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...

Multiple myeloma is an incurable fatal neoplasm of plasma cells affecting mainly elderly people. There are many research laboratories in the world where multiple myeloma is studied. Permanent cell lines are indispensable tools for both basic and applied research. However, the establishment of new cell lines is difficult with poor success. We established 96 primary cultures of bone marrow samples from myeloma patients. Only one culture succeeded in permanet myeloma cell line. The novel plasmacytic cell line ÚHKT-893 was established from bone marrow sample of 57 years old female relapsed with multiple myeloma of IgGκ isotype. The cell line is however dependent on the continuous presence of interleukin-6 in culture media. ÚHKT-893 cells grow continuously already more than 1 year. The growth of cells was regularly monitored according to growth curves and by measurement of cell viability. Surface antigenic profile was repeatedly determined by flow cytometry. The simultaneous expression of both CD138 and CD38 surface molecules confirmed the plasma cell origin of cells. The establishment of the ÚHKT-893 myeloma cell line will extend the panel of existing myeloma cell lines as a new ex vivo model for the study of the etiology and pathogenesis of multiple myeloma. It will also provide the source of new...

<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>

Orientadores : Sonia Maria Alves Bueno, Ricardo de Lima Zollner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T11:50:08Z (GMT). No. of bitstreams: 1 Duarte_IsaSantos_D.pdf: 749498 bytes, checksum: 83d66753c75e7828fd4a8b858aecf473 (MD5) Previous issue date: 2006 / Resumo: A alergia é uma enfermidade do sistema imunológico que afeta aproximadamente de 20 a 30% da população mundial. Dentre as reações alérgicas, a reação de hipersensibilidade imediata é mediada pelas imunoglobulinas E (IgE). Os indivíduos geneticamente predispostos a manifestar reações por hipersensibilidade imediata e polissensibilizados aos alérgenos ambientais são considerados atópicos e, geralmente, possuem teores de IgE total até 10.000 vezes mais elevados do que as pessoas não-atópicas. O conhecimento das interações entre a IgE e ligantes de afinidade pode levar ao desenvolvimento de novos tratamentos da hipersensibilidade imediata, por exemplo a terapia de adsorção seletiva através de circulação extracorpórea, assim como ao desenvolvimento de métodos de obtenção de IgE purificada, para aplicação nas áreas de diagnóstico, pesquisa molecular, dentre outras. Os adsorventes empregados na terapia de adsorção seletiva, bem como na purificação de IgE, geralmente são anticorpos anti-IgE imobilizados em agarose, os quais são de alto custo e difícil obtenção. Este trabalho avaliou o desempenho de adsorventes alternativos ao Sepharose-anti-IgE, visando a remoção de IgE total e específica aos ácaros Dermatophagoides pteronyssinus e Blomia tropicalis de amostras plasmáticas e a preparação de soluções enriquecidas em IgE, como uma das etapas do processo de purificação de IgE. Os adsorventes estudados constituíram-se de lectinas (concanavalina A e Lens culinaris), aminas (poli-L-lisina e aminohexil) e o aminoácido D-triptofano, imobilizados em agarose. Dentre eles, o gel agarose-Lens culinaris mostrou-se o mais promissor para aplicação na terapia de adsorção seletiva de IgE e o gel Sepharose-concanavalina A mostrou-se o mais adequado para ser usado na obtenção de soluções enriquecidas em IgE. Experimentos cromatográficos foram realizados visando estabelecer condições experimentais (velocidade superficial, número de passagens de plasma pela coluna, temperatura e razão entre volume de plasma e volume de leito) mais favoráveis à adsorção de IgE em agarose-Lens culinaris. Posteriormente, essas condições foram utilizadas nos experimentos de simulação in vitro de circulação extracorpórea, nos quais o gel agarose-Lens culinaris removeu de 40,7 a 42,8% de IgE¿s total e específicas. A obtenção da solução enriquecida em IgE foi realizada por meio de duas etapas cromatográficas, empregando-se os princípios de afinidade (colunas agarosejacalina e Sepharose-concanavalina A) e de exclusão por tamanho (permeação em gel). A solução final enriquecida em IgE obtida, continha como principais impurezas, IgA e IgG. Como resultado das duas etapas, 36,6% de IgE foi recuperada e o fator de enriquecimento em IgE, em relação a IgA, IgG, IgM e albumina, foi de 75,8. Apesar do gel Sepharose-anti- IgE apresentar desempenho melhor tanto na remoção quanto na purificação de IgE, os adsorventes agarose-Lens culinaris e Sepharose-concanavalina A apresentam custos mais atrativos / Abstract: Allergy is a disorder of the imune system, affecting approximately 20%-30% of the general population. Among allergic reactions, immediate hypersensitivity is mediated by immunoglobulin E (IgE). Individuals that have a genetic predisposition for responses to immediate hypersensitivity are named atopic and generally have elevated serum IgE concentration, up to 10,000-fold higher than in the normal population. The knowledge of the interactions between IgE and affinity ligands may lead to the development of new methods of treatment for immediate hypersensitivity, for example, IgE selective adsorption therapy through extracorporeal circulation, as well as to new methods for obtaining purified IgE, which is employed in diagnostic and in molecular research. The adsorbents employed in IgE selective adsorption therapy, as well as in IgE purification, are usually antibodies anti-IgE immobilized on agarose, which have high costs and are difficult to obtain. This work assessed the performance of adsorbents (alternative to Sepharose-anti-IgE) for the removal of total IgE and IgE specific for the airbone allergens Dermatophagoides pteronyssinus and Blomia tropicalis from plasma samples, as well as in the production of IgE enriched solutions, considered as a step of IgE purification. The adsorbents studied were lectins (concanavalina A and Lens culinaris), amines (poli-L-lisina e aminohexil) and the aminoacid D-tryptophan, all of them immobilized on agarose. Among them, Lens culinaris-agarose showed the best performance for IgE selective adsorption therapy, and Sepharose¿concanavalin A was considered the most appropriate for the production of IgE enriched solutions. Chromatographic experiments were accomplished in order to determine operating conditions (superficial velocity, number of times the plasma passed through the column, temperature, and ratio of plasma volume to bed volume) more favorable to IgE adsorption on Lens culinaris-agarose. The selected conditions were utilized in in vitro simulation assays of extracorporeal circulation, in which the Lens culinaris-agarose removed from 40.7% to 42.8% of total and specific IgE. The production of IgE enriched solutions was carried out with two chromatographic steps, employing affinity (columns jacalin-agarose and Sepharose-concanavalin A) and size exclusion (gel permeation) principles. The IgE enriched final solution contained IgA and IgG as the major impurities. As a result of both steps, 36.6% of IgE was recovered and the IgE enrichement number concerning IgA, IgG, IgM, and albumin was 75.8. Despite Sepharose-anti-IgE has better performance in removal and purification of IgE, Lens culinaris-agarose and Sepharose-concanavalin A adsorbents have more attractive costs / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutora em Engenharia Quimica

<p>Увод: Хронична бубрежна инсуфицијенција (ХБИ) се дефинише као структурно или функционално оштећење бубрега у трајању од најмање три месеца и/или смањење јачине гломеруларне филтрације (ЈГФ) испод 60 мл/мин/1.73м2. У доступној литератури постоје различити подаци о присуству оралних манифестација код пацијената са ХБИ у квантитативном и квалитативном погледу. Стање бубрежне дисфункције праћено је променама у протоку и саставу пљувачке што је у последњој деценији допринело испитивању клиничких и лабораторијских показатеља бубрежне болести. Циљ: Циљ студије је био да се испита објективно стање оралне слузокоже, вредности рН, сијалометрије, концентрације урее, креатинина и секреторног имуноглобулина А пљувачке као и орални микробиолошки статус код пацијената са ХБИ. Материјал и методе: Узорак је био сачињен од 50 предијализних (31 мушкарца и 19 жена просечне старости 59,06&plusmn;14,30) и 25 хемодијализних пацијената (18 мушкараца и 7 жена просечне старости 54,92&plusmn;13,60) са постављеном дијагнозом ХБИ, заједно са 25 системски здравих испитаника компарибилних по полу и старости. Поред клиничког прегледа усне дупље спроведен је тест витроадхезије, одређивање интензитета саливације, рН вредности пљувачке и индекса крварења из интерденталне папиле (PBI). На узорцима сакупљене пљувачке, уз помоћ аутоматизованог система Beckman Coulter АУ480 спроведено је лабораторијско одређивање урее и креатинина методом спектрофотометрије и секреторног имуноглобулина А методом имунотурбидиметрије. За микробилошко испитивање коришћен је брис језика и техника оралног испирка. Резултати: Нису утврђене статистички значајне разлике између група према демографско-социјалним подацима. Предијализни испитаници су имали значајно веће присуство промена оралне слузокоже и оралних симптома. Просечне вредности клиренса креатинина су биле значајно мање код оболелих испитаника са бледилом оралне слузокоже, уремичним задахом, ксеростомијом и измењеним осећајем укуса у поређењу са испитаницима без наведених промена. Код предијализних су утврђене значајно смањене вредности сијалометрије према контролним групама и повећане pH вредности према групи здравих испитаника. Просечне концентрације урее и креатинина су се статистички значајно разликовале између испитиваних група. Умерена позитивна корелација је утврђена између серумских и пљувачних концентрација урее и креатинина код предијализних и креатинина код хемодијализних. Према просечним вредностима секреторног имуноглобулина А није било разлика између група. Код пацијената са ХБИ утврђено је значајно веће присуство гљива из рода Candida са предоминацијом non-albicans Candida врста. Закључак: Резултати истраживања указују на важност утврђивања клиничких карактеристика усне дупље код предијализних пацијената. Интензитет саливације, pH вредност и пљувачне концентрације уремијских токсина могу бити поуздани маркери бубрежног оштећења. Једноставан и неинвазиван приступ приликом узорковања пљувачке и поузданост лабораторијске анализе треба да допринесу широј примени пљувачке као компетитивним дијагностичким флуидом серуму. Техника оралног испирка је прецизна квантитативна метода за одређивање степена гљивичне колонизације.</p> / <p>Uvod: Hronična bubrežna insuficijencija (HBI) se definiše kao strukturno ili funkcionalno oštećenje bubrega u trajanju od najmanje tri meseca i/ili smanjenje jačine glomerularne filtracije (JGF) ispod 60 ml/min/1.73m2. U dostupnoj literaturi postoje različiti podaci o prisustvu oralnih manifestacija kod pacijenata sa HBI u kvantitativnom i kvalitativnom pogledu. Stanje bubrežne disfunkcije praćeno je promenama u protoku i sastavu pljuvačke što je u poslednjoj deceniji doprinelo ispitivanju kliničkih i laboratorijskih pokazatelja bubrežne bolesti. Cilj: Cilj studije je bio da se ispita objektivno stanje oralne sluzokože, vrednosti rN, sijalometrije, koncentracije uree, kreatinina i sekretornog imunoglobulina A pljuvačke kao i oralni mikrobiološki status kod pacijenata sa HBI. Materijal i metode: Uzorak je bio sačinjen od 50 predijaliznih (31 muškarca i 19 žena prosečne starosti 59,06&plusmn;14,30) i 25 hemodijaliznih pacijenata (18 muškaraca i 7 žena prosečne starosti 54,92&plusmn;13,60) sa postavljenom dijagnozom HBI, zajedno sa 25 sistemski zdravih ispitanika komparibilnih po polu i starosti. Pored kliničkog pregleda usne duplje sproveden je test vitroadhezije, određivanje intenziteta salivacije, rN vrednosti pljuvačke i indeksa krvarenja iz interdentalne papile (PBI). Na uzorcima sakupljene pljuvačke, uz pomoć automatizovanog sistema Beckman Coulter AU480 sprovedeno je laboratorijsko određivanje uree i kreatinina metodom spektrofotometrije i sekretornog imunoglobulina A metodom imunoturbidimetrije. Za mikrobiloško ispitivanje korišćen je bris jezika i tehnika oralnog ispirka. Rezultati: Nisu utvrđene statistički značajne razlike između grupa prema demografsko-socijalnim podacima. Predijalizni ispitanici su imali značajno veće prisustvo promena oralne sluzokože i oralnih simptoma. Prosečne vrednosti klirensa kreatinina su bile značajno manje kod obolelih ispitanika sa bledilom oralne sluzokože, uremičnim zadahom, kserostomijom i izmenjenim osećajem ukusa u poređenju sa ispitanicima bez navedenih promena. Kod predijaliznih su utvrđene značajno smanjene vrednosti sijalometrije prema kontrolnim grupama i povećane pH vrednosti prema grupi zdravih ispitanika. Prosečne koncentracije uree i kreatinina su se statistički značajno razlikovale između ispitivanih grupa. Umerena pozitivna korelacija je utvrđena između serumskih i pljuvačnih koncentracija uree i kreatinina kod predijaliznih i kreatinina kod hemodijaliznih. Prema prosečnim vrednostima sekretornog imunoglobulina A nije bilo razlika između grupa. Kod pacijenata sa HBI utvrđeno je značajno veće prisustvo gljiva iz roda Candida sa predominacijom non-albicans Candida vrsta. Zaključak: Rezultati istraživanja ukazuju na važnost utvrđivanja kliničkih karakteristika usne duplje kod predijaliznih pacijenata. Intenzitet salivacije, pH vrednost i pljuvačne koncentracije uremijskih toksina mogu biti pouzdani markeri bubrežnog oštećenja. Jednostavan i neinvazivan pristup prilikom uzorkovanja pljuvačke i pouzdanost laboratorijske analize treba da doprinesu široj primeni pljuvačke kao kompetitivnim dijagnostičkim fluidom serumu. Tehnika oralnog ispirka je precizna kvantitativna metoda za određivanje stepena gljivične kolonizacije.</p> / <p>Introduction: Chronic kidney disease (CKD) is defined as structural and functional kidney damage for a period of at least three months and/or reduction of glomerular filtration rate (GFR) under 60 ml/min/1.73m2. There are different data in the available literature in term of quantitative and qualitative presence of the oral manifestation in patients with CKD. Kidney dysfunction is accompanied by changes in the salivary flow and composition, which is in the last decade contributed by examination of clinical and laboratory markers of renal disease. Aim: The aim of the study was to examine condition of oral mucosa, pH value, salivary flow rate, concentration of salivary urea, creatinine, secretory immunoglobulin A and oral microbiological status in patients with CKD. Materials and Methods: The sample was consisted of 50 predialysis (31 males and 19 females, mean age 59,06&plusmn;14,30) and 25 hemodialysis patients (18 males and 7 females, mean age 54,92&plusmn;13,60) with a diagnosis of CKD, along with 25 age and gender matched healthy controls. In addition of clinical examination, tongue blade adhesion test, sialometry, salivary pH test and determination of papilla bleeding index (PBI) were conducted. Saliva samples were collected for laboratory analysis performed by automated system Beckman Coulter AU480. Levels of uremic toxins (urea and creatinine) and secretory immunoglobulin A were determinated by spectrophotometric and immunoturbidimetric method, respectively. Oral swab and oral rinse method were used for microbiological examination. Results: The sociodemographic characteristics of the patients with CKD and healthy controls showed no significant differences. Predialysis subjects had significantly higher presence of oral mucosa changes and oral symptoms. Mean values of creatinine clearence were significantly lower in patients with oral mucosa pallor, uremic fetor, xerostomia and disguesia, compared to patients without listed symptoms. Predialysis patients showed significantly decreased salivary flow rate compared to both control groups and significantly increased pH values compared to healthy controls. Mean concentrations of salivary urea and creatinine were statistically different between the groups. Moderate positive correlation was determined between serum and salivary levels of urea and creatinine in predialysis patients and creatinine in hemodialysis patients. Statistical analysis showed no differences between groups in mean concentration of secretory immunoglobulin A. The rate of oral fungal colonisation was significantly higher in CKD patients with predominance of non-albicans Candida species. Conclusion: The results of the present study indicate the importance of determining the clinical characteristics of oral cavity in predialysis patients. Saliva flow rate, pH value and salivary concentration of uremic toxins could be reliable markers of kidney disease. Simple and non-invasive approach due to saliva sampling and reliability of laboratory test should contribute to a wider application of saliva as a competitive diagnostic fluid. Oral rinse technique is an accurate quantitative method for determining the rate of fungal colonization.</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...