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.

The TRAF-binding receptor CD40 belongs to the TNFR superfamily and is broadly expressed on healthy cells, mainly on antigen-presenting cells, but also on other immune cells and non-immune cells. CD40 is bound by its ligand CD40L, which is essential for a wide range of immunological responses by inducing or inhibiting different pathways that are essential for a variety of cellular processes, including immune activation and maturation. (1,2) Dysregulated CD40 signalling has been implicated in inflammatory diseases, such as hyper-IgM syndrome, psoriasis, and cancer. (3–6) Due to its broad expression across various tumour types, it can serve as a tumour-associated antigen and has therefore been proposed as a target for antibodies for cancer treatment. (2,7,8) Agonistic anti-CD40 antibodies have been demonstrated to induce anti-tumoural immune responses as well as therapeutic immunity. (2) Furthermore, prolonged stimulation of CD40 in tumour cells in vitro has been shown to decrease proliferation, increase expression of cytotoxic TNFSFLs and induce apoptosis. (9,10) Their effect on anti-tumoral responses has been well studied and anti-tumoral responses by DC maturation and suppression of malignant growth of B-cells have been confirmed and were found to induce cell death in tumours in vitro. (11–14) Many agonistic anti-CD40 antibodies specifically have been reported to require secondary crosslinking by binding to either activating or inhibitory FcγRs to be agonistic in vitro, while in vivo studies have indicated inhibitory FcƴR2B expression as critical factor. (15–17) However, FcƴR independent agonism has also been reported for anti-CD40 antibodies. (18,19) While agonistic anti-CD40 IgG1, IgG3 and IgG4 antibodies have been shown to display FcƴR dependent agonism, agonistic anti-CD40 IgG2 antibodies have shown to display FcƴR independent agonism. Conversion of anti-CD40 IgG1 antibodies into IgG2 has also been shown to convert the antibody’s agonism into FcƴR independent agonism. (20) To overcome FcƴR dependency, bispecific antibody fusion proteins containing a scFv as anchoring domain allowing for crosslink independent of FcƴR binding have been designed before. This approach has been found to display strong agonism for other antibody fusion proteins when bound to both targets, with response levels resembling that of FcƴR bound antibodies. (21,22) The relevance of antibody isotype and idiotype for FcƴR-dependent agonism as well as the relevance of valency and antibody oligomerization for FcƴR-independent agonism were investigated in this study on a panel of different anti-CD40 antibodies. Several clinically investigated anti-CD40 antibodies (ADC-1013(23), APX005M(24), ChiLob7.4(25) and CP-870,893(26)) and one preclinical antibody (G28.5(27,28)) were considered. Selected antibodies were then cloned onto an IgG1, IgG1(N297A), IgG2 and IgG4 backbone. The IgG1(N297A) isotype is an IgG1 antibody with a point mutation (N297A) that is known to strongly reduce binding to FcƴR1, while reducing the binding affinity to FcƴR2B to undetectable levels. (29,30) In this work it is demonstrated that the investigated anti-CD40 antibody variants across different isotypes activate both the classical and alternative NFκB pathway by stimulating U2OS cells in an FcƴR dependent manner. Stimulation in the presence of both human FcƴRs as well as murine FcƴRs resulted in CD40 stimulation. A difference in binding competition was observed for the various anti-CD40 IgG1 antibodies, but no indication of a CRD-dependent mechanism responsible for their agonistic activity was found. Moreover, this FcƴR dependency could be overcome by creation of tetravalent antibody fusion proteins. / Zusammenfassung Der TRAF-bindende Rezeptor CD40 ist Teil der TNFR-Superfamilie. CD40 wird auf gesunden Zellen, vor allem auf Antigen-präsentierenden Zellen, aber auch auf anderen Immunzellen und Nicht-Immunzellen in großem Umfang exprimiert. Die Aktivierung von CD40 durch seinen Liganden CD40L ist für zahlreiche immunologische Reaktionen von entscheidender Bedeutung, da sie verschiedene Signalwege induziert oder hemmt, die für zahlreiche zelluläre Prozesse, einschließlich Immunaktivierung und -reifung, wichtig sind. (1,2) Defekte CD40-Signalwege werden mit Entzündungskrankheiten wie dem Hyper-IgM-Syndrom, Psoriasis und Krebs in Verbindung gebracht. (3–6) Da CD40 auf verschiedenen Tumorarten weit verbreitet ist, kann es als tumorassoziiertes Antigen verwendet werden, das dann von zytotoxischen Antikörpern angegriffen werden kann, und wurde als Ziel für Antikörper zur Krebsbehandlung vorgeschlagen. (2,7,8) Es hat sich gezeigt, dass agonistische Anti-CD40-Antikörper antitumorale Immunreaktionen und therapeutische Immunität auslösen können. (2) Es hat sich gezeigt, dass die kontinuierliche Stimulation von CD40 in Tumorzellen in vitro die Proliferation reduziert, die Expression zytotoxischer TNFSFLs hochreguliert und Apoptose induziert. (9,10) Ihre Wirkung auf antitumorale Reaktionen wurde gut untersucht, und antitumorale Reaktionen durch DCReifung und Unterdrückung des malignen Wachstums von B-Zellen wurden bestätigt und es wurde festgestellt, dass sie in vitro den Zelltod in Tumoren induzieren. (11–14) Viele agonistische Anti-CD40-Antikörper erfordern Berichten zufolge eine sekundäre Vernetzung durch Bindung an entweder aktivierende oder hemmende FcγRs, um in vitro agonistisch zu wirken, während In-vivo-Studien auf eine hemmende FcƴR2B-Expression als kritischen Faktor hinwiesen. (15–17) Allerdings wurde auch für Anti-CD40-Antikörper ein FcƴR-unabhängiger Agonismus festgestellt. (18,19) Während agonistische Anti-CD40-IgG1-, IgG3- und IgG4-Antikörper nachweislich einen FcƴR-abhängigen Agonismus aufweisen, haben agonistische Anti-CD40-IgG2-Antikörper einen FcƴR-unabhängigen Agonismus gezeigt. Die Umwandlung von Anti-CD40-IgG1-Antikörpern in IgG2-Antikörper hat ebenfalls gezeigt, dass der Agonismus des Antikörpers in einen FcƴR-unabhängigen Agonismus umgewandelt wird. (20) Um die FcƴR-Abhängigkeit zu überwinden, wurden bereits bispezifische AntikörperFusionsproteine entwickelt, die ein scFv als Verankerungsdomäne enthalten und eine von der FcƴR-Bindung unabhängige Vernetzung ermöglichen. Es hat sich gezeigt, dass dieser Ansatz einen starken Agonismus für andere Antikörper-Fusionsproteine aufweist, wenn sie an beide Zielmoleküle gebunden sind, wobei die Ansprechraten denen von FcƴRgebundenen Antikörpern ähneln. (21,22) Die Bedeutung des Antikörper-Isotyps und des Idiotyps für den FcƴR-abhängigen Agonismus sowie die Bedeutung der Valenz und der Antikörper-Oligomerisierung für den FcƴRunabhängigen Agonismus wurden in dieser Studie an einem Panel verschiedener Anti-CD40-Antikörper untersucht. Es wurden mehrere klinisch untersuchte Anti-CD40-Antikörper (ADC1013(23), APX005M(24), ChiLob7.4(25) und CP-870,893(26)) und ein präklinischer Antikörper (G28.5(27,28)) berücksichtigt. Die ausgewählten Antikörper wurden dann auf ein IgG1-, IgG1(N297A)-, IgG2- und IgG4-Backbone kloniert. Beim Isotyp gG1(N297A) handelt es sich um einen IgG1-Antikörper mit einer Punktmutation (N297A), von der bekannt ist, dass sie die Bindung an FcƴR1 stark reduziert, während sie die Bindungsaffinität zu FcƴR2B auf ein nicht nachweisbares Niveau verringert. (29,30)In dieser Arbeit wird gezeigt, dass die Anti-CD40-Antikörper-Varianten verschiedener Isotypen sowohl den klassischen als auch den alternativen NFκB-Signalweg aktivieren, indem sie U2OS-Zellen in einer FcƴR-abhängigen Weise stimulieren. Die Stimulierung in Anwesenheit sowohl von humanen FcƴRs als auch von murinen FcƴRs führte zu einer CD40-Stimulierung. Während für die verschiedenen Anti-CD40-IgG1-Antikörper ein Unterschied in der Bindungskonkurrenz beobachtet wurde, konnten die CRD-Bindungsprofile keine Erkenntnisse über die zugrunde liegenden Mechanismen liefern. Darüber hinaus konnte diese FcƴR-Abhängigkeit durch die Herstellung von tetravalenten AntikörperFusionsproteinen überwunden werden.

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.