Orientador: Sonia Maria Alves Bueno / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-08T05:04:16Z (GMT). No. of bitstreams: 1 Ribeiro_MarianaBorsoi_D.pdf: 5252506 bytes, checksum: b9eb50542c7fa4e04b6b4c83526b4ccc (MD5) Previous issue date: 2006 / Resumo: As imunoglobulinas do isotipo G (IgG) são usadas enOlmemente em aplicações terapêuticas e são requeridas, usualmente, com um elevado grau de pureza. V álias técnicas cromatográficas vêm sendo investigadas para a purificação de IgG a partir do plasma humano. Neste trabalho, investigou-se o efeito dos agentes quelantes ácido iminodiácetico (IDA), ácido aspártico carboxi-metilado (CM-Asp) e TIis-2(aminoetil)amina (TREN) na purificação de IgG a partir do plasma humano utilizando a técnica de cromatografia de afinidade com os íons metálicos imobilizados em membranas de fibras ocas. Para tanto, foram realizados experimentos de adsorção em diferentes sistemas tamponantes em membranas de álcool de poli(etileno) vinílico (PEV A) finamente c011adas e em módulos de filtração derivatizados com IDA, TREN e CM-Asp, utilizando-se os íons metálicos níquel e cobre. A seletividade dos adsorventes foi verificada por eletroforese SDS-P AGE e análise nefelométrica. As melhores condições de purificação foram encontradas utilizando-se as membranas PEV A-CM-Asp-Ni(II) em módulo de filtração, em presença do tampão TrisHCI 25 mM pH 7,0 e eluição por acréscimo da concentração de TIis, sendo obtida IgG com aproximadamente 90% de pureza. As membranas de PEV A-CM-Asp-Ni(II) em módulos de filtraçã? apresentaram melhor seletividade, segundo eletroforese SDS-P AGE, e capacidade dinâmica de adsorção maior comparadas às membranas finamente cortadas (capacidade média de adsorção: 53,0 e 7,7 mg de IgG pOT g seca de membrana, respectivamente). O modelo de LangmUir ajustou-se às isotermas de adsorção, à temperatura ambiente, mostrando alta capacidade de adsorção para os sistemas PEV A-IDA-Ni(II) e PEV A-CM Asp-Ni(II) (204,6 e 302,3 mg/g seca de membrana, respectivamente) e constantes de dissociação característica de sistemas de média afinidade (6, I e 10,1 x 10-6 M, respectivamente). A análise dos parâmetros termodinâmicos encontrados para o sistema modelo PEV A-IDA-Ni(II)-IgG, indicaram a complexidade das interações, indicando a existência de interações hidrofóbicas, eletrostáticas e de ligações de coordenação. Neste trabalho, conseguiu-se purificar IgG a partir do plasma humano em! uma única etapa, utilizando-se a técnica da cromatografia em membranas de afinidade, demonstrando a alta potencialidade da utilização do método desenvolvido em processos industriais / Abstract: The use of immunoglobulin G (lgG) in therapeutic applications has growing vastly and it is used in the treatment of a growing number of indications. Several chromatographic techniques have been investigated for IgG purification. In this work, we investigated the effect of the chelators iminodiacetic acid (IDA), carboxymethylaspartate acid (CM-Asp) and Tris-2(aminoethyl)amine (TREN) for IgG purification, from human plasma using Immobilized Metal-ion Affinity Chromatography (IMAC) technique using hollow fibers membranes as support to the chromatographic experiments. For that, adsorption experiments were done, using different buffers systems, on filtration module and on finely cut poly(ethylene) vinyl alcohol (PEV A) membranes containing chelators IDA, TREN and CM-Asp with the metallic ions nickel and copper. The adsorbent selectivity was verified through electrophoresis SDS-PAGE and nefelometric analysis. Best purification conditions were found with PEV A-CM-Asp-Ni(ll) filtration module in the presence of 25 mM Tris HCI pH 7.0 and elution by increasing Tris concentration. In this case, it was possible to obtain IgG with approximately 90% of purity. According to electrophoresis SDS-P AGE and nefelometric analysis, PEV A-CM-Asp-Ni(ll) filtration modules showed better selectivity and superior adsorption dynamic capacity for IgG than those obtained by finely cut membranes (medium adsorption capacity: 53.0 and 7.7 mg of IgG /g of dry membrane, respectively). Lagmüir model adjusted to adsorption isotherms at room temperature (250 C), showed high adsorption capacity for PEV A-IDA-Ni(ll) and PEV A-CM-Asp-Ni(ll) systems (204.6 and 302.3 mg/g dry of membrane, respectively) and showed dissociation constants characteristic of medium affinity systems (6.1 and 10.1 x 10-6 M, respectively). The thermodynamic parameters analysis obtained for PEV A-IDA-Ni(ll)-lgG system indicated the complexity of the interactions, and the existence of hydrophobic and electrostatics interaction besides the coordination bound. In this work, we could purify IgG from human plasma in a single stage, using the affinity chromatographic membranes technique, allowing in this way, the treatment of great volumes per unit of time, showing the high potentiality of this method in industrial processes / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Kidney transplantation is the best treatment for patients with end-stage renal failure. The main problem of kidney transplantation is however the development of a cellular and antibody-mediated (humoral) rejection. During the last decade, thanks to the advanced immunosuppression, prognosis of survival and function of transplanted organs has significantly improved. Nevertheless, humoral rejection remains very serious obstacle in high-risk patients, because it can permanently damage the graft. Therefore, before transplantation it is necessary to stratify patients into high and low risk groups for development of antibody-mediated rejection. Current immunogenetic tests performed before transplantation include, in addition to HLA typing, detection of panel-reactive antibodies. However, this test does not provide information about B cells which participate in the humoral response of the kidney recipient. Therefore, in the presented thesis we studied B cell reactivity and its regulation in transplanted patients. In this retrospective analysis we measured levels of the B cell activating factor, a cytokine regulating the function of B lymphocytes (BAFF). Current reports suggest that BAFF could serve as a marker of humoral rejection. Furthermore, we focused on B lymphocytes and their capacity to produce...

<p>Kontrola zdravlja vimena krava je bitan element u procesu proizvodnje zdravstveno<br />bezbednog mleka, te se na farmama visokomlečnih krava, kroz program kontrole mastitisa,<br />redovno sprovode mere otkrivanja i prevencije bolesti vimena. Klinički pregled vimena<br />predstavlja osnovni metod koji pruţa korisne informacije o zdravstvenom statusu vimena<br />krava, ali nailazi na pote&scaron;koće u otkrivanju patolo&scaron;kih promena unutar parenhima i papile<br />vimena. U cilju otkrivanja promena u parenhimu vimena moţe se primeniti ultrazvučni<br />pregled koji omogućava vizualizaciju strukturnih promena vimena nastalih kao posledica<br />upalnih procesa i tako olak&scaron;ava dijagnostiku oboljenja.<br />Tokom poslednjih godina, javio se problem povećanja rezistencije bakterija na<br />antimikrobne lekove, &scaron;to oteţava lečenje bolesti, ali i ugroţava zdravlje ţivotinja i ljudi.<br />Najče&scaron;ći uzroci toga su nepravilna upotreba i eventualna zloupotreba antimikrobnih lekova.<br />Mnoga istraţivanja vr&scaron;ena su u in vitro i in vivo uslovima na primeni laktoferina samog ili u<br />kombinaciji sa antibioticima u terapiji i prevenciji mastitisa krava. Laktoferin, gvoţĎe<br />vezujući antimikrobni glikoprotein koji se nalazi u mleku i drugim sekretima, predstavlja<br />bitan deo sistema odbrane mlečne ţlezde.<br />Cilj istraţivanja u okviru ove disertacije je procena dijagnostičke mogućnosti<br />ultrazvučnog pregleda u detekciji subkliničkog mastitisa i poremećene sekrecije vimena,<br />kao i razmatranje opravdanosti primene laktoferina u prevenciji i lečenju mastitisa.<br />Kliničkim pregledom izvr&scaron;ena je procena op&scaron;teg zdravstvenog stanja krava, kao i<br />ispitivanje mlečne ţlezde adspekcijom i palpacijom. Za otkrivanje poremećene sekrecije<br />vimena i subkliničkih mastitisa kori&scaron;ćeni su brzi testovi, Kalifornija mastitis test i<br />Draminski test, kao i ultrazvučni pregled mlečne ţlezde krava. OdreĎivanje broja somatskih<br />ćelija u uzorcima mleka uraĎeno je metodom protočne citometrije. Za identifikaciju<br />uzročnika mastitisa kori&scaron;ćene su klasične mikrobiolo&scaron;ke metode. Krave sa pozitivnim<br />bakteriolo&scaron;kim nalazom podeljene su u dve ogledne grupe. Krave ogledne grupe I su<br />tretirane intramamarnom aplikacijom antibiotika, dok je kravama ogledne grupe II<br />aplikovana kombinacija antibiotika i laktoferina. OdreĎivanje koncentracije imunoglobulina<br />G u mlečnom serumu krava vr&scaron;eno je metodom radioimunodifuzije, a odreĎivanje<br />koncentracije laktoferina u mleku krava ELISA testom.<br />U istraţivanjima u okviru ove disertacije, najče&scaron;će izolovani major mastitis patogeni<br />bile su bakterije Staphylococcus aureus i Streptococcus agalactiae, a najče&scaron;će izolovani<br />minor mastitis patogeni Corynebacterium spp. i koagulaza negativne stafilokoke.<br />Ultrasonografija mlečne ţlezde krava pokazala se kao efikasna metoda u dijagnostici<br />poremećaja sekrecije vimena. Veće vrednosti koncentracije imunoglobulina G u mlečnom<br />serumu krava uočene su tokom perioda predzasu&scaron;enja i zasu&scaron;enja, u odnosu na period rane<br />laktacije. Najveći uticaj na porast koncentracije laktoferina u mleku krava su imali<br />bakterijski uzročnici mastitisa. Efikasnost antibiotske terapije primenjene tokom perioda<br />zasu&scaron;enja kod krava ogledne grupe I iznosila je 52,7%, dok je efikasnost primenjene terapije<br />sa goveĎim laktoferinom i antibiotikom kod krava ogledne grupe II iznosila 60%. Aplikacija<br />laktoferina tokom perioda zasu&scaron;enja doprinela je efikasnosti terapije intramamarnih<br />infekcija, ali nije imala uticaj na koncentraciju laktoferina u mleku tokom perioda rane<br />laktacije.<br />Dalja istraţivanja su neophodna kako bi se u potpunosti potvrdila efikasnost<br />intramamarne terapije sa goveĎim laktoferinom i antibiotikom u cilju eliminacije uzročnika<br />mastitisa i da se precizno odredi terapijska koncentracija laktoferina.</p> / <p>Control of udder health is an essential element in the process of safe milk<br />production, thus, through the mastitis control program, dairy farms regularly conduct<br />measures of detection and prevention of udder diseases. Clinical examination of the udder<br />is a basic method that provides useful information about the health status of cow udders,<br />but this method is limited in its ability to detect pathological changes in the udder<br />parenchyma and teat. In order to detect changes in the parenchyma of the udder, an<br />ultrasound can be applied which allows visualization of udder structural changes incurred<br />as a result of inflammatory processes, thus facilitating diagnosis of diseases.<br />Over the last few years, the problem of increasing resistance to antimicrobial drugs<br />has appeared, making it difficult to treat disease and also threaten the health of animals and<br />humans. The incorrect and widespread use of antimicrobial drugs are the most common<br />cause of this. Many studies were conducted in vitro and in vivo conditions on the use of<br />lactoferrin alone or in combination with antibiotics in the treatment and prevention of<br />mastitis. Lactoferrin, an iron-binding antimicrobial glycoprotein which is found in milk<br />and other secretions, represents an important part of the mammary gland defense system.<br />The aim of the research within this thesis was to evaluate the diagnostic<br />possibilities of udder ultrasonography in the detection of subclinical mastitis and udder<br />secretion disorders, as well as considering the feasibility of lactoferrin application in the<br />prevention and treatment of mastitis.<br />The general condition of the animals was evaluated by clinical examination, as<br />well as udder examination by adspection and palpation. The California mastitis test,<br />Draminski test and ultrasound examination of the cow&#39;s mammary glands were used for<br />detection of udder secretion disorders and subclinical mastitis. Somatic cell count in milk<br />samples was determined by flow cytometry method. Classical microbiological methods<br />were used for the isolation and identification of mastitis causative agents. Cows with<br />positive bacteriological findings were divided into two experimental groups. Cows in<br />experimental group I were treated with intramammary applications of antibiotics, while the<br />cows in experimental group II were treated with a combination of antibiotics and<br />lactoferrin. Immunoglobulin G concentration in bovine milk serum was determined by the<br />immunodiffusion method, while lactoferrin concentration in bovine milk was determined<br />using the ELISA test.<br />In this study, the most common isolated major mastitis pathogens were<br />Staphylococcus aureus and Streptococcus agalactiae, while Corynebacterium spp. and<br />coagulase-negatice staphilococci were the most commonly detected minor mastitis<br />pathogens. Ultrasonography of the bovine mammary gland proved to be an effective<br />method in the diagnosis of udder secretion disorders. Higher immunoglobulin G<br />concentrations were observed in milk serum from cows during pre-dry and dry period,<br />relative to early lactation period. The biggest influence on the increase in the concentration<br />of lactoferrin in the bovine milk had mastitis pathogens. The efficacy of antibiotic therapy<br />during the dry period in cows of experimental group I was 52.7%, while the efficacy of the<br />applied therapy with lactoferrina and antibiotics in cows of experimental group II was<br />60%. Application of lactoferrin during the dry period contributed to the effectiveness of<br />the treatment of intramammary infections, but had no influence on lactoferrin<br />concentration in the milk during the early lactation period.<br />Further studies are necessary to in order to fully confirm the efficacy of<br />intramammary therapy with bovine lactoferrin and antibiotic to eliminate the mastitis<br />pathogens and to determine the therapeutic concentration of lactoferrin.</p>

<p>UVOD:&nbsp; Citopenija je jedna od glavnih&nbsp; karakteristika mnogih hematolo&scaron;kih bolesti. U&nbsp; rutinskoj transfuziolo&scaron;koj upotrebi su metode&nbsp; detekcije prisustva antitela u serumu ili na&nbsp; eritrocitima bolesnika. Primena direktnog testa utro&scaron;ka antihumanog globulina predstavlja&nbsp; efikasan način da se stekne kompletan uvid u imunolo&scaron;ka zbivanja na svim krvnim lozama, prati dinamika razvoja antitela i toka bolesti. MATERIJAL I METODE: Svim pacijentima su se iz uzoraka periferne&nbsp; krvi vr&scaron;ile sledeće analize krvi: 1) direktni antiglobulinski test mikrometodom&nbsp; aglutinacije u gel karticama (LISS)/ Coombs ID. Dobijeni rezultat aglutinacije mikrometodom na gelu moţe biti negativan ili pozitivan i 2) direktni test utro&scaron;ka antihumanog globulina metodom aglutinacije u epruveti. Očitavanje se vr&scaron;ilo određivanjem razlike u titru antihumanog globuli na i očitavanjem postojeće reakcije aglutinacije dobijene u uzorcima pacijenta u odnosu na rezultate reakcije aglutinacije dobijene sa uzorcima zdrave kontrolne osobe. Test se smatra o pozitivnim ukoliko se dobijala razlika u titru AHG - a za bar dva razređen ja sa pacijentovim ćelijama u odnosu na ćelije zdrave kontrolne osobe. Statistička značajnost&nbsp; je analizirana t - testom, Spirmanovim&nbsp; koeficijentom korelacije REZULTATI: Analizirano je 100 pacijenata sa dijagnozom anemije, leukopenije,&nbsp; limfoproliferativnih bolesti,&nbsp; trombocitopenije, trombotične trombocitopenijske purpure, idiopatske trombocitopenične purpure, mijelodisplastičnog sindroma, miastenije gravis i sistemskog eritematoznog lupusa pre i nakon primljene terapije. Direktni antiglobulinski test je biopozitivan u 20% slučajeva dok je direktni test utro&scaron;ka antihumanog globulina bio u 51%, odnosno za 31% vi&scaron;e. Nakon primljene terapije direktni antiglobulinski test je ostao pozitivan u 18% slučajeva a direktni test utro&scaron;ka antihumanog globulina u 46% &scaron;to je za 28% vi&scaron;e. Utvrđivanjem povezanosti između citopenije i stepena utro&scaron;ka antihumanog globulina dokazano je da svi praćeni parametri utiču na stepen utro&scaron;ka AHG-a: hemoglobin (&beta;=-0,579, p=0,000), hematokrit (&beta;=-0,568, p=0,000), eritrociti (&beta;=-0,519, p=0,000), trombociti (&beta;=-0,617, p=0,000) i leukociti(&beta;=-0,119, p=0,237). Takođe je dokazano da &scaron;to su vrednosti posmatranih parametara veće, razlika u titru direktnog testa utro&scaron;ka antihumanog globulina je manja &scaron;to bi i&scaron;lo u prilog boljoj prognozi posmatranog oboljenja. ZAKLJUČAK: Direktni test utro&scaron;ka antihumanog globulina je značajno osetljiviji test u odnosu na direktni antihumani globulinski test. Postoji pozitivna korelacija između citopenije i stepena konzumacije antihumanog globulina. Smanjenje titra antitela direktnog testa utro&scaron;ka antihumanog globulina je jedan od pokazatelja bolje prognoze bolesti.</p> / <p>INTRODUCTION: Cytopenia is one of the main characteristics of many hematologic diseases. In routine use are methods of detecting the presence of antibodies in the serum or on red blood cells of patients. The application of direct consumption test of antihuman globulin is an efficient way to gain complete insight into the immunological events at all bloodlines, monitor the dynamics of the development of antibodies and disease progression. MATERIALS AND METHODS: All patients samples were tested for: 1) direct antiglobulin test by micro agglutination method in the gel card (LISS) / Coombs ID. The result obtained by micro agglutination gel can be negative or positive, 2) direct consumption test of antihuman globulin in a test tube. Interpretation is performed by determining differences in titer of antihuman globulin by reading existing reactions of agglutination in samples of the patient and compare it to the results obtained with the samples of the healthy control persons. The test is considered positive if the difference in titres obtained AHG differs for at least two dilutions of a patient&#39;s cells compared to cells of healthy control persons. Statistical significance was analyzed by t-test, Spearman correlation coefficient. RESULTS: A total of 100 patients diagnosed withanemia, leukopenia, lymphoproliferative disease, thrombocytopenia, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, myelodysplastic syndrome, myasthenia gravis and systemic lupus erythematosus were analyzed before and after receiving treatment. Direct antiglobulin test was positive in 20% cases, while the direct consumption test of anti-human globulin was 51%, that is the difference of 31%. After treatment direct antiglobulin test remained<br />positive in 18% of cases and direct consumption test of antihuman globulin was in 46%, which is 28% higher. Determining the relationship between the degree of cytopenia and consumption of anti-human globulin showed that all monitored parameters affect the level of consumption: hemoglobin (&beta; = -0.579, p = 0.000), hematocrit (&beta; = -0.568, p = 0.000), erythrocytes (&beta; = -0.519 , p = 0.000), platelets (&beta; = -0.617, p = 0.000) and leukocytes (&beta; = -0.119, p = 0.237). It was also proved that if the values of observed parameters are higher, difference in titer of direct consumption test of antihuman globulin is lower, which can indicate better prognosis of disease. CONCLUSION: Direct consumption test of antihuman globulin was significantly more&nbsp; sensitive test than the direct anti-human globulin test. There is a positive correlation between the degree of cytopenia and consumption of anti-human globulin. Decrease in antibody titer in direct consumption test of antihuman globulinis an indicator of a better prognosis of the disease.</p>

Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...