Peritumoural, but not intratumoural, lymphatic vessel density and invasion correlate with colorectal carcinoma poor-outcome markers

To evaluate whether lymphatic vessel density (LVD) and lymphatic vessel invasion (LVI) are useful markers of worse outcome in colorectal carcinoma and if LVD and LVI correlate to the classical clinical-pathological parameters, we analysed 120 cases of colorectal carcinomas selected from the files of Division of Pathology, Hospital das Clinicas, Sao Paulo University, Brazil. Assessment of LVD and LVI was performed by immunohistochemical detection of lymphatic vessels, using the monoclonal antibody D2-40. Higher LVD was found in the intratumoural area, when comparing with normal and peritumoural areas (p < 0.001). However, peritumoural LVD, but not intratumoural, correlated with both colonic-wall-invasion depth (p=0.037) and liver metastasis (p=0.012). Remarkably, LVI was found associated with local invasion (p=0.016), nodal metastasis (p=0.022) and hepatic metastasis (p < 0.001). Peritumoural LVD and LVI are directly related to histopathological variables indicative of poor outcome such as lymph-node status and liver metastasis.

Application of Real-Time PCR Stool Assay for Helicobacter pylori Detection and Clarithromycin Susceptibility Testing in Brazilian Children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real-time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real-time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin-resistant strains is suspected.

Factor XIIIa plus Dermal Dendrocyte Parasitism in American Tegumentary Leishmaniasis Skin Lesions

Dendritic cells belong to a family of antigen-presenting cells that are localized at the entry sites, such as skin and mucosa. Dendritic cells are related to immune surveillance function. The role of Langerhans cells in the pathogenesis of skin infectious diseases is well studied; however, there are few articles addressing involvement of factor XIIIa-positive dermal dendrocytes (FXIIIa+ DD) in such processes. FXIIIa+ DDs are bone marrow-monocytic lineage-derived cells and members of the skin immune system. Due to their immune phenotype and functional characteristics, they are considered complementary cells to Langerhans cells in the process of antigen presentation and inducing immune response. To verify the interaction between FXIIIa+ DD and Leishmania amastigotes, 22 biopsies of American tegumentary leishmaniasis (ATL) skin lesions were subjected to double staining technique with anti-factor XIIIa and anti-Leishmania antibodies. FXIIIa+ DDs were hypertrophic and abundant in the cutaneous reaction of ATL. FXIIIa+ DDs harboring parasites were observed in I I of 22 skin biopsies. The data obtained suggest that FXIIIa+ DD plays a role in the pathogenesis of ATL skin lesion as host cell, immune effector, and/or antigen-presenting cell.

Lymphatic Vessel Density and VEGF-C Expression are Significantly Different Among Benign and Malignant Thyroid Lesions

Thyroid cancer is the most frequent endocrine neoplasia worldwide. The route for metastasis and loco-regional invasion preferentially occurs by lymphatic vessels. For this reason, the assessment of lymphatic vessel density (LVD) is supposed to represent both a prognostic parameter and also a potential therapeutic target. In order to evaluate the value of LVD in benign and malignant thyroid lesions, we analyzed 110 thyroidectomy specimens using D2-40, a specific marker for lymphatic vessels and vascular endothelial growth factor C (VEGF-C), the most potent molecule of lymphatic proliferation. LVD was significantly different between papillary and follicular carcinomas in total (p = 0.045) and peritumoral area (p = 0.042). Follicular adenoma and follicular carcinoma showed an important difference of intra- (p = 0.019) and peritumoral (p = 0.033) LVD. VEGF-C was more markedly expressed in malignancies than in benignant lesions (p = 0.0001). Almost all cancers with high positive VEGF-C expression also exhibited increased peritumoral LVD (p = 0.049) when compared with the benignant lesions. Indeed, the high peritumoral LVD of malignant thyroid lesions is an important finding for surgery planning and supports the practice of total thyroidectomy in malignant thyroid neoplasm`s since the lymphatic peritumoral vessels definitely are an escape path for tumor cells.

Comparative Study of the Standard Fluorescent Antibody to Membrane Antigen (FAMA) Assay and a Flow Cytometry-Adapted FAMA Assay To Assess Immunity to Varicella-Zoster Virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.

Trans-resveratrol inhibits early blood vessel formation (Vasculogenesis) without impairment of embryonic growth

Resveratrol is a stilbene compound found in grapes and other sources. In this study we examined the effects of trans-resveratrol (4.38-438 mu M/implant) in the vasculogenesis of yolk-sac membranes and its capacity to improve chick embryo growth. High concentrations of the stilbene (43.8-438 mu M) significantly inhibited early vessel formation, decreasing the percentage vitelline vessels of 3.5-day embryos by 50% compared to the control. In addition, basic fibroblast growth factor-stimulated vasculogenesis (140% of vessels as compared to control) was partially reversed by t-resveratrol (35% of inhibition) and treatments with cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) as well a protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate) decreased the vessel number to 60%, 50%, and 44%, respectively. Treatments with t-resveratrol (4.38-43.8 mu M/implant) significantly increased the body length of embryos incubated in vitro uncoupled from any impairment in the body shape or detectable embryotoxic effect. We suggest that the antivasculogenic activity and the enhancement in embryonic growth promoted by non acute treatments with t-resveratrol were, at least in part, due to PKC inhibition. We suggest that t-resveratrol can be usable not only as a reliable functional nutriment, but also is useful for the development of prophylactic and/or therapeutic agents for treatment of angiogenic-degenerative diseases.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

Enhancement of diagnostic efficiency by a gamma interferon release assay for pulmonary tuberculosis

This study was designed to examine the use of the QuantiFERON-TB Gold assay as an aid in the diagnosis of active pulmonary tuberculosis (TB) in Brazilian patients. Using the receiver operating characteristic curve, the cutoff was adjusted to >= 0.20 IU/ml. The sensitivity increased to 86%, with 100% specificity. All TB patients with negative sputum smear microscopy and negative culture results were positive using this test.

Increased Vascular Permeability, Angiogenesis and Wound Healing Induced by the Serum of Natural Latex of the Rubber Tree Hevea brasiliensis

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cytokines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Mile`s method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its effects on the wound-healing process was determined by the rabbit ear dermal ulcer model. The serum fraction showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with proteases lost these activities. These results are in accordance with the enhancement of wound healing observed in clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright (C) 2009 John Wiley & Sons, Ltd.

Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Araraquara Virus Recombinant Nucleocapsid Protein

Laboratory diagnosis of hantavirus cardiopulmonary syndrome (HCPS) in Brazil has been performed mostly by a detection of IgM antibodies to recombinant antigen purified from Sin Nombre virus and Andes Virus (ANDV). Recently, a recombinant nucleocapsid (rN) protein of Argentina virus (ARAV), a Brazilian hantavirus, was Obtained in Escherichia coli. To evaluate ARAV rN as antigen for antibody detection, serum samples from 30 patients front Argentina seropositive for hantavirus were tested. All samples were positive for IgG and IgM by enzyme-linked immunosorbent assay (ELISA) using either ARAV rN or ANDV rN antigens. In Brazil, six of 00 serum samples from patients With suspected HCPS (10%) were positive for IgM by ELISA Using ARAV rN antigen and 7 were positive Using ANDV rN antigen. For results obtained with 90 serum samples analyzed by IgM ELISA with ANDV rN antigen, the sensitivity of the IgM ELISA using ARAV rN antigen was 97.2%,, the specificity was 100%, the positive predictive value was 100% and the negative predictive value was 98.1%. The results show that ARAV rN is a Suitable antigen for diagnosis Of hantavirus infection in Brazil and Argentina.

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.

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.

MHM assay: molecular sexing based on the sex-specific methylation pattern of the MHM region in chickens

Bird sex determination using molecular methods has proved to be a valuable tool in different studies. Although it is possible to sex most birds by coupling the CHD assay with others available methods, no sex-determining gene like SRY in mammalians has been identified in birds. The male hypermethylated (MHM) region on the Z chromosome has been found to be hypermethylated in males and hypomethylated in females in birds of the order Galliformes. We analyzed the DNA from feathers of 50 adult chickens to verify the methylation pattern of the MHM region by PCR and the restriction enzyme HpaII (a method named MHM assay). The results, visualized in agarose gel, were compared with PCR amplification of the CHD-Z and CHD-W genes (polyacrylamide gel) and with the birds` phenotype. All males (25) showed hypermethylation of the MHM region, and all females (25) showed hypomethylation. The sexing by MHM assay was in according with phenotype and CHD sexing. To our knowledge, this is the first study that uses the MHM region for sexing birds. Although the real role of the MHM region in the sex determination is still unclear, this could be a universal marker for sexing birds and may be involved in sex determination by its influence on transcriptional processes. The MHM assay could be a good alternative for CHD assay in developmental studies.

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.