STILBENE ST18 AND TERFENYL TR4: IN VITRO ACTIVITY AGAINST TRYPANOSOMA CRUZI

Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted to animals and people by insect vectors that are found only in the Americas. Chagas disease and Leishmaniasis are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as “neglected diseases” due to the lack of effective drug treatments and the scarcity of research work devoted to them. Therefore, the development of novel and effective drugs is an important and urgent need. Natural products are an important source of bioactive molecules for the development of new drugs. Recently, studies showed an interesting cytotoxic action of Stilbene ST18 and the Terphenyl TR4 compounds in Leishmania. In this study, we evaluated the in vitro trypanocidal activity of ST18 and TR4 and Nifurtimox, drug used for the treatment of Chagas disease. In addition, we evaluated the compounds action in infected macrophages with Trypanosoma cruzi. Results showed that three compounds exhibited significant activity against Trypanosoma compared to Nifurtimox. ST18 and TR4 compounds inhibited Trypanosoma growth with IC50 values of 4.5 and 32 μM, respectively. The treatment of infected macrophages with trypanosomes with the IC90 compounds showed a reduction of infection compared to control: ST18 reduced the infected cells to 52 %, TR4 reduced the infected cells to 63%. In conclusion, these news compounds could be considered as promising lead drugs for the development of new therapies for the treatment of Chagas disease

Seroprevalence of and risk factors for Leishmania seropositivity in a sample population of Western Sicily (Italy)

Background: Leishmania is a vector-borne parasite responsible for significant morbidity and mortality worldwide. The aim of this study was to assess the prevalence of and risk factors for Leishmania infantum seropositivity in a sample of Sicilian population.Methods: A total of 260 subjects were interviewed using a standardized questionnaire and requested for an venous blood sample.Results: Overall, 36 subjects (13.8%) were seropositive against L. infantum with a statistically significant higher prevalence of positivity in older subjects (p=0.04). After adjustment for age, a higher risk for Leishmania seropositivity was found in subjects who had pets living outdoors and untreated with anti-pests, and in those who were current smokers (adj-OR = 2.95 and adj-OR = 3.11, respectively; p < 0.05).Conclusions: Our data confirm that Leishmania infections among Sicilian citizens can be considered relatively frequent, suggesting that a percentage of Leishmania seropositivity can be probably attributed to exposure to both old and new risk factors

Microsatellite panel definition to characterize Leishmania strains isolated from human samples in an italian endemic region

The Leishmaniasis affects people, domestic and wild animals in temperature, subtropical and tropical regions. The natural cycle involves phlebotominae sandfly vectors transmitting the parasite to the vertebrate host. The insects influence the epidemiology of the disease by their geographical distribution in the seasons and the specific vectorial capacity. Human Leishmania infections are increasing every year in Sicily, which represent the region with the highest endemic level of the disease in Italy. Among different approaches employed for the diagnostic the parasites isolation remains the gold standard

Deep sequencing targeted for strain identification of trypanosomatids

Trypanosomatidae is a group of kinetoplastid excavates distinguished by having only a single flagellum. All members are exclusively parasitic, found primarily in insects. The custom reads form Ilumina sequencing platform are aligned to reference genomes of different Leishmania species taken from TriTryp dabatase to be used for bioinformatics analysis.peer-reviewe

Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples, must be taken into account in quantitative PCR-based applications; however, it might also be used to differentiate between Leishmania subgenera

Feline leishmaniosis: A case with a high parasitic burden

Leishmaniosis is a widespread zoonotic disease with the potential for significant impact on public health. Although dogs are the main reservoir host, the role of other domestic species in transmission of the disease should be considered, and felids may represent an alternative source of infection.1 We describe a case of Leishmania infantum infection in a cat with oral lesions associated with a high parasitic load detected by a quantitative PCR technique. A 5-year-old male short-haired cat was presented for evaluation of respiratory signs, conjunctivitis and oral lesions. Clinical examination revealed a poor-quality hair coat, bilateral conjunctivitis, nasal exudate and a diffuse hyperplastic and ulcerative stomatitis. Complete blood count and serum protein electrophoresis values were within normal range, and screening tests for feline leukaemia virus and feline immunodeficiency virus were negative. Histopathological evaluation of biopsy tissue from the oral lesion confirmed severe, diffuse granulomatous stomatitis, with numerous macrophages containing intracytoplasmic protozoal organisms (Figure 1). Serology was negative for Toxoplasma and Neospora and positive for Leishmania by an indirect fluorescent antibody titre of 1:320.2 A diagnosis of feline leishmaniosis was confirmed

Feline leishmaniosis: a case with a high parasitic burden

Leishmaniosis is a widespread zoonotic disease with the potential for significant impact on public health. Although dogs are the main reservoir host, the role of other domestic species in transmission of the disease should be considered, and felids may represent an alternative source of infection.1 We describe a case of Leishmania infantum infection in a cat with oral lesions associated with a high parasitic load detected by a quantitative PCR technique. A 5-year-old male short-haired cat was presented for evaluation of respiratory signs, conjunctivitis and oral lesions. Clinical examination revealed a poor-quality hair coat, bilateral conjunctivitis, nasal exudate and a diffuse hyperplastic and ulcerative stomatitis. Complete blood count and serum protein electrophoresis values were within normal range, and screening tests for feline leukaemia virus and feline immunodeficiency virus were negative. Histopathological evaluation of biopsy tissue from the oral lesion confirmed severe, diffuse granulomatous stomatitis, with numerous macrophages containing intracytoplasmic protozoal organisms (Figure 1). Serology was negative for Toxoplasma and Neospora and positive for Leishmania by an indirect fluorescent antibody titre of 1:320.2 A diagnosis of feline leishmaniosis was confirmed

Exosome Secretion by Leishmania infantum: modulate innate and adaptive immune responses and create an environment permissive for early infection

In recent years, several studies demonstrated the role of exosomes in intercellular communications. Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania deliver effectors proteins into host cells; however, the mechanisms involved have remained elusive. The aim of this study was to isolate, characterize Leishmania infantum exosomes and to investigate the biological activity of exosomes in macrophage cultures.Human cell lines used were U937 cells and macrophages derived from U937 after treatment with 25 ng/ml PMA.Western blot assay used antibodies to HSP70, HSP83/90. Exosomes were collected by L. infantum -conditioned medium by ultracentrifugation.IL10, IFN?, IL1?IL1?, IL12, IL4 and IL18 secretion were evaluated by ELISA kit.We first purified extracellular vesicles shed by L. Infantum promastigotes and amastigotes on a sucrose gradient and later we characterized these extracellular vesicles for HSP70, HSP83/90 and acetylcholinesterase. Furthermore we performed a NanoSight nanoparticle tracking analysis revealed an average of the mode value of 76 ± 5 nm for promastigotes and 94± 5 nm for amastigotes exosomes. All these data demonstrated that L. Infantum released exosomes. The treatment of U937 cells with 10 μg/ml of promastigote and amastigote exosomes showed an increase in motility of these cells that can facilitate the progression of infection. We showed also an overproduction of IL10 by macrophages after treatment with exosomes that support parasite persistence and disease establishment, while exosomes limited the production of IFN-? and IL12 that block the parasite killing and host protection, as well as the production of IL1?, IL1? and IL4. Regarding IL18 production,that is involved in the Th1-type immune responses,L. exosomes determined a reduction in the production of IL18 in U937 monocyte culturescompared to control. Value that does not vary instead macrophages cultures