Early prey intake of a short-finned pilot whale (Globicephala macrorhynchus Gray, 1846, Cetacea: Delphinidae) in the Canary Islands.

This study reveals early prey eating by a short-finned pilot whale (Globicephala macrorhynchus Gray, 1846, Cetacea: Delphinidae) in the Canary Islands. Stomach contents, trophic markers, skin isotopic ratios of nitrogen (δ15N:15N/14N) and carbon (δ13C:13C/12C), and fatty acid profiles of the blubber of a short-finned pilot whale of 213 cm size euthanized in free-ranging conditions were analyzed. A total of 15 species of oegopsid squids, mostly diel vertical mesopelagic migrant species of the families Enoploteuthidae, Ommastrephidae, and Histioteuthidae, as well as mother's milk, were identified in the stomach contents. Asperoteuthis acanthoderma (Lu, 1977, Cephalopoda: Chiroteuthidae) was found as first time in this area, suggesting the possibility of its presence on both sides of the subtropical Atlantic, extending its current known distribution. The δ15N value (11.55‰) was higher than expected based on the size range of squid ingested, but lower than that of adult pilot whales, suggesting that mother's milk intake has a significant effect on these values in calves. Similarly, the δ13C values (-17.99‰) were shifted to those of adult pilot whales rather than the ingested squids, also due to the ingestion of high-fat breast milk. The fatty acid (FA) composition of blubber showed a clear stratification. Long-chain polyunsaturated fatty acids (LC-PUFA) were mainly present in the inner layer, while most relevant ≤C20 monounsaturated fatty acids (MUFA) were more abundant in the outer layer.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

Microplastic ingestion and plastic additive detection in pelagic squid and fish: Implications for bioindicators and plastic tracers in open oceanic food webs.

The ubiquitous presence of microplastics (MPs) in the ocean represents a potential threat to marine organisms, with poorly understood long-term adverse effects, including exposure to plastic additives. The present study investigated the ingestion of MPs in two epipelagic fish species (Trachurus picturatus and Scomber colias) and three pelagic squid species (Loligo vulgaris, Ommastrephes caroli and Sthenoteuthis pteropus) from an open oceanic region of the Northeast Atlantic. Seven phthalate esters (PAEs) were also analysed in the organisms' tissue, and the potential correlation between PAEs concentrations and ingested MPs was investigated. Seventy-two fish and 20 squid specimens were collected and analysed. MPs were found in the digestive tract of all species and in the squid species' gills and ink sacs. The highest occurrence of MPs was in the stomach of S. colias (85 %) and the lowest in the stomach and ink sac of O. caroli and L. vulgaris (12 %). Most of the particles identified (>90 %) were fibres. Among all the ecological and biological factors considered (dietary preferences, season, body size, total weight, liver weight, hepatosomatic index and gastrosomatic index), only gastrosomatic index (GSI) and season were significant predictors of MPs ingestion in fish species, with a greater likelihood of ingestion in the cold season and in specimens with higher GSI values (i.e. higher feeding intensity). Four PAEs (DEP, DIBP, BBP, DEHP) were detected in all the species analysed, with average ∑PAEs concentrations ranging between 10.31 and 30.86 ng/g (wet weight). DIBP was positively correlated with ingested MPs, suggesting this compound might represent a "plastic tracer". This study looks into the problem of MPs ingestion for pelagic species in an open oceanic region, highlighting the most suitable bioindicators and providing essential insights into the factors that may influence ingestion rates. Additionally, the detection of PAEs in all species indicates the need for further research on the contamination sources, the effects of these chemicals on marine organisms, and the potential risks to human health through seafood consumption.

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology.

Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.

Towards a Sustainable Vector-Control Strategy in the Post Kala-Azar Elimination Era.

Visceral leishmaniasis (VL) is a potentially deadly parasitic disease. In the Indian sub-continent, VL is caused by Leishmania donovani and transmitted via the bite of an infected Phlebotomus argentipes female sand fly, the only competent vector species in the region. The highest disease burden is in the northern part of the Indian sub-continent, especially in the state of Bihar. India, Bangladesh, and Nepal embarked on an initiative, coordinated by World Health Organization, to eliminate VL as a public health problem by the year 2020. The main goal is to reduce VL incidence below one case per 10,000 people through early case-detection, prompt diagnosis and treatment, and reduction of transmission using vector control measures. Indoor residual spraying, a major pillar of the elimination program, is the only vector control strategy used by the government of India. Though India is close to its VL elimination target, important aspects of vector bionomics and sand fly transmission dynamics are yet to be determined. To achieve sustained elimination and to prevent a resurgence of VL, knowledge gaps in vector biology and behavior, and the constraints they may pose to current vector control methods, need to be addressed. Herein, we discuss the successes and failures of previous and current vector-control strategies implemented to combat kala-azar in Bihar, India, and identify gaps in our understanding of vector transmission towards development of innovative tools to ensure sustained vector control in the post-elimination period.

Heme Oxygenase-1 Induction by Blood-Feeding Arthropods Controls Skin Inflammation and Promotes Disease Tolerance.

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.

In Vitro and In Vivo Characterization of Potent Antileishmanial Methionine Aminopeptidase 1 Inhibitors.

Leishmania major is the causative agent of cutaneous leishmaniasis (CL). No human vaccine is available for CL, and current drug regimens present several drawbacks, such as emerging resistance, severe toxicity, medium effectiveness, and/or high cost. Thus, the need for better treatment options against CL is a priority. In the present study, we validate the enzyme methionine aminopeptidase 1 of L. major (MetAP1Lm), a metalloprotease that catalyzes the removal of N-terminal methionine from peptides and proteins, as a chemotherapeutic target against CL infection. The in vitro antileishmanial activities of eight novel MetAP1 inhibitors (OJT001 to OJT008) were investigated. Three compounds, OJT006, OJT007, and OJT008, demonstrated potent antiproliferative effects in macrophages infected with L. major amastigotes and promastigotes at submicromolar concentrations, with no cytotoxicity against host cells. Importantly, the leishmanicidal effect in transgenic L. major promastigotes overexpressing MetAP1Lm was diminished by almost 10-fold in comparison to the effect in wild-type promastigotes. Furthermore, the in vivo activities of OJT006, OJT007, and OJT008 were investigated in L. major-infected BALB/c mice. In comparison to the footpad parasite load in the control group, OJT008 decreased the footpad parasite load significantly, by 86%, and exhibited no toxicity in treated mice. We propose MetAP1 inhibitor OJT008 as a potential chemotherapeutic candidate against CL infection caused by L. major infection.

Purification of Glycosylphosphatidylinositol-Anchored Mucins from Trypanosoma cruzi Trypomastigotes and Synthesis of α-Gal-Containing Neoglycoproteins: Application as Biomarkers for Reliable Diagnosis and Early Assessment of Chemotherapeutic Outcomes of Chagas Disease.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

A prophylactic α-Gal-based glycovaccine effectively protects against murine acute Chagas disease.

Chagas disease (ChD), caused by the hemoflagellate parasite Trypanosoma cruzi, affects six to seven million people in Latin America. Lately, it has become an emerging public health concern in nonendemic regions such as North America and Europe. There is no prophylactic or therapeutic vaccine as yet, and current chemotherapy is rather toxic and has limited efficacy in the chronic phase of the disease. The parasite surface is heavily coated by glycoproteins such as glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins), which display highly immunogenic terminal nonreducing α-galactopyranosyl (α-Gal)-containing glycotopes that are entirely absent in humans. The immunodominant tGPI-mucin α-Gal glycotope, the trisaccharide Galα1,3Galß1,4GlcNAc (Galα3LN), elicits high levels of protective T. cruzi-specific anti-α-Gal antibodies in ChD patients in both the acute and chronic phases. Although glycoconjugates are the major parasite glycocalyx antigens, they remain completely unexplored as potential ChD vaccine candidates. Here we investigate the efficacy of the T. cruzi immunodominant glycotope Galα3LN, covalently linked to a carrier protein (human serum albumin (HSA)), as a prophylactic vaccine candidate in the acute model of ChD, using the α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mouse, which mimics the human immunoresponse to α-Gal glycotopes. Animals vaccinated with Galα3LN-HSA were fully protected against lethal T. cruzi challenge by inducing a strong anti-α-Gal antibody-mediated humoral response. Furthermore, Galα3LN-HSA-vaccinated α1,3GalT-KO mice exhibited significant reduction (91.7-99.9%) in parasite load in all tissues analyzed, cardiac inflammation, myocyte necrosis, and T cell infiltration. This is a proof-of-concept study to demonstrate the efficacy of a prophylactic α-Gal-based glycovaccine for experimental acute Chagas disease.

Development of Thiophene Compounds as Potent Chemotherapies for the Treatment of Cutaneous Leishmaniasis Caused by Leishmania major.

Leishmania major (L. major) is a protozoan parasite that causes cutaneous leishmaniasis. About 12 million people are currently infected with an annual incidence of 1.3 million cases. The purpose of this study was to synthesize a small library of novel thiophene derivatives, and evaluate its parasitic activity, and potential mechanism of action (MOA). We developed a structure⁻activity relationship (SAR) study of the thiophene molecule 5A. Overall, eight thiophene derivatives of 5A were synthesized and purified by silica gel column chromatography. Of these eight analogs, the molecule 5D showed the highest in vitro activity against Leishmania major promastigotes (EC50 0.09 ± 0.02 µM), with an inhibition of the proliferation of intracellular amastigotes higher than 75% at only 0.63 µM and an excellent selective index. Moreover, the effect of 5D on L. major promastigotes was associated with generation of reactive oxygen species (ROS), and in silico docking studies suggested that 5D may play a role in inhibiting trypanothione reductase. In summary, the combined SAR study and the in vitro evaluation of 5A derivatives allowed the identification of the novel molecule 5D, which exhibited potent in vitro anti-leishmanial activity resulting in ROS production leading to cell death with no significant cytotoxicity towards mammalian cells.

An α-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major.

BACKGROUND: Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL. METHODOLOGY/PRINCIPAL FINDINGS: Here, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galß-BSA (NGP5B), but not Galα(1,4)Galß-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines. CONCLUSIONS/SIGNIFICANCE: We propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.

Ruthenium-Clotrimazole complex has significant efficacy in the murine model of cutaneous leishmaniasis.

In previous studies we reported a novel series of organometallic compounds, RuII complexed with clotrimazole, displaying potent trypanosomatid activity with unnoticeable toxicity toward normal mammalian cells. In view of the promising activity of Ru-clotrimazole complexes against Leishmania major (L. major), the present work sought to investigate the anti-leishmanial activity of the AM162 complex in the murine model of cutaneous leishmaniasis. In addition, to facilitate the design of new therapeutic strategies against this disease, we investigated the mode of action of two Ru-clotrimazole complexes in L. major promastigotes. Overall, we demonstrate that AM162 significantly reduced the lesion size in mice exposed to L. major infection. In addition, Ru-clotrimazole compounds are able to induce a mitochondrial dependent apoptotic-like death in the extracellular form of the parasite based on labeling of DNA fragments, mitochondrial depolarization, cell cycle alteration profile and plasma membrane phospholipid externalization. Our findings reveal a promising efficacy of the Ru-clotrimazole AM162 complex for the treatment of cutaneous leishmaniasis, as well as pro-apoptotic activity and thus guarantees further evaluation in pre-clinical studies.

Novel arylalkylamine compounds exhibits potent selective antiparasitic activity against Leishmania major.

Leishmania major (L. major) is a protozoan parasite causal agent of Leishmaniasis. It is estimated that 12 million people are currently infected and around 2 million infections occur each year. Current treatments suffer of high toxicity for the patient, low efficacy toward the parasite, high cost, and are losing effectiveness due to parasite resistance. Discovering novel small molecule with high specificity/selectivity and drug-like properties for anti-leishmanial activity remains a significant challenge. The purpose of this study is to communicate the design and synthesis strategies of novel chemical compounds based of the arylalkylamine scaffold with selective toxicity towards L. major and less toxicity to human cells in vitro. Here, we have developed a structure activity relationship (SAR) study of arylalkylamine AA1 in order to study their anti-parasitic effect in L. major. Overall, 27 arylalkylamine compounds derived from AA1 were synthesized and purified by silica gel column chromatography. The purity of each analog was confirmed by spectroscopic methods ((1)H, (13)C NMR and LC/MS). Among these analogs, the compound AA9 showed the best toxic activity on L. major (LD50=3.34 µM), which represents a 9 fold higher lethality as compared with its parental AA1 (Fer-1) compound (LD50=28.75 µM). In addition, AA9 showed no significant toxicity at 80 µM on U20S Human Osteoblasts, Raw 264.7 Macrophages or intraperitoneal macrophages. In summary, our combined SAR study and biological evaluation data of AA1-AA27 compounds allow the identification of novel arylalkylamine compound AA9 that exhibits potent cytotoxicity against L. major promastigote with minimum toxic effect on human cells.

Evaluation of α,ß-unsaturated ketones as antileishmanial agents.

In this study, we assessed the antileishmanial activity of 126 α,ß-unsaturated ketones. The compounds NC901, NC884, and NC2459 showed high leishmanicidal activity for both the extracellular (50% effective concentration [EC50], 456 nM, 1,122 nM, and 20 nM, respectively) and intracellular (EC50, 1,870 nM, 937 nM, and 625 nM, respectively) forms of Leishmania major propagated in macrophages, with little or no toxicity to mammalian cells. Bioluminescent imaging of parasite replication showed that all three compounds reduced the parasite burden in the murine model, with no apparent toxicity.

Metal-drug synergy: new ruthenium(II) complexes of ketoconazole are highly active against Leishmania major and Trypanosoma cruzi and nontoxic to human or murine normal cells.

In our ongoing search for new metal-based chemotherapeutic agents against leishmaniasis and Chagas disease, six new ruthenium-ketoconazole (KTZ) complexes have been synthesized and characterized, including two octahedral coordination complexes-cis,fac-[Ru(II)Cl2(DMSO)3(KTZ)] (1) and cis-[Ru(II)Cl2(bipy)(DMSO)(KTZ)] (2) (where DMSO is dimethyl sulfoxide and bipy is 2,2'-bipyridine)-and four organometallic compounds-[Ru(II)(η(6)-p-cymene)Cl2(KTZ)] (3), [Ru(II)(η(6)-p-cymene)(en)(KTZ)][BF4]2 (4), [Ru(II)(η(6)-p-cymene)(bipy)(KTZ)][BF4]2 (5), and [Ru(II)(η(6)-p-cymene)(acac)(KTZ)][BF4] (6) (where en is ethylenediamine and acac is acetylacetonate); the crystal structure of 3 is described. The central hypothesis of our work is that combining a bioactive compound such as KTZ and a metal in a single molecule results in a synergy that can translate into improved activity and/or selectivity against parasites. In agreement with this hypothesis, complexation of KTZ with Ru(II) in compounds 3-5 produces a marked enhancement of the activity toward promastigotes and intracellular amastigotes of Leishmania major, when compared with uncomplexed KTZ, or with similar ruthenium compounds not containing KTZ. Importantly, the selective toxicity of compounds 3-5 toward the leishmania parasites, in relation to human fibroblasts and osteoblasts or murine macrophages, is also superior to the selective toxicities of the individual constituents of the drug. When tested against Trypanosoma cruzi epimastigotes, some of the organometallic complexes displayed activity and selectivity comparable to those of free KTZ. A dual-target mechanism is suggested to account for the antiparasitic properties of these complexes.

Searching for new chemotherapies for tropical diseases: ruthenium-clotrimazole complexes display high in vitro activity against Leishmania major and Trypanosoma cruzi and low toxicity toward normal mammalian cells.

Eight new ruthenium complexes of clotrimazole (CTZ) with high antiparasitic activity have been synthesized, cis,fac-[Ru(II)Cl(2)(DMSO)(3)(CTZ)] (1), cis,cis,trans-[Ru(II)Cl(2)(DMSO)(2)(CTZ)(2)] (2), Na[Ru(III)Cl(4)(DMSO)(CTZ)] (3), Na[trans-Ru(III)Cl(4)(CTZ)(2)] (4), [Ru(II)(η(6)-p-cymene)Cl(2)(CTZ)] (5), [Ru(II)(η(6)-p-cymene)(bipy)(CTZ)][BF(4)](2) (6), [Ru(II)(η(6)-p-cymene)(en)(CTZ)][BF(4)](2) (7), and [Ru(II)(η(6)-p-cymene)(acac)(CTZ)][BF(4)] (8) (bipy = bipyridine; en = ethlylenediamine; acac = acetylacetonate). The crystal structures of compounds 4-8 are described. Complexes 1-8 are active against promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. Most notably, complex 5 increases the activity of CTZ by factors of 110 and 58 against L. major and T. cruzi, with no appreciable toxicity to human osteoblasts, resulting in nanomolar and low micromolar lethal doses and therapeutic indexes of 500 and 75, respectively. In a high-content imaging assay on L. major-infected intraperitoneal mice macrophages, complex 5 showed significant inhibition on the proliferation of intracellular amastigotes (IC(70) = 29 nM), while complex 8 displayed some effect at a higher concentration (IC(40) = 1 µM).