Innate immune activation is thought to play a central role in IBD pathogenesis because genetic polymorphisms in NOD2 and NLRP3, cytosolic innate immune receptors belonging to the NLR family, have been associated with IBD susceptibility. However, the mechanisms through which NLR mutations predispose to IBD remain unclear. The aim of this project was to dissect the functional roles of different NLRs in intestinal inflammation. Using the well-established DSS-induced colitis model as well as experimental models of IBD based on infection with Helicobacter hepaticus, we found that Nod2 expression was significantly increased at the peak of intestinal inflammation, and remained elevated throughout the resolution process. This observation suggests a possible role for Nod2 in the resolution of inflammation. Conversely, upon infection with the acute intestinal pathogen Citrobacter rodentium, Nlrp3-/- mice suffered from increased bacterial colonization as early as 3 days post infection, resulting in exacerbated intestinal inflammation and severe weight loss. Analysis of irradiation bone marrow chimeras showed that the protection required Nlrp3 activation in the non-haematopoietic compartment. Furthermore, this protective mechanism was independent of the inflammasome-associated cytokines IL-1β or IL-18. Therefore, this study implicates Nlrp3 activation in intestinal tissue cells as having a crucial role in controlling pathogenic bacterial colonization, providing a potential mechanism by which NLRP3 polymorphisms could lead to increased susceptibility to IBD.

T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1_156-165 peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1_156-165 peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.

The C-type lectin CD161 is expressed by a large number of T lymphocytes, with approximately a quarter of both T cell receptor (TCR)αβ+ and TCRγδ+ T cells expressing this marker. Within CD8+ T cells, a large proportion of these are comprised of Mucosal Associated Invariant T (MAIT) cells, a novel innate-like lymphocyte subset characterised by expression of a semi-invariant TCR together with high levels of CD161 (CD161++). These cells display a phenotype reflective of type 17 CD4+ helper T cells (Th17), which are also hallmarked by CD161 expression. Both MAIT and Th17 cells arise from preprogrammed progenitors, identifiable within umbilical cord blood by expression of CD161. Thus, CD161 appears to identify cells of a pre-determined and distinct phenotype. Whether this reflects a common transcriptional programme, developmentally induced within these cells, and further whether this extends to other CD161 positive T cells, was examined here by mRNA microarray analysis. This analysis identified a shared transcriptional signature and common innate-like function of all CD161 expressing T lymphocytes, and independent of TCR expression or lineage. Furthermore, a population of CD8+ T lymphocytes expressing lower levels of CD161 which overlap phenotypically with CD161++CD8+ MAIT cells was identified by both mRNA microarray analysis and mass cytometry (CyTOF); the CD161+CD8+ T cell population. TCR repertoire analysis, flow cytometry and cell culture experiments were utilised to investigate the origin of this subset, and its phenotype and function in both health and disease investigated in depth. This revealed a pre-programmed, tissue-resident memory population with potent effector functions. Both CD161++ MAIT and CD161+CD8+ T cells expressed high levels of the drug efflux pump MDR1, previously described to confer drug resistance to certain malignant cells. The significance of expression of this pump was hence investigated to determine its potential affect on the success of a variety of clinical therapies.

Paired receptor families, consisting of multiple genetically and structurally similar but functionally opposite activating and inhibitory cell surface receptors, are among the fine tuners of the immune regulation. Recent studies on the evolutionary origin of these receptor families have suggested links to pathogen driven diversification, according to which activating receptors continuously evolve in order to counterbalance pathogens that try to subvert the immune response by stimulating the inhibitory receptor through their virulence factors. This thesis is about the CD200R paired receptor family. This family consists of an inhibitory receptor CD200R which is expressed on various leukocytes and delivers inhibitory signals upon engagement with its ligand CD200. In this study, the possibility that the activating members of the family evolved under pathogen pressure was investigated. Genomic DNA from twenty two different mice strains was screened for the presence of members of CD200R family. The number of activating receptors varied, CD200RLe and CD200RLc were found to be mutually exclusive and three strains possessed previously unknown members of CD200R family. In addition, the possibility that CD200R family members and other paired receptors interacted directly with bacteria was tested with a new assay but only the interaction of PIR-A1 with <em)S. aureus was found as previously reported. The rabbit CD200R family has been characterized and ligand receptor interaction between rabbit CD200 and rabbit CD200R has been demonstrated. However, no interaction between rabbit CD200R and a candidate viral CD200 homologue, the M141R protein of myxoma viruses, could be shown. This finding suggested a CD200R independent role for M141R molecule and possibly other homologues in pox viruses. Finally, two novel antibodies (OX131 and OX132) were characterized together with formerly generated antibodies against mouse CD200R family. The binding specificities and their effects on the CD200-CD200R interaction have been shown. This will help usage of these antibodies in various studies on the functionality and distribution of these receptors.

A hidrocefalia é uma síndrome complexa caracterizada pelo acúmulo de líquido cérebro-espinal no interior das cavidades ventriculares. Considerando a sua fisiopatologia de caráter multifatorial sendo um dos fatores envolvidos o estresse oxidativo desencadeado pela peroxidação lipídica e formação de radicais livres, este trabalho visa estudar o possível efeito neuroprotetor proveniente do polifenol galato de epigalocatequina (EGCG) na hidrocefalia experimental. Foram utilizados ratos da linhagem Wistar (N=56), com 7 dias de idade. Os filhotes foram submetidos à indução da hidrocefalia pelo método da injeção intracisternal de caulim a 20%. O polifenol foi administrado intraperitonealmente por 9 ou 20 dias consecutivos a partir da indução da hidrocefalia. Aferição do peso corporal diário e testes comportamentais foram realizados. Dez ou 21 dias após a indução da hidrocefalia os animais, profundamente anestesiados, foram sacrificados através da perfusão cardíaca com solução salina. Seus encéfalos foram removidos, fixados com paraformaldeído 3% em tampão fosfato 0,1M e processados para inclusão em parafina. Preparações histológicas foram realizadas para a análise por coloração hematoxilina eosina, solocromo-cianina e imunoistoquímica para GFAP e Ki67. Os diferentes parâmetros de avaliação demonstraram que os animais tratados com o polifenol por 9 dias consecutivos apresentaram redução da atividade astrocitária através da imunomarcação pelo GFAP no corpo caloso, cápsula externa e matriz germinativa; além de apresentarem corpo caloso mais espesso e mielinizado, exibindo uma tonalidade azul mais intensa evidenciada pela coloração solocromocianina. Apesar desses resultados demonstrarem um possível efeito neuroprotetor na fase inicial de instalação da doença, estudos adicionais devem ser realizados para obtenção de uma terapêutica eficiente e segura para o aprofundamento com testes clínicos. / Hydrocephalus is a complex syndrome, characterized by the accumulation of cerebrospinal fluid in cerebral ventricles. Considering its multifactorial pathophysiology, one of the factors being the oxidative stress triggered by lipid peroxidation and free radical formation, this work aims to study the possible neuroprotective effect of the polyphenol epigallocatechin gallate (EGCG) in experimental hydrocephalus. Seven-day old Wistar rats (N=56) were used in this study. The pups were subjected to hydrocephalus induction by kaolin 20% through intracisternal injection. The polyphenol was administered intraperitoneally for 9 or 20 days from the induction of hydrocephalus. Measurement of daily body weight and behavioral tests were performed. The animals, deeply anesthetized, were sacrificed by cardiac perfusion with saline 10 or 21 days after induction of hydrocephalus. Their brains were removed, fixed with 3% paraformaldehyde in 0.1 M phosphate buffer, and processed for paraffin embedding. Preparations were made for histological analysis by hematoxylin and eosin, solochrome-cyanine and immunohistochemistry for GFAP and Ki67. The different evaluated parameters showed that animals treated with the polyphenol for 9 consecutive days displayed reduction on the reactive astrocytes GFAP immunostaining at the corpus callosum, external capsule and germinal matrix, also having thicker and more myelinated corpus callosum exhibiting a more intense blue staining by solocromo-cyanine. Although these results demonstrate a possible neuroprotective effect at the initial onset of the disease, additional studies should be performed to obtain an effective and safe therapy for deeper studies in clinical trials.

The irreversible binding of penicillin to serum protein was investigated by incubating 14C-benzyl-penicillin with reconstituted lyophilized human serum and dialyzing away the non-bound penicillin. Binding appeared in each protein fraction as was observed by polyacrylamide gel electrophoresis and liquid scintillation counting.The nature of penicillin binding was investigated by preincubating reconstituted human serum with specicific blocking reagents.Ball State UniversityMuncie, IN 47306

Dynamic DNA complexes are able to undergo multiple hybridization and dissociation events through a process called strand displacement. This unique property has facilitated the creation of programmable molecular detection systems and chemical logic gates encoded by nucleotide sequence. This work examines whether the ability to selective exchange oligonucleotides among different thermodynamically-stable DNA complexes can be harnessed to create a new class of imaging probes that permit fluorescent reporters to be sequentially activated (“turned on”) and erased (“turned off”). Here, dynamic DNA complexes detect a specific DNA-conjugated antibody and undergo strand displacement to liberate a quencher strand and activate a fluorescent reporter. Subsequently, incubation with an erasing complex allows the fluorophore to be stripped from the target strand, quenched, and washed away. This simple capability therefore allows the same fluorescent dyes to be used multiple times to detect different markers within the same sample via sequential rounds of fluorescence imaging. We evaluated and optimized several DNA complex designs to function efficiently for in situ molecular analyses. We also applied our DNA probes to immunofluorescence imaging using DNA-conjugated antibodies and demonstrated the ability to at least double the number of detectable markers on a single sample. Finally, the probe complexes were reconfigured to act as AND-gates for the detection of co-localized proteins. Given the ability to visualize large numbers of cellular markers using dynamic DNA probe complexes, high-content proteomic analyses can be performed on a single sample, enhancing the power of fluorescence imaging techniques. Furthermore, dynamic DNA complexes offer new avenues to incorporate DNA-based computations and logic for in situ molecular imaging and analyses.

No description available.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.