New approaches to fluorescence-based diagnostics for human African trypanosomiasis

In the absence of any vaccine, prophylactic drug and effective vector control, the fight against human African trypanosomiais (HAT) is based on the the combination of active case-finding and consequent drug treatment of identified positive cases. Unfortunately, low sensitivity and specificity of current diagnostic techniques often result in misdiagnosis, leaving infected patients without cure or exposing them to inappropriate chemotherapy protocols, which use dangerous and expensive drugs. The development of more efficient, simple, cheap and field-robust diagnostic tests is, therefore, urgently needed. In the field, direct observation by light microscopy of trypanosomes in human fluids (blood, lymph node aspirate, cerebrospinal fluid) is considered the ideal way of confirming HAT infection. However, in practice this approach is problematic, especially for the Gambian form of the disease, where patients may present with very low parasitaemia. Detection limits of parasitological techniques can be improved by adding a preliminary step of sample concentration, although this further increases the laboriousness of HAT diagnostic algorithm. Recent advances in fluorescence microscopy could be exploited to facilitate trypanosome detection. The introduction and implementation of fluorescence microscopy in HAT endemic countries would offer the advantages of an increased overall sensitivity of microscopical examination and a more rapid screening of the specimen. In contrast to traditional, expensive and fragile fluorescence microscopes, new LED-illuminated instruments are relatively cheap, very efficient and portable, lending themselves to utilisation in poorly equipped rural settings. In order to design a new diagnostic tool that exploits LED technology, however, selective and reliable fluorescent markers to label trypanosomes in human fluids are needed. The development of new tools to assist in the diagnosis of African trypanosomiasis by use of LED fluorescence microscopy was the overall objective of this project. The work was mainly focused on testing various fluorescent compounds for their ability to selectively stain trypanosomes. Fluorophores were otained from commercial and academic sources, or else directly synthesised during the project. An important requirement evaluated was the compounds’ compatibility with the currently available SMR LED Cytoscience fluorescence microscope, developed and kindly provided by our collaborator Prof. D. Jones (Philipps University, Marburg). The utility of a UV LED-driven microscope in performing the arsenical drug resistance test was also assessed. This assay, developed in our laboratory to detect trypanosome strains resistant to arsenical and diamidine compounds, could represent a useful tool for chemotherapeutic decision making in the field, where resistance to arsenical drugs is a rising problem

EMERGING MODELS FOR CORPORATE WELFARE AND HR MANAGEMENT IN THE SERVICE-DOMINANT LOGIC

large body of research highlights the key-role of human resources in the new service economy. The service-dominant logic considers them as operant resources in value co-creation. Internal marketing policies, total rewards strategies and corporate welfare initiatives underline the importance of getting employees motivated, engaged, and satisfied. By combining HRM, IT and service systems issues, this paper aims to explore the emergence of new professional services providers in the Italian context and their innovative solutions in managing corporate welfare programs through integrated service systems. Through a systematic review, our findings describe the common features of emerging corporate welfare service systems and suggest several future paths of research in order to advance empirical knowledge in this high-potential field

Primary prophylaxis of neutropenia in women affected by breast cancer undergoing adjuvant chemotherapy with fec 100+/- docetaxel. Comparison of efficacy and tolerability between lenograstim and pegfilgrastim

Objectives: evaluate safety and toxicity of a single injection of pegfilgrastim compared to daily administration of lenograstim in breast cancer patient undergoing adjuvant chemotherapy

Traditional decoction and PUAE aqueous extracts of pomegranate peels as potential low-cost anti-tyrosinase ingredients

open6The aim of the study is to evaluate the anti-tyrosinase activity of dierent aqueous extracts obtained from pomegranate juice processing by-products. External pomegranate peels of two certified cultivars (Akko and Wonderful), were extracted using only water as the extraction solvent. A traditional decoction and a pulsed ultrasound-assisted extraction (PUAE), both 10 min long, were performed and compared. All the aqueous extracts proved to be rich in bioactive compounds. In particular, the total phenolic content (TPC) ranged from 148 to 237 mg gallic acid equivalent (GAE)/g of dried peels (DW), the radical-scavenging ability (RSA) ranged from 307 to 472 mg ascorbic acid equivalent (AAE)/g DW, the free ellagic acid content (EA) ranged from 49 to 94 g/mL, and the ellagitannins (ETs) ranged from 242 to 340 g/mL. For both cultivars, PUAE extracts had higher ET content and a lower EC50, while the decoctions had slightly higher TPC, RSA, and free EA amounts. Principal component analysis (PCA) highlighted the direct correlation between the ET content and the tyrosinase enzyme inhibition (lower values of EC50). These findings suggest the potential use of both these natural extracts as low-cost lightening and/or anti-browning ingredients exploitable in several formulations (e.g., cosmetics) or extemporarily usable.openFEDERICA TURRINI; PAOLA MALASPINA; PAOLO GIORDANI; SILVIA CATENA; PAOLA ZUNIN; RAFFAELLA BOGGIA;Turrini, Federica; Malaspina, Paola; Giordani, Paolo; Catena, Silvia; Zunin, Paola; Boggia, Raffaell

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition

Green fluorescent diamidines as diagnostic probes for trypanosomes

LED fluorescence microscopy offers potential benefits to the diagnosis of human African trypanosomiasis, as well as to other aspects of diseases management, such as detection of drug resistant strains. To advance such approaches reliable and specific fluorescent markers to stain parasites in human fluids are needed. Here we report a series of novel green fluorescent diamidines and their suitability as probes to stain trypanosomes

'Transcriptional differentiation of Trypanosoma brucei during in vitro acquisition of resistance to acoziborole

Subspecies of the protozoan parasite Trypanosoma brucei are the causative agents of Human African Trypanosomiasis (HAT), a debilitating neglected tropical disease prevalent across sub-Saharan Africa. HAT case numbers have steadily decreased since the start of the century, and sustainable elimination of one form of the disease is in sight. However, key to this is the development of novel drugs to combat the disease. Acoziborole is a recently developed benzoxaborole, currently in advanced clinical trials, for treatment of stage 1 and stage 2 HAT. Importantly, acoziborole is orally bioavailable, and curative with one dose. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. In this study, an in vitro-derived acoziborole-resistant cell line was generated and characterised. The Aco(R) line exhibited significant cross-resistance with the methyltransferase inhibitor sinefungin as well as hypersensitisation to known trypanocides. Interestingly, transcriptomics analysis of Aco(R) cells indicated the parasites had obtained a procyclic- or stumpy-like transcriptome profile, with upregulation of procyclin surface proteins as well as differential regulation of key metabolic genes known to be expressed in a life cycle-specific manner, even in the absence of major morphological changes. However, no changes were observed in transcripts encoding CPSF3, the recently identified protein target of acoziborole. The results suggest that generation of resistance to this novel compound in vitro can be accompanied by transcriptomic switches resembling a procyclic- or stumpy-type phenotype

Novel minor groove binders cure animal African trypanosomiasis in an in vivo mouse model

Animal African trypanosomiasis (AAT) is a significant socioeconomic burden for sub-Saharan Africa due to its huge impact on livestock health. Existing therapies including those based upon Minor Groove Binders (MGBs), such as the diamidines, which have been used for decades, have now lost efficacy in some places due to the emergence of resistant parasites. Consequently, the need for new chemotherapies is urgent. Here, we describe a structurally distinct class of MGBs, Strathclyde MGBs (S-MGBs), which display excellent in vitro activities against the principal causative organisms of AAT, Trypanosoma congolense and T. vivax. We also show the cure of T. congolense-infected mice by a number of these compounds. In particular, we identify S-MGB-234, compound 7, as curative using 2 applications of 50 mg/kg intraperitoneally. Crucially, we demonstrate that S-MGBs do not show cross-resistance with the current diamidine drugs and are not internalised via the transporters used by diamidines. This study demonstrates that S-MGBs have significant potential as novel therapeutic agents for animal African trypanosomiasis

Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism

Minor groove binders for DNA as antitypanosomal agents : the veterinary context

Animal African trypanosomiasis (or nagana) is a wasting livestock disease found in sub-Saharan Africa and caused by protozoan parasites Trypanosoma congolense, T. vivax and T. brucei. Chemotherapy (mostly with the diamidine diminazene aceturate) and chemoprophylaxis (mostly with the phenanthridine isometamidium chloride) are essential for disease control. However, as current treatments lose efficacy due to increased drug resistance, the need for new veterinary trypanocides becomes a high-priority. Trypanosoma spp. are one of the genera of parasites that are susceptible to DNA minor groove binder drugs synthesised at the University of Strathclyde (S-MGBs). All three of the above African species are susceptible to S-MGBs and in addition, the South American species, T. cruzi has been shown to be similarly susceptible. One of the principal challenges to obtaining compounds useful in the field is to achieve activity across the range of infectious species so that characterisation of the infection is not required at diagnosis. Over 100 S-MGBs have been evaluated at the University of Glasgow and the Swiss Tropical Health Institute, Basel, and compounds with development potential have been identified. As an example, S-MGB 234 has been shown to be curative in in vivo models of trypanosome infection in mice. Importantly S-MGB 234 does not show cross resistance with other antitrypanosomal drugs such as diminazine, isometamidium, or ethidium bromide, which is consistent with a different route into the parasite’s cell. S-MGBs that contain alkene links, such as in S-MGB 234, are the most active sub-class of S-MGB and point the way towards structural optimisation