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

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

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

ALS2-Related Motor Neuron Diseases: From Symptoms to Molecules

Infantile-onset Ascending Hereditary Spastic Paralysis, Juvenile Primary Lateral Sclerosis and Juvenile Amyotrophic Lateral Sclerosis are all motor neuron diseases related to mutations on the ALS2 gene, encoding for a 1657 amino acids protein named Alsin. This ~185 kDa multi-domain protein is ubiquitously expressed in various human tissues, mostly in the brain and the spinal cord. Several investigations have indicated how mutations within Alsin’s structured domains may be responsible for the alteration of Alsin’s native oligomerization state or Alsin’s propensity to interact with protein partners. In this review paper, we propose a description of differences and similarities characterizing the above-mentioned ALS2-related rare neurodegenerative disorders, pointing attention to the effects of ALS2 mutation from molecule to organ and at the system level. Known cases were collected through a literature review and rationalized to deeply elucidate the neurodegenerative clinical outcomes as consequences of ALS2 mutation

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

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

Rapid monitoring of SARS-CoV-2 variants of concern through high-resolution melt analysis

The current global pandemic of COVID-19 is characterized by waves of infection due to the emergence of new SARS-CoV-2 variants carrying mutations on the Spike (S) protein gene. Since autumn 2020 many Variants of Concern (VOC) have been reported: Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, Omicron/B.1.1.529, and sublineages. Surveillance of genomic variants is currently based on whole-genome sequencing (WGS) of viral genomes on a random fraction of samples positive to molecular tests. WGS involves high costs, extended analysis time, specialized staff, and expensive instruments compared to a PCR-based test. To rapidly identify the VOCs in positive samples, six assays based on real-time PCR and high-resolution melting (HRM) were designed on the S gene and applied to 120 oro/nasopharyngeal swab samples collected from October 2020 to June 2022 (106 positive and 14 negative samples). Overall, the assays showed 100% specificity and sensitivity compared with commercial PCR tests for COVID-19. Moreover, 104 samples out of 106 (98.1%) were correctly identified as follows: 8 Wuhan (wild type), 12 Alpha, 23 Delta, 46 Omicron BA.1/BA.1.1, 15 Omicron BA.2/BA.4/BA.5. With our lab equipment, about 10 samples can be processed every 3 h at the cost of less than € 10 ($ 10.60) per sample, including RNA extraction. The implementation of this approach could help local epidemiological surveillance and clinical decision-making

Human Bone RIgeneration in MAXillo-facial area using an innovative medical device for Tissue engineering (BRIMAX)

Bone regeneration today is one of the most important challenges for medicine and the need for this is particularly evident in the maxillo-facial area: our clinical trial will be based on a model of bone defect as in alveolar socket preservation and sinus lift augmentation, well described surgical techniques. The RIGENERA® system permits extraction of stem cells from a small sample of connective tissue obtained from the patient’s lingual mucosa or from a post-extraction surgical site (where an endosseous implant may be inserted), dental pulp or dental follicle. Our project is to demonstrate the efficacy in the maxillo-facial area of an innovative clinical protocol of bone tissue engineering based on a new medical device called Rigeneracons (CE certified Class I). Our clinical trial use already acquired technologies in comparation with new technologies (new selection methods, new Bio-compatible materials etc.) produced by us. Besides, we perform an in-vitro test to quantify the proliferative capacity of a cellular suspension obtained after disaggregation of connective tissue originating from the oral cavity using the RIGENERA® system, a biologic tissue disaggregator (Human Brain Wave–Torino, Italy) that recently came on the market. Evaluation of the histologic characteristics of neo-formed osseous tissue will be shown and discussed

Arthritis in Systemic Lupus Erythematosus: From 2022 International GISEA/OEG Symposium

Musculoskeletal involvement is one of the most common manifestations of systemic lupus erythematosus (SLE), with a negative impact on both quality of life and overall prognosis. SLE arthritis can be classified into three different subtypes, with different prevalence and characteristic biomarkers and MRI findings. Identifying the pathogenetic mechanisms underlying musculoskeletal manifestations' development is crucial to develop therapeutic strategies to suppress synovial inflammation, prevent erosions and deformities, and improve SLE patients' quality of life. Hence, here we discuss the main pathogenetic mechanisms and therapeutic approaches of musculoskeletal manifestations of SLE from the 2022 International GISEA/OEG Symposium