Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 \u3bcM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 \u3bcM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability

Onchocercosis in Red Deer (\u3ci\u3eCervus elaphus\u3c/i\u3e) from Spain

Onchocercosis, caused by Onchocerca flexuosa, was observed in red deer (Cervus elaphus) from Spain for the first time. Adult specimens of O. flexuosa were found in nodules in subcutaneous tissues in 42 of 125 (33%) red deer between October 1994 and September 1995; intensity of infection 6 SD was 3.93 ± 5.26 nodules per infected host. A clear seasonal pattern in the distribution of nodules was observed, with higher values of prevalence and intensity in fall and winter in contrast to spring and summer. Significant differences were found among age groups in prevalence, but not in the mean intensity. No differences in infection were apparent between male and female adult red deer

Age Distribution and Seasonal Dynamics of Abomasal Helminths in Wild Red Deer from Central Spain

A study on age distribution and seasonal dynamics of abomasal helminths in wild red deer was conducted in central Spain, by monthly samplings of fawns (\u3c1 \u3eyr), subadult (1–2 yr), and adult (\u3e2 yr) animals. Both intensity and prevalence of abomasal parasitism were higher in older animals, particularly in males. A bimodal pattern for intensity of infection by gastrointestinal parasites was observed. Maximum values attained in winter and summer may be related to variation in climate and the shifting availability of forage resources. The pattern was largely due to the contribution of Spiculopteragia asymmetrica/Spiculopteragia quadrispiculata, whereas the other species found (Ostertagia leptospicularis/Ostertagia kolchida and Ostertagia drozdzi/Ostertagia ryjikovi) occurred with lower prevalence and intensity of infection. Among these ostertagiines, the ratio for major and minor morphotypes of males of respective species and the relative abundance of males and females were stable through the annual cycle

Abomasal Parasites in Wild Sympatric Cervids, Red Deer, \u3ci\u3eCervus elaphus\u3c/i\u3e, and Fallow Deer, \u3ci\u3eDama dama\u3c/i\u3e, from Three Localities across Central and Western Spain: Relationship to Host Density and Park Management

A survey of abomasal parasites in cervids from Central Spain was conducted at three sites, Quintos de Mora (Toledo), Maluéñez de Arriba (Cáceres), and La Herguijuela (Cáceres). Commonly occurring helminths belonged to three polymorphic species of the Ostertagiinae: Spiculopteragia asymmetrica/S. quadrispiculata, Ostertagia leptospicularis/O. kolchida, and O. drozdzi/O. ryjikovi. Trichostrongylus axei was found in very few cases. Ostertagia drozdzi/O. ryjikovi and the minor male morphotype, S. quadrispiculata, are reported for the first time in red deer from Spain. The three ostertagiine species are also reported for the first time in fallow deer from Spain. These three species of Ostertagiinae are primarily parasites of cervids, and nematode species characteristic of domestic ruminants were not present. Prevalence of infection by gastrointestinal parasites in cervids was high, ranging from 97.5 to 100%, across the three areas sampled. Mean intensity of infection and abundance showed a positive relationship to the population density of red deer. Helminth burdens were higher in fallow deer than in the sympatric red deer and may reflect the gregarious social structure and different foraging patterns of fallow deer

Identical ITS-1 and ITS-2 Sequences Suggest \u3ci\u3eSpiculopteragia asymmetrica\u3c/i\u3e and \u3ci\u3eSpiculopteragia quadrispiculata\u3c/i\u3e (Nematoda: Trichostrongylidae) Constitute Morphologically Distinct Variants of a Single Species (Research Notes)

Sequences of ITS-1 and ITS-2 rDNA for adult males of Spiculopteragia asymmetrica,/i\u3e and Spiculopteragia quadrispiculata in red deer (Cervus elaphus) were determined. They were found to be identical, suggesting that S. asymmetrica and S. quadrispiculata represent a single species and do not refute the concept of dimorphic species in the Spiculopteragia

Vaccination of lambs against Haemonchus contortus with the recombinant rHc23. Effect of adjuvant and antigen dose.

Haemonchus contortus is the most pathogenic gastrointestinal helminth of small ruminants. Natural or experimental repeated infections and several native antigens confer a partially protective immune response but vaccination with subunit antigens has been elusive. Promising results have been obtained with a recombinant form of a somatic antigen (rHc23). In this paper we present the results obtained in vaccination trials in lambs using two dosages of rHc23 and standard adjuvants. Six-months old Manchego females lambs were vaccinated with rHc23 (50 or 200 μg/dose) adjuvanted with 1mL aluminum hydroxide on days -42, -28 and -14 or with 200 μg/dose rHc23 and 5 mg Quil A on days -49, -28 and -7. Control lambs were kept receiving only the adjuvants the same days or no treatment. Moreover one group did not receive any treatment or infection. On day 0 vaccinated lambs, untreated animals and those receiving the adjuvant were infected per os with a monospecific single infection of 4000 L3 of H. contortus. Infection was kept for 58 days and follow-up included the determination of serum specific antibody response (ELISA, WB), hematological parameters (eosinophil counts, hematocrit) and fecal egg counts (epg). Absence of hematocrit alterations, reduction of helminth's eggs output and abomasal parasite burden at the end of the experiment were the efficacy criteria of vaccination with the recombinant. Immunization with both adjuvants and antigen dosages elicited strong antibody responses particularly with Quil A. Vaccinated groups showed significant reduction of fecal egg excretion and abomasal helminth burdens. Highest protection of lambs against challenge was achieved with aluminum hydroxide and 200 μg/dose rHc23 with a reduction of over 70% of the abomasal burden and over 80% of fecal egg output. Results suggest that rHc23 could be a valuable recombinant candidate for vaccination against haemonchosis. No clear relationship was found between antibody levels and protection this pointing towards involvement of both humoral and cellular components in the protective response elicited by rHc23

Infection of dogs by Leishmania infantum elicits a general response of IgG subclasses.

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis. In endemic areas, canine infections are considered the main source of infection for human populations. Therefore, any control of human leishmaniasis must include the control of canine infections. Chemotherapy of leishmaniasis is inadequate and canine immunoprophylaxis has important limitations. Reports on the response of infected dogs are abundant but no clear picture of immune events has emerged. To shed some light on these shortcomings the specific IgG subclass response was followed in 20 Beagle dogs experimentally infected with L. infantum using monoclonal antibodies (MAb) specific for canine IgG1, IgG2, IgG3 and IgG4, along with ELISA and flow cytometry. Results showed that parasitic infection elicits a general response of all IgG subclasses, with a predominant IgG1 response and without any evidence of IgG1/IgG2 dichotomy. These findings suggest that the inconsistent results reported previously could be related to the lack of specific reagents and not to the actual differences in the immune response of infected animals. Differential IgG subclass reactivity in ELISA and cytometry and the analysis of the reacting antigens could facilitate the diagnosis and prognosis of the disease and provide a useful tool for adequate therapeutics and vaccine development against leishmaniasis.This project has received partial funding from the European Union Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 603240 (NMTrypI – New Medicines for Trypanosomatidic Infections).S

Allicin Induces Calcium and Mitochondrial Dysregulation Causing Necrotic Death in Leishmania

BACKGROUND: Allicin has shown antileishmanial activity in vitro and in vivo. However the mechanism of action underlying its antiproliferative effect against Leishmania has been virtually unexplored. In this paper, we present the results obtained in L.infantum and a mechanistic basis is proposed. METHODOLOGY/PRINCIPAL FINDING: Exposure of the parasites to allicin led to high Ca2+ levels and mitochondrial reactive oxygen species (ROS), collapse of the mitochondrial membrane potential, reduced production of ATP and elevation of cytosolic ROS. The incubation of the promastigotes with SYTOX Green revealed that decrease of ATP was not associated with plasma membrane permeabilization. Annexin V and propidium iodide (PI) staining indicated that allicin did not induce phospholipids exposure on the plasma membrane. Moreover, DNA agarose gel electrophoresis and TUNEL analysis demonstrated that allicin did not provoke DNA fragmentation. Analysis of the cell cycle with PI staining showed that allicin induced cell cycle arrest in the G2/M phase. CONCLUSIONS/SIGNIFICANCE: We conclude that allicin induces dysregulation of calcium homeostasis and oxidative stress, uncontrolled by the antioxidant defense of the cell, which leads to mitochondrial dysfunction and a bioenergetic catastrophe leading to cell necrosis and cell cycle arrest in the premitotic phase.This work was supported by Ministerio de Economía y Competitividad, Comisión Interministerial de Ciencia yTecnología, Grant AGL2009-13009, www.mineco.gob.es (JMA), and European Commission COST Action CM1307.www.cost.eu/COST_Actions/cmst/Actions/CM1307 (JMA, MJC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Serum specific IgG response of vaccinated lambs with rHc23 after challenge with 4000 L3 <i>Haemonchus contortus</i>.

<p>Fig 1A: Anti-rHc23 serum IgG response of experimental lambs. Fig 1B: Anti-Adult Soluble Extract of <i>H</i>. <i>contortus</i> (ASE) serum IgG levels along the experiment. Sera were diluted 1/200 and data are mean±standard deviation of the optical density (OD) values found before infection (weeks -7 and -3), the day of the infection (week 0) and weekly afterwards up to week 7 post challenge.</p