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

none8noBackground 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). Results 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. Conclusions 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.Buffi, Gloria; Diotallevi, Aurora; Ceccarelli, Marcello; Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Magnani, Mauro; Galluzzi, LucaBuffi, Gloria; Diotallevi, Aurora; Ceccarelli, Marcello; Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Magnani, Mauro; Galluzzi, Luc

In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment’s onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy

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

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

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

none10noThe 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.openCeccarelli, Marcello; Buffi, Gloria; Diotallevi, Aurora; Andreoni, Francesca; Bencardino, Daniela; Vitale, Fabrizio; Castelli, Germano; Bruno, Federica; Magnani, Mauro; Galluzzi, LucaCeccarelli, Marcello; Buffi, Gloria; Diotallevi, Aurora; Andreoni, Francesca; Bencardino, Daniela; Vitale, Fabrizio; Castelli, Germano; Bruno, Federica; Magnani, Mauro; Galluzzi, Luc

Development and Application of a High Resolution Melt (HRM) based test for the Rapid Screening of Leishmania infantum Genotypes

INTRODUCTION Leishmaniasis includes anthropo zoonotic infectious diseases caused by a protozoan of the Leishmania genus, associated with different clinical manifestations, affecting both humans and other vertebrates, including dogs The Mediterranean basin, including Italy, is considered an endemic area for both visceral and cutaneous leishmaniasis caused by L infantum The multi locus enzyme electrophoresis ( based on the electrophoretic mobility of several enzymes from promastigotes cultures, is considered the reference method for parasite typing Through this method, about 45 L infantum zymodemes (also termed MON) have been identified in humans in the Mediterranean basin 1 Among these, L infantum MON 1 is the most widespread, representing about 70 of all identified strains In Italy, canine infections showed a high prevalence of MON 1 91 with the remaining composed almost exclusively of MON 72 2 Since MLEE technique is cumbersome, time consuming and requires parasites isolation, several biomolecular approaches have been developed In particular, we identified the SNP 390 T>G in malic enzyme ( gene as a potential marker to differentiate the most common L infantum genotype, i e 390 T (corresponding to zymodemes MON 1 72 201 from all others. AIMS This study aimed to develop a Rapid Genotype Screening ( assay for L infantum genetic characterization in clinical samples using high resolution melt ( analysis, exploiting the polymorphism 390 T>G in the ME gene

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

none11noWe 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.openBarocci, Simone; Orlandi, Chiara; Diotallevi, Aurora; Buffi, Gloria; Ceccarelli, Marcello; Vandini, Daniela; Carlotti, Eugenio; Galluzzi, Luca; Rocchi, Marco Bruno Luigi; Magnani, Mauro; Casabianca, AnnaBarocci, Simone; Orlandi, Chiara; Diotallevi, Aurora; Buffi, Gloria; Ceccarelli, Marcello; Vandini, Daniela; Carlotti, Eugenio; Galluzzi, Luca; Rocchi, Marco Bruno Luigi; Magnani, Mauro; Casabianca, Ann

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

none11sìWe 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.openDiotallevi, Aurora; Scalvini, Laura; Buffi, Gloria; Pérez-Pertejo, Yolanda; De Santi, Mauro; Verboni, Michele; Favi, Gianfranco; Magnani, Mauro; Lodola, Alessio; Lucarini, Simone; Galluzzi, LucaDiotallevi, Aurora; Scalvini, Laura; Buffi, Gloria; Pérez-Pertejo, Yolanda; De Santi, Mauro; Verboni, Michele; Favi, Gianfranco; Magnani, Mauro; Lodola, Alessio; Lucarini, Simone; Galluzzi, Luc

Development and application of a MLST panel for the identification of informative polymorphisms in Leishmania infantum strains in the Mediterranean region

Background. Leishmaniasis is a zoonotic disease endemic in the Mediterranean region, where the causative agent of human and canine infection is Leishmania infantum. The spread of leishmaniasis is associated with population movements, ecology of phlebotomine vectors, and reservoir host. We used multilocus sequence typing (MLST) to explore the genetic variability of L. infantum strains in the Mediterranean region, including the borderline territory of Pantelleria island, and identify informative polymorphisms for rapid identification of genotypes through high-resolution melt (HRM)-based assays. Material and Methods. A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed using the Ion Torrent S5 on 9 L. infantum strains/isolates: 5 canine isolates (3 from Pantelleria Island and 2 from central Italy), and 4 human isolates/strains from Tunisia, France, central and southern Italy. MLST results and in silico analysis of sequences available in Genbank allowed to select two informative polymorphisms on ME and GPI genes (390T/G and 1834A/G, respectively) used to develop two HRM-based assays for fast screening of 28 clinical samples. Results. The MLST analysis identified a single L. infantum clonal complex regardless of the geographic origin or host (human or canine), except for the human isolate from central Italy that showed polymorphisms in 11 out of 14 housekeeping genes, and clustered independently in a molecular phylogenetic analysis. Successively, the screening through HRM-based assays of 28 clinical samples from central/south Italy and Pantelleria island allowed to identify 6 diploid sequence types (DSTs). Interestingly, the sequence type 390T/1834A was found only in Pantelleria island (prevalence 75%). Conclusion. This study represents a description of the genetic variability of L. infantum through a first approach based on MLST and then by HRM analysis on selected polymorphisms. The HRM assays could be used as fast and cheap tools for epidemiological surveillance of L. infantum

Phenotype screening of a bisindole chemical library identifies URB1483 as a new Antileishmanial agent with Topoisomerase IB as possible molecular target

Leishmaniasis is one of the most dangerous neglected tropical diseases, second only to malaria in parasitic causes of death. [1] Caused by global warming, the endemic regions of leishmaniasis are continuously spreading to non-tropical areas, including Europe. So far, no vaccine against leishmaniasis is on the market. [2] Approved drugs are currently limited and/or exorbitantly priced (i.e. amphothericin B; paromomycin; miltefosine; pentamidine) with few of them effective on antimonial-resistant Leishmania strains. Moreover, the use of these agents on infected patients is seriously hampered by the insurgence of acute and/or chronic toxicity. Therefore, there is an urgent need for developing safe, effective, and affordable drugs for the treatment of leishmaniasis. To fill this therapeutic gap, several research groups introduced new classes of active compounds against leishmaniasis, including bisindole derivatives. [3] As part of our ongoing investigations on the biological activities and applications of bisindole derivatives [4] and leishmaniasis [3,5], in this contribution, a phenotypic screening of a small library of azole-bisindoles against several human and canine L. infantum strains was performed. Most of the tested compounds showed good activity against the promastigotes (IC50 values 50. Biochemical studies based on DNA-plasmid relaxation assay point at Leishmania topoisomerase IB as molecular-target for URB1483. Docking studies together with metadynamics simulations support the hypothesis that URB1483 might undertake interactions within the topoisomerase IB similar to that of the topoisomerase poison topotecan [3]. Taken together, these data indicate that URB1483 may represent a promising lead compound for the generation of new Leishmania topoisomerase IB poisons with low toxicity on host cells. [1] A. S. Nagle, V. Molteni, et al., Chem. Rev. 2014, 114, 11305; [2] S. Srivastava, P. Shankar, J. Mishra, S. Singh, Parasites & Vectors 2016, 9, 277; [3] A. Roy, A. Desideri, H. K. Majumder, et al., PLoS ONE 2011, 6 (12), e28493; [4] R. Campana, S. Lucarini, et al., Pharmaceuticals 2020, 13 (9), 210; [5] A. Diotallevi, G. Buffi, L. Galluzzi, et al., Acta Tropica 2020, 201, 105178