Hyaluronic acid-amphotericin B nanocomplexes: a promising anti-leishmanial drug delivery system.

The development of an effective amphotericin B (AmB) formulation to replace actual treatments available for leishmaniasis, which present serious drawbacks, is a challenge. Here we report the development of hyaluronic acid-amphotericin B self-assembled nanocomplexes (HA-AmB), processed by freeze-drying (FD) or nano spray-drying (SD), using a simple process that favors the non-covalent drug-polysaccharide association in an amorphous state. These water-soluble formulations, which presented a nanometric size (300-600 nm), high colloidal stability (zeta potential around -39 mV) and an AmB loading (15-18%) in aggregated and super aggregated states, demonstrated less in vitro cytotoxic and hemolytic effects compared to the free-drug. A significant decrease in the number of intramacrophagic L. infantum amastigotes upon treatment (IC50 of 0.026 and 0.030 µM for HA-AmB FD and HA-AmB SD, respectively) was also observed, and the best selectivity index (SI) was observed for the HA-AmB SD nanocomplex (SI of 651). Intravenous administration of the HA-AmB SD nanocomplex for 3 alternate days showed an effective parasite reduction in the spleen and liver of C57BL/6 mice without signs of toxicity commonly observed upon free-AmB treatment. Although lower than that achieved with AmBisome® in the liver, the observed parasite reduction for the nanocomplex was of a similar order of magnitude. The efficacy, stability, safety and low cost of the HA-AmB SD nanocomplex highlight its potential as an alternative treatment for leishmaniasis.

Development of dextrin-amphotericin B formulations for the treatment of Leishmaniasis.

The most effective medicines available for the treatment of leishmaniasis, a life-threatening disease, exhibit serious toxicological issues. To achieve better therapeutic efficiency while decreasing toxicity associated with amphotericin B (AmB), water-soluble dextrin-AmB (Dex-AmB) formulations were developed. Self-assembled nanocomplexes were formed by dissolving Dex and AmB in alkaline borate buffer, followed by dialysis and either freeze-drying (FD) or nano spray-drying (SD), yielding water dispersible particles with a diameter of 214 nm and 347 nm, respectively. The very simple production process allowed the formation of amorphous inclusion complexes containing 14% of AmB in the form of monomers and water-soluble aggregates. Nanocomplexes were effective against parasites in axenic culture (IC50 of 0.056 and 0.096 µM for L. amazonensis and 0.030 and 0.044 µM for L. infantum, respectively for Dex-AmB FD and Dex-AmB SD) and in decreasing the intramacrophagic infection with L. infantum (IC50 of 0.017 and 0.023 µM, respectively for Dex-AmB FD and Dex-AmB SD). Also, the formulations were able to significantly reduce the cytotoxicity of AmB. Overall, this study demonstrates the suitability of dextrin as an AmB carrier and the facile and inexpensive development of a delivery system for the treatment of leishmaniasis.

Dog hepatocytes are key effector cells in the liver innate immune response to Leishmania infantum.

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

Leishmania infantum exerts immunomodulation in canine Kupffer cells reverted by meglumine antimoniate.

Kupffer cells (KC) are the liver macrophage population that resides in the hepatic sinusoids and efficiently phagocyte pathogens by establishing an intimate contact with circulating blood. KC constitute the liver host cells in Leishmania infection, nevertheless little is described about their role, apart from their notable contribution in granulomatous inflammation. The present study aims to investigate how canine KC sense and react to the presence of Leishmania infantum promastigotes and amastigotes by evaluating the gene expression of specific innate immune cell receptors and cytokines, as well as the induction of nitric oxide and urea production. Complementarily, the impact of a leishmanicidal drug - meglumine antimoniate (MgA) - in infected KC was also explored. KC revealed to be susceptible to both parasite forms and no major differences were found in the immune response generated. L. infantum parasites seem to interact with KC innate immune receptors and induce an anergic state, promoting immune tolerance and parasite survival. The addition of MgA to infected KC breaks the parasite imposed silence and increased gene expression of Toll-like receptors (TLR) 2 and TLR4, possibly activating downstream pathways. Understanding how KC sense and react to parasite presence could bring new insights into the control or even elimination of canine leishmaniasis.

Influence of schooling and age on cognitive performance in healthy older adults.

Few studies have examined the influence of a low level of schooling on age-related cognitive decline in countries with wide social and economic inequalities by using the Cambridge Automated Neuropsychological Test Battery (CANTAB). The aim of the present study was to assess the influence of schooling on age-related cognitive decline using unbiased cognitive tests. CANTAB allows cognitive assessment across cultures and education levels with reduced interference of the examiner during data acquisition. Using two-way ANOVA, we assessed the influences of age and education on test scores of old adults (61-84 years of age). CANTAB tests included: Visual Sustained Attention, Reaction Time, Spatial Working Memory, Learning and Episodic Memory. All subjects had a minimum visual acuity of 20/30 (Snellen Test), no previous or current history of traumatic brain/head trauma, stroke, language impairment, chronic alcoholism, neurological diseases, memory problems or depressive symptoms, and normal scores on the Mini Mental State Examination (MMSE). Subjects were grouped according to education level (1 to 7 and ≥8 years of schooling) and age (60-69 and ≥70 years). Low schooling level was associated with significantly lower performance on visual sustained attention, learning and episodic memory, reaction time, and spatial working memory. Although reaction time was influenced by age, no significant results on post hoc analysis were detected. Our findings showed a significantly worse cognitive performance in volunteers with lower levels of schooling and suggested that formal education in early life must be included in the preventive public health agenda. In addition, we suggest that CANTAB may be useful to detect subtle cognitive changes in healthy aging.

Leishmania infantum antigens modulate memory cell subsets of liver resident T lymphocyte.

In the recent years, the liver has been recognized as an important immune organ with major regulatory functions and immune memory, adding to the well-described vital metabolic functions. There are evidences from experimental infections performed with visceral Leishmania species that immune responses to parasite infection can be organ-specific. The liver is the compartment of acute resolving infection, with minimal tissue damage and resistance to reinfection, whereas the spleen is the compartment of parasite persistence. Control of hepatic infection in mice requires a coordinated immune response that involves the development of inflammatory granulomas. It is also described that the liver harbors populations of resident lymphocytes, which may exhibit memory characteristics. Therefore, the present study aims to address the role of the liver as an immune memory organ in the context of Leishmania infantum infection, by characterizing phenotypically resident liver T lymphocytes. The dynamics of memory T cells in L. infantum infected BALB/c mice and the effect of anti-leishmanial treatment in the differentiation of memory cell subsets were analyzed. The potential of recognition, differentiation and selection of memory lymphocytes by three L. infantum recombinant proteins were also explored. L. infantum infection generates effector and central memory T cells, but the cells did not expand when recalled, demonstrating a possible parasite silencing effect. The treatment with a leishmanicidal drug (antimoniate meglumine) increases the levels of memory and effector T cells, eliciting a more robust hepatic immune response. L. infantum parasites with a decreased sensitivity to the leishmanicidal drug favor the expansion of memory CD8+ T cell subset, but inhibit the proliferation of CD8+ T effector cells, possibly assuring their own survival. The recombinant proteins LirCyp1 and LirSOD are strongly recognized by memory cells of treated mice, indicating that these proteins might be used in a prophylactic or therapeutic vaccine formulation. Thus, L. infantum released antigens induce the development of immune memory subsets in the liver resident T cell population that specifically recognized parasite antigens, including recombinant proteins.

Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells.

Control of zoonotic visceral leishmaniosis can be achieved using several available drugs. These drugs present high toxicity and require longer treatment regimens which complicate compliance to the treatment. Other control measures directed to the vector or the reservoirs are useful tools to restrain the spreading of this disease but the effects are transitory. A safe, affordable and efficient vaccine conferring long lasting immunity should be the most cost effective way of controlling zoonotic visceral leishmaniosis. The present study aims at characterizing a cyclophilin protein 1 of Leishmania infantum (LiCyP1) and investigating whether recombinant LiCyP1 (LirCyP1) is able to confer protection against infection by evaluating viable parasite load and the generation of specific CD4(+) and CD8(+) effector and central memory T cells in rodent model. LiCyP1 is present in the cytoplasm of L. infantum amastigotes and promastigotes. Immunization of BALB/c mice with LirCyP1 confers high protection to L. infantum infection, causing a marked reduction in parasite replication in the liver and spleen. Furthermore, helper and cytotoxic memory T cell subsets able to specifically recognize parasite antigens expanded in immunized and in challenged mice. CD4(+) T cell subpopulation of intermediate phenotype (CD62L(high)CD127(low)) of challenging mice also presented an accentuated expansion after the recall. This study demonstrated that LirCyP1 confers partial protection to L. infantum infection, promoting the generation of a desired long lasting immunity. LirCyP1 can be considered a potential candidate for the design of a vaccine against zoonotic visceral leishmaniosis.

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.

Transfection systems for animal models of malaria.

Transfection of malaria parasites is a rapidly emerging technology that offers great promise for the investigation of many aspects of infection. Animal models of malaria have always played an important role in the investigation of the disease. In this article, two of the Dutch groups that have been involved in combining transfection with animal models describe the relevant techniques and recent vector developments for the expression of transgenes, giving examples of their application.

Transfection of the primate malaria parasite Plasmodium knowlesi using entirely heterologous constructs.

The recently developed transfection systems for Plasmodium berghei and Plasmodium falciparum offer important new tools enabling further insight into the biology of malaria parasites. These systems rely upon artificial parasite-host combinations which do not allow investigation into the complex interactions between parasites and their natural hosts. Here we report on stable transfection of Plasmodium knowlesi (a primate malaria parasite that clusters phylogenetically with P. vivax) for which both natural and artificial experimental hosts are available. Transfection of this parasite offers the opportunity to further analyze the biology of antigens not only in a natural host but also in hosts that are closely related to humans. To facilitate future development of integration-dependent transfection in P. knowlesi, completely heterologous plasmids that would reduce homologous recombination at unwanted sites in the genome were constructed. These plasmids contained the pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase (dhfr-ts) from Toxoplasma gondii or P. berghei, under control of either (a) P. berghei or (b) P. falciparum promoters. Plasmids were electroporated into mature P. knowlesi schizonts and these cells were injected into rhesus monkeys (Macaca mulatta). After pyrimethamine treatment of these monkeys, resistant parasites were obtained that contained the plasmids. Promoter regions of both P. berghei and P. falciparum controlling dhfr-ts expression were effective in conferring pyrimethamine resistance in P. knowlesi, indicating that common signals control gene expression in phylogenetically distant Plasmodium species.

Overexpression of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, is associated with enhanced metacyclogenesis.

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected T. cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandemly repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteine proteinase inhibitor Cbz-Phe-Phe-CHN2 (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.

Stage-regulated expression of cruzipain, the major cysteine protease of Trypanosoma cruzi is independent of the level of RNA1.

The genes that encode cruzipain, the major cysteine protease of Trypanosoma cruzi are known to be arranged in tandem arrays. To gain a detailed insight into how these arrays are organised at the chromosomal level we have isolated clones from a cosmid library constructed with DNA from the X10.6 strain. In this strain we found that cruzipain is encoded by two allelic clusters composed of approximately 14 and 23 tandemly repeated genes which are located on homologous chromosomes of 650 and 670 kb. With the exception of the 3'-proximal genes, the cruzipain genes were all of identical or very similar sequence. An unusual feature of the 3'-proximal genes is that they lack the sequences that encode the 130 amino acid carboxyl terminal extension which is characteristic of cruzipain. Both gene clusters are situated in a similar chromosomal environment and are flanked by sequences which have the potential to form Z-DNA. In other eukaryotes, these motifs have been associated with recombinational hotspots and have been demonstrated to enhance gene conversion. The cruzipain genes are transcribed to produce a 1.8-kb transcript which is present at the same steady-state level in each of the parasite life cycle stages. However, protein levels and activity are 4-5-times higher in the insect epimastigote stage than in the trypomastigote and amastigote stages. By implication developmental regulation of cruzipain expression occurs predominantly at the translational and/or post-translational levels.

An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector.

To extend the range of genetic tools available for the functional analysis of trypanosomatid genes we have constructed a cosmid shuttle vector (pcosTL) which facilitates the introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani. The vector contains several features to simplify library construction and insert mapping and transformed cells can be selected on the basis of G418 resistance. To evaluate the vector and to determine the fidelity of replication we first constructed cosmid libraries and isolated clones containing the T. cruzi major cysteine protease genes (a tandemly repeated array) and the L. donovani trypanothione reductase gene (a single copy gene). T. cruzi and L. donovani cells transfected with their respective cosmids were characterised by the presence of multiple copies of cosmid DNA and by a considerable over-expression of the corresponding enzyme activity. Rearrangements or deletions of insert sequences were not detected. These findings and the observation that cosmid DNA can be rescued unaltered from transformed parasites suggest that the pcosTL vector will be ideally suited for studies involving functional complementation.

[The prevalence of the surface antigen of the hepatitis B virus (HBsAg) in pregnant women in Alicante]. / Prevalencia del antígeno de superficie del virus de la hepatitis B (HBsAg) en gestantes de Alicante.

To assess the prevalence of the hepatitis B virus carrier status in pregnant women from the population of Alicante and to evaluate the need of screening studies in future programs of attention to pregnant women, 550 women at the final stage of their pregnancies who were admitted to the Alicante Hospital were evaluated with a questionnaire and the determination of specific markers. Our results disclose a lower prevalence (0.54%) than other studies; it was always associated with risk groups and indicators of high infectiousness were absent. Therefore, before other results are available, screening for the carrier state in all pregnant women does not appear justified except in risk groups. These results are limited to the city of Alicante, where most women attend the Hospital at the final stage of their pregnancies.