Proefschrift Universiteit Maastricht. / Auteursnaam op omslag: Joep Brinkmann. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Thesis (Ph.D.)--Boston University / The purpose of this investigation was to study the metabolism of normal human leukocytes. Leukocytes were incubated in vitro with c14-labeled acetate, and the fate of the radioactive carbon was determined by fractionation and analysis of the major, cell components. Leukocytes were obtained from whole blood by fibrinogen sedimentation and differential centrifugation. Optimal conditions for isolation and incubation of viable cells were developed and assessed using phase microscopy for direct observation of their morphological integrity and by their oxidative metabolism. Control experiments to determine the possible effect of erythrocyte utilization of acetate were run using twice the number of cells ordinarily found in leukocyte suspensions. No utilization of acetate by red blood cells was observed. Respiration studies were performed using standard Warburg manometry. Otherwise, incubations were carried out in modified Erlenmeyer flasks equipped with center wells for C02 collection and stoppered with serum bottle caps. Flasks were equilibrated and various additions were made to the suspensions using hypodermic needles. Total cells per flask varied from 1 to 5 X 108 with concentrations ranging from 60 to 80 X 106 cells per ml. of suspension. Response of respiration and combustion of acetate were directly proportional to cell number and no detrimental effects due to cell crowding were detectable within this range. [TRUNCATED]

CD23, the low affinity IgE receptor, is expressed by various cell types and has numerous functions depending on the form of the protein, its interaction with various ligands and the type of cell involved. CD23 is pivotal in the regulation of IgE, with the soluble form involved in up-regulation, while the membrane bound form is involved in the down-regulation. It is clear why it is believed to be a central molecule in allergic responses, and a therapeutic target for the treatment of allergic disease. In this study a recombinant form of the entire extracellular domain of the protein, exCD23, was produced by PCR cloning and expressed in E. coli. His•Tag™s were introduced onto the C-terminus and N-terminus, respectively, in order to simplify the purification procedure. After renaturation and purification, the recombinant exCD23 bound IgE, indicating its activity. From the IgE binding studies it was established that the position of the tag did not influence the binding. GST•Tagged™ exCD23 was also produced in an attempt to increase the solubility of the recombinant protein, but this proved unsuccessful. Butyrate differentiated EoL-1 cells were treated with the Nterminal His•Tagged™ exCD23, and the protein appeared to suppress the secretion of the constitutively expressed cytokines, especially IL-8 and IFN- , when compared to untreated cells. In addition, treatment of the EoL-1 cells with exCD23 had a significant proliferative effect, but could not induce differentiation of this cell line into mature eosinophilic-like cells.

Orientador: Lilian Maria de Castilho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T12:16:53Z (GMT). No. of bitstreams: 1 Melo_Laerciode_D.pdf: 15534575 bytes, checksum: 0d830bc1ab58a6f7460b5beacb32557c (MD5) Previous issue date: 2006 / Resumo: O Programa de Controle de Qualidade Externo em Imunohematologia foi introduzido com o objetivo de avaliar a qualidade do diagnóstico em Imunohematologia. Foram realizadas 41 avaliações em 223 instituições no período de 1992 a 2003 que incluíram testes de proficiência para determinação ABO e RhD, fenotipagem Rh e K, teste direto da antiglobulina humana, pesquisa de anticorpos irregulares e identificação de anticorpos. No período de 12 anos o programa incluiu 8014 determinações de grupo sanguíneo ABO, 8000 classificações RhD, 5193 fenotipagens Rh, 5101 fenotipagens K, 7939 pesquisas de anticorpos irregulares, 4533 identificações de anticorpos e 7912 testes diretos da antiglobulina humana. Respostas incorretas foram classificadas como erros clericais, técnicos, ou não determinados. Ocorreu um número elevado de erros clericais na determinação do grupo sanguíneo ABO (76/76 erros), classificação RhD (34/58 erros) e na fenotipagem Rh (50/73 erros). Erros técnicos ocorreram predominantemente na determinação do antígeno D fraco (91/95 erros), na pesquisa de anticorpos irregulares (252/301 erros) e na identificação de anticorpos (321/335 erros) e estavam associados à sensibilidade das técnicas e dos reagentes utilizados. As avaliações teóricas, como incentivo à educação continuada, auxiliaram na diminuição dos erros, estimularam o treinamento dos participantes e o trabalho em equipe. A detecção dos erros foi importante para propor melhoria técnica com reavaliação dos protocolos bem como ações corretivas visando à melhoria da qualidade. A participação em um programa de controle de qualidade externo bem organizado mostrou ser fundamental na melhoria da qualidade dos testes em imunohematologia, reduzindo a freqüência dos erros e no potencial risco de reações hemolíticas garantido uma melhoria na qualidade das transfusões / Abstract: The Brazilian External Quality Assessment Program in Immunohematology (BEQAPI) was introduced with the objective of evaluating the quality of diagnosis in Immunohematology. From 1992 to 2003, proficiency tests for ABO grouping, Rh (D, C, c, E, e), K phenotyping, Direct Antiglobulin Testing (DAT), Antibody Screening (AS) and Antibody Identification (AI) were performed. Forty-one evaluations were carried out in 223 institutions. Over the period of 12 years, the program included 8,014 ABO typing, 8,000 RhD typing, 5,193 Rh (C, c, E, e), 5,101 K phenotyping, 7,939 AS, 4,533 AI and 7,912 DATs. Erroneous responses were classified as clerical, technical or undetermined. A substantial proportion of erroneous responses due to clerical errors occurred in ABO typing (76/76 errors), RhD typing (34/58 errors) and Rh phenotyping (50/73 errors). Technical errors occurred predominantly for weak D (91/95 errors), AS (252/301 errors) and AI (321/335 errors). Based on these results, since 1996, participants have received ¿Questions and Case Studies¿ as an incentive for training and education. The results of the present study show an improvement in the performance of participants in the course of the program. We found that a well-organized external proficiency program can contribute to the improvement of quality of testing in Immunohematology / Doutorado / Clinica Medica / Doutor em Clínica Médica

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that can be transmitted to humans through direct contact with infected animals or consumption of contaminated food products. Current serological tests used to identify infected animals rely on the detection of antibodies to O-antigens of the smooth lipopolysaccharide (sLPS) of B. abortus. Due to the presence of structurally similar O-antigens of bacteria such as E. coli O:157 and Yersinia enterocolitica O:9, these tests can produce false-positive results, which requiring alternative protein target antigens. We hypothesize that through comparative genomics and bioinformatics analysis of all ORFs within the B. abortus genome, followed by profiling of the humoral immune responses to surface and extracellular proteins, novel protein antigens with diagnostic potential may be discovered. In this study, the genomes of thirteen strains were analyzed using a subcellular localization prediction database (PSORTb) to identify proteins in the outer membrane and extracellular space. A total of 100 ORFs coding for such proteins including known immunogenic proteins reported in literature were identified and selected for recombinant protein expression using high-throughput in vivo cloning and in vitro transcription/translation strategies. The in vitro expression of 67 of these candidates has been successfully demonstrated. These recombinant B. abortus proteins were subsequently probed with E. coli pre-adsorbed sera from infected animals for the identification of immunoreactive protein antigens. Ten unique candidates were demonstrated to be antigenic and have the potential for diagnostic applications. This study illustrates a unique, high throughput strategy to express and screen proteins of a bacterial pathogen for novel diagnostic antigen discovery.

I. A number of commercial enzyme immunoassay (EIA) tests are available for the diagnosis of giardiasis. In a time of rising health-care costs, there is a need for diagnostic tests that are rapid, specific, sensitive and inexpensive. In the first phase of this study, I developed a monoclonal-based EIA, the GIARDIA TEST, with these qualities in mind. This assay's performance characteristics were determined by a comparison study using conventional ova and parasite examination, immunofluorescence antibody test (IFA) and other commercial EIA tests. Studies were done in-house at TechLab, Inc. and at various U.S. medical facilities. Results were statistically analyzed to determine sensitivity (ability of the assay to detect a positive result), specificity (amount of cross-reactivity), predictive positive value (the confidence in a positive result), predictive negative value (the confidence in a negative result) and overall correlation with the reference assay. II. There remain many questions to be answered about the various antigens produced by Giardia lamblia and how they can be utilized as diagnostic markers. In the second phase of this study, I identified and partially characterized the antigen (Ct7 Ag) that reacts with the Ct7 monoclonal antibody (MAb). This MAb is an IgM class mouse immunoglobin that is utilized by the GIARDIA TEST and by an immunofluorence antibody test (IFA) which detects Giardia cysts in water and feces. The results of this study will provide physicians and researchers with detailed information about the Ct7 Ag and why it is a useful marker for giardiasis. / Master of Science

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No description available.

Hepatitis B virus is a member of the hepadnavirus family. The hepatitis B virus core gene codes for two proteins viz. core protein and pre-core protein. These proteins assemble to form particles viz. HBcAg and HBeAg respectively. The structure of the HBcAg has been widely studied but very little is known about the structure of HBeAg. Therefore, the aim of this study was to identify the disulfide bonding patterns in HBeAg. Recombinant HBeAg was isolated from E.coli and used for this study along with various mutants of HBeAg. There are four cysteines present in HBeAg each at position -7, 48, 61 and 107. From this study it can be inferred that the cysteine at 61 and 48 were found to be involved in inter-molecular disulfide bonds between the cysteine at 61 and 48 of other identical monomers. These di-mers were further inter-molecularly linked with cysteine at -7 to form chains. Moreover, the cysteine at -7 and cysteine at 107 were sometimes involved in intra-molecular disulfide bond formation. Thus, the HBeAg in a solution was found be particulate with a heterogeneous pattern of inter chain disulfide bonds.

Ein Tumor stellt nicht nur eine Ansammlung entarteter Zellen dar, sondern ist vielmehr ein komplexes Pseudoorgan, das aus Tumorzellen und aus mit ihnen assoziierten „normalen“ Zelltypen, wie Fibroblasten, Endothelzellen und Makrophagen, den sogenannten Tumorstromazellen, besteht. Die Tumorstromazellen wurden von den Tumorzellen dahingehend konditioniert, dass sie das Tumorwachstum und -progression fördern. Im Rahmen der vorliegenden Arbeit wurde die Bedeutung von zwei Oberflächenmolekülen, nämlich CD147 und CD28, für solche im Tumorstroma stattfindenden Interaktionen im syngenen murinen B16 Melanommodell untersucht. Rolle von CD147 für MMP Expression, Neoangiogenese und Metastasierung CD147, das von Tumorzellen exprimiert wird, wird als ein Faktor angesehen, der auf benachbarten Stromazellen die Expression von MMPs induziert. MMPs sind essentiell für den Umbau der extrazellulären Matrix und der Basalmembranen und somit für die Invasion und Metastasierung des Tumors essentiell. Daneben gibt es erste Hinweise, dass CD147 auch die Induktion von vascular endothelial growth factor (VEGF) vermittelt und damit die Tumorangiogenese fördert. In dem eingesetzten Melanommodell war überraschenderweise kein Unterschied hinsichtlich der Expression von MMP-2, MMP-9 und MT1-MMP in Abhängigkeit von der CD147 Expression nachweisbar. Die in vitro Kokultur der Melanomzellen mit unterschiedlichen murinen Fibroblasten zeigte zudem, dass weder CD147+ noch CD147- Melanomzellen die Expression von MMP-2 oder MMP-9 in den Fibroblasten veränderten. Als eindeutige Effekte des CD147 knock downs wurde aber eine reduzierte VEGF Expression in vivo einhergend mit einer gehemmten Tumorangiogenese, sowie einer reduzierten Metastasierung festgestellt. Es konnte somit die Funktion von CD147 in dem gewählten Modell als angiogenetischer, jedoch als MMP unabhängiger, Metastasierungsfaktor demonstriert werden. Einfluss von CD28 auf antitumorale Immunantworten CD28 ist ein kostimulatorisches Molekül, das zusammen mit dem TCR für eine effiziente Stimulation von T-Lymphozyten wesentlich ist. In CD28 k.o Mäusen fand sich im Vergleich zu Wildtyp Kontrolltieren eine verminderte Effektivität von prophylaktischen anti-Tumor Impfungen, die sich in einem beschleunigten Tumorwachstum sowie einer erhöhten Tumorlast auswirkten. Die Frequenz von Vakzine induzierten TRP-2180-188 /Kb reaktiven CD8+ T-Zellen in TIL von Tumoren war aber in beiden Genotypen gleich. Dagegen war die Anzahl IFN- produzierender TRP-2180-188 /Kb reaktiver T-Zellen sowie die Fähigkeit der TRP-2180-188 /Kb reaktiven T-Zellen zu lysieren, in den CD28-defizienten Mäusen deutlich geringer. Diese Beobachtungen legen nahe, dass CD28-vermittelte kostimulatorische Signale im gewählten Modell weniger für die initiale Expansion als für die Differenzierung funktioneller tumorspezifischer CD8+ T-Effektorzellen eine wesentliche Funktion einzunehmen scheinen. / Malignant tumors not only represent an accumulation of neoplastic cells but should be seen as a complex pseudoorgan, which consists on tumor cells and tumor-associated normal cell types, e.g. fibroblasts, endothelial cells and macrophages, the so-called tumorstroma cells. Tumorstroma cells were unprogrammed from tumor cells, facilitating both tumor growth and progression. In the present work, the significance of two cell surface molecules, CD147 and CD28, was investigated in a syngeneic melanoma model. Role of CD147 in MMP induction, neoangiogenesis and metastasis formation CD147, which is expressed by tumor cells, is regarded as a key factor, which induces the expression of MMPs in adjacent stroma cells. MMPs are essentiell for degradation of the extracellular matrix and basal membranes and consequently important for invasion and metastasis formation of tumors. Furthermore, there are some indices, that CD147 can mediate also the induction of vascular endothelial growth factor (VEGF) and therefore promote neoangiogenesis. In the B16 melanoma model, surprisingly no differences were detectable, regarding the expression of MMP-2, MMP-9 and MT1-MMP in dependence of CD147 expression. Co-culture of melanoma cells with different fibroblast cell lines demonstrated that neither CD147+ nor CD147- melanoma cells altered the expression of MMP-2 or MMP-9 in the fibroblasts. As distinct results of the CD147 knock down a reduced VEGF expression in vivo accompanied by inhibited angiogenesis as well as reduced metastasis was observed. Thus, in the used model, CD147 can be regarded as angiogenic, however, MMP independent metastasis formation factor. Influence of CD28 on anti-tumoral immune responses CD28 is a costimulatory molecule which is essential in conjunction with the TCR for efficient stimulation of T lymphocytes. In CD28 k.o. mice a reduced efficacy of prophylactic anti-tumor vaccinations, which resulted both in an accelerated tumor development and an increased tumor load compared with wildtyp mice was detected. The frequency of TRP-2180-188/Kb -reactive CD8+ T cells among TILs, however, was similar in both genotypes. In contrast, the number of IFN-γ -producing TRP-2180-188/Kb -reactive T cells and the efficiency of the TRP-2180-188/Kb -reactive T cells lysing target cells, was reduced in CD28-deficient mice. These results suggest that CD28-mediated costimulatory signals, at least in the used model of CD28 k.o. mice, seem to be less essential for the initial expansion than for the differentiation of functional tumor-specific CD8+ T-effector cells.