Intimina é o principal fator de virulência envolvido na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecção de EHEC e EPEC típica ou atípica é de fundamental importância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos países desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentaram 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 µg desse anticorpo reconheceu 0,6 µg de intimina purificada com uma constante de dissociação de 1.3 x 10-8 M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isso, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplificadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressão pAE. Linhagem de E. coli BL21(DE3)pLys foi transformada com o plasmídeo pAE-scFv antiintimina e submetida à indução protéica. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteína por 100 mL de cultivo bacteriano. Para testar a funcionalidade do scFv, foram realizados ensaios de ELISA de captura e imunofluorescência. Os resultados mostraram que 275 ng de scFv reagiram com 2 µg de intimina purificada a uma absorbância de aproximadamente 0,75 e por imunofluorescência mostrou uma forte reatividade ao isolado de EPEC típica E2348/69. Este estudo demonstrou que o anticorpo recombinante anti-intimina obtido foi capaz de reconhecer a região conservada de intimina (Int388-667) na forma purificada e a intimina α no isolado de EPEC típica, e se mostrou mais eficiente que o anticorpo monoclonal nativo. / Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections caused by E. coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificity of polyclonal and monoclonal antibodies against intimin in the detection of EPEC and EHEC by immunoblotting. All employed antibodies showed 100% specificity and the sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, respectively. This anti-intimin monoclonal was characterized as IgG2b and 1 mg recognized 0.6 µg of purified intimin with a dissociation constant of 1.3 x 10-8 M. The less extent reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFv). Thus, the anti-intimin hybridoma mRNA was extracted, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in a strategy to amplify and link the chains, obtaining the scFv, which was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction of protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed. The results showed that 275 ng of scFv reacted with 2 µg of purified intimin resulting in an absorbance of 0.75. By immunofluorescence it was observed a strong reactivity to the typical EPEC isolate E2348/69. This study demonstrated that the recombinant anti-intimin antibody obtained was able to recognize the conserved region of intimin (Int388-667) in its purified form and α intimin in a typical EPEC isolate, and was more efficient than the native monoclonal antibody.

Made available in DSpace on 2014-08-20T13:33:00Z (GMT). No. of bitstreams: 1 tese_marcelo_mendonca.PDF: 4204978 bytes, checksum: e41a9490cdb350a5e7add8e129afdd82 (MD5) Previous issue date: 2011-12-01 / The conventional methods used to detect the Listeria monocytogenes in foods are laborious and expensive, requiring several days for final identification. Monoclonal antibody (MAb) based immunoassays are highly specific and rapid to perform, especially when MAbs are raised to conserved virulence factors in the pathogen. Among diverse virulence factors of L. monocytogenes, the surface protein internalin A (InlA) is one of the most well-known and characterized protein, being an excellent target as it is highly exposed on the surface and exclusive of pathogenic species. In this work we report the production, characterization and use of a panel of MAbs against InlA (2D12, 3B7, 4E4), and a MAb (3F8) which specifically recognizes all bacteria belonging the genus Listeria. MAbs were produced by the immunization of BALB/c mice with a recombinant InlA together with heat killed L. monocytogenes. The MAbs produced showed excellent reativities by indirect ELISA, Western blot and immunofluorescence. A Cy5 conjugated anti-InlA MAb-2D12 was used as detection antibody for L. monocytogenes in a sandwich-like fiber optic immunoassay. Using MAb-2D12 as capture antibody on the waveguides, the limit of detection was ~3 x 102 CFU.mL-1, and when MAb-3F8 was used for capture the limit of detection was ~1 x 105 CFU.mL-1. Furthermore, MAbs 2D12 and 3F8 were used to coat paramagnetic beads and tested in the immunomagnetic separation (IMS) of L. monocytogenes from pure cultures, and artificially contaminated cheeses and hotdogs. After IMS capture, bacteria were released from the beads, used in the fiber optic assay or plated on agar for counting. In parallel, the capture of L. monocytogenes was confirmed by real-time qPCR and light-scattering technology (BARDOT). Using IMS to concentrate and separate L. monocytogenes, followed by a fiber optic platform, it was possible to detect in less than 22 h, approximately 40 CFU/g of L. monocytogenesi, even in the presence of L. innocua in cheese and hot dogs artificially contaminated. In addition, using mass spectrometry (MALDI-TOF-MS) the protein to which MAb-3F8 binds, was identified as fructose 1,6-bisphosphate aldolase (FBA). The results presented in this work indicate that using both systems together, the IMS and fiber optic immunosensor, were more reliable and faster, and could be applied in the routinely for detection of L. monocytogenes in food. Moreover, both MAbs have the potential to useful in others biosensor platforms, as well as in other detection and functionality immunoassays for InlA and FBA in Listeria. / Os métodos convencionais empregados para detecção de Listeria monocytogenes em alimentos são laboriosos e onerosos, requerendo vários dias para sua identificação final. A utilização de anticorpos monoclonais (MAbs) em imunoensaios para detecção rápida de bactérias tem como vantagem a alta especificidade e rapidez, principalmente quando direcionados para fatores de virulência conservados. Dentre os diversos fatores de virulência de Listeria, a proteína de membrana internalina A (InlA), é umas das mais bem caracterizadas, sendo um excelente alvo por ser altamente exposta na superfície e exclusiva de espécies patogênicas. Neste trabalho é relatado a produção, caracterização e utilização em métodos de diagnósticos de um painel de MAbs contra a InlA (2D12, 3B7, 4E4), e de um MAb (3F8) que reconhece especificamente todas as bactérias do gênero Listeria. Na produção dos MAbs, camundongos BALB/c foram imunizados com uma proteína recombinante InlA (rInlA) concomitantemente com L. monocytogenes inativadas por fervura. Os MAbs gerados demonstraram excelente reatividade por ELISA indireto, Western blot e imunofluorescência. O MAb anti-InlA 2D12 marcado com Cy5 foi usado como anticorpo de detecção de L. monocytogenes, no sistema tipo sanduíche de sensor de fibra óptica. Usando MAb-2D12 como anticorpo de captura nas fibras ópticas, obteve-se um limite de detecção de ~3 x 102 CFU.mL-1, e um limite de detecção de ~1 x 105 CFU.mL-1 foi visualizado com MAb-3F8 como captura. Os MAbs anti-InlA 2D12 e anti-Listeria 3F8 foram posteriormente utilizados para sensibilizar esferas paramagnéticas e testados na separação imunomagnética (IMS) de L. monocytogenes em culturas puras, e em queijo e salsichas tipo hotdog artificialmente contaminados. Após a captura por IMS, as bactérias foram liberadas, incubadas com a fibra óptica ou plaqueadas em agares para contagem. Em paralelo, a confirmação da captura de L. monocytogenes foi realizada por PCR quantitativo em tempo real e por light-scattering technology (BARDOT). Utilizando IMS para separar e concentrar L. monocytogenes, seguido da utilização em plataforma de fibra óptica, foi possível realizar a detecção em menos de 22 horas, de aproximadamente 40 UFC/g de L. monocytogenes em presença de L. innocua, em queijo e salsicha artificialmente contaminados. Além disso, a proteína alvo do MAb3F8 foi identificado como frutose 1,6-bifosfato aldolase através de espectrometria de massa (MALDI-TOF-MS). Os resultados obtidos nesse trabalho indicam que a utilização em conjunto dos sistemas de IMS e fibra óptica com os MAb-2D12 e MAb3F8, foram confiáveis e rápidos, e assim, podendo ser empregados em imunoensaios de rotina para detecção de L. monocytogenes em alimentos. Contudo, ambos MAbs possuem ainda grande potencial para serem mais explorados em outras plataformas de biossensores, assim como, em outros imunoensaios de detecção e funcionalidade de InlA e FBA em Listeria

Dans l’approche expérimentale de l’immunothérapie des tumeurs solides, les modèles murins sont utilisés pour des raisons de rapidité et de reproductibilité. Le plus souvent, les modèles tumoraux murins sont ectopiques ce qui constitue un modèle artificiel qui ne reflète qu’une partie de la réalité biologique de tumeurs provenant de diverses origines.Mon projet de thèse a consisté à mettre au point de nouveaux modèles pré-cliniques murins permettant de mieux mimer les situations pathologiques des tumeurs solides chez l’homme. Pour cela, je me suis notamment intéressée à un modèle orthotopique de cancer du rein (implantation de cellules RenCa ou RenCa-MUC1 dans la capsule rénale) et à un modèle spontané de cancer du sein (souris MMTV-PyMT). Ces modèles ont ensuite permis l’évaluation de trois différentes approches d’immunothérapie à savoir la thérapie virale oncolytique, la vectorisation d’antigènes tumoraux au moyen d’un vecteur viral ainsi que la thérapie via un anticorps monoclonal. / In experimental approaches to immunotherapy of cancer, mouse tumor models are used for reasons of speed and reproducibility. Ectopic mouse tumor models are the most often used, but they constitute artificial models that reflect only a part of the biological reality of tumors from various origins.The aim of my thesis project was to develop new mouse preclinical tumor models to better mimic the pathological situations of solid tumors in humans. First, I developed an orthotopic model of kidney cancer (subcapsular kidney implantation of a renal carcinoma cell line which either expressed or did not express the human xeno-antigen, MUC1). In addition to this, I also studied a spontaneous model of breast cancer (MMTV-PyMT).These models enabled us to evaluate the efficacy of three different immunotherapy approaches namely oncolytic virus strategy, tumor antigen vectorization by using a viral vector, and monoclonal antibody.

In Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), aggregated α-synuclein deposit inside cells within the brain. Smaller soluble α-synuclein aggregates, oligomers, are present both intra- and extracellularly. The α-synuclein oligomers are known to be particularly harmful, although the underlying neurotoxic mechanisms are not fully understood. The aim of this thesis was to investigate the pathogenic roles of α-synuclein oligomers and the possibility to target such species with antibody treatment. Passive immunotherapy with α-synuclein antibodies can lead to reduced pathology and ameliorated symptoms in transgenic mice. However, it remains unknown whether the antibodies are taken up by cells or whether they act extracellularly. In Paper I, we assessed cellular internalization of various α-synuclein monoclonal antibodies. The oligomer selective mAb47 displayed the highest uptake, which was promoted by the extracellular presence of α-synuclein. Alpha-synuclein aggregates can be found in both neurons and glial cells, but the pathogenic role of glial deposits has only been sparsely investigated. In Paper II, co-cultures of neurons and glia were exposed to α-synuclein oligomers. The astrocytes in the cultures rapidly accumulated oligomers, which were only partially degraded by lysosomes. The sustained intracellular α-synuclein deposits were associated with mitochondrial stress reactions in the astrocytes.  In Paper III, we sought to explore whether the astrocytic pathology induced by α-synuclein oligomers could be ameliorated by antibody treatment. Pre-incubation of oligomers with mAb47 promoted α-synuclein clearance, reduced astrocytic accumulation and rescued cells from mitochondrial stress. We could demonstrate that binding of the antibody to its antigen in the extracellular space was crucial for these effects to occur. The progressive pathology in PD is believed to be driven by cell-to-cell spreading of α-synuclein aggregates, potentially via exosomes and other extracellular vesicles (EVs). In Paper IV, we found that either fusing α-synuclein to a non-physiological protein tag or introducing the PD-causing A53T mutation directed α-synuclein towards EV secretion. Also, EV-associated α-synuclein was particularly prone to induce toxicity in recipient cells. In conclusion, this thesis sheds new light on the cellular dysfunction related to α-synuclein pathology and on how the underlying pathogenic processes may be targeted by antibody treatment.

En Afrique, les fièvres récurrentes causées par différentes espèces bactériennes du genre Borrelia sont des infections négligées transmises par les arthropodes et sont responsables de manifestations cliniques variant d’une septicémie mortelle à des formes plus bénignes et d'autres manifestations cliniques, en particulier d'avortement chez les femmes enceintes. Quatre espèces différentes de Borrelia, initialement séparées les unes des autres sur la base de leur répartition géographique et de leur vecteur, sont actuellement cultivées de prélèvements cliniques et de vecteurs : Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. Ces différentes espèces circulent sur le continent africain en parallèle avec au moins six espèces non encore cultivées et détectées dans des vecteurs. Notre travail est une contribution à l’investigation des fièvres récurrentes à Borrelia en Afrique. Dans cette perspective, nous avons mis au point la détection rapide en spectrométrie de masse MALDI-TOF des Borrelia dans les tiques en créant au préalable une base de données Borrelia MALDI-TOF-MS. La base de données de Borrelia et un logiciel de soustraction IHU ont été utilisés pour détecter B. crocidurae dans 20 tiques Ornithodoros sonrai, y compris huit tiques qui ont été testées positives pour B. crocidurae par PCR-séquençage, ce qui ouvre la voie à l'utilisation du MALDI-TOF-MS pour la double identification des vecteurs et des agents pathogènes vectorisés, dont il s’agissait du premier exemple maintenant étendu à d’autres modèles dans notre laboratoire. / In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and other clinical manifestations, particularly abortion in pregnant women. Four different species of Borrelia, initially distinguished one from another on the basis of geography and vector, are currently cultured causative agents in Africa: Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. These different species are circulating in parallel to at least six not-yet cultured species in vectors. Our work consisted in the investigation of recurrent fevers borreliosis in Africa. We have developed rapid detection in MALDI-TOF mass spectrometry of Borrelia in ticks by creating a prior a Borrelia MALDI-TOF-MS database. The Borrelia database and a custom software program that subtracts the uninfected O. sonrai profile were used to detect B. crocidurae in 20 O. sonrai ticks, including eight ticks that tested positive for B. crocidurae by PCR-sequencing; which paves the way for the use of MALDI-TOF-MS for the dual identification of vectors and vectorized pathogens. We have also illustrates a non-specialized circulation of B. crocidurae borreliae within a collection of 35 O. sonrai ticks in West Africa. These ticks were genotyped by 16S rRNA mitochondrial gene sequencing while B. crocidurae was genotyped by Multispacer Sequence Typing (MST). The 35 ticks were grouped into 12 genotypes strong geographic structuring and 35 B. crocidurae into 29 genotypes without strict geographic structure. One O. sonrai genotype carried several B. crocidurae genotypes and one B. crocidurae genotype was found in different O. sonrai genotypes.

Type II Ribosome Inactivating Proteins (RIPs), commonly known as A/B toxins are heterodimers comprising of a catalytically active A chain, an RNA N-glycosidase which inhibits protein synthesis and a lectin-like B chain required for the binding of the toxin to the cell surface and internalization of the same. Abrin is a type II RIP obtained from the mature seeds of Abrus precatorius plant that is extremely toxic and has been shown to be 75 times more potent than its well studied sister toxin, ricin. The LD50 dose for abrin is only 2.8 µg/kg body weight of mice and its potential use in bio-warfare is a cause of major concern. Abrin has been classified as a select agent by the Centre for Disease Control and Prevention, U.S.A., because it is stable, effective at very low concentrations and easy to purify and disseminate in large amounts. In spite of abrin being a potential bio-warfare agent, there is no antidote or vaccine available against this toxin till date. The first and only neutralizing monoclonal antibody (mAb) against abrin, namely D6F10, was reported from our laboratory and has been shown to rescue toxicity of abrin in cells as well as in mice. The study reported in the thesis focuses on understanding the mechanism of neutralization of abrin by the mAb D6F10 and development of a potential vaccine candidate against the toxin. In order to map the epitope corresponding to the antibody, first, overlapping gene deletion constructs spanning the entire length, 251 amino acids, of ABA were generated and checked for binding to the mAb. Fragments shorter than 1-175 did not show immuoreactivity. Analysis of the crystal structure of abrin A chain revealed that a helix spanning the amino acids 148-167 was present at the core of the protein structure and truncation in this region of the protein possibly results in loss of conformation leading to abrogation of antibody binding. Therefore, a novel strategy of epitope mapping was adopted. Abrus precatorius agglutinin (APA) is a homologue of abrin obtained from the same plant source. The A chains of abrin and APA share 67% sequence identity and their crystal structures superimpose very well but unlike abrin the APA A chain does not bind the mAb D6F10. Chimeric constructs were generated within the region 1-175 of A chains of both ABA and APA and deletions and mutations of the ABA was then made on the APA as scaffold. It could be concluded that the amino acids of the region 75¬123 are involved in the formation of the epitope. Further, based on sequence alignment of ABA and APA A chain 13 residues in the chimera ABA1-123APA124-175 were mutated and it was found that the mutation of the residues Thr 112, Gly 114 and Arg 118 resulted in loss of binding to the antibody. Furthermore, the mAb D6F10 rescues inhibition of protein synthesis by abrin in HeLa cells by internalizing in cells along with abrin and possibly occluding the active site cleft of ABA. The antibody prevents cell attachment of abrin at higher concentrations. The observations provide novel insights into mechanisms of many known neutralizing antibodies against A/B toxins. The study also highlights that chimeric protein constructs could possibly be developed as potential vaccine candidates for neutralization of abrin intoxication.

INTRODUÇÃO: A deficiência de IgA (DIgA) é a imunodeficiência primária mais comum e caracteriza-se pela presença de concentrações de IgA sérica abaixo de 7 mg/dL e níveis normais de IgM e IgG. A maioria dos indivíduos acometidos não apresenta doença aparente embora alguns possam apresentar infecções recorrentes ou crônicas de mucosas, atopia e/ou doenças autoimunes (DAIs). Presumivelmente, a doença resulta de um defeito na troca de isótipo para IgA ou de falha na maturação de linfócitos produtores de IgA. A imunodeficiência comum variável (ICV) constitui uma deficiência primária de anticorpos caracterizada por níveis séricos baixos de IgG, IgA e/ou IgM, ao lado de valores normais ou diminuídos de linfócitos B e/ou T, levando a infecções crônicas ou recorrentes principalmente dos tratos respiratório e gastrintestinal. Embora a fisiopatologia da ICV ainda não esteja esclarecida, em muitos pacientes ela pode ser decorrente de algum defeito intrínseco de linfócitos B. De modo especial, as células B de memória (CD27+) têm sido correlacionadas com alguns aspectos clínicos da doença. Números elevados de células B de memória com persistência de IgM (CD27+IgM+) parecem estar correlacionados com a presença de infecções, enquanto valores diminuídos de células B de memória clássicas ou class-switched (CD27+IgG-IgM-) parecem estar associados a baixos níveis de IgG e presença de autoimunidade. A progressão de DIgA para ICV tem sido descrita em alguns pacientes embora não constitua regra geral. Uma hipótese é a de que uma base genética comum e a associação com DAIs possam constituir fatores de risco para a progressão de DIgA para ICV. Há relato anterior de que a persistência de células B imaturas IgM+ IgD+ em alguns pacientes com DIgA estava associada à progressão para ICV. Adicionalmente, há evidências de que a diminuição de células B de memória em uma proporção de pacientes com ICV esteja associada à presença de autoimunidade. OBJETIVOS: comparar em pacientes com DIgA e ICV várias subpopulações de células B e analisar a relação entre estas populações celulares e a presença de DAIs em ambos grupos. MÉTODO: Este estudo incluiu 56 pacientes adultos de ambos sexos com DIgA e ICV, distribuídos em grupos de acordo com a associação com DAI: grupo DIgA sem DAI (14 pacientes), grupo DIgA com DAI (14 pacientes), grupo ICV sem DAI (14 pacientes) e grupo ICV com DAI (14 pacientes). As seguintes subpopulações de células B foram determinadas por citometria de fluxo de quatro-cores: células B naive (CD19+IgM+), células B de memória clássicas ou class-switched (CD27+IgM-IgD-) e células B de memória imaturas (CD27+IgM+ or CD27+IgD+). Na análise estatística foi aplicado o teste de ANOVA; valores significativos foram determinados pela correção de Bonferoni. RESULTADOS: os grupos analisados foram homogêneos quanto à idade e distribuição de gêneros. Os valores de linfócitos totais e de células B naive foram similares nos quatro grupos estudados. Os pacientes com deficiência de IgA e ICV com DAIs associadas apresentaram valores igualmente aumentados de células B de memória imaturas CD27+IgM+ e CD27+IgD+ quando comparados a pacientes sem doenças autoimunes. CONCLUSÕES: estes resultados sugerem que a persistência de células B de memória imaturas possa estar relacionada à presença de autoimunidade em pacientes com DIgA e ICV. Especula-se se a persistência destas células em pacientes com DIgA e DAI associada possa constituir fator preditivo da progressão de DIgA para ICV. / INTRODUCTION: IgA deficiency (IgAD) is the most common primary immunodeficiency disorder and is characterized by serum IgA concentration below 7 mg/dL and normal serum IgM and IgG levels. Most of the affected individuals have no apparent disease, whereas selected patients suffer from recurrent or chronic mucosal infections, atopy and/or autoimmune diseases (AIDs). This defect is presumed to result from impaired class-switching to IgA or from maturational failure of IgA-producing lymphocytes. Common variable immunodeficiency (CVID) is a primary antibody deficiency disease characterized by low serum levels of IgG, IgA and/or IgM, and normal or decreased B and/or T cell numbers, leading to chronic or recurrent infections, noted mostly in the respiratory and gastrointestinal tract. While the pathophysiology of CVID remains elusive, in many patients it may be due to an intrinsic B cell defect. Memory B cells (CD27+) in particular, have been noted to correlate with certain clinical aspects of the disease. High numbers of IgM+ memory B cells (CD27+IgM+) appear to correlate with the presence of infections, whereas decreased numbers of classic (class-switched) memory B cells (CD27+IgG-IgM- ) correlate with lower serum IgG levels and increased rates of autoimmune features. Progression from IgAD to common variable immunodeficiency (CVID) has been reported in some patients, but is not a general rule. It is postulated that a common genetic base and association with AIDs could be risk factors for progression from IgAD to CVID. OBJECTIVES: The aim of this study was to compare B cell subpopulations of patients with IgAD and with CVID, and to assess the relationship between these populations and the presence of autoimmune diseases in both group of patients: . METHOD: The study included 56 adult patients of both genders with IgAD or CVID. Patients were grouped, according to the association with autoimmune disease,as follows: group IgAD with AID (14 patients), group IgAD without AID (14 patients), group CVID with AID (14 patients) and group CVID without AID (14 patients). We determined by immunophenotyping of lymphocytes by four-colour cytometry the following subpopulations of B cells: naïve B cells (CD19+IgM+), class-switched memory B cells (CD27+IgM-IgD-) and immature B memory cells (CD27+IgM+ or CD27+IgD+). Statistical analysis was performed by the ANOVA test; significant P-values were determined by means of Bonferonis correction. RESULTS: there is no statistically significant difference between the average ages and the gender of patients between the groups. the distribution of the sample values seem to indicating that, there is no statistically significant difference in the CD19 levels between the groups. patients with AID represent greater values of CD27 IgM+ and CD27+ IgD+ than patients without AID, independent of the group studied. CONCLUSIONS: These results suggest that the persistence of immature memory B cells in patients with IgAD and CVID can be related to autoimmune diseases. We speculate if the persistence of immature B cells can constitute risk factor to progression of IgAD for CVID.

Angiogenese ist an physiologischen Vorgängen wie der Embryogenese und der Wundheilung, aber auch bei pathologischen Abläufen wie bei Neoplasien und der Makula Degeneration beteiligt. Im Bereich des Tissue Engineering ist sie ebenfalls unersetzlich und ausschlaggebend für den Erfolg einer Gewebetransplantation. In dieser Studie wurde an 40 männliche Charles Lewis-Ratten das arteriovenöse (AV-) Loop-Modell angewandt, um spontane Angiogenese unter Einfluss wachstumshemmender Faktoren in vivo zu untersuchen. Der AV-Loop wurde in einer mit Fibrin gefüllten Teflonkammer gebettet. Für die statistische Auswertung wurde der Student t-Test mit ungepaarten Stichproben angewandt und das Signifkanzniveau betrug α=0,05. Multiple Testungen wurden nach der Bonferroni-Holm-Methode angepasst. In der Anfangsphase der Studie wurde der zeitliche Verlauf der AV-Loop assoziierten Angiogenese an 16 Tieren untersucht. Es wurde ein signifikanter Anstieg der Gefäßfläche über einen Zeitraum von 5 (n=2), 10 (n=3) und 15 Tagen (n=3) beobachtet und ebenfalls eine signifikant höhere Gefäßanzahl an Tag 15 im Vergleich zu Tag 5. Acht Tiere konnten nicht in die Studie miteingeschlossen werden infolge von Thrombosierungen des Loops. Diese erwartete Verlustrate trat aufgrund Lernkurve dieses komplexen mikrochirurgischen Modells, insbesondere zu Beginn des Projektes, auf. In der zweiten Phase der Studie wurde die Neoangiogenese auf drei unterschiedliche Verfahren gehemmt. Die Implantationszeit betrug bei allen Gruppen 15 Tage. In der ersten Gruppe (n = 6) wurde ein Inhibitor des Wachstumfaktors VEGF (vascular endothelial growth factor) intravenös appliziert, nämlich der monoklonale Antikörper Bevacizumab. Hier konnte ein signifikanter Unterschied zur Kontrollgruppe (n = 6) bei der Gefäßfläche (94 432 ± 17 903 μm2 gegenüber 268 682 ± 63 575 μm2) und ebenfalls bei der Gefäßdichte (18 ± 5 Gefäße pro mm2 versus 40 ± 9 Gefäße pro mm2 in der Kontrollgruppe) gemessen werden. Dieser Befund ließ darauf schließen, dass die Neovaskularisation durch VEGF vermittelt wurde. Die direkte Bestrahlung von 2 Gy auf den venösen Graft in der zweiten Versuchsgruppe (n = 7) löste eine signifikante Verringerung von Gefäßanzahl (311 ± 73), -fläche (43 137 ± 10 225 μm2) und –dichte (15 ± 7 Gefäße pro mm2) im Vergleich zur Kontrollgruppe (776 ± 123, 268 682 ± 63 575 μm2 und 40 ± 9 Gefäße pro mm2) aus. Dieses Verfahren hatte somit starken Einfluss auf den Reifeprozess der Neoangiogenese. Bei der Kombinationsgruppe (Bevacizumab und Bestrahlung, n = 5) konnte nur bei der Gefäßfläche ein signifikant geringerer Unterschied in der Angiogenese erhoben werden. Dies ließ vermuten, dass das hier zu findende physiologische und somit geordnete Gefäßwachstum nicht auf diese hemmende Methode anspricht, wie es bei chaotischen Tumorgefäßsystemen der Fall ist und der vermutete Synergismus ausbleibt. Zusätzlich wurde im Zuge dieser Studie ein standardisiertes Auswertungsprogramm etabliert. Dabei handelt es sich um ein selbstentwickeltes Computerprogramm, das nicht nur die hier gesammelten aber auch 2-D-Aufnahmen anderer Angiogenese-Modelle benutzerunabhängig und standardisiert evaluieren kann. Zusammenfassend kann gesagt werden, dass das AV-Loop-Modell sich ausgezeichnet für die Untersuchung der Angiogenese im gesunden Gewebe eignet. Es bietet die Möglichkeit verschiedene angiogene und anti-angiogene Faktoren zu applizieren sowie deren Einfluss auf eine physiologische Neovaskularisation zu beobachten.:1 EINLEITUNG 1 1.1 ZIELSETZUNG DIESER ARBEIT 3 2 LITERATURÜBERSICHT 5 2.1. ANGIOGENESE 5 2.1.1 PHYSIOLOGIE 5 2.1.1.1 Bildung von Blutgefäßen 5 2.1.1.2 Embryogenese 8 2.1.1.3 Angiogenese und Wundheilung 9 2.1.2 TUMOR-ASSOZIIERTE ANGIOGENESE 11 2.2 TISSUE ENGINEERING 22 2.3 DAS ARTERIOVENÖSE (AV) LOOP-MODELL 22 2.4 AUSWERTUNG VON ANGIOGENESEPROZESSEN 23 3 MATERIAL UND METHODEN 24 3.1 TIERE UND HALTUNG 24 3.2 OPERATIVE EINGRIFFE 24 3.2.1 KAMMER UND MATRIX 24 3.2.2 HERSTELLUNG DES AV-LOOPS 25 3.2.3 BESTRAHLUNG 29 3.3 EXPLANTATION 29 3.3.1 PERFUSION MIT INDIA INK 29 3.3.2 PERFUSION MIT MICROFIL® 31 3.4. HISTOLOGISCHE UND IMMUNHISTOLOGISCHE METHODEN 32 3.4.1 VORBEREITUNG DER SCHNITTE 32 3.4.2 HÄMATOXYLIN-EOSIN-FÄRBUNG 33 3.4.3 LEKTINFÄRBUNG 34 3.5 DATENVERARBEITUNG 36 3.5.1 MIKROSKOPISCHE AUFZEICHNUNGEN 36 3.5.2 AUSWERTUNG DER HISTOLOGISCHEN SCHNITTE 36 3.5.3 AUFNAHMEN DER MIKRO-CT-BILDER 41 3.5.4 AUSWERTUNG DER MIKRO-CT-BILDER 42 3.5.5 POWER ANALYSE 43 3.5.6 STATISTISCHE AUSWERTUNGEN 43 3.6 VERWENDETES MATERIAL 44 4 ERGEBNISSE 47 4.1 AV-LOOP-ASSOZIIERTE ANGIOGENESE 48 4.2 ZEITLICHER VERLAUF DER AV-LOOP-ASSOZIIERTEN ANGIOGENESE 50 4.3 HISTOMORPHOMETRISCHE ANGIOGENESE-CHARAKTERISIERUNG MITTELS HE- UND LEKTINFÄRBUNG 52 4.4 AUTOMATISCHE, COMPUTERGESTÜTZTE UND UNTERSUCHERUNABHÄNGIGE ANGIOGENESE- QUANTIFIZIERUNG 53 4.5 EINFLUSS VON VEGF AUF AV-LOOP-ANGIOGENESE 58 4.6 EINFLUSS VON BESTRAHLUNG AUF DIE NEOVASKULARISATION IM AV-LOOP-MODELL 62 4.7 WECHSELWIRKUNG VON VEGF UND IONISIERENDER BESTRAHLUNG AUF DIE ANGIOGENESE IM AV-LOOP-MODELL 65 5 DISKUSSION 71 6 ZUSAMMENFASSUNG 81 7 SUMMARY 83 8 LITERATURVERZEICHNIS 85 9 ANHANG 95 9.1 PROTOKOLL ZUR HERSTELLUNG DER PUFFER 95 9.1.1 CITRATPUFFER 95 9.1.2 TRISPUFFER 95 9.2 TABELLEN 95 9.3 ABBILDUNGEN 95 10 DANKSAGUNG 98 / Angiogenesis is evident in both physiological and pathological processes in the body. It is involved in events of embryogenesis and in wound healing as well as in neoplastic growth and macula degeneration. In the field of Tissue Engineering neovascularisation plays an irreplaceable role and determines the result of the transplantation. Here, an arterio-venous loop (AV-loop) model embedded in fibrin- filled teflon chambers in 40 Charles Lewis rats was applied to conduct in vivo investigations of the physiological processes of vessel growth in healthy tissue and to understand neovascularisation under the impact of anti-angiogenic factors such as monoclonal anti-bodies and ionizing radiation (IR). For statistical analysis the unpaired t-test was applied with a significance level of α = 0,05. Multiple testing was adapted according to the Bonferroni-Holm method. At the beginning of the study the AV-loop induced angiogenesis was examined on 16 animals and consecutively characterized. A significant increase in vessel area was observed over a time frame of 5 (n=2), 10 (n=3) and 15 days (n=3). Additionally the vessel count has increased significantly at day 15 in comparison to day 5. Eight of the animals had to be excluded due to thrombosis of the loop, which was expected due to the complex microsurgical model, especially at the onset of the project. In the second phase of the study three different anti-angiogenic procedures were investigated. Time of implantation was 15 days. In group one (n = 6) the monoclonal antibody of VEGF (vascular endothelial growth factor) named Bevacizumab was applied intravenously. As a result a significant lower vessel area (94 432 ± 17 903 μm2) and density (18 ± 5 vessels per mm2) could be measured in comparison to the control group (n = 6; 268 682 ± 63 575 μm2 respectively 40 ± 9 vessels per mm2). We concluded from these results that angiogenesis was mediated by VEGF. In comparison to the controlgroup direct IR of 2 Gy led in group two (n = 7) to a significant decrease in vessel number (311 ± 73 versus 776 ± 123), area (43137±10225μm2 versus 268682±63575μm2) and density (15±7 versus 40±9 vessels per mm2). Therefore this procedure has an obvious impact on the vessel maturation. In the combined group (n = 5) of both anti-angiogenic procedures (anti- VEGF and IR) a significant decrease was only evident in the vessel area. We assumed that this physiological and accordingly organized angiogenesis does not respond to the applied inhibiting methods, as it is observed in tumor vessel growth. Additionally, an evaluation program was established with the goal of designing a user-independent and standardized computer program to measure 2-D-images of both this and other angiogenesis models. In summary the AV-loop presents a proficient model to investigate angiogenesis in healthy tissue. It offers a variety of possibilities to apply pro- and anti-angiogenic factors and to examine their impact in vivo.:1 EINLEITUNG 1 1.1 ZIELSETZUNG DIESER ARBEIT 3 2 LITERATURÜBERSICHT 5 2.1. ANGIOGENESE 5 2.1.1 PHYSIOLOGIE 5 2.1.1.1 Bildung von Blutgefäßen 5 2.1.1.2 Embryogenese 8 2.1.1.3 Angiogenese und Wundheilung 9 2.1.2 TUMOR-ASSOZIIERTE ANGIOGENESE 11 2.2 TISSUE ENGINEERING 22 2.3 DAS ARTERIOVENÖSE (AV) LOOP-MODELL 22 2.4 AUSWERTUNG VON ANGIOGENESEPROZESSEN 23 3 MATERIAL UND METHODEN 24 3.1 TIERE UND HALTUNG 24 3.2 OPERATIVE EINGRIFFE 24 3.2.1 KAMMER UND MATRIX 24 3.2.2 HERSTELLUNG DES AV-LOOPS 25 3.2.3 BESTRAHLUNG 29 3.3 EXPLANTATION 29 3.3.1 PERFUSION MIT INDIA INK 29 3.3.2 PERFUSION MIT MICROFIL® 31 3.4. HISTOLOGISCHE UND IMMUNHISTOLOGISCHE METHODEN 32 3.4.1 VORBEREITUNG DER SCHNITTE 32 3.4.2 HÄMATOXYLIN-EOSIN-FÄRBUNG 33 3.4.3 LEKTINFÄRBUNG 34 3.5 DATENVERARBEITUNG 36 3.5.1 MIKROSKOPISCHE AUFZEICHNUNGEN 36 3.5.2 AUSWERTUNG DER HISTOLOGISCHEN SCHNITTE 36 3.5.3 AUFNAHMEN DER MIKRO-CT-BILDER 41 3.5.4 AUSWERTUNG DER MIKRO-CT-BILDER 42 3.5.5 POWER ANALYSE 43 3.5.6 STATISTISCHE AUSWERTUNGEN 43 3.6 VERWENDETES MATERIAL 44 4 ERGEBNISSE 47 4.1 AV-LOOP-ASSOZIIERTE ANGIOGENESE 48 4.2 ZEITLICHER VERLAUF DER AV-LOOP-ASSOZIIERTEN ANGIOGENESE 50 4.3 HISTOMORPHOMETRISCHE ANGIOGENESE-CHARAKTERISIERUNG MITTELS HE- UND LEKTINFÄRBUNG 52 4.4 AUTOMATISCHE, COMPUTERGESTÜTZTE UND UNTERSUCHERUNABHÄNGIGE ANGIOGENESE- QUANTIFIZIERUNG 53 4.5 EINFLUSS VON VEGF AUF AV-LOOP-ANGIOGENESE 58 4.6 EINFLUSS VON BESTRAHLUNG AUF DIE NEOVASKULARISATION IM AV-LOOP-MODELL 62 4.7 WECHSELWIRKUNG VON VEGF UND IONISIERENDER BESTRAHLUNG AUF DIE ANGIOGENESE IM AV-LOOP-MODELL 65 5 DISKUSSION 71 6 ZUSAMMENFASSUNG 81 7 SUMMARY 83 8 LITERATURVERZEICHNIS 85 9 ANHANG 95 9.1 PROTOKOLL ZUR HERSTELLUNG DER PUFFER 95 9.1.1 CITRATPUFFER 95 9.1.2 TRISPUFFER 95 9.2 TABELLEN 95 9.3 ABBILDUNGEN 95 10 DANKSAGUNG 98

La manipulation des anticorps monoclonaux thérapeutiques (Acm) reconstitués avant administration aux patients est susceptible d’entraîner leurs dégradations physiques (e.g. dénaturation, agrégation). Ceci peut avoir un impact sur leur efficacité et sécurité. Afin d’étudier la conformation et la stabilité des Acms reconstitués, nous avons développé des méthodes séparatives couplées avec la spectrométrie de masse (MS) en conditions non-dénaturantes (« native »). Une méthode de CZE-native MS utilisant un revêtement constitué d’une triple-couche ionique a été développée pour séparer et détecter les différentes conformations (monomère natifs, dénaturés, dimères) d’Infliximab. Une étude approfondie réalisée en analysant de l’infliximab digéré a permis d’établir que la formation du dimère était liée à la dénaturation du fragment Fab. L’intérêt des revêtements statiques (commerciaux et préparés in situ) ont été étudiés pour analyser les Acms par CZE-UV et CZE-MS. Nos résultats ont montré l’intérêt d’un nouveau revêtement monolithique. Un couplage simultané de la SEC avec la MS et un détecteur de fluorescence a été développé. Nous avons ainsi identifié les conditions expérimentales qui entraînaient des dénaturations et des dimérisations artificielles. Cette méthode a ensuite été également appliquée avec succès pour la caractérisation d’un échantillon de Trastuzumab stressé. Une méthode orthogonale en utilisant la SEC-mobilité ionique-MS a été employé pour évaluer la proportion de monomères dénaturés par rapport aux monomères natifs. La méthodologie ainsi développée permettra la détection de très faibles taux d'anticorps dégradés dans des poches d'infusion. Ceci permettra de définir les paramètres critiques à maîtriser lors de la reconstitution et la manipulation d’anticorps à usage hospitalier. / Manufacturing and manipulation of therapeutic monoclonal antibodies (mAb) in the hospital before administration to patient is prone to induce their physical degradations (e.g., denaturation, aggregation). This may impact their efficacy and safety. To study the stability of mAbs, capillary zone electrophoresis (CZE) and size exclusion chromatography (SEC), coupled to native mass spectrometry (MS) have been developed. CZE-native MS method using a triple-layer coating was developed to detect and separate different conformational states (unfolded monomer, dimer) of Infliximab in a single analysis. In-depth study with digested infliximab confirmed that dimer formation was related to the Fab fragment. We also focused on covalent coatings in order to find the more adapted coating to analyze mAbs by CZE-UV and CZE-MS. We also developed for SEC a simultaneous coupling with MS and a fluorescence detector to detect the degraded mAbs. We have identified the biases inducing conformational changes (e.g. dimerization, denaturation) that may arise during native MS. We also successfully characterized aggregates and denatured monomer in stressed Trastuzumab sample. In addition, the orthogonal method SEC-ion mobility-MS has been employed to separate and measure the denatured monomers compared to their related native conformations. Moreover, the developed system enables the detection of a very low levels of degraded mAbs in infusion bags. It allows to define the critical parameters to be controlled during the reconstitution and manipulation of therapeutic mAbs in hospital.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18186号 / 医科博第51号 / 新制||医科||4(附属図書館) / 31044 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 松岡 雅雄, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM