Julio López-Abán

Systematic Review and Meta-Analysis of Artemisinin Based Therapies for the Treatment and Prevention of Schistosomiasis

Background Chemotherapy based on repeated doses of praziquantel is still the most effective control strategy against Schistosomiasis, however artemisinin derivatives emerged as a family of compounds with schistomicide activity. The aim of the present work is to compare the efficacy of artemisinin-based therapies in the treatment and prophylaxis of human schistosomiasis. The design of this work involved a quantitative systematic review and meta-analysis. Methodology/Principal Findings Retrieval of published studies was carried out through an electronic search of the PubMed (MEDLINE), EMBASE, Cochrane Library and CINAHL databases. This included reports comparing the therapeutic efficacy of artesunate alone, artesunate plus sulfadoxine-pyrimethamine and a combination of artemisinin derivatives plus praziquantel against praziquantel alone on different types of schistosomiasis. Moreover, studies on artesunate and artemether used as preventive drugs were also analyzed against placebo. The primary outcome measure for schistosomiasis treatment was “parasitological cure”, whereas for the prophylaxis the outcome evaluated was “infection rate”. Our results show that patients treated with artesunate alone have significantly lower cure rates than those treated with praziquantel (OR = 0.27 (95% C.I. 0.13–0.53; p<0.001)) and that the combined therapy of artesunate plus sulfadoxine-pyrimethamine is also significantly less effective than praziquantel treatment (OR = 0.14 (95% C.I. 0.02–0.92; p = 0.04)). However, the combination of an artemisinin derivatives plus praziquantel showed a higher cure rate than praziquantel monotherapy with OR = 2.07 (95% C.I. 1.27–3.36; p = 0.003). Finally, chemoprophylaxis with either artesunate (RR = 0.11 (95% C.I. 0.06–0.22; p<0.001)) or artemether (RR = 0.25 (95% C.I. 0.16–0.40; p<0.001)) was significantly better than a placebo in both cases. Conclusions/Significance This meta-analysis confirms that artemisinin derivatives used in combination with praziquantel have the potential to increase the cure rates in schistosomiasis treatment, but not artesunate alone. It is also confirmed that repeated doses of artemisinin derivatives play a prophylactic role, significantly reducing the incidence of Schistosoma japonicum infections compared with placebo.

In Vitro and In Vivo Efficacy of Ether Lipid Edelfosine against Leishmania spp. and SbV-Resistant Parasites

Background The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro. Methodology/Principal Findings We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance. Conclusions/Significance Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.

Antigens from Ascaris suum trigger in vitro macrophage NO production

We investigated the in vitro effect of total excretory/secretory and somatic antigens from Ascaris suum adults (ESA and SA) and larvae 3 (ESL3 and SL3), and of 10 purified protein fractions from ESA components on rat alveolar macrophage nitric oxide (NO) production. Our results showed that in vitro incubation of macrophages with SA and SL3 antigens of A. suum did not result in NO release from cells, whereas incubation with ESA or ESL3 antigens resulted in the stimulation of NO production by these cells, both in a specific (inhibited by l‐NAME and l‐canavanine) and dose‐dependent manner. In addition, we could demonstrate that a purified ESA fraction consisting of three Coomassie‐stained bands of approximately 37, 44 and 46 kDa is involved in the in vitro triggering of NO production by host cells. These three bands were subjected to MALDI‐peptide mass fingerprint, showing similarities with phosphoglycerate kinase, elongation factor Tu and enolase molecules, respectively. Future studies will focus on the characterization of these parasite‐derived molecules.

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

Background Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites. Methodology/Principal Findings We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis. Conclusions/Significance Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

The addition of a new immunomodulator with the adjuvant adaptation ADAD system using fatty acid binding proteins increases the protection against Fasciola hepatica.

Fatty acid binding proteins (FABP) have shown protective immune response against Fasciola hepatica infection. We evaluated the protection induced by the Fh12 FABP from F. hepatica (Fh12) combined with the new immunomodulator the lipidic aminoalcohol OA0012 in the ADAD system in mice and sheep. In this work we introduced a lipidic aminoalcohol OA0012 as immunomodulator alone or in combination with the hydroalcoholic extract of Phlebodium pseudoaureum; PAL. Mice vaccinated with ADAD containing OA0012+Fh12 or OA0012+Qs+Fh12 had survival rates of 40-50%. Sheep ADAD-vaccinated with OA0012+Qs+Fh12 showed lower fluke recovery, less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Sheep ADAD-vaccinated with OA0012 combined PAL and Qs+Fh12 showed lower fluke recovery (42%), lower adult worms count (57%) lower faecal egg count (38%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the addition of a new immunomodulator of synthesis to ADAD system with FABPs increased the protection against F. hepatica.

Helmintos y aparato respiratorio

El término “parasitismo” indica un tipo de asociación entre 2 seres vivos que tiene consecuencias nocivas para uno de ellos (hospedador) y aporta beneficios al otro (parásito). Aunque que el término “parásito” incluye conceptualmente a todos los seres vivos capaces de causar perjuicio a otros, tradicionalmente en medicina este nombre se aplica de forma exclusiva a los protozoos, helmintos y artrópodos que viven temporal o permanentemente en el ser humano1. Desde un punto de vista práctico, el parasitismo puede ocasionar manifestaciones clínicas (enfermedades parasitarias) o cursar como una infección asintomática1. Con algunas excepciones (principalmente la amebosis y el paludismo grave2), la mayor parte de parásitos que afectan al pulmón son helmintos, es decir, animales invertebrados que tienen aspecto de gusano (vermes). En este trabajo revisaremos de forma conjunta las enfermedades del aparato respiratorio producidas por estos seres vivos en humanos, actualizando varios aspectos ya considerados en ARCHIVOS DE BRONCONEUMOLOGÍA por Cremades3 en 1998. Inicialmente se indicará de forma breve cuáles son los principales géneros y especies implicadas, así como el momento del ciclo biológico en que tiene lugar el contacto con las estructuras respiratorias. En un segundo apartado se revisarán las principales características epidemiológicas, haciendo hincapié en la distribución geográfica de estas enfermedades. A continuación se indicarán los mecanismos de lesión pulmonar (agresión por el parásito y defensa del hospedador) para señalar posteriormente las manifestaciones clínicas. Los 2 últimos bloques se dedicarán a los métodos diagnósticos y a las posibilidades terapéuticas. Agentes causales

Evaluation of the role of angiogenic factors in the pathogenesis of schistosomiasis

Schistosomiasis is one disease produced by helminths, which affect many people in tropical areas. Granuloma formation is the main mechanism involved in the pathogenesis of this disease. Experimental studies have demonstrated angiogenesis (blood vessels formation from pre-existing vessels) in the initial phase of granuloma formation. In the present work, VEGF (vascular endothelial growth factor) levels were analyzed in sera from people diagnosed with different helminthic infections. Patients with schistosomiasis and filariasis had significantly high VEGF levels in compared with healthy people and patients diagnosed with hookworms. In addition, the effects of angiogenesis inhibition using anti-angiogenic factors (endostatin) were evaluated in a schistosomiasis murine model. A lesion decrease was observed in mice infected with Schistosoma mansoni and treated with endostatin. Finally, mechanisms of angiogenesis induction were studied and observed that cercariae antigens stimulated the angiogenic factors by host alveolar macrophages.

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

Background Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis. Methodology/Principal Findings A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%). Conclusions/Significance We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas.

In Vitro and In Vivo Studies for Assessing the Immune Response and Protection-Inducing Ability Conferred by Fasciola hepatica-Derived Synthetic Peptides Containing B- and T-Cell Epitopes

Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L+ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.

IDENTIFICATION OF FASCIOLA HEPATICA RECOMBINANT 15-KDA FATTY ACID–BINDING PROTEIN T-CELL EPITOPES THAT PROTECT AGAINST EXPERIMENTAL FASCIOLIASIS IN RABBITS AND MICE

Vaccination with fatty acid–binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Th1/Th2 response to vaccination.