Gloria Buffi

University of Urbino

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Luca Galluzzi

University of Urbino

Aurora Diotallevi

University of Urbino

Mauro Magnani

Istituto Ortopedico Gaetano Pini

Marcello Ceccarelli

University of Urbino

Mauro De Santi

University of Urbino

Fabrizio Vitale

Istituto Zooprofilattico Sperimentale Della Sicilia

Chiara Orlandi

Universidade Estadual Paulista (Unesp)

Federica Bruno

Istituto Zooprofilattico Sperimentale Della Sicilia

Germano Castelli

Istituto Zooprofilattico Sperimentale Della Sicilia

Orazio Cantoni

University of Urbino

Cooperative Institutions

University of Urbino

University of Milan

University of Bologna

Ospedali Riuniti di Ancona

Istituti di Ricovero e Cura a Carattere Scientifico

University of Genoa

European Institute of Oncology

University of Parma

University of Helsinki

Universidad Nacional Autónoma de México

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The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis

BMC Veterinary Research (2022)

Gloria Buffi Aurora Diotallevi Marcello Ceccarelli Federica Bruno Germano Castelli

Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18).The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs.Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host-pathogen interaction in leishmaniasis.

10.1186/s12917-022-03359-5

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Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species

Microorganisms (2020)

Marcello Ceccarelli Gloria Buffi Aurora Diotallevi Francesca Andreoni Daniela Bencardino

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10−4–1 × 10−6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

Minicircle

Amastigote

10.3390/microorganisms8122006

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Comparing Heterologous and Homologous COVID-19 Vaccination: A Longitudinal Study of Antibody Decay

Viruses (2023)

Chiara Orlandi Giuseppe Stefanetti Simone Barocci Gloria Buffi Aurora Diotallevi

The humoral response after vaccination was evaluated in 1248 individuals who received different COVID-19 vaccine schedules. The study compared subjects primed with adenoviral ChAdOx1-S (ChAd) and boosted with BNT162b2 (BNT) mRNA vaccines (ChAd/BNT) to homologous dosing with BNT/BNT or ChAd/ChAd vaccines. Serum samples were collected at two, four and six months after vaccination, and anti-Spike IgG responses were determined. The heterologous vaccination induced a more robust immune response than the two homologous vaccinations. ChAd/BNT induced a stronger immune response than ChAd/ChAd at all time points, whereas the differences between ChAd/BNT and BNT/BNT decreased over time and were not significant at six months. Furthermore, the kinetic parameters associated with IgG decay were estimated by applying a first-order kinetics equation. ChAd/BNT vaccination was associated with the longest time of anti-S IgG negativization and with a slow decay of the titer over time. Finally, analyzing factors influencing the immune response by ANCOVA analysis, it was found that the vaccine schedule had a significant impact on both the IgG titer and kinetic parameters, and having a Body Mass Index (BMI) above the overweight threshold was associated with an impaired immune response. Overall, the heterologous ChAd/BNT vaccination may offer longer-lasting protection against SARS-CoV-2 than homologous vaccination strategies.

Heterologous

10.3390/v15051162

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ERO1α primes the ryanodine receptor to respond to arsenite with concentration dependent Ca2+ release sequentially triggering two different mechanisms of ROS formation

Chemico-Biological Interactions (2023)

Andrea Guidarelli Andrea Spina Gloria Buffi Giulia Blandino Mara Fiorani

A 6 h exposure of U937 cells to 2.5 μM arsenite stimulates low Ca2+ release from the inositol 1, 4, 5-triphosphate receptor (IP3R), causing a cascade of causally connected events, i.e., endoplasmic reticulum oxidoreductin-1α (ERO1α) expression, activation of the ryanodine receptor (RyR), mitochondrial Ca2+ accumulation, mitochondrial superoxide formation and further ERO1α expression. At greater arsenite concentrations, the release of the cation from the IP3R and the ensuing ERO1α expression remained unchanged but were nevertheless critical to sequentially promote concentration-dependent increases in Ca2+ release from the RyR, NADPH oxidase activation and a third mechanism of ERO1α expression which, in analogy to the one driven by mitochondrial superoxide, was also mediated by reactive oxygen species (ROS) and devoid of effects on Ca2+ homeostasis. Thus, concentration-independent stimulation of Ca2+ release from the IP3R is of pivotal importance for the effects of arsenite on Ca2+ homeostasis. It stimulates the expression of a fraction of ERO1α that primes the RyR to respond to the metalloid with concentration-dependent Ca2+-release, triggering the formation of superoxide in the mitochondrial respiratory chain and via NADPH oxidase activation. The resulting dose-dependent ROS formation was associated with a progressive increase in ERO1α expression, which however failed to affect Ca2+ homeostasis, thereby suggesting that ROS, unlike IP3R-dependent Ca2+ release, promote ERO1α expression in sites distal from the RyR.

Homeostasis

Mitochondrial ROS

10.1016/j.cbi.2023.110694

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Activation of NRF2 and ATF4 Signaling by the Pro-Glutathione Molecule I-152, a Co-Drug of N-Acetyl-Cysteine and Cysteamine

Antioxidants (2021)

Rita Crinelli Carolina Zara Luca Galluzzi Gloria Buffi Chiara Ceccarini

I-152 combines two pro-glutathione (GSH) molecules, namely N-acetyl-cysteine (NAC) and cysteamine (MEA), to improve their potency. The co-drug efficiently increases/replenishes GSH levels in vitro and in vivo; little is known about its mechanism of action. Here we demonstrate that I-152 not only supplies GSH precursors, but also activates the antioxidant kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (KEAP1/NRF2) pathway. The mechanism involves disulfide bond formation between KEAP1 cysteine residues, NRF2 stabilization and enhanced expression of the γ-glutamil cysteine ligase regulatory subunit. Accordingly, a significant increase in GSH levels, not reproduced by treatment with NAC or MEA alone, was found. Compared to its parent compounds, I-152 delivered NAC more efficiently within cells and displayed increased reactivity to KEAP1 compared to MEA. While at all the concentrations tested, I-152 activated the NRF2 pathway; high doses caused co-activation of activating transcription factor 4 (ATF4) and ATF4-dependent gene expression through a mechanism involving Atf4 transcriptional activation rather than preferential mRNA translation. In this case, GSH levels tended to decrease over time, and a reduction in cell proliferation/survival was observed, highlighting that there is a concentration threshold which determines the transition from advantageous to adverse effects. This body of evidence provides a molecular framework for the pro-GSH activity and dose-dependent effects of I-152 and shows how synergism and cross reactivity between different thiol species could be exploited to develop more potent drugs.

KEAP1

Cysteamine

ATF4

Mechanism of Action

Thiol

10.3390/antiox10020175

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Histone H3 K4 trimethylation occurs mainly at the origins of polycistronic transcription in the genome of Leishmania infantum promastigotes and intracellular amastigotes

BMC Genomics (2025)

Aurora Diotallevi Stefano Amatori Giuseppe Persico Gloria Buffi Enrica Sordini

Trypanosomatids include the genera Trypanosoma and Leishmania, which are the etiological agents of important human diseases. These pathogens present unique mechanisms of gene expression characterized by functionally unrelated genes positioned in tandem and organized into polycistronic transcription units transcribed in a large pre-mRNA by RNA Polymerase II. Since most of the genome is constitutively transcribed, gene expression is primarily controlled by post-transcriptional processes. As in other organisms, histones in trypanosomatids contain a considerable number of post-translational modifications, highly conserved across evolution, such as the acetylation and methylation of some lysines on histone H3 and H4. These modifications have been mainly studied in Trypanosoma spp. The aim of this work was to elucidate the distribution of histone H3 lysine 4 trimethylation (H3K4me3) over the chromatin landscape of Leishmania infantum, the causative agent of canine and human leishmaniasis in the Mediterranean region. To this end, we investigated by chromatin immunoprecipitation (ChIP)-sequencing either the promastigotes (the flagellated motile form) and the amastigotes (the intracellular form) in an in vitro infection model. The chromatin was prepared from THP-1 cells non infected, THP-1 cells infected with L. infantum MHOM/FR/78/LEM75, and THP-1 cells non infected and mixed with L. infantum MHOM/FR/78/LEM75 promastigotes. ChIP was conducted using anti-H3K4me3 or anti-H3K27me3 antibodies and ChIP-seq was performed on an Ion S5 sequencer. We showed that histone H3K4me3 is mainly enriched at transcription start sites (67%) or internally within the polycistronic transcription units (30%), with no differences between L. infantum promastigotes and amastigotes. Moreover, the enriched regions co-localize with another hallmark of transcriptional activation (histone H3 acetylation) in L. major, a species characterized by a high degree of synteny with L. infantum. These findings expand our knowledge of the epigenomics of Leishmania parasites, focusing on epigenetic markers associated with transcription in L. infantum, and will contribute to elucidate the transcriptional mechanisms in these pathogens.

Amastigote

Transcription

Kinetoplastida

10.1186/s12864-025-11350-1

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Citations (0)

Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: Application on Brazilian clinical samples

Acta Tropica (2019)

Aurora Diotallevi Gloria Buffi Marcello Ceccarelli Herintha Coeto Neitzke-Abreu Laísa Vieira Gnutzmann

Subgenus

Canine leishmaniasis

10.1016/j.actatropica.2019.105178

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Unveiling the Link Between NADPH Oxidase 2 Activation and Mitochondrial Superoxide Formation in Leukemic Cell Killing Induced by Arsenic Trioxide

Pharmacological Research (2024)

Andrea Spina Andrea Guidarelli Gloria Buffi Mara Fiorani Orazio Cantoni

Arsenic Trioxide

10.1016/j.phrs.2024.107554

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Evaluation of Two-Month Antibody Levels after Heterologous ChAdOx1-S/BNT162b2 Vaccination Compared to Homologous ChAdOx1-S or BNT162b2 Vaccination

Vaccines (2022)

Simone Barocci Chiara Orlandi Aurora Diotallevi Gloria Buffi Marcello Ceccarelli

We evaluated the post-vaccination humoral response of three real-world cohorts. Vaccinated subjects primed with ChAdOx1-S and boosted with BNT162b2 mRNA vaccine were compared to homologous dosing (BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S). Serum samples were collected two months after vaccination from a total of 1248 subjects. The results showed that the heterologous vaccine schedule induced a significantly higher humoral response followed by homologous BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S vaccines (p < 0.0001). Moreover, analyzing factors (i.e., vaccine schedule, sex, age, BMI, smoking, diabetes, cardiovascular diseases, respiratory tract diseases, COVID-19 diagnosis, vaccine side effects) influencing the IgG anti-S response, we found that only the type of vaccine affected the antibody titer (p < 0.0001). Only mild vaccine reactions resolved within few days (40% of subjects) and no severe side effects for either homologous groups or the heterologous group were reported. Our data support the use of heterologous vaccination as an effective and safe alternative to increase humoral immunity against COVID-19.

Heterologous

Vaccination schedule

10.3390/vaccines10040491

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Citations (6)

Phenotype Screening of an Azole-bisindole Chemical Library Identifies URB1483 as a New Antileishmanial Agent Devoid of Toxicity on Human Cells

ACS Omega (2021)

Aurora Diotallevi Laura Scalvini Gloria Buffi Yolanda Pérez‐Pertejo Mauro De Santi

We report the evaluation of a small library of azole-bisindoles for their antileishmanial potential, in terms of efficacy on Leishmania infantum promastigotes and intracellular amastigotes. Nine compounds showed good activity on L. infantum MHOM/TN/80/IPT1 promastigotes with IC50 values ranging from 4 to 10 μM. These active compounds were also tested on human (THP-1, HEPG2, HaCaT, and human primary fibroblasts) and canine (DH82) cell lines. URB1483 was selected as the best compound, with no quantifiable cytotoxicity in mammalian cells, to test the efficacy on intracellular amastigotes. URB1483 significantly reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. URB1483 emerges as a new anti-infective agent with remarkable antileishmanial activity and no cytotoxic effects on human and canine cells.

Amastigote

HaCaT

IC50

10.1021/acsomega.1c05611

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Citations (7)