Microbial community composition in fresh water at different stations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea. Cells were concentrated from water samples (1 to 100 ml) on white polycarbonate filters (diameter, 47 mm; pore size, 0.2 mm; type GTTP 4700 [Millipore, Eschborn, Germany]) by applying a vacuum of <25 kPa. They were subsequently fixed by covering the filter with 3 ml of a freshly prepared, phosphate-buffered saline (pH 7.2)-4% paraformaldehyde (Sigma, Deisenhofen, Germany) solution for 30 min at room temperature. Airdried filters are ready for hybridization and can be stored at 220°C or room temperature for several months without showing apparent changes. Probes BET42a, GAM42a, and PLA886 were used with competitor oligonucleotides as described previously amongst others in Manz et al., (1992; doi:10.1016/S0723-2020(11)80121-9). The filters were transferred to a vial containing 50 ml of prewarmed (48°C) washing solution (70 mM NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.01% sodium dodecyl sulfate) and incubated freely floating without shaking at 48°C for 15 min. The filter sections were dried on Whatman 3M paper (Whatman Ltd., Maidstone, United Kingdom) and covered with 50 ml of DAPI solution (1 mg/ml in distilled water filtered through at 0.2-mm filter) for 5 min at room temperature in the dark. For each sample and probe, more than 500 cells were enumerated; for the DAPI examination, more than 1,500 cells were counted per sample. All probe-specific cell counts are presented as the percentage of cells visualized by DAPI. The mean abundances and standard deviations were calculated from the counts of 10 to 20 randomly chosen fields on each filter section. All counts were corrected by subtracting the counts obtained with the negative control NON338. Mean and standard deviation were calculated from the counts of 10 to 20 randomly chosen fields on each filter section.

Optimization of Dengue immunoassay by label-free interferometric optical detection method

In this communication we report a direct immunoassay for detecting dengue virus by means of a label-free interferometric optical detection method. We also demonstrate how we can optimize this sensing response by adding a blocking step able to significantly enhance the optical sensing response. The blocking reagent used for this optimization is a dry milk diluted in phosphate buffered saline. The recognition curve of dengue virus over the proposed surface sensor demonstrates the capacity of this method to be applied in Point of Care technology.

Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind α-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, d-Phe7–α-MSH4-13 analog. Structural analysis of the Re–peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate–metal–thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re–peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re–α-MSH analog. Characterization of the second-generation Re–peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of α-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties.

Avaliação in vivo e in vitro de diferentes protocolos no tratamento da hipersensibilidade dentinária cervical

Este estudo possui duas partes distintas: 1. in vivo (randomizado e longitudinal) que teve como objetivo avaliar protocolos de tratamento para hipersensibilidade dentinária com laser de baixa potência (com diferentes dosagens), laser de alta potência e agente dessensibilizante, por um período de 12 e 18 meses; e 2. in vitro que teve como objetivo analisar a perda de estrutura de dois dentifrícios distintos (Colgate Total 12 e Colgate Pró Alívio) e analisar a permeabilidade dentinária dos tratamentos da etapa 01, associados aos dentifrícios, após diferentes ciclos de abrasão. Na parte in vivo, as lesões cervicais não cariosas de 32 voluntários, previamente submetidos aos critérios de elegibilidade ou exclusão, foram divididas em nove grupos (n=10): G1: Gluma Desensitizer (Heraeus Kulzer), G2: Laser de baixa potência com baixa dosagem (Photon Lase, DMC) (três pontos de irradiação vestibulares e um ponto apical: 30 mW, 10 J/cm2, 9 seg por ponto com o comprimento de onda de 810nm). Foram realizadas três sessões com um intervalo de 72 horas), G3: Laser de baixa potência com alta dosagem (um ponto cervical e um ponto apical: 100 mW, 90 J/cm2, 11 seg por ponto com o comprimento de onda de 810nm. Foram realizadas três sessões com um intervalo de 72 horas), G4: Laser de baixa potência com baixa dosagem + Gluma Desensitizer, G5: Laser de baixa potência com alta dosagem + Gluma Desensitizer, G6: Laser de Nd:YAG (Power LaserTM ST6, Lares Research®), em contato com a superfície dental: 1,0W, 10 Hz e 100 mJ, ? 85 J/cm2, com o comprimento de onda de 1064nm, G7: Laser de Nd:YAG + Gluma Desensitizer, G8: Laser de Nd:YAG + Laser de baixa potência com baixa dosagem, G9: Laser de Nd:YAG + Laser de baixa potência com alta dosagem. O nível de sensibilidade de cada voluntário foi avaliado através da escala visual analógica de dor (VAS) com auxílio do ar da seringa tríplice e exploração com sonda após 12 e 18 meses do tratamento. Na parte 02, in vitro, foram utilizados terceiros molares humanos não irrompidos e recém-extraídos. Todos foram limpos e tiveram suas raízes separadas das coroas. As raízes foram seccionadas em quadrados de dentina com dimensões de 4x4x2 mm, os quais foram embutidos em resina Epoxi e devidamente polidos até uma curvatura de 0,3 ?m, analisados em perfilometria ótica. Estes foram imersos em solução de EDTA 17% por 2min para abertura dos túbulos e armazenados em uma solução de Soro Fetal Bovino diluído em salina tamponada com fosfato. Os espécimes foram divididos aleatoriamente em 12 grupos (n=10) G1: Sem tratamento de superfície, sem dentifrício; G2: Nd:YAG/sem dentifrício; G3: Gluma/sem dentifrício; G4: Nd:YAG + Gluma/sem dentifrício; G5: Sem tratamento de superfície/Colgate Total 12; G6: Nd:YAG/Colgate Total 12; G7: Gluma/Colgate Total 12; G8: Nd:YAG + Gluma/Colgate Total 12; G9: Sem tratamento de superfície/Colgate Pró Alívio; G10: Nd:YAG/Colgate Pró Alívio; G11: Gluma/Colgate Pró Alívio; G12: Nd:YAG + Gluma/Colgate Pró Alívio. Em seguida, as superfícies receberam a aplicação de fitas adesivas nas duas margens, mantendo uma área central de teste exposta de 4 x 1 mm, onde foram realizados os tratamentos de superfície e os ciclos de abrasão correspondentes a 1, 7, 30 e 90 dias de escovação (52 ciclos, 210 segundos de contato com o slurry; 361 ciclos, 1470 segundos de contato com o slurry; 1545 ciclos, 6300 segundos de contato com o slurry; 4635 ciclos, 18900 segundos de contato com o slurry, respectivamente). A cada etapa de abrasão, foi realizada análise em Perfilometria Ótica. Para as analises de permeabilidade e Microscopia Eletrônica de Varredura, foram utilizadas amostras circulares de 6 mm de diâmetro e 1 mm de espessura de dentina obtidas das coroas dentais. Estas foram divididas aleatoriamente nos mesmos grupos já descritos anteriormente, sendo que 120 espécimes foram utilizados para permeabilidade (n=10) e 36 para MEV (n=3). Ambas as análises foram realizadas após imersão no EDTA; após tratamentos para a sensibilidade; pós 1 dia, 7 dias, 30 dias e 90 dias de escovação. Após análise estatística pode-se concluir que, in vivo, todos os tratamentos foram eficazes para a redução da hipersensibilidade dentinária. Ainda que o nível da sensibilidade dos pacientes aumentou numericamente, estes não são considerados estatisticamente diferentes a partir de 12 meses. Portanto, até a avaliação de 18 meses, podemos concluir que não houve um aumento na sensibilidade dentinária desde a sua diminuição pós-tratamento. In vitro, pode-se concluir que todos os tratamentos foram capazes de diminuir a permeabilidade dentinária. O dentifrício Total 12 apresentou-se como o mais abrasivo em comparação com o dentifrício Pro Alivio, pois este último promoveu uma perda de estrutura menor, porém ambos não apresentaram aumento na permeabilidade nos tempos de escovação. As microscopias eletrônicas de varredura mostram a formação da smear layer, obliterando os túbulos para ambos os dentifricios. Como conclusão, pode-se afirmar que todos os agentes dessensibilizantes foram efetivos, mesmo apresentando estratégias de ação diferentes. Os dentifrícios são igualmente interessantes para o uso caseiro por ocasionarem oclusão tubular e a associação de tratamentos (caseiro e de consultório) parece ser uma alternativa eficaz no tratamento da hipersensibilidade dentinária.

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Triscitrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P< 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa ( P< 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol ( P< 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.

Polymer film formulations for the preparation of enteric pharmaceutical capsules

Objectives. Standard pharmaceutical capsules are designed to dissolve in the acidic environment of the stomach releasing the encapsulated contents for absorption. When release is required further along the gastrointestinal tract capsules can be coated with acid insoluble polymers to enable passage through the stomach and dissolution in the intestine. This paper describes formulations that have the potential to be used to produce two-piece hard capsules for post-gastric delivery without the requirement of an exterior coat. Methods. The formulation uses three polysaccharides: sodium alginate, hypromellose and gellan gum to provide acid insolubility and the ability to form capsules using standard industrial equipment. Key findings. The rheological profile, on cooling, of the base material, water content and thickness of the films were shown to be comparable with those of commercial capsules. The capsules remained intact for 2 h in 100 mm HCl at pH 1.2, and within 5 min of being removed from the acid and submerged in phosphate-buffered saline at pH 6.8 were ruptured. Conclusions. Selected formulations from this study have potential for use as delayed release capsules.

Studies on the antiobesity effect of zinc-α2-glycoprotein in the ob/ob mouse

OBJECTIVE: To investigate the mechanism of the lipid depletion by zinc-a(2)-glycoprotein (ZAG). DESIGN: Studies were conducted in the ob/ob mouse, or on isolated adipocytes from these animals or their lean counterparts. RESULTS: Treatment of these animals for 15 days with ZAG (100? µg, intravenously, daily) resulted in a reduction of body weight of 6.55? g compared with phosphate-buffered saline-treated controls, without a change in food or water intake, but with a 0.4?°C rise in rectal temperature. ZAG-treated mice had a 30% reduction in carcass fat mass and a twofold increase in weight of brown adipose tissue. Epididymal adipocytes from ZAG-treated mice showed an increased expression of ZAG and hormone-sensitive lipase (HSL), and this was maintained for a further 3 days in the absence of ZAG. There was an increased lipolytic response to isoproterenol, which was retained for 3 days in vitro in the absence of ZAG. Expression of HSL was also increased in subcutaneous and visceral adipose tissue, as was also adipose triglyceride lipase (ATGL). There was a rapid loss of labelled lipid from epididymal adipose tissue of ZAG-treated mice, but not from the other depots, reflecting the difference in sensitivity to lipolytic stimuli. The increased expression of HSL and ATGL may involve the extracellular signal-regulated kinase (ERK) pathway, as the active (phospho) form was upregulated in all adipose depots after ZAG administration, whereas in vitro studies showed induction of HSL and ATGL by ZAG to be attenuated by PD98059, an inhibitor of the ERK pathway. CONCLUSION: These results suggest that ZAG not only induces direct lipolysis, but also sensitizes adipose tissue to other lipolytic stimuli.

Effect of a tumour-derived lipid-mobilising factor on glucose and lipid metabolism in vivo

Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of cachectic cancer patients, caused a significant increase in glucose oxidation to CO2, compared with control mice receiving phosphate buffered saline. Glucose utilisation by various tissues was determined by the 2-deoxyglucose tracer technique and shown to be elevated in brain, heart, brown adipose tissue and gastrocnemius muscle. The tissue glucose metabolic rate was increased almost three-fold in brain, accounting for the ability of lipid mobilising factor to decrease blood glucose levels. Lipid mobilising factor also increased overall lipid oxidation, as determined by the production of 14CO2 from [14C carboxy] triolein, being 67% greater than phosphate buffered saline controls over a 24 h period. There was a significant increase in [14C] lipid accumulation in plasma, liver and white and brown adipose tissue after administration of lipid mobilising factor. These results suggest that changes in carbohydrate metabolism and loss of adipose tissue, together with an increased whole body fatty acid oxidation in cachectic cancer patients, may arise from tumour production of lipid mobilising factor. © 2002 Cancer Research UK.

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house amino-propyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37°C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P <0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 μm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P <0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications. © VSP 2005.

Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

NEW MICROFLUIDIC SYSTEM TO INCREASE ROBUSTNESS OF ELECTRODE PERFORMANCE AND DEVELOP POINT-OF-CARE HEMATOCRIT DEVICE

The present dissertation aimed to develop a new microfluidic system for a point-of-care hematocrit device. Stabilization of microfluidic systems via surfactant additives and integration of semipermeable SnakeSkin® membranes was investigated. Both methods stabilized the microfluidic systems by controlling electrolysis bubbles. Surfactant additives, Triton X-100 and SDS stabilized promoted faster bubble detachment at electrode surfaces by lowering surface tension and decreased gas bubble formation by increasing gas solubility. The SnakeSkin® membranes blocked bubbles from entering the microchannel and thus less disturbance to the electric field by bubbles occurred in the microchannel. Platinum electrode performance was improved by carbonizing electrode surface using red blood cells. Irreversibly adsorbed RBCs lysed on platinum electrode surfaces and formed porous carbon layers while current response measurements. The formed carbon layers increase the platinum electrode surface area and thus electrode performance was improved by 140 %. The microfluidic system was simplified by employing DC field to use as a platform for a point-of-care hematocrit device. Feasibility of the microfluidic system for hematocrit determination was shown via current response measurements of red blood cell suspensions in phosphate buffered saline and plasma media. The linear trendline of current responses over red blood cell concentration was obtained in both phosphate buffered saline and plasma media. This research suggested that a new and simple microfluidic system could be a promising solution to develop an inexpensive and reliable point-of-care hematocrit device.

A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

Quantification of pyridoxal-5´-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was > 92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

In Vitro Degradation of Poly(caprolactone)/nHA Composites

The degradation behavior and mechanical properties of polycaprolactone/nanohydroxyapatite composite scaffolds are studied in phosphate buffered solution (PBS), at 37 degrees C, over 16 weeks. Under scanning electron microscopy (SEM), it was observed that the longer the porous scaffolds remained in the PBS, the more significant the thickening of the pore walls of the scaffold morphology was. A decrease in the compressive properties, such as the modulus and the strength of the PCL/nHA composite scaffolds, was observed as the degradation experiment progressed. Samples with high nHA concentrations degraded more significantly in comparison to those with a lower content. Pure PCL retained its mechanical properties comparatively well in the study over the period of degradation. After the twelfth week, the results obtained by GPC analysis indicated a significant reduction in their molecular weight. The addition of nHA particles to the scaffolds accelerated the weight loss of the composites and increased their capacity to absorb water during the initial degradation process. The addition of these particles also affected the degradation behavior of the composite scaffolds, although they were not effective at compensating the decrease in pH prompted by the degradation products of the PCL.

Degradation of poly-L-lactide. Part2 : increased temperature accelerated degradation

Poly-L-lactide (PLLA) is one of the most significant members of a group of polymers regarded as bioresorbable. The degradation of PLLA proceeds through hydrolysis of the ester linkages in the polymer's backbone; however, the time for the complete resorption of orthopaedic devices manufactured from PLLA is known to be in excess of five years in a normal physiological environment. To evaluate the degradation of PLLA in an accelerated time period, PLLA pellets were processed by compression moulding into tensile test specimens, prior to being sterilized by ethylene oxide gas (EtO) and degraded in a phosphate-buffered solution (PBS) at both 50°C and 70°C. On retrieval, at predetermined time intervals, procedures were used to evaluate the material's molecular weight, crystallinity, mechanical strength, and thermal properties. The results from this study suggest that at both 50°C and 70°C, degradation proceeds by a very similar mechanism to that observed at 37°C in vitro and in vivo. The degradation models developed also confirmed the dependence of mass loss, melting temperature, and glass transition temperature (Tg) on the polymer's molecular weight throughout degradation. Although increased temperature appears to be a suitable method for accelerating the degradation of PLLA, relative to its physiological degradation rate, concerns still remain over the validity of testing above the polymer's Tg and the significance of autocatalysis at increased temperatures.

Toxoplasma gondii: Evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n = 11) received 12.5 mu g of each recombinant protein plus 0.5 mu g of cholera toxin, group 2 (G2, n = 11) received phosphate buffer saline (PBS) plus 0.5 mu g of cholera toxin, and group 3 (G3, n = 11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRAT Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P < 0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P > 0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T gondii. (c) 2007 Elsevier B.V. All rights reserved.