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1. Anisakis extracellular vesicles elicit immunomodulatory and potentially tumorigenic outcomes on human intestinal organoids

Author

Ilaria Bellini, Daniela Scribano, Cecilia Ambrosi, Claudia Chiovoloni, Silvia Rondón, Annamaria Pronio, Anna Teresa Palamara, Agostina Pietrantoni, Anna Kashkanova, Vahid Sandoghdar, Stefano D’Amelio, and Serena Cavallero

Subjects
Anisakiasis, Extracellular vesicles, Tumorigenic potential, Immunomodulation, Human intestinal organoids, Inflammation, Infectious and parasitic diseases, RC109-216
Abstract

Abstract Background Anisakis spp. are zoonotic nematodes causing mild to severe acute and chronic gastrointestinal infections. Chronic anisakiasis can lead to erosive mucosal ulcers, granulomas and inflammation, potential tumorigenic triggers. How Anisakis exerts its pathogenic potential through extracellular vesicles (EVs) and whether third-stage infective larvae may favor a tumorigenic microenvironment remain unclear. Methods Here, we investigated the parasite's tumorigenic and immunomodulatory capabilities using comparative transcriptomics, qRT-PCR and protein analysis with multiplex ELISA on human intestinal organoids exposed to Anisakis EVs. Moreover, EVs were characterized in terms of shape, size and concentration using classic TEM, SEM and NTA analyses and advanced interferometric NTA. Results Anisakis EVs showed classic shape features and a median average diameter of around 100 nm, according to NTA and iNTA. Moreover, a refractive index of 5–20% of non-water content suggested their effective biological cargo. After treatment of human intestinal organoids with Anisakis EVs, an overall parasitic strategy based on mitigation of the immune and inflammatory response was observed. Anisakis EVs impacted gene expression of main cytokines, cell cycle regulation and protein products. Seven key genes related to cell cycle regulation and apoptosis were differentially expressed in organoids exposed to EVs. In particular, the downregulation of EPHB2 and LEFTY1 and upregulation of NUPR1 genes known to be associated with colorectal cancer were observed, suggesting their involvement in tumorigenic microenvironment. A statistically significant reduction in specific mediators of inflammation and cell-cycle regulation from the polarized epithelium as IL-33R, CD40 and CEACAM1 from the apical chambers and IL-1B, GM-CSF, IL-15 and IL-23 from both chambers were observed. Conclusions The results here obtained unravel intestinal epithelium response to Anisakis EVs, impacting host’s anthelminthic strategies and revealing for the first time to our knowledge the host-parasite interactions in the niche environment of an emerging accidental zoonosis. Use of an innovative EV characterization approach may also be useful for study of other helminth EVs, since the knowledge in this field is very limited. Graphical Abstract

Published
2024
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2. A Case of Gastroallergic and Intestinal Anisakiasis in Italy: Diagnosis Based on Double Endoscopy and Molecular Identification

Author

Stefano D’Amelio, Ilaria Bellini, Claudia Chiovoloni, Cristina Magliocco, Annamaria Pronio, Arianna Di Rocco, Ilaria Pentassuglio, Marco Rosati, Gianluca Russo, and Serena Cavallero

Subjects
gastroallergic anisakiasis, intestinal anisakiasis, gastric endoscopy, colon endoscopy, molecular diagnosis, Medicine
Abstract

Nematodes of the genus Anisakis (Rhabditida, Anisakidae) are zoonotic fish-borne parasites and cause anisakiasis, a disease with mild to severe acute or chronic gastrointestinal and allergic symptoms and signs. Anisakiasis can potentially lead to misdiagnosis or delay in diagnosis, and it has been suggested as a risk factor for gastrointestinal tumors. Here, we describe a case report of a 25-year-old woman who presented with gastrointestinal (abdominal pain, nausea, diarrhea) and allergic (diffuse skin rash) symptoms and reported ingestion of raw fish contaminated by worms. Gastro and colon endoscopy allowed the visualization and removal of nematodes and collection of bioptic tissue from ulcers and polyps. The removed nematodes were molecularly identified as Anisakis pegreffii. The patient was treated with chlorphenamine maleate, betamethasone, omeprazole, paracetamol, albendazole. We conclude that an upper endoscopy matched with a colonoscopy and molecular characterization of the pathogen yields the most reliable diagnosis and treatment for human anisakiasis, enabling the complete removal of the larvae and preventing chronic inflammation and damage.

Published
2023
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3. Inflammatory Response in Caco-2 Cells Stimulated with Anisakis Messengers of Pathogenicity

Author

Ilaria Bellini, Daniela Scribano, Meysam Sarshar, Cecilia Ambrosi, Antonella Pizzarelli, Anna Teresa Palamara, Stefano D’Amelio, and Serena Cavallero

Subjects
Anisakis, Caco-2, crude extract, extracellular vesicles, inflammation, Medicine
Abstract

Background: Anisakis spp. third-stage larvae (L3) are the causative agents of human zoonosis called anisakiasis. The accidental ingestion of L3 can cause acute and chronic inflammation at the gastric, intestinal, or ectopic levels. Despite its relevance in public health, studies on pathogenetic mechanisms and parasite-human interplay are scarce. The aim of this study was to investigate the human inflammatory response to different Anisakis vehicles of pathogenicity. Methods: Human colorectal adenocarcinoma (Caco-2) cells were exposed to Anisakis L3 (the initial contact with the host), extracellular vesicles (EVs, Anisakis–host communication), and crude extract (CE, the larval dying). The protein quantity and gene expression of two pro-inflammatory cytokines (IL-6 and IL-8) were investigated using an ELISA test (6 h and 24 h) and a qReal-Time PCR (1 h, 6 h, and 24 h), respectively. Results: The L3 and EVs induced a downregulation in both the Il-6 and Il-8 gene expression and protein quantity. On the contrary, the CE stimulated IL-6 gene expression and its protein release, not affecting IL-8. Conclusions: The Caco-2 cells seemed to not react to the exposure to the L3 and EVs, suggesting a parasite’s immunomodulating action to remain alive in an inhospitable niche. Conversely, the dying larva (CE) could induce strong activation of the immune strategy of the host that, in vivo, would lead to parasite expulsion, eosinophilia, and/or granuloma formation.

Published
2022
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4. What Do In Vitro and In Vivo Models Tell Us about Anisakiasis? New Tools Still to Be Explored

Author

Serena Cavallero, Ilaria Bellini, Antonella Pizzarelli, and Stefano D’Amelio

Subjects
anisakiasis, in vitro model, in vivo model, immune response, host–parasite interplay, Medicine
Abstract

Anisakiasis is a zoonosis caused by the ingestion of raw or undercooked seafood infected with third-stage larvae (L3) of the marine nematode Anisakis. Based on L3 localization in human accidental hosts, gastric, intestinal or ectopic (extra-gastrointestinal) anisakiasis can occur, in association with mild to severe symptoms of an allergic nature. Given the increasing consumption of fish worldwide, the European Food Safety Authority declared Anisakis as an emerging pathogen. Despite its importance for public health and economy, the scientific literature is largely characterized by taxonomic, systematic and ecological studies, while investigations on clinical aspects, such as the inflammatory and immune response during anisakiasis, using a proper model that simulates the niche of infection are still very scarce. The aims of this review are to describe the clinical features of anisakiasis, to report the main evidence from the in vivo and in vitro studies carried out to date, highlighting limitations, and to propose future perspectives in the study field of anisakiasis.

Published
2022
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5. Genetic polymorphisms associated with outcome in multiple myeloma patients receiving high-dose melphalan

Author

Federica Gemignani, Adriana Plesa, Roberto Barale, Sean V. Tavtigian, Ilaria Bellini, Charles Dumontet, J Troncy, Troy Perry, Tony Reiman, Lm Pilarski, and Stefano Landi

Subjects
Adult, Male, Melphalan, Oncology, medicine.medical_specialty, DNA Repair, Genotype, Single-nucleotide polymorphism, TP53BP2, Polymorphism, Single Nucleotide, XRCC1, Internal medicine, medicine, Mucositis, Humans, Genotyping, Multiple myeloma, Aged, Retrospective Studies, Transplantation, Polymorphism, Genetic, Hematology, business.industry, Middle Aged, medicine.disease, Treatment Outcome, Pharmaceutical Preparations, Pharmacogenetics, Immunology, Female, Multiple Myeloma, business, medicine.drug
Abstract

High-dose melphalan (HDM) is an essential component in the treatment of patients with multiple myeloma (MM). Few data are available regarding genetic polymorphisms associated with patient outcome or toxicity in this setting. To identify such polymorphisms, we performed a retrospective analysis, genotyping single nucleotide polymorphisms (SNPs) with the arrayed primer extension (APEX) technology in 169 patients having received HDM for MM. We analyzed 209 SNPs in 95 genes involved in drug metabolism, DNA repair, cell cycle and apoptosis. SNPs in ABCB1, CYP3A4 and TP53BP2 were associated with response to VAD induction therapy (P

Published
2009
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6. Development of lung cancer before the age of 50: the role of xenobiotic metabolizing genes

Author

Ilaria Bellini, Lenka Foretova, Stefano Landi, Jolanta Lissowska, Federica Gemignani, Vladimir Bencko, Valerie Gaborieau, David Zaridze, Rayjean J. Hung, Janet Hall, Dana Mates, Neonilia Szeszenia-Dabrowska, Federico Canzian, Peter Rudnai, Roberto Barale, Vladimir Janout, Paul Brennan, Lydie Gioia-Patricola, Eleonora Fabianova, Paolo Boffetta, Gemignani, F., Landi, S., Szeszenia-Dabrowska, N., Zaridze, D., Lissowska, J., Rudnai, P., Fabianova, E., Mates, D., Foretova, L., Janout, V., Bencko, V., Gaborieau, V., Gioia-Patricola, L., Bellini, I., Barale, R., Canzian, F., Hall, J., Boffetta, P., Hung, R.J., and Brennan, P.

Subjects
Adult, Male, Cancer Research, Lung Neoplasms, Development lung cancer before age of 50 role xenobiotic metabolizing gene, Single-nucleotide polymorphism, EPHX1, Biology, medicine.disease_cause, Polymorphism, Single Nucleotide, Xenobiotics, medicine, Humans, Age of Onset, Lung cancer, CYP2A6, Oligonucleotide Array Sequence Analysis, Genetics, Haplotype, Heterozygote advantage, General Medicine, Middle Aged, medicine.disease, Metabolic Detoxication, Phase II, Phenotype, Haplotypes, Case-Control Studies, Methylenetetrahydrofolate reductase, biology.protein, Female, Metabolic Detoxication, Phase I, Carcinogenesis
Abstract

The role of genes coding for xenobiotic metabolizing enzymes (XMEs) and the risk of lung cancer is unclear. Under the assumption that these genes may be more important among people having a diagnosis of lung cancer at younger ages, we have investigated the role of single-nucleotide polymorphisms (SNPs) within phase I and phase II XME genes, and also genes involved in the metabolism of nucleic acids in a series of young onset patients and matched controls. We genotyped 299 lung cancer cases diagnosed before the age of 50 and 317 controls, from six countries of Central and Eastern Europe, by use of an oligonucleotide microarray and arrayed primer extension technique for 45 SNPs in 15 phase I XME genes, 46 SNPs in 17 phase II genes and 9 SNPs in 4 genes related to metabolism of nucleic acids. Heterozygote carriers of SNPs in CYP1A2 1545T>C, -164C>A and -740T>G; CYP2A6 -47A>C; MDR1 3435T>C; NAT1 1088T>A and 1095A>C; GSTA2 S112T; GSTM3 V224I and MTHFR A222V had altered risk of developing lung cancer. Phenotypes reconstructed after haplotype analyses showed that the carriers of the combined NAT1 fast+ NAT2 fast phenotypes were at lower risk when compared with those with the combined NAT1 slow + NAT2 slow acetylator phenotypes. Finally, extensive EPHX1 metabolizers showed an increased risk as compared with the poor metabolizers. © The Author 2007. Published by Oxford University Press. All rights reserved.

Published
2007
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7. DNA microarray based on arrayed-primer extension technique for identification of pathogenic fungi responsible for invasive and superficial mycoses

Author

Ilaria Bellini, Daniele Campa, Arianna Tavanti, Federica Gemignani, Crocifissa S. Mogavero, Fabio Bottari, Roberto Barale, Sonia Senesi, and Stefano Landi

Subjects
Microbiology (medical), Trichophyton rubrum, Mycology, Candida parapsilosis, Sensitivity and Specificity, Microbiology, Candida tropicalis, Species Specificity, Candida krusei, DNA, Ribosomal Spacer, Aspergillosis, Humans, Candida albicans, skin and connective tissue diseases, DNA, Fungal, Mycological Typing Techniques, Candida, Oligonucleotide Array Sequence Analysis, biology, Candida glabrata, Candida lusitaniae, Candidiasis, Fungi, Reproducibility of Results, biology.organism_classification, bacterial infections and mycoses, Aspergillus, Mycoses, Oligonucleotide Probes, Candida dubliniensis
Abstract

An oligonucleotide microarray based on the arrayed-primer extension (APEX) technique has been developed to simultaneously identify pathogenic fungi frequently isolated from invasive and superficial infections. Species-specific oligonucleotide probes complementary to the internal transcribed spacer 1 and 2 (ITS1 and ITS2) region were designed for 24 species belonging to 10 genera, including Candida species ( Candida albicans , Candida dubliniensis , Candida famata , Candida glabrata , Candida tropicalis , Candida kefyr , Candida krusei , Candida guilliermondii , Candida lusitaniae , Candida metapsilosis , Candida orthopsilosis , Candida parapsilosis , and Candida pulcherrima ), Cryptococcus neoformans , Aspergillus species ( Aspergillus fumigatus and Aspergillus terreus ), Trichophyton species ( Trichophyton rubrum and Trichophyton tonsurans ), Trichosporon cutaneum , Epidermophyton floccosum , Fusarium solani , Microsporum canis , Penicillium marneffei , and Saccharomyces cerevisiae . The microarray was tested for its specificity with a panel of reference and blinded clinical isolates. The APEX technique was proven to be highly discriminative, leading to unequivocal identification of each species, including the highly related ones C. parapsilosis , C. orthopsilosis , and C. metapsilosis . Because of the satisfactory basic performance traits obtained, such as reproducibility, specificity, and unambiguous interpretation of the results, this new system represents a reliable method of potential use in clinical laboratories for parallel one-shot detection and identification of the most common pathogenic fungi.

Published
2007

8. Genetic Polymorphisms Correlated with Outcome in Multiple Myeloma Patients Receiving High Dose Melphalan

Author

Tony Reiman, Sean V. Tavtigian, Linda M. Pilarski, Ilaria Bellini, Adriana Plesa, Troy Perry, Charles Dumontet, Stefano Landi, Roberto Barale, and Federica Gemignani

Subjects
Oncology, Melphalan, medicine.medical_specialty, business.industry, Immunology, Single-nucleotide polymorphism, Cell Biology, Hematology, CYP2C19, medicine.disease, Biochemistry, Internal medicine, Toxicity, medicine, Mucositis, ERCC1, business, Multiple myeloma, ERCC4, medicine.drug
Abstract

High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However few data are available regarding genetic polymorphisms possibly correlated with patient outcome or toxicity in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a retrospective single nucleotide polymorphism (SNP) analysis using APEX technology in 169 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 108 males and 61 females and the median age was 55 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 58 patients (34%) received two high dose melphalan courses. A complete/very good response was observed in 43 of 103 evaluable patients (42%). Forty patients among 107 evaluable (37%) experienced high grade (III–IV) mucositis after high dose melphalan. The median freedom from progression was 45 months and the overall survival was 91 months. SNPs in SULT1A1, MDR, ERCC5, GSTP1, NQO1, CYP1A2, CCND1, PARP, RAD51, DRD4, P53 or associated proteins were found to be correlated with response to VAD therapy. SNPs in GRPR, ERCC5, GSTM3, GSTP1, XRCC1, ERCC4, CYP1B1, BARD1, MDR1, P53BP1, TERT, DRD4 were found to be correlated with response to high dose melphalan. Polymorphisms in CYP2E1, CYP2C9, CYP1A1, CYP1B1, TMPT, OGG1, PCNA, DRD2, XPC were correlated with disease progression while polymorphisms in ERCC1, DRD4, CYP2D6, CYP2C9, CYP1A1, CYP1A2, CYP1B1, ALDH, BARD1, BRCA1, MYH, TP53BP1 were correlated with death. Polymorphisms in XRCC1, SNP103, CYP2C9, CYP2C19, BRCA1, BRCA2, NBS1, CDKN1A were correlated with severe mucositis. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan.

Published
2007
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9. Genetic Polymorphisms Correlated with Outcome in Multiple Myeloma Patients Receiving High Dose Melphalan

Author

Charles Dumontet, Stefano Landi, Tony Reiman, Adriana Plesa, Ilaria Bellini, Roberto Barale, Linda Pilarski, Sean Tavtigian, and Federica Gemignani

Subjects
hemic and lymphatic diseases, Immunology, Cell Biology, Hematology, Biochemistry
Abstract

High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However little is known regarding genetic polymorphisms possibly correlated with patient outcome in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a SNP analysis in 83 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 48 males and 35 females and the median age was 58 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 49 patients received two high dose melphalan courses. The best response to melphalan was CR in 22 pts, very good response in 14 pts, partial response in 37 patients and stable or progressive disease in 10 patients. 37 patients had experienced disease progression and 8 patients had died at last follow-up. 29 patients experienced high grade (III-IV) mucositis after high dose melphalan. The median freedom from progression was 40 months. CYP2E1 polymorphism 7632 was correlated with freedom from progression (FFP) after high dose melphalan (p=0.02). SULT1A2 704 polymorphism was correlated both with FFP (p=0.002) and with overall survival (p=0.001). Occurrence of severe mucositis after high dose melphalan therapy was correlated with XRCC1 polymorphism 1198 (24% vs 51% of high grade mucositis) as well as ATM polymorphisms. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan. OS according to SULT1A2 N235T OS according to SULT1A2 N235T

Published
2006
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10. Development of lung cancer before the age of 50: the role of xenobiotic metabolizing genes.

Author

Federica Gemignani, Stefano Landi, Neonilia Szeszenia-Dabrowska, David Zaridze, Jolanta Lissowska, Peter Rudnai, Eleonora Fabianova, Dana Mates, Lenka Foretova, Vladimir Janout, Vladimir Bencko, Valérie Gaborieau, Lydie Gioia-Patricola, Ilaria Bellini, Roberto Barale, Federico Canzian, Janet Hall, Paolo Boffetta, Rayjean J. Hung, and Paul Brennan

Subjects
CANCER treatment, LUNG cancer, XENOBIOTICS, MEDICAL research, THERAPEUTICS
Abstract

The role of genes coding for xenobiotic metabolizing enzymes (XMEs) and the risk of lung cancer is unclear. Under the assumption that these genes may be more important among people having a diagnosis of lung cancer at younger ages, we have investigated the role of single-nucleotide polymorphisms (SNPs) within phase I and phase II XME genes, and also genes involved in the metabolism of nucleic acids in a series of young onset patients and matched controls. We genotyped 299 lung cancer cases diagnosed before the age of 50 and 317 controls, from six countries of Central and Eastern Europe, by use of an oligonucleotide microarray and arrayed primer extension technique for 45 SNPs in 15 phase I XME genes, 46 SNPs in 17 phase II genes and 9 SNPs in 4 genes related to metabolism of nucleic acids. Heterozygote carriers of SNPs in CYP1A2 1545T>C, −164C>A and −740T>G; CYP2A6 −47A>C; MDR1 3435T>C; NAT1 1088T>A and 1095A>C; GSTA2 S112T; GSTM3 V224I and MTHFR A222V had altered risk of developing lung cancer. Phenotypes reconstructed after haplotype analyses showed that the carriers of the combined NAT1 fast+ NAT2 fast phenotypes were at lower risk when compared with those with the combined NAT1 slow + NAT2 slow acetylator phenotypes. Finally, extensive EPHX1 metabolizers showed an increased risk as compared with the poor metabolizers. [ABSTRACT FROM AUTHOR]

Published
2007
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