María Jesús Corral

Reuse of Liver Grafts after Early Death of the First Recipient

Abstract. Three cases are reported of reuse of a transplanted liver graft after early death of the first recipient due to cerebral hemorrhage. The good condition of the donors; the excellent biochemical evolution of the graft in the first recipients; total ABO compatibility and donor-recipient crossmatch; the absence of positivity to hepatitis B virus (HBV), hepatitis C virus (HCV), and bacteriologic cultures; and early death made reuse possible. The shortage of donors in relation to patients on the waiting list and the poor clinical condition of the second recipients made it necessary to adopt the decision to reuse the graft in an attempt to save their lives. The evolution of the patients and the reused grafts was satisfactory, and there were no complications that could be attributed to the fact that the graft had been transplanted before.

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths.

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5-6 months old Assaf lambs using Al(OH)(3) or Escherichia coli lipopolysaccharide+inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%-85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.

Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue

The value of resazurin-based Alamar Blue redox indicator to determine multiplication of Leishmania promastigotes in 96-well microtiter plates was examined. In addition, assay was validated with amphotericin B (AmB) and allicin. The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variable air-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed that the gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating cell densities of 2.5 × 10⁵ promastigotes/well, up to 72 h incubation and 5% CO₂ atmosphere or reduced air availability (sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results could be obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results. Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration (IC₅₀) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditions this one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferation rates of Leishmania isolates or strains in a 96-well format.

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani.

The toxicity and antileishmanial activity of 20 betulin derivatives were studied. The toxicity of betulin and synthesized compounds was determined using a bacterial test (Microtox) and two mammalian cell lines (CHO-K1 and J774). The antileishmanial activity of compounds (50 μM) was examined in both the promastigote and intracellular amastigote stages of Leishmania infantum and L. donovani. No correlation was found among the toxicity tests. All the compounds showed significant antipromastigote activity. The antiproliferative capacity of derivatives was dependent on the parasite stage studied, and no substantial differences were found between Leishmania species. Betulin, 3,28-di-O-acetylbetulin and L-aspartyl amide of betulonic acid showed moderate activity against amastigotes. The highest inhibition of intracellular amastigote multiplication was achieved with a low micromolar concentration (IC50 ca 9 μM) of heterocyclic betulin derivative 3,28-di-O-acetyllup-13(18)-ene with N-ethyltriazolo moiety 16, without significant toxicity for mammalian cells. These results point to the interest of this lead compound for further in vitro and in vivo tests.

In Vitro Synergistic Effect of Amphotericin B and Allicin on Leishmania donovani and L. infantum

ABSTRACT Current monotherapy against visceral leishmaniasis has serious side effects, and resistant Leishmania strains have been identified. Amphotericin B (AmB) has shown an extraordinary antileishmanial efficacy without emergence of resistance; however, toxicity has limited its general use. Results obtained showed, using a fixed-ratio analysis, that the combination of diallyl thiosulfinate (allicin) and AmB ranged from moderately synergic to synergic at low concentrations (0.07 μM AmB plus 35.45 μM allicin induced 95% growth inhibition). None of the treatments, alone or in combination, had noticeable adverse effects on macrophages (Mϕ) in the concentration range examined (allicin, 0.5, 1, 5 and 10 μM; AmB, 0.05, 0.075, and 0.1 μM). Allicin, AmB, or the combination did not affect the infection rate (percentage of infected Mϕ) of Leishmania. Allicin enhanced the activity of AmB on intracellular amastigotes of Leishmania donovani and L. infantum (ca. 45% reduction of amastigote burden with 0.05 μM AmB plus 10 μM allicin); this represented nearly a 2-fold reduction in the 50% inhibitory concentration (IC50) of the antibiotic added alone. Results point toward the possible utility of testing this combination in vivo to reduce the toxicity associated with monotherapy with AmB.

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 μM. 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 μM, 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.

Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge

Gene encoding a somatic protein of Haemonchus contortus (Hc23) known to confer significant protection against experimental haemonchosis has been cloned and expressed in a prokaryotic system. A cDNA library of H. contortus using the vector λ ZAP II was obtained. Full-length gene was amplified, cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in Ni-NTA column. Recombinant protein (rHc23) had 203 aminoacids and a molecular mass of 24.15 kDa. Recombinant protein (100 μg/dose) with aluminum hydroxide as adjuvant was administered to 5-6 months age female Assaf lambs on days -42, -28 and -14. On day 0 animals were infected with 15,000 L3 of H. contortus. Vaccination with rHc23 elicited a significant protection against challenge, with >80% reductions in both fecal egg counts and average abomasal parasite burdens at the end of the experiment (45 days post challenge) besides lack of variations in packed cell volume. Results support the feasibility of vaccination against lamb haemonchosis with a recombinant product from an exposed antigen.

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.

In-Vitro Activity of Silybin and Related Flavonolignans against Leishmania infantum and L. donovani

Flavonolignans from the seeds of the milk thistle (Silybum marianum) have been extensively used in folk medicine for centuries. Confirmation of their properties as hepatoprotective, antioxidant and anticancer has been obtained using standardized extracts and purified flavonolignans. Information on their potential effect on Leishmania is very scarce. We have investigated the effect of silymarin, silybin and related flavonolignans on the multiplication of promastigotes in vitro and ex vivo on intracellular amastigotes of L. infantum (Li) and L. donovani (Ld), causative agents of human and canine visceral leishmaniasis (VL). In addition, the potential synergistic effect of the most active molecule and well-established antileishmanial drugs against promastigotes was explored. Dehydroisosilybin A elicited the highest inhibition against Ld and Li promastigotes with an approximate IC50 of 90.23 µM. This molecule showed a moderate synergism with amphotericin B (AmB) but not with SbIII or paromomycin, although it was ineffective against amastigotes. Antileishmanial activity on intracellular amastigotes of the two diastereoisomers of dehydrosilybin (10 µM) was comparable to that elicited by 0.1 µM AmB. Antiproliferative activity and safety of flavonolignans suggest the interest of exploring their potential value in combination therapy against VL.

Pharmacokinetics and disposition of miltefosine in healthy mice and hamsters experimentally infected with Leishmania infantum

&NA; Miltefosine is the only currently available oral drug for treatment of leishmaniasis. However, information on the pharmacokinetics (PK) of miltefosine is relatively scarce in animals. PK parameters and disposition of the molecule was determined in healthy NMRI mice and Syrian hamsters infected and treated with different miltefosine doses and regimens. Long half‐life of the molecule was confirmed and differential pattern of accumulation of the drug was observed in analyzed organs in mice and hamster. Long treatment schedules produced miltefosine levels over IC50 value against L. infantum intracellular amastigotes for at least 24 days in spleen and liver of infected hamsters. The observed differential pattern of organ accumulation of the drug in mice and hamster supports the relevance of both species for translational research on chemotherapy of leishmaniasis. Graphical abstract Figure. No caption available.