Daniel R. Abánades

Immunohistological features of visceral leishmaniasis in BALB/c mice

It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10 3 , 10 5 and 10 6 parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti‐Leishmania antibodies, up‐regulation of spleen arginase activity, balance between IFN‐γ and IL‐10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 105 parasites should prove useful for vaccine studies against VL.

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.

Protein haptenation by amoxicillin: High resolution mass spectrometry analysis and identification of target proteins in serum

Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.

Turn-on fluorescent probes for nitric oxide sensing based on the ortho-hydroxyamino structure showing no interference with dehydroascorbic acid

A new family of fluorescent synthetic molecular probes for nitric oxide sensing based on ortho-hydroxyamino-triarylpyrylium salts is presented.

Heme Oxygenase-1 Promotes the Persistence of Leishmania chagasi Infection

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.

Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system

The study reported here aimed to develop an optimized nanoparticle delivery system for amphotericin B (AmpB) using a polyelectrolyte complexation technique. For this, two oppositely charged polymers presenting anti-leishmanial activity – chitosan (Cs) and chondroitin sulfate (ChS) – were used: Cs as a positively charged polymer and ChS as a negatively charged polymer. The chitosan (NQ) nanoparticles, chitosan-chondroitin sulfate (NQC) nanoparticles, and chitosan-chondroitin sulfate-amphotericin B (NQC-AmpB) nanoparticles presented a mean particle size of 79, 104, and 136 nm, respectively; and a polydispersity index of 0.2. The measured zeta potential of the nanoparticles indicated a positive charge in their surface, while scanning and transmission electron microscopy revealed spherical nanoparticles with a smooth surface. Attenuated total reflectance-Fourier transform infrared spectroscopy analysis showed an electrostatic interaction between the polymers, whereas the release profile of AmpB from the NQC-AmpB nanoparticles showed a controlled release. In addition, the Cs; ChS; and NQ, NQC, and NQC-AmpB nanoparticles proved to be effective against promastigotes of Leishmania amazonensis and Leishmania chagasi, with a synergistic effect observed between Cs and ChS. Moreover, the applied NQ, NQC, and NQC-AmpB compounds demonstrated low toxicity in murine macrophages, as well as null hemolytic activity in type O+ human red blood cells. Pure AmpB demonstrated high toxicity in the macrophages. The results show that cells infected with L. amazonensis and later treated with Cs, ChS, NQ, NQC, NQC-AmpB nanoparticles, or pure AmpB presented with a significant reduction in parasite number in the order of 24%, 31%, 55%, 66%, 90%, and 89%, respectively. The data presented indicate that the engineered NQC-AmpB nanoparticles could potentially be used as an alternative therapy to treat leishmaniasis, mainly due its low toxicity to mammals’ cells.

A DNA aptamer population specifically detects Leishmania infantum H2A antigen.

Aptamers are short single-stranded DNA or RNA oligonucleotides that are selected in vitro by their affinity and specificity for the target. Binding is a consequence of the particular tertiary structure that they are able to acquire, depending on their sequence. Parasites of the genus Leishmania belongs to the lower eukaryote order Kinetoplastida that causes leishmaniosis in man and animals. Histone genes in Leishmania are of considerable interest because these flagellates do not condense their chromatin during mitosis. Thus, the study of the structural features of histones has been considered of particular interest and, as a result, in recent years a great number of histone genes have been characterized in trypanosomatids. Histones are extremely conserved proteins, reflecting their apparent universality of function. Sequence similarity of kinetoplastid core histones those of higher eukaryotes is found predominantly in the globular region with high sequence divergences in the N- and in the C-terminal domains. These divergences indicate that they may be potential diagnostic and/or therapeutics targets. We have successfully isolated a pool of DNA sequences, named SELH2A, which specifically binds to Leishmania infantum H2A. When tested in an enzyme-linked oligonucleotide assay, slot blot and Western blot analysis, the aptamer pool exhibited specificity in its ability to bind only to H2A antigen but not to other proteins from L. infantum including other histones. Thus, it appears that this novel anti-H2A aptamer population may be of potential application as a diagnostic system for leishmaniosis.

The immunodominant T helper 2 (Th2) response elicited in BALB/c mice by the Leishmania LiP2a and LiP2b acidic ribosomal proteins cannot be reverted by strong Th1 inducers

The search for disease‐associated T helper 2 (Th2) Leishmania antigens and the induction of a Th1 immune response to them using defined vaccination protocols is a potential strategy to induce protection against Leishmania infection. Leishmania infantum LiP2a and LiP2b acidic ribosomal protein (P proteins) have been described as prominent antigens during human and canine visceral leishmaniasis. In this study we demonstrate that BALB/c mice infected with Leishmania major develop a Th2‐like humoral response against Leishmania LiP2a and LiP2b proteins and that the same response is induced in BALB/c mice when the parasite P proteins are immunized as recombinant molecules without adjuvant. The genetic immunization of BALB/c mice with eukaryotic expression plasmids coding for these proteins was unable to redirect the Th2‐like response induced by these antigens, and only the co‐administration of the recombinant P proteins with CpG oligodeoxynucleotides (CpG ODN) promoted a mixed Th1/Th2 immune response. According to the preponderance of a Th2 or mixed Th1/Th2 responses elicited by the different regimens of immunization tested, no evidence of protection was observed in mice after challenge with L. major. Although alterations of the clinical outcome were not detected in mice presensitized with the P proteins, the enhanced IgG1 and interleukin (IL)‐4 response against total Leishmania antigens in these mice may indicate an exacerbation of the disease.

Role of prostaglandin F2α production in lipid bodies from Leishmania infantum chagasi: insights on virulence.

Lipid bodies (LB; lipid droplets) are cytoplasmic organelles involved in lipid metabolism. Mammalian LBs display an important role in host-pathogen interactions, but the role of parasite LBs in biosynthesis of prostaglandin F2α (PGF2α) has not been investigated. We report herein that LBs increased in abundance during development of Leishmania infantum chagasi to a virulent metacyclic stage, as did the expression of PGF2α synthase (PGFS). The amount of parasite LBs and PGF2α were modulated by exogenous arachidonic acid. During macrophage infection, LBs were restricted to parasites inside the parasitophorous vacuoles (PV). We detected PGF2α receptor (FP) on the Leishmania PV surface. The blockage of FP with AL8810, a selective antagonist, hampered Leishmania infection, whereas the irreversible inhibition of cyclooxygenase with aspirin increased the parasite burden. These data demonstrate novel functions for parasite-derived LBs and PGF2α in the cellular metabolism of Leishmania and its evasion of the host immune response.