Remoção da biomassa algal e determinação da concentração de microcistina pelo Método ELISA em ensaios de coagulação, sedimentação, filtração e adsorção

Nessa pesquisa são relatados os resultados da determinação das concentrações de microcistina e de biomassa algal após as várias etapas de tratamento de amostras de água coletadas junto ao reservatório de Barra Bonita-SP visando obtenção de água potável. O tratamento foi realizado em escala de laboratório com e sem aplicação de carvão ativado em pó (CAP) e as etapas foram: coagulação com aplicação de cloreto férrico, sedimentação, filtração em papel de filtro. Foi possível observar que a pré-clarificação desse tipo de água por coagulação seguida de sedimentação requereu dosagens relativamente elevadas de cloreto férrico (80 mg/L), tendo sido verificada eficiência muito baixa de remoção de microcistina nas etapas de tratamento por sedimentação seguida de filtração, quando não foi aplicado CAP. Apenas com a aplicação de CAP a microcistina foi reduzida à níveis que atendessem os padrões de potabilidade previstos na Portaria 518/04 (concentração menor que 1 μg/L). A determinação de microcistina pelo método que utiliza Imunoadsorventes Ligados à Enzima (ELISA) mostrou-se uma ferramenta útil e confiável para detectar e quantificar essa toxina, embora ainda apresente custo relativamente elevado.

Clinical pathway intervention compliance and effectiveness when used in the treatment of patients with severe sepsis and septic shock at an Intensive Care Unit in Spain

The purpose of this quasi-experimental study was to assess levels of compliance with the intervention bundles contained in a clinical pathway used in the treatment of patients with severe sepsis and septic shock, and to analyze the pathway’s impact on survival and duration of hospital stays. We used data on 125 patients in an Intensive Care Unit, divided into a control group (N=84) and an intervention group (N=41). Levels of compliance increased from 13.1% to 29.3% in 5 resuscitation bundle interventions and from 14.3% to 22% in 3 monitoring bundle interventions. In-hospital mortality at 28 days decreased by 11.2% and the duration of hospital stay was reduced by 5 days. Although compliance was low, the intervention enhanced adherence to the instructions given in the clinical pathway and we observed a decline in mortality at 28 days and shorter hospital stays.

Short Peptide Vaccine Induces CD4+ T Helper Cells in Patients with Different Solid Cancers

Previous cancer vaccination trials often aimed to activate CD8(+) cytotoxic T-cell (CTL) responses with short (8-10mer) peptides and targeted CD4(+) helper T cells (TH) with HLA class II-binding longer peptides (12-16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8(+) T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4(+) TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4- and HLA-DR-restricted TH1 cells. Some short peptide-reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4(+) T-cell repertoire in many patients, facilitating a strong combined CD4(+)/CD8(+) T-cell response. Cancer Immunol Res; 4(1); 18-25. ©2015 AACR.

Performance of commercial enzyme-linked immunosorbent assays (ELISA) for diagnosis of HSV-1 and HSV-2 infection in a clinical setting

Thesis (Master's)--University of Washington, 2016-06

Serum antibodies to oral anaerobic bacteria in patients with rheumatoid arthritis

BACKGROUND: This study was conducted to determine the component that causes the disease in rheumatoid arthritis (RA), which shows great resemblance to periodontitis in a pathologic context. MATERIALS AND METHODS: Within this study, the pathogen-specific IgG levels formed against Porphyromonas gingivalis FDC 381, Prevotella melaninogenica ATCC 25845, Actinobacillus actinomycetemcomitans Y4, Bacteroides forsythus ATCC 43047, and Prevotella intermedia 25611 oral bacteria were researched from the blood serum samples of 30 RA patients and 20 healthy controls with the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The IgG levels of P gingivalis, P intermedia, P melaninogenica, and B forsythus were found to be significantly higher in RA patients when compared with those of the controls. Of the other bacteria antibodies, A actinomycetemcomitans was not found at greater levels in RA serum samples in comparison with the healthy samples. CONCLUSION: The antibodies formed against P gingivalis, P intermedia, P melaninogenica, and B forsythus could be important to the etiopathogenesis of RA.

Semi-global techniques in image matching and change detection with applications to civil and environmental engineering.

This work of thesis wants to present a dissertation of the wide range of modern dense matching algorithms, which are spreading in different application and research fields, with a particular attention to the innovative “Semi-Global” matching techniques. The choice of develop a semi-global numerical code was justified by the need of getting insight on the variables and strategies that affect the algorithm performances with the primary objective of maximizing the method accuracy and efficiency, and the results level of completeness. The dissertation will consist in the metrological characterization of the proprietary implementation of the semi-global matching algorithm, evaluating the influence of several matching variables and functions implemented in the process and comparing the accuracy and completeness of different results (digital surface models, disparity maps and 2D displacement fields) obtained using our code and other commercial and open-source matching programs in a wide variety of application fields.

Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies

Antibodies reactive with native double stranded DNA are characteristic of the chronic inflammatory disease systemic lupus erythematosus. Native DNA is however, a poor immunogen and the mechanism of anti-DNA antibody production is incompletely understood. Modification of DNA can increase its immunogenicity and in inflammatory disease states reactive oxygen species produced from phagocytic cells have been shown to thus modify DNA. In this study, monoclonal antibodies produced spontaneously by two mice strains with lupus-like disease were used in a competition ELISA to monitor changes to DNA induced by reactive oxygen species. Different procedures for reactive oxygen species generation were found to cause distinct and characteristic changes to DNA involving modifications of base residues, the sugar-phosphate backbone and the gross conformational structure of double-stranded DNA. In view of this, it may be possible to use these antibodies further to probe DNA and infer the source and nature of the reactive oxygen species it has been exposed to, particularly in vivo.

Oxygen free radicals denature human IgG and increase its reactivity with rheumatoid factor antibody

Rheumatoid inflammation is characterised by the production of rheumatoid factor antibodies directed against denatured IgG. Oxygen free radicals have the potential to denature all manner of proteins and can be generated by activated phagocytic cells in the inflamed joint. By modifying routine ELISA and nephelometric procedures for measuring rheumatoid factor, (i.e. substituting free radical altered IgG for rabbit and heat aggregated IgG as antigens) we have observed that oxygen radicals, generated by (1) UV light and (2) PMA-activated neutrophils, give rise to monomeric and polymeric forms of IgG which have increased reactivity towards IgM and IgA polyclonal rheumatoid factor antibodies. We conclude that free radical alteration of IgG may be a stimulus to the formation of immune complexes with rheumatoid factor antibody, thereby promoting and amplifying tissue damage during rheumatoid inflammation.

Validation of a novel ELISA for measurement of MDA-LDL in human plasma

The involvement of oxidatively modified low density lipoprotein (LDL) in the development of CHD is widely described. We have produced two antibodies, recognizing the lipid oxidation product malondialdehyde (MDA) on whole LDL or ApoB-100. The antibodies were utilized in the development of an ELISA for quantitation of MDA-LDL in human plasma. Intra- and inter-assay coefficients of variation (% CV) were measured as 4.8 and 7.7%, respectively, and sensitivity of the assay as 0.04 μg/ml MDA-LDL. Recovery of standard MDA-LDL from native LDL was 102%, indicating the ELISA to be specific with no interference from other biomolecules. Further validation of the ELISA was carried out against two established methods for measurement of lipid peroxidation products, MDA by HPLC and F2-isoprostanes by GC-MS. Results indicated that MDA-LDL is formed at a later stage of oxidation than either MDA or F2- isoprostanes. In vivo analysis demonstrated that the ELISA was able to determine steady-state concentrations of plasma MDA-LDL (an end marker of lipid peroxidation). A reference range of 34.3 ± 8.8 μg/ml MDA-LDL was established for healthy individuals. Further, the ELISA was used to show significantly increased plasma MDA-LDL levels in subjects with confirmed ischemic heart disease, and could therefore possibly be of benefit as a diagnostic tool for assessing CHD risk. © 2003 Elsevier Inc.

On the decline of 1st and 2nd order sensitivity with eccentricity

We studied the relationship between the decline in sensitivity that occurs with eccentricity for stimuli of different spatial scale defined by either luminance (LM) or contrast (CM) modulation. We show that the detectability of CM stimuli declines with eccentricity in a spatial frequency-dependent manner, and that the rate of sensitivity decline for CM stimuli is roughly that expected from their 1st order carriers, except, possibly, at finer scales. Using an equivalent noise paradigm, we investigated the possible reasons for why the foveal sensitivity for detecting LM and CM stimuli differs as well as the reason why the detectability of 1st order stimuli declines with eccentricity. We show the former can be modeled by an increase in internal noise whereas the latter involves both an increase in internal noise and a loss of efficiency. To encompass both the threshold and suprathreshold transfer properties of peripheral vision, we propose a model in terms of the contrast gain of the underlying mechanisms.

Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

Oxidative impairment of plasma and skeletal muscle sarcoplasmic reticulum in rats with adjuvant arthritis - effects of pyridoindole antioxidants

OBJECTIVES: To study possible oxidation of proteins and lipids in plasma and sarcoplasmic reticulum (SR) from skeletal muscles and to assess the effects of pyridoindole antioxidants in rats with adjuvant arthritis (AA) and to analyze modulation of Ca-ATPase activity from SR (SERCA). METHODS: SR was isolated by ultracentrifugation, protein carbonyls in plasma and SR were determined by ELISA. Lipid peroxidation was analyzed by TBARS determination and by mass spectrometry. ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Tryptophan fluorescence was used to analyze conformational alterations. RESULTS: Increase of protein carbonyls and lipid peroxidation was observed in plasma of rats with adjuvant arthritis. Pyridoindole antioxidant stobadine and its methylated derivative SMe1 decreased protein carbonyl formation in plasma, effect of stobadine was significant. Lipid peroxidation of plasma was without any effect of pyridoindole derivatives. Neither protein oxidation nor lipid peroxidation was identified in SR from AA rats. SERCA activity from AA rats increased significantly, stobadine and SMe1 diminished enzyme activity. Ratio of tryptophan fluorescence intensity in SR of AA rats increased and was not influenced by antioxidants. CONCLUSION: Plasma proteins and lipids were oxidatively injured in rats with AA; antioxidants exerted protection only with respect to proteins. In SR, SERCA activity was altered, apparently induced by its conformational changes, as supported by study of tryptophan fluorescence. Stobadine and SMe1 induced a decrease of SERCA activity, elevated in AA rats, but they did not affect conformational changes associated with tryptophan fluorescence.

Receptor Activity Modifying Proteins (RAMPs) interact with the VPAC2 Receptor and CRF1 receptors and modulate their function

Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-Activating peptide 2 receptor (VPAC) and the type 1 corticotrophin releasing factor receptor (CRF) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca mobilization and GTPγS binding to G, G, G and G were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2 mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC enhanced the cell surface expression of all three RAMPs. CRF enhanced the cell surface expression of RAMP2; the cell surface expression of CRF was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF: RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2 mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC and CRF receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF, coupling to RAMP2 may be of physiological significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Rotational co-culture of clonal β-cells with endothelial cells:effect of PPAR-γ agonism in vitro on insulin and VEGF secretion

Aim: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co-culture of insulin secreting ß-cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator-activated receptor gamma (PPAR-?) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function. Methods: Clonal BRIN-BD11 (D11) cells were maintained in static culture (SC) and rotational culture (RC) ± HUVEC and ± the TZD (thiazolidinedione) rosiglitazone (10 mmol/l) as a specific PPAR-? agonist. HUVECs were cultured in SC and RC ± D11 and ± TZD. D11 insulin secretion was induced by static incubation with low glucose (1.67 mmol/l), high glucose (16.7 mmol/l) and high glucose with 10 mmol/l theophylline (G+T) and assessed by enzyme-linked immunosorbent assay (ELISA). HUVEC proliferation was determined by ATP luminescence, whereas VEGF secretion was quantified by ELISA. Co-cultured cells were characterized by immunostaining for insulin and CD31. Results: D11 SC and RC showed enhanced insulin secretion in response to 16.7 mmol/l and G+T (p <0.01); without significant alteration by the TZD. Co-culture with HUVEC in SC and RC also increased D11 insulin secretion when challenged with 16.7 mmol/l and G+T (p <0.01), and this was slightly enhanced by the TZD. The presence of HUVEC increased D11 SC and RC insulin secretion in response to high glucose and G+T, respectively (p <0.01). Addition of the TZD increased SC and RC HUVEC ATP content (p <0.01) and VEGF production (p <0.01) in the presence and absence of D11 cells. Conclusions: Rotational co-culture of insulin secreting cells with endothelial cells, and exposure to a PPAR-? agonist may improve the prospects for graft revascularization and function after implantation. © 2011 Blackwell Publishing Ltd.