Targeting lung macrophages for fungal and parasitic pulmonary infections with innovative amphotericin B dry powder inhalers.

The incidence of fungal pulmonary infections is known to be on the increase, and yet there is an alarming gap in terms of marketed antifungal therapies that are available for pulmonary administration. Amphotericin B (AmB) is a highly efficient broad-spectrum antifungal only marketed as an intravenous formulation. Based on the lack of effective antifungal and antiparasitic pulmonary treatments, the aim of this study was to develop a carbohydrate-based AmB dry powder inhaler (DPI) formulation, prepared by spray drying. Amorphous AmB microparticles were developed by combining 39.7 % AmB with 39.7 % γ-cyclodextrin, 8.1 % mannose and 12.5 % leucine. An increase in the mannose concentration from 8.1 to 29.8 %, led to partial drug crystallisation. Both formulations showed good in vitro lung deposition characteristics (80 % FPF < 5 µm and MMAD < 3 µm) at different air flow rates (60 and 30 L/min) when used with a DPI, but also during nebulisation upon reconstitution in water.

Immunoproteomic analysis of Trichinella spiralis larval crude antigens recognized by sera from patients with trichinellosis after treatment with albendazole.

Antibody responses and antigen recognition were monitored during and after treatment with albendazole (ABZ) in nine patients selected from a trichinellosis outbreak that occurred in north-west Poland in 2007. Seven out of the nine patients yielded positive serum IgG response during treatment. One month after treatment, the IgG response decreased in most patients. Serum levels of ABZ and main metabolites greatly varied among patients without correlation with the IgG response. Two-dimensional electrophoresis and western blot with serum from each patient showed highly immunoreactive spots located between 3- 10 pI and 45-97 kDa in all patients. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/time-of-flight mass spectrometry (TOF MS) analysis identified actine, enolase, p49 protein, Caenorhabditis elegans-targeted antigen, and serine protease as the most reactive proteins. A minor spot located at 6 pI and 26 kDa identified as annexin I failed recognition in most patients showing decline in IgG response and clinical improvement after treatment. This protein could constitute a sensitive marker for the effectiveness of ABZ against trichinellosis.

Immunoproteomic analysis of Trichinella spiralis larval crude antigens recognized by sera from patients with trichinellosis after treatment with albendazole

Antibody responses and antigen recognition were monitored during and after treatment with albendazole (ABZ) in nine patients selected from a trichinellosis outbreak that occurred in north-west Poland in 2007. Seven out of the nine patients yielded positive serum IgG response during treatment. One month after treatment, the IgG response decreased in most patients. Serum levels of ABZ and main metabolites greatly varied among patients without correlation with the IgG response. Two-dimensional electrophoresis and western blot with serum from each patient showed highly immunoreactive spots located between 3– 10 pI and 45–97 kDa in all patients. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF) and MALDI–TOF/time-of-flight mass spectrometry (TOF MS) analysis identified actine, enolase, p49 protein, Caenorhabditis elegans-targeted antigen, and serine protease as the most reactive proteins. A minor spot located at 6 pI and 26 kDa identified as annexin I failed recognition in most patients showing decline in IgG response and clinical improvement after treatment. This protein could constitute a sensitive marker for the effectiveness of ABZ against trichinellosis.

Identification of proteins interacting with HSP70 mRNAs in Leishmania braziliensis.

HSP70 protein is involved in Leishmania differentiation, apoptosis, antimony-resistance and host-immune response. Therefore, this protein and the regulatory mechanisms of HSP70 gene expression are promising targets for therapeutic intervention against leishmaniasis. The regulation of mRNA expression in trypanosomatids operates mostly through the interaction of trans-acting proteins, and elements located in the untranslated regions of mRNAs. The aim of this work was to identify protein factors interacting specifically with the Leishmania braziliensis HSP70 mRNAs. Thus, the 5' UTR and the two types of 3' UTRs (UTR-I and UTR-II) from L. braziliensis HSP70 genes were used as baits in pull down assays using total protein extracts from parasites cultured at 26 or 35°C. The captured proteins were resolved by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS) analysis. As a result, 52 different proteins were identified based on their binding to the L. braziliensis HSP70-mRNAs. As expected, several of the identified proteins were related to RNA metabolism (27%) and translation process (7%). In addition, five hypothetical conserved proteins having motifs related with RNA interaction were also identified (9.6%). Nevertheless, unexpected proteins, apparently unrelated to the mRNA expression, were also identified. The biological significance of these and others L. braziliensis detected proteins, including the HSP70 itself, is discussed. BIOLOGICAL SIGNIFICANCE: For the first time, a riboproteomic analysis of the proteins interacting with the untranslated regions of the heat shock protein 70 (HSP70) mRNA from Leishmania braziliensis was carried out. This work provides new insights related to protein factors putatively involved in the regulation of HSP70 gene expression in L. braziliensis, and thereby, contributes to a better understanding of the parasite biology, and ultimately to the development of novel therapeutic interventions for controlling the important diseases caused by this parasite.

The kinetoplastid chemotherapy revisited: current drugs, recent advances and future perspectives.

Leishmaniasis, African sleeping sickness and Chagas disease, caused by the kinetoplastid parasites Leishmania spp, Trypanosoma brucei and Trypanosoma cruzi, respectively, are among the most important parasitic diseases, affecting millions of people and considered to be within the most relevant group of neglected tropical diseases. The main alternative to control such parasitosis is chemotherapy. Nevertheless, the current chemotherapeutic treatments are far from being satisfactory. This review outlines the current understanding of different drugs against leishmaniasis, African sleeping sickness and Chagas disease, their mechanism of action and resistance. Recent approaches in the area of anti-leishmanial and trypanocidal therapies are also enumerated, new modulators from the mode of action, development of new formulations of old drugs, therapeutic switching and "in silico" drug design.

3D entropy and moments prediction of enzyme classes and experimental-theoretic study of peptide fingerprints in Leishmania parasites.

The number of protein 3D structures without function annotation in Protein Data Bank (PDB) has been steadily increased. This fact has led in turn to an increment of demand for theoretical models to give a quick characterization of these proteins. In this work, we present a new and fast Markov chain model (MCM) to predict the enzyme classification (EC) number. We used both linear discriminant analysis (LDA) and/or artificial neural networks (ANN) in order to compare linear vs. non-linear classifiers. The LDA model found is very simple (three variables) and at the same time is able to predict the first EC number with an overall accuracy of 79% for a data set of 4755 proteins (859 enzymes and 3896 non-enzymes) divided into both training and external validation series. In addition, the best non-linear ANN model is notably more complex but has an overall accuracy of 98.85%. It is important to emphasize that this method may help us to predict not only new enzyme proteins but also to select peptide candidates found on the peptide mass fingerprints (PMFs) of new proteins that may improve enzyme activity. In order to illustrate the use of the model in this regard, we first report the 2D electrophoresis (2DE) and MADLI-TOF mass spectra characterization of the PMF of a new possible malate dehydrogenase sequence from Leishmania infantum. Next, we used the models to predict the contribution to a specific enzyme action of 30 peptides found in the PMF of the new protein. We implemented the present model in a server at portal Bio-AIMS (http://miaja.tic.udc.es/Bio-AIMS/EnzClassPred.php). This free on-line tool is based on PHP/HTML/Python and MARCH-INSIDE routines. This combined strategy may be used to identify and predict peptides of prokaryote and eukaryote parasites and their hosts as well as other superior organisms, which may be of interest in drug development or target identification.

Generalized lattice graphs for 2D-visualization of biological information.

Several graph representations have been introduced for different data in theoretical biology. For instance, complex networks based on Graph theory are used to represent the structure and/or dynamics of different large biological systems such as protein-protein interaction networks. In addition, Randic, Liao, Nandy, Basak, and many others developed some special types of graph-based representations. This special type of graph includes geometrical constrains to node positioning in space and adopts final geometrical shapes that resemble lattice-like patterns. Lattice networks have been used to visually depict DNA and protein sequences but they are very flexible. However, despite the proved efficacy of new lattice-like graph/networks to represent diverse systems, most works focus on only one specific type of biological data. This work proposes a generalized type of lattice and illustrates how to use it in order to represent and compare biological data from different sources. We exemplify the following cases: protein sequence; mass spectra (MS) of protein peptide mass fingerprints (PMF); molecular dynamic trajectory (MDTs) from structural studies; mRNA microarray data; single nucleotide polymorphisms (SNPs); 1D or 2D-Electrophoresis study of protein polymorphisms and protein-research patent and/or copyright information. We used data available from public sources for some examples but for other, we used experimental results reported herein for the first time. This work may break new ground for the application of Graph theory in theoretical biology and other areas of biomedical sciences.

Contrasting effects of Trichinella spiralis and Trichuris muris antigens on the infection by Leishmania infantum in BALB/c mice.

The interaction of Trichinella spiralis and Trichuris muris derived antigens with the infection by Leishmania infantum was investigated in BALB/c mice. Infection with 10(6) promastigotes of L. infantum did not induce relevant serum antibody (IgG subclasses), nor cytokine (IFN-gamma, IL-4) responses despite that mice could partially control the infection. Immunization with T. spiralis activated a moderate IgG1 and secondarily an IgG2a anti-leishmanial response whereas immunization with T. muris elicited only a weak and late activation of IgG1 anti-leishmanial response. Immunization with T. muris caused an elevation of serum IFN-gamma levels which was drastically reinforced by the L. infantum infection, and that was accompanied by almost complete parasitological cure of infected mice. Immunization with T. spiralis induced an elevation of serum IL-4 levels but this response was greatly (about 60%) neutralized by the infection with L. infantum, and this was associated to exacerbation of the infection.

Setting new immunobiological parameters in the hamster model of visceral leishmaniasis for in vivo testing of antileishmanial compounds.

To establish suitable immunobiological parameters for in vivo testing of new antileishmanial compounds in the golden hamster model of visceral leishmaniasis, two groups of 8 animals were infected each with 10(5) or 10(7) stationary promastigotes by the intracardiac route and the clinical and immunoparasitological features were monitored up to day 155 after infection. All animals became infected at both doses, although significant differences were observed between parasite burdens in liver and spleen. The mean number of parasites in animals infected with 10(7) promastigotes increased by 9.5 times in liver and by 43.1 times in spleen compared with those infected with 10(5) promastigotes. In animals given the higher dose, the outcome of the disease occurred between days 75 and 90 after infection, whereas no signs of disease were apparent in those given the lower infecting dose. Positive antibody (IgG) responses were detected earlier (week 5-7 after infection) in animals infected with the higher dose than in those infected with the lower dose (week 8-10 after infection), but these responses did not correlate with individual parasitological loads in liver and spleen. An inverse correlation was observed between infecting doses and in vitro spleen lymphocyte proliferation against mitogens (ConA). The proportion of CD4(+) and CD19(+) spleen cell increased in animals given the higher infection, whereas it decreased in those given the lower infection compared to naive controls.

Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation.

A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 10(7) metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-gamma) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-gamma and TNF-alpha, with no effect on the deactivating cytokine TGF-beta. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-gamma and TNF-alpha and strong suppression of TGF-beta. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-beta, which in turn results in up-regulation of the Th1 cytokines IFN-gamma and TNF-alpha.

Two-dimensional electrophoresis and mass spectrometry for the identification of species-specific Trichinella antigens.

A proteomic approach was utilized for fine antigenic characterization of the closely related Trichinella genotypes Trichinella britovi T3 and Trichinella T8. Crude extract of muscle larvae L1 (LCE) from both isolates were analyzed by 2D-PAGE. Over 500 protein spots were reproducibly separated in both genotypes. These separated proteins were identified in Western blot with IgG1 and IgG3 from homologous and heterologous hyperimmune sera raised in BALB/c mice. A group of 20 and 15 spots migrating at 50--60 k Da and pH 5.5--6.5 in T. britovi and Trichinella T8 maps, respectively, reacted with the IgG1 from heterologous sera whereas a group of minor spots of similar migration patterns did not. Low cross-reactivity occurred for IgG3. MALDI-TOF and MALDI-TOF/TOF analysis of these antigens identified a predicted enolase, the protein P 49 and a predicted actin among the cross-reactive proteins and two hypothetical actins among the non cross-reactive proteins.

Anti-leishmanial activity of a new formulation of amphotericin B.

The effectiveness of albumin microspheres loaded with amphotericin B was tested in an in vivo model of visceral leishmaniasis using the golden hamster. Free and encapsulated amphotericin B was tested at the dose of 1 mg/kg given by the intracardiac route on days 25, 26 and 27 post-infection (p.i.) to treat animals previously infected with 10(7) stationary promastigotes by the intracardiac route. Encapsulated amphotericin was highly effective against infection causing a reduction of 88.8% and 87.2% in the early stage of infection (day 32 p.i.) and of 66.7% and 54% in a later stage of infection (day 135 p.i.) in liver and spleen parasite load respectively, compared with untreated animals, whereas free amphotericin was inactive. Lymphocyte proliferation was restored together with an increase in CD4(+) subsets in animals treated with encapsulated amphotericin B, but not in those treated with the non-encapsulated compound. Antibody responses did not increase after treatment with encapsulated amphotericin B with antibody levels remaining at base levels for most animals in contrast to those of untreated or treated with free amphotericin, where in most animals the antibody levels sharply increased. This new formulation could be a more economical alternative to liposomes for the treatment of visceral leishmaniasis with amphotericin B.

Heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53.

This study investigates the heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53. Western blotting analysis of several Trichinella isolates with the gp53-specific monoclonal antibodies (mAbs) US5 and US9, produced in Btkxid mice, revealed that gp53 from the species T. britovi, T. murrelli and genotype T8 had higher MW (60 kDa) than gp53 from T. spiralis, T. nelsoni and genotype T6 (53 kDa) and from T. nativa (55 kDa). mAb US5 reacted only with gp53 from T. spiralis. Experiments including immunoassays of gp53 binding by sera from T. spiralis-infected mice, in the presence of different potential inhibitors (recombinant gp53, US5, T. britovi-crude larval extract (CLE), and CLE N- and O-glycans), indicate (i) that gp53 from T. spiralis bears specific epitopes that induce antibody formation during infection; (ii) that the protein epitopes of gp53 are much more important (76 or 68% of total antibody reactivity in BALB/c and Swiss CD-1 mice, respectively) than the corresponding glycan epitopes including tyvelose (11 or 32% of total reactivity) for the induction of anti-gp53 circulating antibodies; and (iii) that the species-specific epitopes present on gp53 are differentially recognized in different mouse strains. Whereas in BALB/c mice US5- and non-US5-recognized species-specific epitopes on gp53 bind about 84% of circulating antibodies on day 80 post-infection, this percentage was only 38% in Swiss CD-1 mice. These data on the antigenicity of gp53 contrast with data for Trichinella CLE antigens, in that most circulating antibodies reactive with CLE antigens recognized tyvelose-containing epitopes (57% and 58% of circulating antibodies in BALB/c and Swiss CD-1 mice, respectively). Together these results demonstrate that gp53 is recognized during infection but is antigenically different from other Trichinella TSL-1 antigens.

A comparison of antigenic peptides in muscle larvae of several Trichinella species by two-dimensional western-blot analysis with monoclonal antibodies.

The antigens recognised by mAb US5 specific to 53 kDa glycoprotein (gp 53) in T. spiralis L-1 muscle larvae (TSL1) antigens, mAb US9 specific to gp 53 in TSL1 from all encapsulated species and mAb US4 specific to a tyvelose containing tetrasaccharide present in TSL1, were investigated in crude extracts from muscle larvae of T. spiralis, T. nativa and T. britovi by 2D-electrophoresis and western-blot. At least four proteins of different p1 were recognised by mAb US5 on T. spiralis antigens. Recognition profile of mAb US9 on T. spiralis antigens exhibited some variation with regard to that of the US5. Polymorphism was apparent in gp 53. High reactivity was shown by the mAb US4 with the three species.

Possible presence of common tyvelose-containing glycans in Trichinella L1 larvae and embryonated eggs of several nematodes.

A monoclonal antibody (mAb US4) recognising an epitope containing tyvelose within the T. spiralis L-1 muscle larvae (TSL-1) antigens was tested in western-blot against various antigenic preparations from different stages of the following nematodes: T. spiralis (L1, adult), T. muris (egg, L1, L3, adult), Ascaris suum (egg, adult), Toxocara canis (egg, adult), Anisakis simplex (L3) and Haemochus contortus (egg). Positive reaction was present in antigen preparations from L1 larvae of T. spiralis and T. muris and from embryonated eggs of T. muris, A. seum, T. canis and H. contortus.

Comparison of IgG3 responses to carbohydrates following mouse infection or immunization with six species of Trichinella.

The IgG3 antibody responses to carbohydrate epitopes were compared in BALB/c mice infected or immunized with six species of Trichinella: T. spiralis (T1), T. nativa (T2), T. britovi (T3), T6, T. nelsoni (T7), and T8. The dynamics of IgG3 responses and antigen recognition following infection or immunization were measured by ELISA and Western blot respectively, using glycosylated and deglycosylated larval crude extracts (LCE) prepared from homologous isolates. A high degree of protein glycosylation was found in all species and with similar profiles. Deglycosylation was completely achieved only in LCE from T1 and T6 isolates. The dynamics of IgG3 responses following infection or immunization significantly differed whereas the antigen recognition profiles appeared similar. Variations in the levels and antigen recognition patterns of IgG3 among the different species were apparent. The highest IgG3 levels were recorded in infections by the T8 isolate and the lowest in infections by the T6 isolate, whereas for immunization the highest IgG3 response was induced by T7 and the lowest by T8. Following antigen deglycosylation, the IgG3 responses were significantly reduced or abrogated and the recognition patterns markedly modified or suppressed in the different species of Trichinella.

Trichinella antigens: a review.

This paper presents a review of the Trichinella antigens within the context of species variation. As with other parasites, Trichinella antigens can be classified according to their localisation as surface, excretory/secretory (ES) and somatic components. Surface antigens are mainly constituents of the outer cuticle although secretions from inner parts of the body wall as well as from the oesophagus can temporarily accumulate in the surface. ES antigens come mainly from the excretory granules of the stichosome, whereas somatic constitutive antigens come from the internal parts of the worms. ES products are considered very important from an immunological point of view as they are easily targeted by the immune system, whereas parasite death is required for exposure of somatic products. Some of the antigenic components have been characterised chemically. Phosphorylcholine is an important hapten that modulates the immune responses in Trichinella infections. Glycoproteins are the major components of surface and excretory/secretory products. A 43-kDa glycoprotein has been regarded as a good candidate for diagnosis and vaccination purposes. Recently some glycans have received special attention either as relevant epitopes or as parasite evasion strategies.