A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: Comparison with the in vivo Draize rabbit test

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (131); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

eNOS haplotype associated with hypertension in obese children and adolescents

Objective: The aim of our study is to investigate whether genetic polymorphisms in the endothelial nitric oxide synthase (eNOS) gene (in the promoter region T(-786)C, in exon 7 (Glu298Asp) and in intron 4 (4b/4a)) or eNOS haplotypes are associated with hypertension in obese children and adolescents. Methods: We genotyped 175 healthy (controls), 110 normotensive obese and 73 hypertensive obese children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR, and by PCR followed by fragment separation by electrophoresis. We compared the distribution of eNOS genotypes, alleles and haplotypes in the three study groups of subjects. We have also measured whole-blood nitrite concentrations. Results: The 4a4a genotype for the intron 4 polymorphism was more common in normotensive obese and hypertensive obese (P < 0.01). The AspAsp genotype for Glu298Asp polymorphism was less common in normotensive obese (P < 0.02). No significant differences were found in allele distributions for the three eNOS polymorphisms. However, the haplotype combining the C, 4b and Glu variants for the three polymorphisms was more common in hypertensive obese than in normotensive obese or control children and adolescents (odds ratio = 2.28 and 2.79, respectively; 95% confidence interval: 1.31-4.31 and 1.39-5.64, respectively; both P < 0.00625). This haplotype was not associated with significantly different nitrite concentrations (P > 0.05). Conclusions: Our findings suggest that the eNOS haplotype, C b Glu, is associated with hypertension in obese children and adolescents. Further studies examining the possible interactions of eNOS haplotypes with environmental factors and other genetic markers involved in the development of obesity and its complications are warranted. International Journal of Obesity (2011) 35, 387-392; doi:10.1038/ijo.2010.146; published online 27 July 2010

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 degrees C, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.

Chemokines expression during Leptospira interrogans serovar Copenhageni infection in resistant BALB/c and susceptible C3H/HeJ mice

The role of innate immune responses in protection against leptospirosis remains unclear. We examined the expression of the chemokines CCL2/JE (MCP-1), CCL3/MIP-1 alpha (MIP-1 alpha) and CXCL1/KC (IL-8) regarding resistance and susceptibility to leptospirosis in experimental mice models BALB/c and C3H/HeJ, respectively. A virulent strain of Leptospira interrogans serovar Copenhageni was used in this study. Twenty-five animals of each mouse strain of C3H/HeJ and BALB/c, were infected intraperitoneally with 106 cells. Five un-infected animals of each strain were kept as control. Mortality of C3H/HeJ mouse was observed while BALB/c mice were asymptomatic. The presence of leptospire DNA in tissues of infected animals was demonstrated by PCR. Chemokines were measured in serum, spleen, liver, kidney and lung of both strains of animals using immunoenzymatic assay (ELISA). Elevations in the levels of chemokines MCP-1 and IL-8 occurred in all organs and sera of C3H/HeJ and BALB/c infected mice. The levels of MIP-1 alpha were lower when compared to MCP-1 and IL-8 in all analyzed organs, with a slight increase in liver and kidney. Our results indicate that the expression of inflammatory mediators can vary greatly, depending on the tissue and mouse strains. It is possible that the resistance to Leptospira can be partially correlated to the increase of MIP-1 alpha observed in BALB/c mice, while an increasing and a sustained expression of MCP-1 and IL-8 in the lungs of C3H/HeJ mice can be correlated to the severity and progression of leptospirosis. (C) 2009 Elsevier Ltd. All rights reserved.

Experimental infection of capybaras Hydrochoerus hydrochaeris by Rickettsia rickettsii and evaluation of the transmission of the infection to ticks Amblyomma cajennense

The present study evaluated the infection of capybaras (Hydrochoerus hydrochaeris) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Two groups of two capybaras each were evaluated: on day 0, group 1 (G1) was infested by R. rickettsii-infected ticks, and group 2 (G2) was inoculated intraperitoneally with R. rickettsii. Two additional groups were control groups, not exposed to R. rickettsii, being CG1 group the control of G1, and CG2 group the control of G2. Capybara rectal temperature was measured daily. Blood samples were collected every 3 days during 30 days, and used to (i) inoculate guinea pigs intraperitoneally; (ii) DNA extraction followed by real-time PCR targeting the rickettsial gene gltA; (iii) hematology; (iv) detection of R. rickettsii-reactive antibodies by indirect immunofluorescence assay (IFA). Blood was also collected from G I capybaras every approximate to 10-30 days till the 146th day, to be tested by serology. Capybaras were infested by uninfected A. cajennense nymphs from the 3rd to the 18th day. Engorged nymphs were collected, allowed to molt to adults in an incubator. Thereafter, the subsequent flat ticks were tested by PCR. All G1 and G2 capybaras became infected by R. rickettsii, as demonstrated by guinea pig inoculation and seroconversion, but they showed no fever. Rickettsemia was continually detected from the 6th (G2 capybaras) or 9th (G1 capybaras) to the 18th day post inoculation or infestation with R. rickettsii-infected ticks. A total of 20-25% and 30-35% of the flat ticks previously fed on G1 and G2 capybaras, respectively, became infected by R. rickettsii. The study demonstrated that R. rickettsii was capable to infect capybaras without causing clinical illness, inducing rickettsemia capable to cause infection in guinea pigs and ticks. Our results indicate that capybaras act as amplifier host of R. rickettsii for A. cajennense ticks in Brazil. (c) 2008 Elsevier B.V. All rights reserved.

Use of the real time RT-PCR for immune related gene expression quantitation in experimentally infected Neospora caninum bovine calves

Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-gamma and TNF-alpha genes showed increased expression in some samples. iNOS and TGF-beta 1 genes had some non-significant variations and IL-4 and IL-10 stayed pratically inaltered.

Differential diagnosis of oocysts of Hammondia-like organisms of dogs and cats by PCR-RFLP analysis of 70-kilodalton heat shock protein (HSP70) gene

Organisms of the genera Toxoplasma, Hammondia and Neospora, the Hammondia-like organisms, are closely related coccidian with similarly sized oocysts. Therefore, a diagnosis based on microscopy of oocysts in feces is not a method of choice for species identification of these important parasites. In this paper, we present a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method to differentially diagnose oocysts of Toxoplasma gondii from oocyst of Hammondia hammondi. Another PCR-RFLP was designed to differentiate oocysts of Hammondia heydorni from oocysts of Neospora spp. Both PCR-RFLP are based on nucleotide sequences of the Hsp70 coding gene. In conclusion, we presented two alternative molecular diagnostic assays that can be successfully applied for the differentiation of oocysts of Hammondia-like organisms shed by felids and canids.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G I (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r = 0.62, P = 0.05), MAT(s) x MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r = 0. 14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. (c) 2007 Elsevier Ltd. All rights reserved.

Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii from Rhipicephalus sanguineus ticks

The present study was performed in an area endemic for Brazilian spotted fever (BSF) in Juiz de Fora, state of Minas Gerais, Brazil, during the years 2007 and 2008, when fatal cases of BSF (caused by Rickettsia rickettsii) were reported. Adult ticks (Acari: Ixodidae) identified as Rhipicephalus sanguineus (Latreille) and Amblyomma cajennense (Fabricius) were collected from dogs and horses, respectively, and tested by polymerase chain reaction (PCR). Overall, 13.1% of the Rh. sanguineus ticks and none of the A. cajennense were found to be infected with R. rickettsii. Two isolates of R. rickettsii were successfully established in Vero cell culture from two Rh. sanguineus ticks. An indirect immunofluorescence assay (IFA) using R. rickettsii antigens detected blood serological reaction to R. rickettsii in 67.9% (53/78) of dogs and 41.0% (16/39) of horses living in the study area. Larval offspring from two Rh. sanguineus engorged females, naturally infected by R. rickettsii, were reared to adult stage in the laboratory. All active stages (larvae, nymphs, adults) remained 100% infected by R. rickettsii, which was efficiently transmitted to naive rabbits. Overall, the results of the present study indicate a potential risk for transmission of R. rickettsii to humans by Rh. sanguineus, an occurrence yet to be documented in Brazil.

Rickettsial infection in domestic mammals and their ectoparasites in El Valle de Anton, Cocle, Panama

The present research evaluated the presence of Rickettsia spp. on ectoparasites of horses and dogs (using PCR techniques), and their sera (using immunofluorescence assay) in El Valle de Anton town in Panama. A total of 20 horses and 20 dogs were sampled, finding four species of ectoparasites on dogs (the ticks Rhipicephalus sanguineus, Amblyomma ovale, Amblyomma oblongoguttatum, and the flea Ctenocephalides felis), and two tick species on horses (Amblyomma cajennense and Dermacentor nitens). DNA of Rickettsia amblyommii was found in pools of A. cajennense, D. nitens, and R. sanguineus, while Rickettsia fells was detected in C. felis pools. Overall, 70% (14/20) and 65% (13/20) of the horses and dogs, respectively, were seroreactive (titer >= 64) to spotted fever group rickettsiae. Sera from six dogs and five horses reacted to R. amblyommii antigens with titers at least four-fold higher than those for the other antigens tested (Rickettsia bellii, Rickettsia parked, Rickettsia rhipicephali, R. felis, and R. rickettsii). These serological results, coupled with our molecular findings, suggest that these dogs and horses were infected by Rickettsia amblyommii. More studies need to be realized afford to identify the Rickettsia species responsible for other serological and molecular positive results, and their ecological importance. (C) 2010 Elsevier B.V. All rights reserved.

A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay

At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme Alwl. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RI-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RI-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. (C) 2010 Elsevier B.V. All rights reserved.

Evaluation of DNA damage by the alkaline comet assay of the olfactory and respiratory epithelia of dogs from the city of Sao Paulo, Brazil

Animals kept as pets may be considered sentinels for environmental factors to which humans could be exposed. Olfactory and respiratory epithelia are directly subjected to airborne factors, which could cause DNA lesions, and the alkaline comet assay is considered a reliable tool for the assessment of DNA damage. The objective of this work is to evaluate the extent of DNA damage by the comet assay of the olfactory and respiratory epithelia of dogs from different regions of the city of sao Paulo, Brazil. Thirty-three clinically healthy dogs, aged 5 years or more, were used in the study, with 7 from the North region of Sao Paulo, 7 from the South region, 3 dogs from the East region, and 16 dogs from the West city region. Three dogs younger than 6 months were used as controls. DNA damage was analyzed by the alkaline comet assay. We observed no difference in histopathological analysis of olfactory and respiratory epithelia between dogs from different regions of Sao Paulo. Dogs older than 5 years presented significantly higher comet length in both olfactory and respiratory epithelia, when compared with controls, indicating DNA damage. When separated by regions, olfactory and respiratory epithelia presented similar DNA damage in dogs from different regions of Sao Paulo, corroborating with similar levels of particulate matter index (PM10) in all regions of the city. In this study, we report for the first time that the comet assay can be used to quantify the extent of DNA damage in dog olfactory and respiratory epithelia, and that comet length (DNA damage) increases with age, probably due to environmental factors. Air pollution, as measured by PM 10, can be responsible for this DNA damage. (C) 2009 Elsevier GmbH. All rights reserved.