IgG functionalized polymeric nanoparticles for oral insulin administration.

The oral route is the best way to administer a drug; however, fitting peptide drugs in this route is a major challenge. In insulin cases, less than 0.5% of the administered dose achieves systemic circulation. Oral delivery by nanoparticles can increase insulin permeability across the intestinal epithelium while maintaining its structure and activity until release in the gut. This system can be improved to increase permeability across intestinal cells through active delivery. This study aimed to improve a nanoparticle formulation by promoting functionalization of its surface with immunoglobulin G to increase its absorption by intestinal epithelium. The characterization of formulations showed an adequate size and a good entrapment efficiency. Functionalized nanoparticles led to a desirable increase in insulin release time. Differential scanning calorimetry, infrared spectroscopy and paper chromatography proved the interactions of nanoparticle components. With immunoglobulin G, the nanoparticle size was slightly increased, which did not show aggregate formation. The developed functionalized nanoparticle formulation proved to be adequate to carry insulin and potentially increase its internalization by epithelial gut cells, being a promising alternative to the existing formulations for orally administered low-absorption peptides.

Ultrasonication of insulin-loaded microgel particles produced by internal gelation: impact on particle's size and insulin bioactivity.

Alginate-dextran sulfate (ADS) microgel has been used to protect insulin from gastrointestinal attack and as a carrier to promote insulin permeation through intestinal epithelium. The throughput of ADS submicron particles generation by emulsification/internal gelation is limited by its wide size distribution. The aim of this work was to study the recovery protocol influence on ADS particles through the determination of its impact on particles' size distribution and bioactivity. ADS particles showed a wide and multimodal distribution, characterized by a high aggregation phenomenon. In an attempt to reverse particles' tendency to aggregate and to homogenize particle size ADS populations were submitted to ultrasonication, while particle size distribution, physical and chemical stability, and the bioactivity of entrapped insulin were investigated. After ultrasonication a narrower particle population shifted to the nanoscale, with higher physical stability and significant insulin bioactivity was obtained. Emulsification internal/gelation followed by ultrasonication constituted a valid strategy to obtain ADS particles at the submicron range, with high stability and without significantly compromising insulin bioactivity, so offering promises, under previously well established conditions, to evaluate impact of ADS particle's size on biopharmaceutical and pharmacokinetics phases.

Solid lipid nanoparticles as intracellular drug transporters: an investigation of the uptake mechanism and pathway.

The aim of this work was to develop a systematic analysis of the cellular internalisation mechanism and pathway of solid lipid nanoparticles (SLN) internalisation. To evaluate if SLN show cell uptake and to understand the mechanism of internalisation, four human glioma cell lines (A172, U251, U373 and U87) and a human macrophage cell line (THP1) were used. For this purpose rhodamine 123 (R123) was loaded into SLN coated with polysorbate 60 and 80. Fluorescence microscopy and flow cell cytometry techniques were assessed to study internalisation of these systems within the cells. MTT studies were performed to evaluate the cytotoxicity of the R123-loaded SLN. To assess the SLN internalisation mechanism and intracellular pathway, excluding endocytosis mechanisms were applied. Our results revealed that R123-loaded SLN with mean size below 200 nm and slight negative surface charge (around -20 mV) have the ability to be internalised by gliomas in a higher amount than by macrophages. The mechanism of internalisation was found to be mainly through a clathrin-dependent endocytic pathway. In addition, the cytotoxicity of SLN was higher for gliomas than for macrophages. These results suggest that SLN can be a promising alternative in brain tumours treatment.

Characterization of Leishmania infantum thiol-dependent reductase 1 and evaluation of its potential to induce immune protection.

The need to develop an effective vaccine against leishmaniasis to prevent the 2 million new cases each year led to the search for antigens able to elicit protection against infection with Leishmania. In this study, we have characterized a parasite-specific protein of Leishmania infantum named thiol-dependent reductase 1 (TDR1). The protein is present in both life cycle stages of L. infantum with a notable higher expression in the amastigote forms, suggesting a role in the interaction between the parasite and the mammalian host. Thiol-dependent reductase 1 is localized in the cytosol, although we were able to detect the protein in the culture medium of both promastigotes and axenic amastigotes, and consequently, TDR1 is considered an excreted/secreted molecule of the parasite. Therefore, we have evaluated the potential of TDR1 recombinant protein to protect against experimental challenge with L. infantum parasites using a murine model. Despite a reduction in spleen parasite load in the chronic phase of disease, TDR1 administration was not effective in the protection of Balb/c mice against visceral leishmaniasis and thus TDR1 do not have a crucial role in the modulation of mammalian host immune response, as observed with its protein counterpart Tc52 of Trypanosoma cruzi.

Modulation of mammalian apoptotic pathways by intracellular protozoan parasites.

During intracellular parasitic infections, pathogens and host cells take part in a complex web of events that are crucial for the outcome of the infection. Modulation of host cell apoptosis by pathogens attracted the attention of scientists during the last decade. Apoptosis is an efficient mechanism used by the host to control infection and limit pathogen multiplication and dissemination. In order to ensure completion of their complex life cycles and to guarantee transmission between different hosts, intracellular parasites have developed mechanisms to block apoptosis and sustain the viability of their host cells. Here, we review how some of the most prominent intracellular protozoan parasites modulate the main mammalian apoptotic pathways by emphasizing the advances from the last decade, which have begun to dissect this dynamic and complex interaction.

The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.

Characterization of the anti-Leishmania effect induced by cisplatin, an anticancer drug.

The cis-diamminedichloroplatinum(II), known as cis-DDP or cisplatin is a widely used drug in cancer chemotherapy. Although a recent study has shown the anti-Leishmania activity of some cis-DDP derivatives, the cytotoxic properties were measured only on promastigotes, the insect vector form of the parasite. In this study the effect of cis-DDP on promastigotes and amastigotes, the vertebrate stage of the parasite is reported. The IC50, determined by flow cytometry, after 72 h of drug incubation was four times higher, 7.73+/-1.03 microM in the case of promastigotes compared to axenic amastigotes, 1.88+/-0.10 microM. In intracellular amastigotes the IC50, determined by counting the parasite index was 1.85+/-0.22 microM. By using flow cytometry, two patterns of cell cycle changes was observed: cis-DDP treated promastigotes and amastigotes accumulated in S phase and G2 phase, respectively. The cis-DDP response was also found to involve an "apoptosis-like" death of both promastigotes and amastigotes. However, DNA fragmentation was only detected in promastigote forms. In contrast mitochondrial transmembrane potential loss was observed for both stages of the parasite. Upon incubation of parasites with the drug an increase on GSH and GSSG levels and reactive oxygen species could be detected in the case of promastigote. Moreover, a slight increase of GSH level was detected on amastigote form. Taken together, these observations indicate that amastigotes are more sensitive to cis-DDP when compared to promastigotes. However, the signaling pathways leading to cell death could be different.

Anti-Leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs.

Canine infections with Leishmania infantum represent a considerable veterinary medical and public health problem. In this study, immunoglobulin G1 (IgG1) and IgG2 specific humoral responses were measured and compared with the delayed type hypersensitivity (DTH) cellular response to a leishmanin, in three groups of dogs clinically and serologically characterised as: (I) asymptomatic and direct agglutination test (DAT)-seronegative; (II) asymptomatic and DAT-seropositive; (III) DAT-seropositive and symptomatic. IgG2 was regarded as a marker of disease, since significantly higher levels of this subclass were recorded in the symptomatic dogs. In contrast, the IgG1 response could not be related to clinically relevant infection. A high correlation was observed between IgG2 level and DAT titre; the correlations between IgG1 and IgG2 levels, and between IgG1 level and DAT titre were lower. This may indicate that IgG2 is the main subclass in the specific humoral response which is detected by the DAT. A reduced IgG2 response, albeit not significantly different, was recorded among dogs with clear cellular immune responses detected by a DTH positive reaction. Furthermore, no correlations were observed between cellular response measured by DTH and humoral responses quantified by DAT titre or IgG1 and IgG2 levels. Combining serology and DTH skin test is a practical procedure to assess anti-Leishmania immune responses in dogs.

Advances and perspectives in Leishmania cell based drug-screening procedures.

Efforts for the development of new therapeutics, essential for the control of leishmaniasis rely mainly on screening of potentially effective compounds in pathogen growth/multiplication assays, both in vitro and in vivo. Screenings designed to closely reflect the situation in vivo are currently labor-intensive and expensive, since they require intracellular amastigotes and animal models. Screenings designed to facilitate rapid testing of a large number of drugs are not performed on the clinically relevant parasite stage, but the promastigotes. The ability to select transgenic Leishmania expressing reporter proteins, such as the green fluorescent protein (GFP) or the luciferase, opened up new possibilities for the development of drug screening tests. In this review we will focus on available methodologies for direct drug screening purposes against the mammalian stage of the parasite, with emphasis on the future developments that could improve sensitivity, reliability, versatility and the throughput of the intracellular model screening.

Looking for putative functions of the Leishmania cytosolic SIR2 deacetylase.

During the past few years, the silent information regulator SIR2 protein family has attracted great interest due to its implication in an organism's life span extension. They bear diverse subcellular localization and play a role in transcriptional silencing and DNA repair. The biochemical reaction catalysed by these enzymes (nicotinamide adenine dinucleotide-dependant deacetylase/adenosine diphosphate-ribosyl transferase) is supposed to be linked to metabolism. Members of this protein family were described in parasitic organisms, but little information is available on potential functions of such enzymes in these organisms. In this article, we review recent information on structure and peculiar functions of SIR2s in eukaryotes, with emphasis on parasitic protozoa, particularly the Trypanosomatidae. Through the enzyme localization and the diverse substrates and by-products of the enzymatic reactions, we approach the potential pathways in which the Leishmania cytosolic SIR2 protein can be involved.

Conversion of Trypanosoma cruzi Tc52 released factor to a protein inducing apoptosis.

In this study Tc52, a Trypanosoma cruzi released protein, which exerts an immunoregulatory activity, was converted to a molecular form with altered biological function. Indeed, the genetic fusion of Tc52 to a carrier protein, the Shistosoma japonicum glutathione S-transferase (Tc52-Sj26), was shown to induce apoptosis in spleen cells from BALB/c or CBA mice and the human T-cell leukemic cell line (CEM). Cell death by apoptosis was evidenced by the following criteria: (1) increased binding of Annexin V to rTc52-treated spleen cells; (2) the presence of an ordered cleavage of the DNA backbone; (3) double labeling showed increased number of T cells undergoing apoptosis upon incubation with rTc52; (4) the use of a CEM cell line and TUNEL assay allowed to show in situ DNA fragmentation. Surprisingly, intraperitoneal injections of rTc52 to BALB/c mice, which were then infected with T. cruzi, resulted in increased parasiteamia levels and is congruent to 2.5 times increase of macrophages number. Since native Tc52 could not trigger, apoptosis of T cells we could hypothesized that the fusion of Tc52 with Sj26 led to conformational changes resulting in apoptosis inducing properties of rTc52. The possible in vivo physiopathological implications of these finding were discussed.

Differential effects of polyamine derivative compounds against Leishmania infantum promastigotes and axenic amastigotes.

The natural polyamines are ubiquitous polycationic compounds that play important biological functions in cell growth and differentiation. In the case of protozoan species that are causative agents of important human diseases such as Leishmaniasis, an exogenous supply of polyamines supports parasite proliferation. In the present study, we have investigated the effect of three polyamine derivatives, (namely bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd) and spermine (BNIPSpm)), on the proliferative stages of Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean basin. A significant reduction of promastigotes and axenic amastigotes growth was observed in the presence of increasing concentrations of the drugs, although the mechanisms leading to the parasite growth arrest seems to be different. Indeed, by using a number of biochemical approaches to analyse the alterations that occurred during early stages of parasite-drug interaction (i.e. membrane phosphatidylserine exposure measured by annexin V binding, DNA fragmentation, deoxynucleotidyltranferase-mediated dUTP end labelin (TUNEL), mitochondrial transmembrane potential loss), we showed that the drugs had the capacity to induce the death of promastigotes by a mechanism that shares many features with metazoan apoptosis. Surprisingly, the amastigotes did not behave in a similar way to promastigotes. The drug inhibitory effect on amastigotes growth and the absence of propidium iodide labelling may suggest that the compounds are acting as cytostatic substances. Although, the mechanisms of action of these compounds have yet to be elucidated, the above data show for the first time that polyamine derivatives may act differentially on the Leishmania parasite stages. Further chemical modifications are needed to make the polyamine derivatives as well as other analogues able to target the amastigote stage of the parasite.

Effect of abietane diterpenes from Plectranthus grandidentatus on T- and B-lymphocyte proliferation.

Five known abietane diterpenes of the royleanone and coleon type, namely, fatty acid esters of 7alpha-acyloxy-6beta-hydroxyroyleanone (1), grandidone A (2), 7alpha-acetoxy-6beta-hydroxyroyleanone (3), 6beta,7alpha-dihydroxyroyleanone (4) and coleon U (5), isolated from Plectranthus grandidentatus, were evaluated for their effect on the proliferation of human lymphocytes induced by the mitogen PHA. All except 4, showed a dose-dependent suppressor effect, with 3 yielding the most potent antiproliferative activity, followed by 5. These two compounds, that represent diterpenes of the royleanone and coleon type respectively, were also shown to be potent inhibitors of mouse splenocyte proliferation induced by ConA or LPS mitogens. However, the sensitivity of ConA-stimulated splenocytes to their suppressive effect was higher, suggesting a preferential inhibition of T-lymphocyte proliferation. The antiproliferative activity of 3 seemed to be exerted without affecting the expression of the lymphocyte activation marker CD69. On the contrary, 5 was shown to reduce the expression of CD69 of TCD8(+) and B-cells, suggesting a relationship between its antiproliferative effect and the expression of this early marker of activation on these cell populations. The capacity of 5 to induce apoptosis on ConA-stimulated splenocytes could also be related with its antiproliferative activity.

Inhibition of lymphocyte proliferation by prenylated flavones: artelastin as a potent inhibitor.

Eight natural prenylated flavones, previously isolated from Artocarpus elasticus, were evaluated for their effect on the mitogenic response of human lymphocytes to PHA. They all exhibited a dose-dependent suppression effect. An interesting relationship was observed between their antiproliferative activity and their chemical structure. Indeed, the most potent flavones possessed a 3,3-dymethylallyl group (prenyl) at C-8, such as artelastin, which exhibited the highest antiproliferative activity. Studies of the mechanism underlying its effect revealed that artelastin had an irreversible inhibitory effect on the PHA-induced lymphocyte proliferation and could affect the course of the ongoing mitogenic response either at the initial induction phase or at the late phase of proliferation. This prenylated flavone was also shown to be a potent inhibitor of both T- and B-lymphocyte mitogen induced proliferation although B-mitogenic response was the more sensitive one. Artelastin did not affect either the basal levels of the early marker of activation CD69 on non-stimulated splenocytes or its expression on ConA- or LPS-stimulated splenocytes. However, it decreased the production of IFN-gamma, IL-2, IL-4 and IL-10 in ConA-stimulated splenocytes. Furthermore, artelastin had no effect on apoptosis of splenocytes.

Identification of antibodies to Leishmania silent information regulatory 2 (SIR2) protein homologue during canine natural infections: pathological implications.

Dogs are the domestic reservoir of zoonotic visceral Leishmaniasis caused by Leishmania infantum in the Mediterranean basin and thus constitute an important health problem in both human and veterinary medicine. Until vaccines become available, conventional measures such as epidemiological surveillance including reservoir control will be among the practical options for prevention and containment of the disease. We have recently characterised novel Leishmania sp. genes encoding parasite proteins named (LmS3a: homologous to mammalian ribosomal protein S3a; LmSIR2: homologous to the silent information regulatory 2 protein family; LimTXNPx: homologous to the peroxiredoxin family with N-terminal mitochondrial leader sequence) that may contribute to the host immune dysfunction in murine experimental Leishmaniasis. In the present study we have investigated the humoral responses against the parasite antigens in groups of L. infantum-infected dogs with different clinical status: symptomatic and asymptomatic with DTH positive or negative test. The determination of immunoglobulin (Ig) isotypes revealed high levels of total IgG in both symptomatic and asymptomatic animals when compared to IgM. Furthermore, the IgG2 appeared to be the predominant subclass of Ig present in the sera of infected animals particularly in the case of symptomatic dogs. The IgG subclass reactivity analysis revealed a broad specific recognition range of parasite recombinant antigens. Interestingly, differential profiles of IgG1 and IgG2 antibody reactivity were observed in asymptomatic and symptomatic dogs. The LmSIR2 protein was found to be a highly reactive molecule with IgG2 from most of the asymptomatic and symptomatic animals. Considering the fact that LmSIR2 secreted by the parasites can be bound and taken up by neighbouring cells, the latter could be a target for anti-LmSIR2 antibodies and this may contribute to the immunopathological alterations and host tissue damage. The implications of these observations in the pathogenesis of Leishmaniasis are discussed.

Leishmania infantum MON-98: infection in a dog from Alto Douro, Portugal.

We report the isolation of Leishmania infantum zymodeme MON-98 in Portugal, its first known occurrence outside the focus of El Agamy, Egypt. One dog found to be infected with Acanthocheilonema dracunculoides during a survey of canine filariosis in the Alto Douro region, north-east Portugal, was also discovered to have Leishmania in bone marrow. The isolated strain was identified by isoenzyme analysis. A search for other possible cases of L. infantum MON-98 infection in dogs, vectors and particularly humans is necessary to establish the real epidemiological importance of this zymodeme in the endemic region of Alto Douro.

Dual role of the Leishmania major ribosomal protein S3a homologue in regulation of T- and B-cell activation.

We have recently characterized a novel Leishmania major gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). In the present study we have extended our approach to the analysis of LmS3arp activity on T- and B-cell functions in a murine model. The results presented in this report show that LmS3arp plays a dual role in the regulation of T- and B-cell reactivity. Indeed, we found that injection of the LmS3arp recombinant protein (rLmS3arp) into BALB/c mice induces preferential activation of B cells, as shown by the following criteria: (i) increased expression of CD69 molecules on immunoglobulin M (IgM)-secreting spleen cells, (ii) a considerable increase of IgM-secreting B cells, and (iii) elevated levels of IgM antibodies in the sera of injected animals. Moreover, the IgM antibodies are not specific to the Leishmania antigens but preferentially recognize heterologous antigens like myosin, thyroglobulin, DNA, and keyhole limpet hemocyanin. Furthermore, the strong polyclonal expansion of nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is concomitant with a marked inhibition of T-cell proliferation. Analysis of cytokine production revealed a significant downregulation of gamma interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together, our data suggest that rLmS3arp, through direct or indirect action toward B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response.

Significant association between the skewed natural antibody repertoire of Xid mice and resistance to Trypanosoma cruzi infection.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self-antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre- and post-infected Xid mice. In particular, relatively increased natural (pre-existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivities to self and non-self proteins. These results suggest that self-reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self-aggressive components responsible for pathology.

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase.

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.