Development of Eosinophilia in dogs intradermically inoculated with sand fly saliva and Leishmania (Leishmania) chagasi stationary-phase promastigotes

Salivary glad lysates of the sand fly Lutzomia longipalpis have been shown to enhance the infectivity of Leishmania in mice. As shown herein, the simultaneous inoculation of Leishmania chagasi stationary-phase promastigotes and L. longipalpis salivary gland by the intradermal route in a group of mongrel dogs induced a statistically significant eosinophilia, in relation to dogs inoculated with Leishmania or with salivary gland lysate only. These dogs had no evidence of infection, in spite of the infectivity of the promastigotes when inoculated by the intravenous route.

Construção de câmara de luz ultravioleta para fotopolimerização de fases estacionárias monolíticas

The assembly of a photochemical reactor with six fluorescent lamps, used for photopolymerizations is described. This chamber presents a mobile support, allowing the placement of samples at different heights and a safety lock that interrupts the radiation, if it is opened during operation. The mirrored internal walls avoid the dispersion and non-uniform distribution of light. There is no high heating because the own character of the used lamps. All parts could be purchased in commerce with less than U$ 150,00. This reactor was successfully used for monolithic stationary phase photopolymerization.

CONSTRUÇÃO DE UM DISPOSITIVO DE PRESSURIZAÇÃO A VÁCUO PARA A PREPARAÇÃO DE FASES ESTACIONÁRIAS MONOLÍTICAS VIA PROCESSO SOL-GEL

The filling of capillaries via the sol-gel process is growing. Therefore, this technical note focuses on disseminating knowledge acquired in the Group of Analytical Chemistry and Chemometrics over seven years working with monolithic stationary phase preparation in fused silica capillaries. We believe that the detailed information presented in this technical note concerning the construction of an alternative high pressurization device, used to fill capillary columns via the sol-gel process, which has promising potential for applications involving capillary electrochromatography and liquid chromatography in nano scale, may be enlightening and motivating for groups interested in developing research activities within this theme.

Novas fases estacionárias à base de sílica para cromatografia líquida de alta eficiência

The present work reviews recent advances in the preparation of new reversed phase packing materials such as sterically protected, bidentate, hybrid organic-inorganic and monolithic phases and phases containing embedded polar groups. The bonding chemistry involved in the preparation of these phases as well as their advantages over conventional C8 and C18 reversed phases are discussed. Understanding the reasons behind the development of these newer column packings helps analysts select the best stationary phase for a given application.

Fases estacionárias modernas para cromatografia líquida de alta eficiência em fase reversa

This review first discusses the limitations of many of the supports and stationary phases used in reversed phase high performance liquid chromatography and then describes those, developed more recently, that present better stabilities and more versatile selectivities. Emphases will be given to stationary phases that use higher purity silicas, hybrid silicas, monolithic silicas, metallic oxides and mixed oxides as supports and those that have embedded polar groups or contain phenyl or fluoro groups as the stationary phase as well as the phases used for mixed mode or hydrophilic interaction separations. These modern stationary phases facilitate the analysis of complex mixtures.

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

In Amazonian Brazil, the Cebus apella monkey (Primates: Cebidae) has been associated with the enzootic cycle of Leishmania (V.) shawi, a dermotropic parasite causing American cutaneous leishmaniasis (ACL). It has also been successfully used as animal model for studying cutaneous leishmaniasis. In this work, there has been investigated its susceptibility to experimental Leishmania (L.) infantum chagasi-infection, the etiologic agent of American visceral leishmaniasis (AVL). There were used ten C. apella specimens, eight adult and two young, four males and six females, all born and raised in captivity. Two experimental infection protocols were performed: i) six monkeys were inoculated, intra-dermal via (ID), into the base of the tail with 2 x 10(6) promastigotes forms from the stationary phase culture medium; ii) other four monkeys were inoculated with 3 x 10(7) amastigotes forms from the visceral infection of infected hamsters by two different via: a) two by intravenous via (IV) and, b) other two by intra-peritoneal via (IP). The parameters of infection evaluation included: a) clinical: physical exam of abdomen, weigh and body temperature; b) parasitological: needle aspiration of the bone-marrow for searching of amastigotes (Giemsa-stained smears) and promastigotes forms (culture medium); c) immunological: Indirect fluorescence antibody test (IFAT) and, Delayed-type hypersensitivity (DTH). In the six monkeys ID inoculated (promastigotes forms) all parameters of infection evaluation were negative during the 12 months period of follow-up. Among the four monkeys inoculated with amastigotes forms, two IV inoculated showed the parasite in the bone-marrow from the first toward to the sixth month p.i. and following that they cleared the infection, whereas the other two IP inoculated were totally negative. These four monkeys showed specific IgG-antibody response since the third month p.i. (IP: 1/80 and IV: 1/320 IgG) toward to the 12th month (IP: 1/160 and IV: 1/5120). The DTH-conversion occurred in only one IV inoculated monkey with a strong (30 mm) skin reaction. Considering these results, we do not encourage the use of C. apella monkey as animal model for studying the AVL.

In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB/c mice

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be 2.28 ± 0.24 µg/mL while those of Dim and Art were 9.16 ± 0.3 µg/mL and 4.64 ± 0.48 µg/mL respectively. The IC50 for Amphot B was 0.16 ± 0.32 µg/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly (p < 0.001) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower (p < 0.05) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

Apoptotic-like activity of staurosporine in axenic cultures of Trypanosoma evansi

Trypanosoma evansi is a blood protozoan parasite of the genus Trypanosoma which is responsible for surra (Trypanosomosis) in domestic and wild animals. This study addressed apoptotic-like features in Trypanosoma evansi in vitro. The mechanism of parasite death was investigated using staurosporine as an inducing agent. We evaluated its effects through several cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, maintenance of plasma membrane integrity, and mitochondrial trans-membrane potential. For access to these features we have used the flow cytometry and fluorescence microscopy with cultures in the stationary phase and adjusted to a density of 10(6) cells/mL. The apoptotic effect of staurosporine in T. evansi was evaluated at 20 nM final concentration. There was an increase of phosphatidylserine exposure, whereas mitochondrial potential was decreased. Moreover, no evidence of cell permeability increasing with staurosporine was observed in this study, suggesting the absence of a necrotic process. Additional studies are needed to elucidate the possible pathways associated with this form of cell death in this hemoparasite.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Action of pentoxifylline on experimental cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis

In the animal model of leishmaniasis caused by Leishmania (Leishmania) amazonensis there is a complex mechanism of the host-parasite interaction. The present study was performed to interfere with the inflammatory reaction to the parasites, through immune modulation. Female C5BL/6 isogenic mice were used, some of which were inoculated on the right ear and others on the right footpad with 3.10(6) stationary phase promastigotes of the MHOM/BR/PH8 strain of L. (L.) amazonensis, and were allocated in three groups: the first received pentoxifylline 8mg/kg every 12 h, since the first day; the second one received the same dose since the 40th day of infection and a control group that did not receive any treatment. All the ears excised were analyzed to determine the variation in weight between both ears and for histopathological analyses. A quantification of the parasites was done using the limiting dilution assay. A significant reduction of the number of parasites, was observed among the animals treated which had an accordingly significant reduction on the weight of the ears. Pentoxifylline reduced the macrophages propensity to vacuolation and induced a more effective destruction of the parasites by these cells. Moreover, the group that began the treatment later did not show the same effectiveness.

A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp. medellin Cry 11Bb toxin deduced by homology modelling

Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this ¶-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata

Candida glabrata is an opportunistic fungal pathogen that can cause severe invasive infections and can evade phagocytic cell clearance. We are interested in understanding the virulence of this fungal pathogen, in particular its oxidative stress response. Here we investigated C. glabrata, Saccharomyces cerevisiae and Candida albicans responses to two different oxidants: menadione and cumene hydroperoxide (CHP). In log-phase, in the presence of menadione, C. glabrata requires Cta1p (catalase), while in a stationary phase (SP), Cta1p is dispensable. In addition, C. glabrata is less resistant to menadione than C. albicans in SP. The S. cerevisiae laboratory reference strain is less resistant to menadione than C. glabrata and C. albicans; however S. cerevisiaeclinical isolates (CIs) are more resistant than the lab reference strain. Furthermore, S. cerevisiae CIs showed an increased catalase activity. Interestingly, in SP C. glabrata and S. cerevisiae are more resistant to CHP than C. albicans and Cta1p plays no apparent role in detoxifying this oxidant.

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.

Discriminação quiral por CLAE em carbamatos de polissacarídeos: desenvolvimento, aplicações e perspectivas

The importance of chromatographic separation of enantiomers has been acknowledged. This review surveys the development, application and perspectives of polysaccharide carbamates as chiral stationary phase for HPLC.

Estado da arte da cromatografia gasosa de alta resolução e alta temperatura

The developments in stationary phase synthesis and capillary column technology, have opened new perspectives in analysis of high molecular mass compounds (³600 daltons) and thermolabile organic compounds by High Temperature High Resolution Gas Chromatography (HT-HRGC). HT-HRGC is a new analytical borderline and its application to the analysis of high molecular mass compounds is still in its infancy. The apolar and medium polar gum phases can now be operated at temperatures up to 400-480ºC, being used for the analysis of n-alcanes up to C-100, lipids, oligosaccharides, industrial resins, polyglycerols, cyclodextrins, porphyrins, etc. This technique should play a leading role as a powerful tool, for many different analysis types, in multidisciplinary fields of Science.