bFGF) by fructose, glucose-6-phosphate (G6P), or glyceraldehyde-3-phosphate (G3P) reduced high affinity heparin-binding activity of recombinant bFGF by 73, 77, and 89%, respectively. Mitogenic activity was reduced 40, 50, and 90%. To investigate the effects of bFGF glycosylation in GM7373 endothelial cells, we first demonstrated that GLUT-1 transporters were not downregulated by increased glucose concentration. In 30 mM glucose, the rate of glucose transport increased 11.6-fold, and the intracellular glucose concentration increased sixfold at 24 h and fivefold at 168 h. The level of total cytosolic protein modified by advanced glycosylation endproducts (AGEs) was increased 13.8-fold at 168 h. Under these conditions, mitogenic activity of endothelial cell cytosol was reduced 70%. Anti-bFGF antibody completely neutralized the mitogenic activity at both 5 and 30 mM glucose, demonstrating that all the mitogenic activity was due to bFGF. Immunoblotting and ELISA showed that 30 mM glucose did not decrease detectable bFGF protein, suggesting that the marked decrease in bFGF mitogenic activity resulted from posttranslational modification of bFGF induced by elevated glucose concentration. Cytosolic AGE-bFGF was increased 6.1-fold at 168 h. These data are consistent with the hypothesis that nonenzymatic glycosylation of intracellular protein alters vascular cell function. (J. Clin.

Journal of Clinical Investigation, 1998

Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, for... more Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D -lactoylglutathione, which in turn is converted to D -lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo -transfected control cells (21.80 Ϯ 0.1 vs. 0.76 Ϯ 0.02 mol/min/mg protein, n ϭ 3, P Ͻ 0.001). In neo -transfected cells, 30 mM glucose incubation increased MG and D -lactate concentration approximately twofold above 5 M M (35.5 Ϯ 5.8 vs. 19.6 Ϯ 1.6, P Ͻ 0.02, n ϭ 3, and 21.0 Ϯ 1.3 vs. 10.0 Ϯ 1.2 pmol/ 10 6 cells, n ϭ 3, P Ͻ 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D -lactate by Ͼ 10-fold (18.9 Ϯ 3.2 vs. 18.4 Ϯ 5.8, n ϭ 3, P ϭ NS, and 107.1 Ϯ 9.0 vs. 9.4 Ϯ 0 pmol/10 6 cells, n ϭ 3, P Ͻ 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58 Ϯ 0.15 vs. 0.19 Ϯ 0.03 total absorbance units, n ϭ 3, P Ͻ 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis. ( J. Clin.

Journal of Biological Chemistry, 2007

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates d... more Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

Diabetes, 1998

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induce... more Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the e ffects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H 2 O 2 , there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 µmol/l H 2 O 2 (+AG 0 vs. 10 µmol/l, NS), and reduced the percentage of apoptotic cells in response to 50 µmol/l H 2 O 2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dosedependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H 2 O 2induced benzoate hydroxylation in a dose-dependent m a n n e r. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.

Gut