Clinical Evaluation of the NS1 Antigen-Capture ELISA for Early Diagnosis of Dengue Virus Infection in Brazil

The fact that the diagnosis of infection with dengue virus is usually made by detecting IgM antibodies during the convalescent phase of the disease interferes with disease management and, consequently, with reducing mortality rates. This study evaluated the sensitivity and specificity of detection of NS1 in samples of patients suspected of acute dengue virus infection in Brazil. The results were used to institute treatment and the sensitivity and specificity of detection of NS1 were compared to the results of detection of IgM, virus isolation, and RT-PCR. Detection of NS1 yielded better results than RTPCR and virus isolation. When considering IgM detection and RT-PCR positive results as ""gold standards,"" the sensitivity and specificity of the NS1 assay were 95.9% and 81.1%, respectively. All patients enrolled in the study were treated promptly and had an uneventful course of the disease. The detection of NS1 provided better results than the diagnostic techniques used currently during the acute phase of disease (RT-PCR and virus isolation). Detection of NS1 is an important tool for the diagnosis of acute dengue infection, particularly in highly endemic areas, allowing for rapid treatment of patients and reduction of disease burden. J. Med. Virol. 82: 1400-1405, 2010. (C) 2010 Wiley-Liss, Inc.

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

Targeting endothelin ET(A) and ET(B) receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

CXCR2-specific chemokines mediate leukotriene B-4-dependent recruitment of neutrophils to inflamed joints in mice with antigen-induced arthritis

Objective. To investigate the mechanism underlying neutrophil migration into the articular cavity in experimental arthritis and, by extension, human-inflammatory synovitis. Methods. Antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Migration assays and histologic analysis were used to evaluate neutrophil recruitment to knee joints. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay. Antibodies and pharmacologic inhibitors were used in vivo to determine the role of specific disease mediators. Samples of synovial tissue and synovial fluid from rheumatoid arthritis (RA) or osteoarthritis patients were evaluated for CXCL1 and CXCL5 expression. Results. High levels of CXCL1, CXCL5, and leukotriene B-4 (LTB4) were expressed in the joints of arthritic mice. Confirming their respective functional roles, repertaxin (a CXCR1/CXCR2 receptor antagonist), anti-CXCL1 antibody, anti-CXCL5 antibody, and MK886 (a leukotriene synthesis inhibitor) reduced mBSA-induced neutrophil migration to knee joints. Repertaxin reduced LTB4 production in joint tissue, and neutrophil recruitment induced by CXCL1 or CXCL5 was inhibited by MK886, suggesting a sequential mechanism. Levels of both CXCL1 and CXCL5 were elevated in synovial fluid and were released in vitro by RA synovial tissues. Moreover, RA synovial fluid neutrophils stimulated with CXCL1 or CXCL5 released significant amounts of LTB4. Conclusion. Our data implicate CXCL1, CXCL5, and LTB4, acting sequentially, in neutrophil migration in AIA. Elevated levels of CXCL1 and CXCL5 in the synovial compartment of RA patients provide robust comparative data indicating that this mechanism plays a role in inflammatory joint disease. Together, these results suggest that inhibition of. CXCL1, CXCL5, or LTB4 may represent a potential therapeutic strategy in RA.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Chemokines and chemokine receptors coordinate the inflammatory immune response in human cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) includes different clinical manifestations displaying diverse intensities of dermal Inflammatory infiltrate Diffuse CL (DCL) cases are hyporesponsive and lesions show very few lymphocytes and a predominance of macrophages In contrast localized CL (LCL) cases are responsive to leishmanial antigen and lesions exhibit granulocytes and mononuclear cell infiltration in the early phases changing to a pattern with numerous lymphocytes and macrophages later in the lesion Therefore different chemokines may affect the predominance of cell infiltration in distinct clinical manifestations In lesions from LCL patients we examined by flow cytometry the presence of different chemokines and their receptors in T cells and we verified a higher expression of CXCR3 in the early stages of LCL (less than 30 days of infection) and a higher expression of CCR4 in the late stages of disease (more than 60 days of infection) We also observed a higher frequency of T cells producing IL-10 in the late stage of LCL Using immunohistochemistry we observed a higher expression of CCL7 CCL17 in lesions from late LCL as well as CCR4 suggesting a preferential recruitment of regulatory T cells in the late LCL Comparing lesions from LCL and DCL patients we observed a higher frequency of CCL7 in DCL lesions These results point out the Importance of the chemokines defining the different types of cells recruited to the site of the infection which could be related to the outcome of infection as well as the clinical form observed (C) 2010 American Society for Histocompatibility and Immunogenetics Published by Elsevier Inc All rights reserved

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [plate I et-derived growth factor-BB, transforming growth factor (TGF)-beta 1, TGF-beta 2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.

Patterns of Antibodies Against Latent and Lytic Antigens of Human Herpesvirus 8 in an Endemic Population and Patients With Kaposi`s Sarcoma in Mozambique

The patterns of antibodies against latent and lytic antigens of human herpesvirus 8 (HHV-8) were assessed using immunofluorescence assays of samples from 155 persons seropositive for HHV-8 seen at public health centers and 24 patients with Kaposi`s sarcoma (KS) from Mozambique. Of the 155 persons without KS, 48(31%) had antibodies against latent antigens only, 29 (18.7%) had antibodies against lytic antigens only, and 78 (50.3%) had antibodies against both types of antigen. The HHV-8 antibody titer tended to increase with age until age 40, after which it began to decrease. High titers of antibodies against latent and lytic antigens of HHV-8 were detected mostly in persons co-infected with HIV, and these increased titers could have a predictive value. All patients with KS except four patients who were seronegative for HHV-8 had elevated titers of HHV-8 antibodies, predominantly against latent antigens. The data suggest the potential for an increase in the development of KS in this endemic area for HHV-8. J. Med. Virol. 82:1576-1581, 2010. (C) 2010 Wiley-Liss, Inc.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Anti-inflammatory effect of parenteral fish oil lipid emulsion on human activated mononuclear leukocytes

Background & aim: To compare the effect of fish oil-based (FO) lipid emulsions (LE) for parenteral administration with standard LE and a new FO containing LE composed of four different oils on the antigen presentation and inflammatory variables. Methods: Phytohemagglutinin (PHA) activated human mononuclear leukocytes were cultured with different LE - Control: without LE; SO: soybean oil; SO/FO: soybean and FO (4:1); MCT/SO: medium chain triglycerides and SO (1:1); MCT/SO/FO: MCT/SO and FO (4:1) and SMOF: a new LE containing FO. Cytokine production was evaluated by ELISA, the expression of antigen-presenting and co-stimulatory surface molecules were analyzed by flow cytometry and lymphocyte proliferation was assessed by H(3)-Thymidine incorporation, after tetanus toxoid-induced activation. Results: All LE decreased the HLA-DR and increased CD28 and CD152 expression on monocytes/macrophages and lymphocytes surface (p < 0.05). SO/FO and MCT/SO/FO decreased lymphocyte proliferation (p<0.05). All LE decreased IL-2 product ion, but this effect was enhanced with MCT/SO/FO and SMOF (p < 0.05). MCT/SOTO decreased IL-6 and increased IL-10, whereas SO had the opposite effect (p < 0.05). Conclusion: FO LE inhibited lymphocyte proliferation and had an anti-inflammatory effect. These effects seem to be enhanced when FO is mixed with MCT/SO. SMOF had a neutral impact on lymphocyte proliferation and IL-6 and IL-10 production.

Human monocytes but not dendritic cells are killed by blocking of autocrine cyclooxygenase activity

Dendritic cells (DCs), in peripheral tissues, derive mostly from blood precursors that differentiate into DCs under the influence of the local microenvironment. Monocytes constitute the main known DC precursors in blood and their infiltration into tissues is up-regulated during inflammation. During this process, the local production of mediators, like prostaglandins (PGs), influence significantly DC differentiation and function. In the present paper we show that treatment of blood adherent mononuclear cells with 10 mu M indomethacin, a dose achieved in human therapeutic settings, causes monocytes` progressive death but does not affect DCs viability or cell surface phenotype. This resistance of DCs was observed both for cells differentiated in vitro from blood monocytes and for a population with DCs characteristics already present in blood. This phenomenon could affect the local balance of antigen-presenting cells, influence the induction and pattern of immune responses developed under the treatment with non-steroidal anti-inflammatory drugs and, therefore, deserves further investigation. (C) 2009 Elsevier Inc. All rights reserved.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Human toxocariasis in rural Brazilian Amazonia: Seroprevalence, risk factors, and spatial distribution

This population-based cross-sectional study of 403 rural settlers in Brazilian Amazonia revealed an overall rate of IgG seropositivity to Toxocara canis excretory-secretory larval antigen of 26.8% (95% confidence interval [CI], 22.5-31.4%). Multilevel logistic regression analysis identified current infection with hookworm (odds ratio [OR], 2.32; 95% CI, 1.11-4.86) and residence in the most recently occupied sectors of the settlement (OR, 1.81.; 95%CI, 1.3-2.52) as significant risk factors for Toxocara seropositivity; age > 14 years (OR, 0.46; 95% CI, 0.28-0.73) and the presence of cats in the household (OR, 0.57; 95% CI, 0.32-1.02) appeared to be protective. Two significant high-prevalence clusters were detected in the area, together comprising 38.9% of the seropositive subjects; households in the clusters had slightly lower socioeconomic status and were less likely to have cats as pets. The obstacles for controlling human toxocariasis in this and other tropical rural settings are discussed.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

Quality evaluation of DNA obtained from stored human saliva and its applicability to identification in Forensic Dentistry

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.