Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Enhanced T cell activation in Plasmodium falciparum malaria-infected human immunodeficiency virus-1 patients from Mozambique

Human immunodeficiency virus (HIV)-1 infection has an important impact on malaria. Plasmodium falciparum and HIV-1 co-infected patients (Pf/HIV) present with a high degree of anaemia, enhanced parasitaemia and decreased CD4+ T cell counts, which increase the risk of developing severe malaria. In addition, infection with either Pf or HIV-1 alone causes extensive immune activation. Our hypothesis was that lymphocyte activation is potentiated in Pf/HIV co-infected patients, consequently worsening their immunosuppressed state. To test this hypothesis, 22 Pf/HIV patients, 34 malaria patients, 29 HIV/AIDS patients and 10 healthy controls without malaria or HIV/acquired immune deficiency syndrome (AIDS) from Maputo/Mozambique were recruited for this study. As expected, anaemia was most prevalent in the Pf/HIV group. A significant variation in parasite density was observed in the Pf/HIV co-infected group (110-75,000 parasites/µL), although the median values were similar to those of the malaria only patients. The CD4+ T cell counts were significantly lower in the Pf/HIV group than in the HIV/AIDS only or malaria only patients. Lymphocyte activation was evaluated by the percentage of activation-associated molecules [CD38 expression on CD8+ and human leukocyte antigen-DR expression on CD3+ T cells]. The highest CD38 expression was detected in the Pf/HIV co-infected patients (median = 78.2%). The malaria only (median = 50%) and HIV/AIDS only (median = 52%) patients also exhibited elevated levels of these molecules, although the values were lower than those of the Pf/HIV co-infected cases. Our findings suggest that enhanced T-cell activation in co-infected patients can worsen the immune response to both diseases.

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

Studies on the acid activation of Brazilian smectitic clays

Fuller's earth and acid activated smectitic clays are largely used as bleaching earth for the industrial processing of vegetable, animal and mineral oils and waxes. The paper comments about the nomenclature used for these materials, the nature of the acid activation of smectitic clays (bentonites), activation laboratory procedures and presents a review of the acid activation of bentonites from 20 deposits from several regions of Brazil. The activated clays were tested and show good decolorizing power for soybean, castor, cottonseed, corn and sunflower oils.

MICROWAVE ACTIVATION OF IMMOBILIZED LIPASE FOR TRANSESTERIFICATION OF VEGETABLE OILS

This work investigated the effect of microwave irradiation (MW) on the ethanolysis rate of soybean and sunflower oils catalyzed by supported Novozyme 435 (Candida antarctica). The effects of tert-butanol, water addition and oil:ethanol molar ratio on transesterification were evaluated under conventional heating (CH), and under optimum reaction conditions (with no added water in the system, 10% tert-butanol and 3:1 ethanol-to-oil molar ratio). The reactions were monitored up to 24 h to determine the conditions of initial reaction velocity. The investigated variables under MW (50 W) were: reaction time (5.0-180 min) and mode of reactor operation (fixed power, dynamic and cycles) in the absence and presence of tert-butanol (10% (w/w). The measured response was the reaction conversion in ethyl esters, which was linked to the enzyme catalytic activity. The results indicated that the use of microwave improved the activity at fixed power mode. A positive effect of the association of tert-butanol and MW irradiation on the catalytic activity was observed. The reaction rate improved in the order of approximately 1.5 fold compared to that under CH with soybean oil. Using soybean oil, the enzymatic transesterification under MW for conversion to FAEE (fatty acid ethyl esters) reached >99% in 3h, while with the use of CH the conversions were about 57% under similar conditions.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Immunolocalisation of secreted-excreted products of Meloidogyne spp. using polyclonal and monoclonal antibodies

Molecules expressed at the surface cuticle (SC) of plant parasitic nematodes represent the primary plant-nematode interface, and together with secreted-excreted (S-E) products are probably the first signals perceived by the host. These molecules, which are released into plant tissue, probably play important roles in the host-parasite interactions. Characterisation of these antigens will help in the identification of nematode targets useful for novel control strategies, which interfere with the nematode infection of plants. Three monoclonal (MAbs) and three polyclonal (PAbs) antibodies produced to S-E products of Meloidogyne spp. and Heterodera avenae were used to examine their reactivity towards M. incognita and/or M. arenaria second stage juveniles and adult females. The three PAbs showed cross-reactivity with M. incognita and M. arenaria. Antibody Roth-PC 373 strongly recognised molecules present in the SC, amphids and intestine, antibody Roth-PC 389 recognised the nematode amphids and metacorpus, while antibody Roth-PC 419 bound to molecules present in the subventral glands. Reactivity of the MAbs was only tested against M. arenaria. Monoclonal antibody Roth-MAb T116C1.1 showed intense reactivity with molecules present in the amphidial and phasmidial glands. Monoclonal antibodies Roth-MAb T46.2 and T42D.2 labeled the nematode amphids and molecules present in the nematode oesophagus (metacorpus), respectively.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Loss of PTEN expression and AKT activation in HER2-positive breast carcinomas

PURPOSE: To examine the expression of AKT and PTEN in a series of HER2-positive primary invasive breast tumors using immunohistochemistry, and to associate these expression profiles with classic pathologic features such as tumor grade, hormone receptor expression, lymphatic vascular invasion, and proliferation.METHODS: A total of 104 HER2-positive breast carcinoma specimens were prepared in tissue microarrays blocks for immunohistochemical detection of PTEN and phosphorylated AKT (pAKT). Original histologic sections were reviewed to assess pathological features, including HER2 status and Ki-67 index values. The associations between categorical and numeric variables were identified using Pearson's chi-square test and the Mann-Whitney, respectively.RESULTS: Co-expression of pAKT and PTEN was presented in 59 (56.7%) cases. Reduced levels of PTEN expression were detected in 20 (19.2%) cases, and these 20 tumors had a lower Ki-67 index value. In contrast, tumors positive for pAKT expression [71 (68.3%)] were associated with a higher Ki-67 index value.CONCLUSION: A role for AKT in the proliferation of HER2-positive breast cancers was confirmed. However, immunohistochemical detection of PTEN expression did not correlate with an inhibition of cellular proliferation or control of AKT phosphorylation, suggesting other pathways in these mechanisms of control.

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10% calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49% of the platelets were classified as state of activation uncertain, 41% as resting, 9% as fully activated and 1% as irreversibly damaged. Treatment with 10% CaCl2 provided a distribution of 54% platelets in state of activation uncertain, 24% as fully activated, 20% as resting, and 2% as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10% CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC). The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Complement activation-related pseudoallergy in dogs following intravenous administration of a liposomal formulation of meglumine antimoniate

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

Anhydrobiosis in yeast: activation effect

Intracellular substances released into the medium during rehydration of dry yeast cells can significantly improve the quality of a synthetic medium. Acceleration of yeast growth in this medium and increased yield of biomass are observed simultaneously. The change in the molecular arrangement of intracellular membranes as a result of the strong dehydration of live organisms is a negative phenomenon that reduces the level of cell viability. However, this phenomenon also represents an adaptive mechanism which facilitates the maintenance of population viability as a whole under extreme environmental conditions