Publications by authors named "Giuseppina Ruggiero"

35 Publications

Effect of a Weight Loss Program on Biochemical and Immunological Profile, Serum Leptin Levels, and Cardiovascular Parameters in Obese Dogs.

Front Vet Sci 2020 6;7:398. Epub 2020 Aug 6.

Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy.

This study aimed to investigate the effects of a weight loss program (WLP) on biochemical and immunological profile, and cardiovascular parameters in a cohort of dogs with naturally occurring obesity. Eleven obese dogs [body condition scoring (BCS), ≥7/9] were enrolled into the study and underwent clinical and cardiovascular examination, and blood testing before (T0) and after 6 months (T1) of WLP. Eleven normal weight (BCS, 4/5) healthy dogs were used as a control (CTR) group. Compared to the CTR group, at T0 obese dogs expressed higher serum leptin concentrations ( < 0.0005) that significantly decreased after weight loss ( < 0.005) but remained higher than the CTR group. Furthermore, obese dogs showed considerably lower levels ( < 0.0005) of regulatory T cell (Treg) compared to the CTR group, but they did not change after weight loss at T1. In obese dogs, tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations were substantially reduced at T1 ( < 0.0001 and < 0.005). Regarding the cardiovascular parameters, only one obese dog was hypertensive at T0, and systolic blood pressure values showed no significant differences at the end of the WLP. The ratio of interventricular septal thickness in diastole to left ventricle internal diameter in diastole (IVSd/LVIDd) was significantly greater in obese dogs at T0 than in the CTR group ( < 0.005). It decreased after weight loss ( < 0.05). In obese dogs, troponin I level significantly reduced with weight loss ( < 0.05), while endothelin-1 level did not differ statistically. The results suggest that the immune dysregulation in the presence of high leptin levels and reduced number of Treg could affect obese dogs as well as humans. Based on our findings, we may speculate that a more complete immune-regulation restore could be obtained by a greater reduction in fat mass and a longer-term WLP. Finally, left ventricular remodeling may occur in some obese dogs. However, in canine species, further studies are needed to investigate the impact of obesity and related WLP on cardiovascular system.
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http://dx.doi.org/10.3389/fvets.2020.00398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424025PMC
August 2020

T1D progression is associated with loss of CD3CD56 regulatory T cells that control CD8 T cell effector functions.

Nat Metab 2020 02 17;2(2):142-152. Epub 2020 Feb 17.

Laboratorio di Immunologia, Istituto per l'Endocrinologia e l'Oncologia Sperimentale G. Salvatore, Consiglio Nazionale delle Ricerche, Naples, Italy.

An unresolved issue in autoimmunity is the lack of surrogate biomarkers of immunological self-tolerance for disease monitoring. Here, we show that peripheral frequency of a regulatory T cell population, characterized by the co-expression of CD3 and CD56 molecules (T), is reduced in subjects with new-onset type 1 diabetes (T1D). In three independent T1D cohorts, we find that low frequency of circulating T cells is associated with reduced β-cell function and with the presence of diabetic ketoacidosis. As autoreactive CD8 T cells mediate disruption of insulin-producing β-cells, we demonstrate that T cells can suppress CD8 T cell functions by reducing levels of intracellular reactive oxygen species. The suppressive function, phenotype and transcriptional signature of T cells are also altered in T1D children. Together, our findings indicate that T cells constitute a regulatory cell population that controls CD8 effector functions, whose peripheral frequency may represent a traceable biomarker for monitoring immunological self-tolerance in T1D.
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http://dx.doi.org/10.1038/s42255-020-0173-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272221PMC
February 2020

Serafino Zappacosta: An Enlightened Mentor and Educator.

Front Immunol 2020 13;11:217. Epub 2020 Feb 13.

Dipartimento di Scienze Mediche Traslazionali, Università di Napoli "Federico II", Naples, Italy.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the "." The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists.
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http://dx.doi.org/10.3389/fimmu.2020.00217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031500PMC
March 2021

Knockdown of PTGS2 by CRISPR/CAS9 System Designates a New Potential Gene Target for Melanoma Treatment.

Front Pharmacol 2019 5;10:1456. Epub 2019 Dec 5.

Department of Pharmacy, University of Naples Federico II, Naples, Italy.

CRISPR/Cas9 has become a powerful method to engineer genomes and to activate or to repress genes expression. As such, in cancer research CRISPR/Cas9 technology represents an efficient tool to dissect mechanisms of tumorigenesis and to discover novel targets for drug development. Here, we employed the CRISPR/Cas9 technology for studying the role of prostaglandin-endoperoxide synthase 2 (PTGS2) in melanoma development and progression. Melanoma is the most aggressive form of skin cancer with a median survival of less than 1 year. Although oncogene-targeted drugs and immune checkpoint inhibitors have demonstrated a significant success in improving overall survival in patients, related toxicity and emerging resistance are ongoing challenges. Gene therapy appears to be an appealing option to enhance the efficacy of currently available melanoma therapeutics leading to better patient prognosis. Several gene therapy targets have been identified and have proven to be effective against melanoma cells. Particularly, PTGS2 is frequently expressed in malignant melanomas and its expression significantly correlates with poor survival in patients. In this study we investigated on the effect of knockdown in B16F10 murine melanoma cells. Our results show that reduced expression of in melanoma cells: ) inhibits cell proliferation, migration, and invasiveness; ) modulates immune response by impairing myeloid derived suppressor cell differentiation; ) reduces tumor development and metastasis . Collectively, these findings indicate that could represent an ideal gene to be targeted to improve success rates in the development of new and highly selective drugs for melanoma treatment.
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http://dx.doi.org/10.3389/fphar.2019.01456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915044PMC
December 2019

Modulation of the functions of myeloid-derived suppressor cells : a new strategy of hydrogen sulfide anti-cancer effects.

Br J Pharmacol 2020 02 27;177(4):884-897. Epub 2019 Nov 27.

Department of Pharmacy, University of Naples Federico II, Naples, Italy.

Background And Purpose: Myeloid-derived suppressor cells (MDSCs) represent a major obstacle to cancer treatment, as they negatively regulate anti-tumour immunity through the suppression of tumour-specific T lymphocytes. Thus, the efficacy of immunotherapies may be improved by targeting MDSCs. In this study, we assessed the ability of hydrogen sulfide (H S), a gasotransmitter whose anti-cancer effects are well known, to inhibit the accumulation and immunosuppressive functions of MDSCs in melanoma.

Experimental Approach: Effects of H S on the host immune response to cancer were evaluated using an in vivo syngeneic model of murine melanoma. B16F10-melanoma-bearing mice were treated with the H S donor, diallyl trisulfide (DATS) and analysed for content of MDSCs, dendritic cells (DCs) and T cells. Effects of H S on expression of immunosuppressive genes in MDSCs and on T cell proliferation were evaluated.

Key Results: In melanoma-bearing mice, DATS inhibited tumour growth, and this effect was associated with a reduction in the frequency of MDSCs in the spleen, in the blood as well as in the tumour micro-environment. In addition, we found that CD8 T cells and DCs were increased. Furthermore, DATS reduced the immuno-suppressive activity of MDSCs, restoring T cell proliferation.

Conclusions And Implications: The H S donor compound, DATS, inhibited the expansion and the suppressive functions of MDSCs, suggesting a novel role for H S as a modulator of MDSCs in cancer. Therefore, H S donors may provide a novel approach for enhancing the efficacy of melanoma immunotherapy.

Linked Articles: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.
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http://dx.doi.org/10.1111/bph.14824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024705PMC
February 2020

Clinical and Immunological Response in Dogs Naturally Infected by Treated with a Nutritional Supplement.

Animals (Basel) 2019 Jul 30;9(8). Epub 2019 Jul 30.

Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, 80138 Napoli NA, Italy.

The use of nutraceuticals as immunomodulators in the treatment of visceral leishmaniasis has generated interest in the current approaches to treat the disease. In this clinical and immunological study, we investigated whether the administration of a nutritional supplement mediates the immune-modulatory response in canine leishmaniosis (CL) and improves the clinical outcome of the disease. With this purpose, we analysed T lymphocyte subsets in peripheral blood (PB) of 12 dogs naturally infected by , following treatment with a nutritional supplement. The regulatory T (Treg) cells and the T helper (Th) 1 population were specifically evaluated. The animals underwent complete clinical examination and blood sample collection for haematological, biochemical, serological and immunological analysis before treatment (T0), one month (T30) and 3 months (T90) after the onset of the nutraceutical supplementation. We observed that nutraceutical supplementation was associated with immunomodulation of Th1 response and significant clinical improvement of the animals. No side effects were observed. Therefore, a potential supportive role for the nutraceutical supplement during canine leishmaniasis is proposed.
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http://dx.doi.org/10.3390/ani9080501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721201PMC
July 2019

Circulating regulatory T cells (Treg), leptin and induction of proinflammatory activity in obese Labrador Retriever dogs.

Vet Immunol Immunopathol 2018 Aug 7;202:122-129. Epub 2018 Jul 7.

Dipartimento di Scienze, Università della Basilicata, Via Nazario Sauro 85, 85100, Potenza, Italy; Dipartimento di Scienze Mediche Traslazionali, Università Federico II, Via Pansini 5, 80131, Napoli, Italy.

Over-nutrition and obesity have been associated with impaired immunity and low-grade inflammation in humans and mouse models. In this context, a causal role for unbalanced T regulatory cell (Treg)-dependent mechanisms has been largely suggested. Obesity is the most common nutritional disorder in dogs. However, it is not defined whether canine obesity may influence circulating Treg as well as if their number variation might be associated with the occurrence of systemic inflammation. The present study investigated the immune profile of healthy adult obese dogs belonging to the Labrador Retriever breed, in comparison with the normal weight counterpart. Indeed, obesity has been described as particularly evident in this dogs. With this purpose, 26 healthy dogs were enrolled and divided into two groups based on body condition score (BCS): controls (CTR: BCS 4-5) and obeses (OB: BCS ≥ 7). Our data indicate that adult obese Labrador Retrievers are characterised by the inverse correlation between leptin serum concentration and circulating Treg (CD4CD25Foxp3) levels. In addition, an increased number of cytotoxic T cell effectors (CD3CD8) and a higher IFN-γ production by cytotoxic T lymphocytes were observed in OB group. These results may provide new insights into the immunological dysregulation frequently associated to obesity in humans and still undefined in dogs.
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http://dx.doi.org/10.1016/j.vetimm.2018.07.004DOI Listing
August 2018

Mechanical phenotyping of K562 cells by the Micropipette Aspiration Technique allows identifying mechanical changes induced by drugs.

Sci Rep 2018 01 19;8(1):1219. Epub 2018 Jan 19.

Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, Via G. Campi 213/A, 41125, Modena, Italy.

Mechanical properties of living cells can be used as reliable markers of their state, such as the presence of a pathological state or their differentiation phase. The mechanical behavior of cells depends on the organization of their cytoskeletal network and the main contribution typically comes from the actomyosin contractile system, in both suspended and adherent cells. In the present study, we investigated the effect of a pharmaceutical formulation (OTC - Ossitetraciclina liquida 20%) used as antibiotic, on the mechanical properties of K562 cells by using the Micropipette Aspiration Technique (MAT). This formulation has been shown to increase in a time dependent way the inflammation and toxicity in terms of apoptosis in in vitro experiments on K562 and other types of cells. Here we show that by measuring the mechanical properties of cells exposed to OTC for different incubation times, it is possible to infer modifications induced by the formulation to the actomyosin contractile system. We emphasize that this system is involved in the first stages of the apoptotic process where an increase of the cortical tension leads to the formation of blebs. We discuss the possible relation between the observed mechanical behavior of cells aspirated inside a micropipette and apoptosis.
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http://dx.doi.org/10.1038/s41598-018-19563-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775209PMC
January 2018

Oxytetracycline induces DNA damage and epigenetic changes: a possible risk for human and animal health?

PeerJ 2017 27;5:e3236. Epub 2017 Apr 27.

Department of Translational Medical Sciences, University of Naples Federico II, Naples, Italy.

Background: Oxytetracycline (OTC), which is largely employed in zootechnical and veterinary practices to ensure wellness of farmed animals, is partially absorbed within the gastrointestinal tract depositing in several tissues. Therefore, the potential OTC toxicity is relevant when considering the putative risk derived by the entry and accumulation of such drug in human and pet food chain supply. Despite scientific literature highlights several OTC-dependent toxic effects on human and animal health, the molecular mechanisms of such toxicity are still poorly understood.

Methods: Here, we evaluated DNA damages and epigenetic alterations by quantitative reverse transcription polymerase chain reaction, quantitative polymerase chain reaction, chromatin immuno-precipitation and Western blot analysis.

Results: We observed that human peripheral blood mononuclear cells (PBMCs) expressed DNA damage features (activation of ATM and p53, phosphorylation of H2AX and modifications of histone H3 methylation of lysine K4 in the chromatin) after the exposure to OTC. These changes are linked to a robust inflammatory response indicated by an increased expression of Interferon (IFN)- and type 1 superoxide dismutase (SOD1).

Discussion: Our data reveal an unexpected biological activity of OTC able to modify DNA and chromatin in cultured human PBMC. In this regard, OTC presence in foods of animal origin could represent a potential risk for both the human and animal health.
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http://dx.doi.org/10.7717/peerj.3236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410137PMC
April 2017

In Vitro Effects of Some Botanicals with Anti-Inflammatory and Antitoxic Activity.

J Immunol Res 2016 15;2016:5457010. Epub 2016 Aug 15.

Department of Translational Medical Sciences, University of Naples Federico II, 80131 Naples, Italy; Department of Science, University of Basilicata, 85100 Potenza, Italy.

Several extrinsic factors, like drugs and chemicals, can foster autoimmunity. Tetracyclines, in particular oxytetracycline (OTC), appear to correlate with the emergence of immune-mediated diseases. Accumulation of OTC, the elective drug for gastrointestinal and respiratory infectious disease treatment in broiler chickens, was reported in chicken edible tissues and could represent a potential risk for pets and humans that could assume this antibiotic as residue in meat or in meat-derived byproducts. We investigated the in vitro anti-inflammatory properties of a pool of thirteen botanicals as a part of a nutraceutical diet, with proven immunomodulatory activity. In addition, we evaluated the effect of such botanicals in contrasting the in vitro proinflammatory toxicity of OTC. Our results showed a significant reduction in interferon- (INF-) γ production by human and canine lymphocytes in presence of botanicals ((⁎) p < 0.05). Increased INF-γ production, dependent on 24-hour OTC-incubation of T lymphocytes, was significantly reduced by the coincubation with Haematococcus pluvialis, with Glycine max, and with the mix of all botanicals ((⁎) p < 0.05). In conclusion, the use of these botanicals was shown to be able to contrast OTC-toxicity and could represent a new approach for the development of functional foods useful to enhance the standard pharmacological treatment in infections as well as in preventing or reducing the emergence of inflammatory diseases.
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http://dx.doi.org/10.1155/2016/5457010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002466PMC
March 2017

Novel STAT1 gain-of-function mutation and suppurative infections.

Pediatr Allergy Immunol 2016 Mar 15;27(2):220-3. Epub 2015 Dec 15.

Department of Translational Medical Sciences, Federico II University, Naples, Italy.

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http://dx.doi.org/10.1111/pai.12496DOI Listing
March 2016

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori.

BMC Microbiol 2014 Aug 27;14:228. Epub 2014 Aug 27.

Department of Molecular Medicine, Human Physiology Section, University of Pavia Medical School, Pavia, Italy.

Background: Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world's population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection.

Results: In the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD50 lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes.

Conclusions: G. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients' disease status.
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http://dx.doi.org/10.1186/s12866-014-0228-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148543PMC
August 2014

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization, production and secretion.

Biochim Biophys Acta 2014 Feb 31;1843(2):265-74. Epub 2013 Oct 31.

Dipartimento di Scienze Mediche Traslazionali, Università di Napoli "Federico II", 80131 Napoli, Italy. Electronic address:

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. Copper-zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the "non-T" cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.
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http://dx.doi.org/10.1016/j.bbamcr.2013.10.020DOI Listing
February 2014

Regulatory T cells, Cytotoxic T lymphocytes and a T(H)1 cytokine profile in dogs naturally infected by Leishmania infantum.

Res Vet Sci 2013 Dec 22;95(3):942-9. Epub 2013 Aug 22.

Department of Veterinary Medicine and Animal Productions, Division of Internal Medicine, University of Naples Federico II, Via Delpino, 1, 80137 Naples, Italy.

Canine leishmaniasis caused by the protozoan parasite Leishmania infantum is a chronic systemic disease endemic in Mediterranean basin. The aim of the study is to investigate the immune profile of dogs naturally infected by Leishmania infantum. In order to address such issue, CD4(+) and CD8(+) lymphocyte T cell subsets, peripheral CD4(+)CD3(+)Foxp3(+) (Treg) levels and the presence of pro-inflammatory T cells have been assessed, in 45 infected dogs and in 30 healthy animals, by using immunofluorescence and flow cytometry detection. Animals were categorised according to their clinical-pathological status and their antibody titer at diagnosis. Results showing a significant increase of CD8(+)CD3(+) T lymphocytes, a reduced percentage of the T regulatory CD4(+)CD3(+)Foxp3(+) subset and a significant increase of T(H)1 cells, characterise the infected dogs, regardless of their antibody titer or the occurrence of clinical symptomatic disease. These data may provide new insights into the pathogenesis of immune-mediated alterations associated with canine leishmaniasis.
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http://dx.doi.org/10.1016/j.rvsc.2013.08.005DOI Listing
December 2013

Natural killer expansion, human leukocyte antigens-E expression and CD14(+) CD56(+) monocytes in a myelodysplastic syndrome patient.

Eur J Haematol 2013 Sep 28;91(3):265-9. Epub 2013 Jun 28.

Department of Science, University of Basilicata, Potenza, Italy; Department of Translational Medical Sciences, University of Naples "Federico II", Naples, Italy.

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and possible evolution to acute leukemia. Occurrence of stem cell defects and of immune-mediated mechanisms was evidenced as relevant for pathophysiology of MDS. Here, we described one case of MDS patient carrying CD14(+) CD56(+) monocytes in bone marrow (BM), in the presence of a defective human leukocyte antigen (HLA)-E expression on peripheral blood (PB) cells and of natural killer (NK) cell expansion in PB and BM. The defective HLA-E expression and the NK expansion are proposed to be relevant for the pathogenesis of myelodysplasia in those patients showing CD14(+) CD56(+) monocytes in BM.
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http://dx.doi.org/10.1111/ejh.12152DOI Listing
September 2013

HLA-E and HLA class I molecules on bone marrow and peripheral blood polymorphonuclear cells of myelodysplatic patients.

Leuk Res 2013 Feb 3;37(2):169-74. Epub 2012 Oct 3.

Dipartimento di Scienze, Università della Basilicata, Potenza, Italy.

Relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones in myelodysplastic syndromes (MDS) was suggested, but valuable or predictive criteria on this involvement are lacking. We previously reported that reduced T-regulatory cells (Treg) and high CD54 expression on T cell identify a sub-group of patients in whom an immune-pathogenesis might be inferred. Here, we suggest the occurrence of immune-selection of dysplastic clones in a subgroup of MDS patients, with reduced HLA-I and HLA-E on PMN, and propose that an altered immune profile might represent a valuable criterion to classify Low/Int-1 patients on the basis of immune-pathogenesis of MDS.
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http://dx.doi.org/10.1016/j.leukres.2012.09.015DOI Listing
February 2013

Eculizumab treatment modifies the immune profile of PNH patients.

Immunobiology 2012 Jul 3;217(7):698-703. Epub 2011 Dec 3.

Department of Biochemistry and Biomedical Technologies, University of Naples Federico II, Italy.

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced. Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged.
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http://dx.doi.org/10.1016/j.imbio.2011.11.009DOI Listing
July 2012

Prevalence of anti-platelet antibodies in dogs naturally co-infected by Leishmania infantum and Ehrlichia canis.

Vet J 2011 Apr 24;188(1):118-21. Epub 2010 Apr 24.

Department of Veterinary Clinical Science, Section of Internal Medicine, University of Naples Federico II, Via Delpino, 1, 80137 Naples, Italy.

Thirty naturally infected dogs were divided into three groups (10 animals each): group I (canine leishmaniosis), group II (canine ehrlichiosis) and group III (dogs co-infected by both infectious agents). Ten clinically healthy dogs were used as controls. A haematological profile was obtained and dog sera were assessed for the presence of platelet-bound IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. Eight of the naturally co-infected dogs (80%), five of the leishmaniotic dogs (50%) and six of the ehrlichiotic dogs (60%) had detectable levels of platelet-bound antibodies (PBIg). No PBIg were observed in the control group. The hypothesis that Leishmania and Ehrlichia co-infection could more severely affect the immune response of infected dogs is discussed.
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http://dx.doi.org/10.1016/j.tvjl.2010.03.015DOI Listing
April 2011

Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes.

Br J Haematol 2010 Jan 29;148(1):90-8. Epub 2009 Sep 29.

Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Napoli, Italy.

The myelodysplastic syndromes (MDS) are clonal disorders characterised by ineffective haematopoiesis with high risk of leukaemia progression. The relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones has been suggested, but valuable criteria to obtain insight into these connections are lacking. This study showed significant increase of CD8 lymphocytes and mature B cells in the bone marrow (BM) compared to peripheral blood (PB) of low risk MDS patients. Different BM levels of Regulatory T cells (Treg) identified two sub-groups in these patients; only the sub-group with lower Treg percentage showed BM recruitment of CD8 lymphocytes. Different levels of CD54 on BM CD8 cells revealed two sub-groups of Intermediate-1 (Int-1) patients. The sub-group with higher CD54 expression on BM CD8 showed high levels of this molecule also on CD4 cells. BM recruitment of CD8 lymphocytes in the low risk group and/or the presence of high CD54 expression on BM CD8 in Int-1 patients were associated with more pronounced dyserythropoiesis and erythropoietin treatment. Our data shed light on the involvement of immune-mediated mechanisms in Low and Int-1 risk MDS patients and suggest that BM versus PB levels of immune effectors could represent useful criteria for a more homogeneous grouping of MDS patients.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07921.xDOI Listing
January 2010

Novel mutations in GCK and HNF1A genes in Italian families with MODY phenotype.

Diabetes Res Clin Pract 2009 Mar 15;83(3):e72-4. Epub 2009 Jan 15.

U.O. Laboratorio Analisi Cliniche e Microbiologiche, Settore di Medicina Molecolare ASUR ZT13, Ascoli Piceno, Italy.

Analysis of GCK and HNF1A genes in 32 MODY families identified three novel mutations: the missense mutation G170D and the deletion/insertion P432Xfs in GCK and the splicing mutation IVS4nt-1G>T, in HNF1A. For IVS4nt-1G>T the sequence analysis of RT-PCR products demonstrated exon skipping with the use of a cryptic splicing site.
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http://dx.doi.org/10.1016/j.diabres.2008.12.007DOI Listing
March 2009

Paroxysmal nocturnal hemoglobinuria: significant association with specific HLA-A, -B, -C, and -DR alleles in an Italian population.

Hum Immunol 2008 Mar 10;69(3):202-6. Epub 2008 Mar 10.

Dipartimento di Oncologia Sperimentale, Istituto Nazionale Tumori di Napoli, Napoli, Italy.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a PIG-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the linked DQB1*0602 (p
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http://dx.doi.org/10.1016/j.humimm.2008.02.001DOI Listing
March 2008

Glycosyl-phosphatidyl-inositol-defective granulocytes from paroxysmal nocturnal haemoglobinuria patients show increased bacterial ingestion but reduced respiratory burst induction.

Am J Hematol 2007 Feb;82(2):98-107

Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Naples, Italy.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the emergence of a GPI-defective clonal hematopoiesis. Its clinical features are hemolytic anemia, cytopenia, and thrombosis. Circulating monocytes and granulocytes are largely GPI-defective in PNH patients. This study aims to investigate the granulocyte functional properties in PNH. We analyzed bacterial-dependent intracellular ingestion and the consequent activation of oxidative burst in GPI-defective granulocytes from four neutropenic PNH patients. Our data show a significant increase in the ability of GPI-defective granulocytes to ingest opsonized bacteria. In addition, an impaired respiratory burst effectiveness in response to two independent bacterial stimuli, the N-formyl-MetLeuPhe (fMLP) synthetic bacterial peptide and E. coli, was revealed. The occurrence of neutropenia and the severe impairment of oxidative burst, occurring in chronic granulomatosis disease, were unable to significantly affect phagocytosis. Thus, additional mechanisms, able to differentially affect ingestion ability and respiratory burst effectiveness, have to be hypothesized. The reduced burst effectiveness of GPI-defective granulocytes was maintained after treatment with phorbol 12-myristate 13-acetate, a pharmacological stimulus able to extensively recruit and to trigger intracellular protein kinase C (PKC). Moreover, blocking of PKC has been observed to severely affect granulocyte respiratory burst with a mild effect on the phagocytosis. These data suggest a role for a modulation of intracellular PKC in the pathogenesis of the impaired granulocyte oxidative burst.
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http://dx.doi.org/10.1002/ajh.20779DOI Listing
February 2007

Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2.

J Cell Sci 2005 Sep 9;118(Pt 17):3917-24. Epub 2005 Aug 9.

Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini 5, Istituto di Endocrinologia ed Oncologia Sperimentale Centro Nazionale delle Ricerche, Università Federico II, Napoli, Italy.

Friedreich's ataxia is a recessive neurodegenerative disease due to insufficient expression of the mitochondrial protein frataxin. Although it has been shown that frataxin is involved in the control of intracellular iron metabolism, by interfering with the mitochondrial biosynthesis of proteins with iron/sulphur (Fe/S) clusters its role has not been well established. We studied frataxin protein and mRNA expression and localisation during cellular differentiation. We used the human colon adenocarcinoma cell line Caco-2, as it is considered a good model for intestinal epithelial differentiation and the study of intestinal iron metabolism. Here we report that the protein, but not the mRNA frataxin levels, increase during the enterocyte-like differentiation of Caco-2 cells, as well as in in-vivo-differentiated enterocytes at the upper half of the crypt-villus axis. Furthermore, subcellular fractionation and double immunostaining, followed by confocal analysis, reveal that frataxin localisation changes during Caco-2 cell differentiation. In particular, we found an extramitochondrial localisation of frataxin in differentiated cells. Finally, we demonstrate a physical interaction between extramitochondrial frataxin and IscU1, a cytoplasmic isoform of the human Fe/S cluster assembly machinery. Based on our data, we postulate that frataxin could be involved in the biosynthesis of iron-sulphur proteins not only within the mitochondria, but also in the extramitochondrial compartment. These findings might be of relevance for the understanding of both the pathogenesis of Friedreich's ataxia and the basic mechanism of Fe/S cluster biosynthesis.
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http://dx.doi.org/10.1242/jcs.02516DOI Listing
September 2005

T cells from paroxysmal nocturnal haemoglobinuria (PNH) patients show an altered CD40-dependent pathway.

J Leukoc Biol 2005 Jul 7;78(1):27-36. Epub 2005 Apr 7.

Cattedra de Immunologia, Dipartimento de Biologia e Pathologia Cellulare e Molecolare, Naples, Italy.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoiesis disorder characterized by the expansion of a stem cell bearing a somatic mutation in the phosphatidylinositol glycan-A (PIG-A) gene, which is involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. A number of data suggest the inability of the PIG-A mutation to account alone for the clonal dominance of the GPI-defective clone and for the development of PNH. In this context, additional immune-mediated mechanisms have been hypothesized. We focused on the analysis of T lymphocytes in three PNH patients bearing a mixed GPI(+) and GPI(-) T cell population and showing a marked cytopenia. To analyze the biological mechanisms underlying the control of T cell homeostasis in PNH, we addressed the study of CD40-dependent pathways, suggested to be of crucial relevance for the control of autoreactive T cell clones. Our data revealed significant, functional alterations in GPI(+) and GPI(-) T cell compartments. In the GPI(-) T cells, severe defects in T cell receptor-dependent proliferation, interferon-gamma production, CD25, CD54, and human leukocyte antigen-DR surface expression were observed. By contrast, GPI(+) T lymphocytes showed a significant increase of all these parameters, and the analysis of CD40-dependent pathways revealed a functional persistence of CD154 expression on the CD48(+)CD4(+) lymphocytes. The alterations of the GPI(+) T cell subset could be involved in the biological mechanisms underlying PNH pathogenesis.
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http://dx.doi.org/10.1189/jlb.0105026DOI Listing
July 2005

Cu,Zn superoxide dismutase increases intracellular calcium levels via a phospholipase C-protein kinase C pathway in SK-N-BE neuroblastoma cells.

Biochem Biophys Res Commun 2004 Nov;324(2):887-92

Division of Physiology, Department of Neuroscience, School of Medicine, University of Naples Federico II, Via S. Pansini, 5, 80131 Naples, Italy.

The superoxide dismutase isoenzymes (SOD) play a key role in scavenging, O*2- radicals. In contrast with previous studies, recent data have shown that human neuroblastoma cells are able to export the cytosolic Cu,Zn superoxide dismutase (SOD1), thus suggesting a paracrine role exerted by this enzyme in the nervous system. To evaluate whether SOD1 could activate intracellular signalling pathways, the functional interaction between SOD1 and human neuroblastoma SK-N-BE cells was investigated. By analyzing the surface binding of biotinylated SOD1 on SK-N-BE cells and by measuring intracellular calcium concentrations and PKC activity, we demonstrated that SOD1 specifically interacts in a dose-dependent manner with the cell surface membrane of SK-N-BE. This binding was able to activate a PLC-PKC-dependent pathway that increased intracellular calcium concentrations mainly deriving from the intracellular stores. Furthermore, we showed that this effect was independent of SOD1 dismutase activity and was totally inhibited by U73122, the PLC blocker. On the whole, these data indicate that SOD1 carries out a neuromodulatory role affecting calcium-dependent cellular functions.
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http://dx.doi.org/10.1016/j.bbrc.2004.09.131DOI Listing
November 2004

GPI-defective monocytes from paroxysmal nocturnal hemoglobinuria patients show impaired in vitro dendritic cell differentiation.

J Leukoc Biol 2004 Sep 14;76(3):634-40. Epub 2004 Jun 14.

Cattera di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitá Frederico II, Naples, Italy.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, acquired hematopoietic disorder characterized by a phosphatidylinositol (PI) glycan-A gene mutation, which impairs the synthesis of the glycosyl-PI (GPI) anchor, thus causing the absence of all GPI-linked proteins on the membrane of the clonal-defective cells. The presence of a consistent GPI-defective monocyte compartment is a common feature in PNH patients. To investigate the functional behavior of this population, we analyzed its in vitro differentiation ability toward functional dendritic cells (DCs). Our data indicate that GPI-defective monocytes from PNH patients are unable to undergo full DC differentiation in vitro after granulocyte macrophage-colony stimulating factor and recombinant interleukin (IL)-4 treatment. In this context, the GPI-defective DC population shows mannose receptor expression, high levels of the CD86 molecule, and impaired CD1a up-regulation. The analysis of lipopolysaccharide and CD40-dependent, functional pathways in these DCs revealed a strong decrease in tumor necrosis factor alpha and IL-12 production. Finally, GPI-defective DCs showed a severe impairment in delivering accessory signals for T cell receptor-dependent T cell proliferation.
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http://dx.doi.org/10.1189/jlb.1203607DOI Listing
September 2004