Polyelectrolytic gelatin nanoparticles as a drug delivery system for the promastigote form of Leishmania amazonensis treatment.

In this study, phthalocianato[bis(dimethylaminoethanoxy)] silicon (NzPC) was loaded onto gelatin nanoparticles functionalized with polyelectrolytes (polystyrene sulfonate/polyallylamine hydrochloride) by layer-by-layer (LbL) assembly for photodynamic therapy (PDT) application in promastigote form of Leishmania amazonensis treatment. The process yield, and encapsulation efficiency were 80.0% ± 1.8 and EE = 87.0% ± 1.1, respectively. The polyelectrolytic gelatin nanoparticles (PGN) had a mean diameter of 437.4 ± 72.85 nm, narrow distribution size with a polydispersity index of 0.086. The obvious switching of zeta potential indicates successful alternating deposition of the polyanion PSS and polycation PAH directly on the gelatin nanoparticles. Photosensitizer photophysical properties were shown to be preserved after gelatin nanoparticle encapsulation. The impact of the PDT in the viability and morphology of Leishmania amazonensis promastigote in culture medium was evaluated. The PGN-NzPc presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 80% in 0.1 µmol.L-1 concentration tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. These results reinforce the idea that the use of PGN as photosensitizers carriers is useful for PDT of Leishmania promastigotes.

Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide.

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC50 and IC90, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC50 > 4.7 µM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC50 < 0.2 µM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC50 between 0.4 and 0.9 µM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC50 between 1.0 and 1.2 µM). In cluster 4 were species with moderate to high tolerance (IC50 between 1.3 and 1.6 µM). Cluster 5 contained the species with high tolerance (IC50 between 1.9 and 4.0 µM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.

Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis.

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500µg/ml, in serial dilution, until 7,8µg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost.

Differentiation of Leishmania species by FT-IR spectroscopy.

UNLABELLED: Leishmaniasis is a parasitic infectious disease caused by protozoa that belong to the genus Leishmania. It is transmitted by the bite of an infected female Sand fly. The disease is endemic in 88 countries Desjeux (2001) [1] (16 developed countries and 72 developing countries) on four continents. In Brazil, epidemiological data show the disease is present in all Brazilian regions, with the highest incidences in the North and Northeast. There are several methods used to diagnose leishmaniasis, but these procedures have many limitations, are time consuming, have low sensitivity, and are expensive. In this context, Fourier Transform Infrared Spectroscopy (FT-IR) analysis has the potential to provide rapid results and may be adapted for a clinical test with high sensitivity and specificity. In this work, FT-IR was used as a tool to investigate the promastigotes of Leishmaniaamazonensis, Leishmaniachagasi, and Leishmaniamajor species. The spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. RESULTS: cluster analysis found four specific regions that are able to identify the Leishmania species. The dendrogram representation clearly indicates the heterogeneity among Leishmania species. The band deconvolution done by the curve fitting in these regions quantitatively differentiated the polysaccharides, amide III, phospholipids, proteins, and nucleic acids. L. chagasi and L. major showed a greater biochemistry similarity and have three bands that were not registered in L. amazonensis. The L. amazonensis presented three specific bands that were not recorded in the other two species. It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. The high sensitivity and specificity of this technique opens up the possibilities for further studies about characterization of other microorganisms.

Photodynamic effects of zinc phthalocyanines on intracellular amastigotes of Leishmania amazonensis and Leishmania braziliensis.

This study investigated the photoactivity of four zinc phthalocyanines (PcZns) on a murine macrophage cell line infected with Leishmania amazonensis or Leishmania braziliensis. Infected and uninfected cells were incubated with PcZns at different concentrations (1-10 µM) for 3 h and then exposed to an LED device in continuous wave mode at 660 nm with a fluency of 50 J/cm(2) (25 mV). Enzymatic activity was determined by MTT assay 24 h after light treatment. The results demonstrated that all PcZns exhibited high photoactivity, particularly when used at 10 µM. The photodynamic effects were different for uninfected cells versus parasite-infected cells and among the four PcZns. Uninfected cells were more sensitive to photoactivity than infected cells. Although PcZns photodynamic therapy provided promising results, further studies are necessary to better understand its mechanism of action in the treatment of leishmaniasis.

Cytokine mRNA expression is decreased in the subplantar muscle of rat paw subjected to carrageenan-induced inflammation after low-level laser therapy.

OBJECTIVE: The objective of this work was to investigate the anti-inflammatory effects of low-level laser therapy, applied at different wavelengths (660 and 684 nm), on cytokine mRNA expression after carrageenan-induced acute inflammation in rat paw. BACKGROUND DATA: Low-level laser therapy (LLLT) has been observed to reduce pain in inflammatory disorders. However, little is known about the mechanisms behind this effect or whether it is wavelength-specific. MATERIALS AND METHODS: The test sample consisted of 32 rats divided into four groups: A(1) (control-saline), A(2) (carrageenan-only), A(3) (carrageenan + 660 nm laser therapy), and A(4) (carrageenan + 684 nm laser therapy). The animals from groups A(3) and A(4) were irradiated 1 h after induction of inflammation by carrageenan injection. Continuous-wave red lasers with wavelengths of 660 and 684 nm and dose of 7.5 J/cm(2) were used. RESULTS: Both the 660 nm and 684 nm laser groups had 30%-40% lower mRNA expression for cytokines TNF-alpha, IL-1beta, and IL-6 in the paw muscle tissue than the carrageenan-only control group. Cytokine measurements were made 3 h after laser irradiation of the paw muscle, and all cytokine differences between the carrageenan-only control group and the LLLT groups were statistically significant (p < 0.001). CONCLUSIONS: LLLT at the 660-nm and 684-nm wavelengths administered to inflamed rat paw tissue at a dose of 7.5 J/cm(2) reduce cytokine mRNA expression levels within 3 h in the laser-irradiated tissue.

Suitability of a recombinant Staphylococcus aureus enterotoxin C bovine variant for immunodiagnostics and therapeutic vaccine development.

Recombinant bovine variant of staphylococcal enterotoxin C (SECbovine), produced as a NH2-terminal histidine hexamer fusion protein (His6-tagged SECbovine), expressed at high levels (25%) in Escherichia coli and affinity purified to homogeneity (99.9%), was tested for its diagnostic and therapeutic potentials. His6-tagged SECbovine is antigenically authentic to native SECbovine across host species, as confirmed by antibody-based capture detection assays using human, mouse, rabbit and chicken hyperimmune sera. His6-tagged SECbovine showed significant T-cell stimulation activity in vitro. His6-tagged SECbovine was immunogenic for IgG in mice (intragastric and intravenous routes) and rabbits (intramuscular and subcutaneous routes), dispensing immunoadjuvant coadministration. The formation of neutralizing antibodies reduced the severity of intoxication symptoms in immunized rabbits. Purified anti-recombinant SECbovine rabbit polyclonal IgG neutralized the pyrexic and diarrhoeagenic effects of native SEC/SED and recombinant SEC, tested by the kitten and rabbit bioassays, respectively.

Differentiation of mouse embryonic stem cells and their hybrids during embryoid body formation

We studied the karyotypes of three hybrid clones of mouse embryonic stem cells and murine splenocytes (two having near diploid and one having near tetraploid chromosome numbers) and the characteristics of their differentiation during the formation of embryoid bodies. The X chromosome originating from embryonic stem cells may be lost in hybrids with a near diploid chromosome number and reprogramming of the "somatic" X may occur. The morphological data we obtained using light and electron microscopy revealed a correlation between the karyotype constitution of hybrid cells and their differentiation during the formation of embryoid bodies. At the beginning of development, the embryoid bodies derived from hybrid cells already showed an advanced degree of differentiation. The production of significant quantities of cartilage was typical for hybrid cells with near tetraploid chromosome numbers. The hybrid cells showed restricted pluripotent capacity and were already committed when they started to differentiate into embryoid bodies

Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes

The properties of embryonic hybrid cells obtained by fusion of embryonic stem (ES) or teratocarcinoma (TC) cells with differentiated cells are reviewed. Usually, ES-somatic or TC-somatic hybrids retain pluripotent capacity at high levels quite comparable or nearly identical with those of the pluripotent partner. When cultured in vitro, ES-somatic- and TC-somatic hybrid cell clones, as a rule, lose the chromosomes derived from the somatic partner; however, in some clones the autosomes from the ES cell partner were also eliminated, i.e. the parental chromosomes segregated bilaterally in the ES-somatic cell hybrids. This opens up ways for searching correlation between the pluripotent status of the hybrid cells and chromosome segregation patterns and therefore for identifying the particular chromosomes involved in the maintenance of pluripotency. Use of selective medium allows to isolate in vitro the clones of ES-somatic hybrid cells in which "the pluripotent" chromosome can be replaced by "the somatic" counterpart carrying the selectable gene. Unlike the TC-somatic cell hybrids, the ES-somatic hybrids with a near-diploid complement of chromosomes are able to contribute to various tissues of chimeric animals after injection into the blastocoel cavity. Analysis of the chimeric animals showed that the "somatic" chromosome undergoes reprogramming during development. The prospects for the identification of the chromosomes that are involved in the maintenance of pluripotency and its cis- and trans-regulation in the hybrid cell genome are discussed. Key words: ES cells, EG cells, hybrid cells, pluripotency, reprogramming