CD8 wird von thymisch gereiften a/b T Zellen als CD8ab-Heterodimer exprimiert und dient als Korezeptor bei der MHC Klasse I (MHC I) restringierten Antigenerkennung. In dieser Funktion stabilisiert CD8 die Bindung des T Zellrezeptors (TCR) an seinen Liganden (MHC I mit antigenem Peptid) und vermittelt darüber hinaus kostimulatorische Signale an die T Zelle. Die Antigene der g/d T Zellen sind bis auf einige Ausnahmen unbekannt, aber g/d T Zellen von Maus und Mensch weisen im allgemeinen höchstwahrscheinlich keine MHC-restringierte Antigenerkennung auf. Dementsprechend findet man auf den meisten g/d T Zellen dieser Spezies keinen der MHC-spezifischen Korezeptoren CD4 oder CD8. In auffälligem Gegensatz dazu exprimieren in der Milz der Ratte bis zu 80 Prozent der g/d T Zellen das CD8ab-Heterodimer. Um die mögliche Funktion von CD8ab auf g/d T Zellen besser zu verstehen, wurden zunächst die CD8-vermittelten Signale untersucht. Eine Schlüsselrolle bei der Initiierung der TCR-Signalkaskade kommt der mit CD8a assoziierten Proteintyrosinkinase p56lck zu. Diese ist, wie durch Kopräzipitationsexperimente gezeigt wurde, bei a/b und g/d T Zellen gleichermaßen mit CD8 assoziiert und zeigt dieselbe Kinaseaktivität. Weiterhin konnte in vitro ein kostimulatorisches Potential von CD8-spezifischen Antikörpern für a/b und g/d T Zellen gezeigt werden. Demnach besitzt CD8 auf a/b und g/d T Zellen prinzipiell eine vergleichbare Fähigkeit zur Übertragung kostimulatorischer Signale. Es war folglich naheliegend, einen Zusammenhang zwischen der CD8ab-Expression der g/d T Zellen mit einer möglichen MHC I estringierten Antigenerkennung zu prüfen. Da bisher keine Antigene für g/d T Zellen der Ratte bekannt sind, wurden die Antigen bindenden Regionen des g/d TCR analysiert. Bei Antigenrezeptoren von Mensch und Maus bestehen nämlich charakteristische Längenunterschiede zwischen den mit dem Antigen interagierenden CDR3-Proteinschleifen (englisch: complementarity determining regions), die in der Verknüfungsregion der V-, D- und J-Gensegmente codiert sind. Bei Maus und Mensch sind die CDR3-Längen der TCRd-Kette ähnlich variabel und etwa so lang wie bei der schweren Kette des B Zellrezeptors. Dagegen sind die CDR3-Regionen der TCRb-Kette sehr kurz, was wahrscheinlich durch die notwendige Interaktion des MHC-Peptid-Komplexes mit allen drei CDR-Schleifen bedingt wird. Daraus wurde geschlossen, daß die Bestimmung der CDR3d-Längen Aussagen über eine mögliche MHC-Restriktion von g/d T Zellen der Ratte erlauben sollte. Zur Durchführung der CDR3d-Längenanalysen wurde die Methode des Spektratyping etabliert. Hierfür wurden in der Milz häufig vorkommende TCRd V-Gensegmente (DV105 und fünf Mitglieder der ADV7-Familie) partiell kloniert und sequenziert. In CD8 positiven und negativen g/d T Zellen wurden sowohl DV105 als auch Mitglieder der ADV7-Familie jeweils etwa gleich häufig exprimiert. Spektratyp-Analysen ergaben in g/d T Zellen der Ratte für beide V-Genfamilien etwas längere CDR3d-Schleifen als bei der Maus; CD8ab positive, CD8aa positive und CD8 negative g/d T Zellen der Ratte besaßen identische Längenspektren. Demnach kann man die CD8ab-Expression von g/d T Zellen der Ratte nicht als Hinweis auf eine der klassischen MHC-Restriktion ähnliche Antigenerkennung werten. Als spezifische Eigenschaft von g/d T Zellen der Ratte wurde eine Modulation der CD8b-Genexpression nachgewiesen, die mit der Stärke des Aktivierungssignals zunahm. Nach Aktivierung in vitro exprimierte ein großer Teil zuvor CD8ab positiver g/d T Zellen nur noch das CD8aa-Homodimer, das auf MHC I restringierten a/b T Zellen deutlich schlechtere Korezeptoreigenschaften aufweist als das CD8ab-Heterodimer. Die Modulation war irreversibel, fand prätranslational statt und zeigte keine Einflüsse auf Effektorfunktionen der g/d T Zellen wie Interferon-g-Produktion oder zytotoxische Fähigkeiten. Über die physiologische Bedeutung dieser Modulation kann nur spekuliert werden, doch könnte sie ein Mechanismus sein, um den Schwellenwert für Signale einer erneuten T Zellaktivierung zu erhöhen. Darüber hinaus limitiert sie den Nutzen der CD8ab-Expression als Marker für die thymisch gereifte Linie von g/d T Zellen. / CD8 is expressed on thymus derived a/b T cells and serves as the co-receptor for MHC class I (MHC I) restricted antigen recognition. In this role CD8 stabilises the binding of the T cell receptor (TCR) to its ligand (MHC with antigenic peptide) and moreover transduces co-stimulatory signals to the T cell. Aside from few examples the antigens of g/d T cells are unknown, but mouse and human g/d T cells most probably do not possess a MHC restricted way of antigen recognition. Correspondingly most g/d T cells of these species do not express one of the MHC specific co-receptors CD4 or CD8. In striking contrast up to 80 per cent of splenic rat g/d T cells express the CD8ab heterodimer. To understand the putative function of CD8ab expressed by rat g/d T cells, CD8 signalling was investigated first. The protein tyrosine kinase p56lck plays a key role at the initiation of the TCR signalling cascade. This kinase is equally associated with CD8 on a/b and g/d T cells as found by co-precipitations, and it shows the same kinase activity there. Furthermore, in vitro a co-stimulatory potential of CD8 specific antibodies could be demonstrated for a/b and g/d T cells likewise. Therefore in principle, CD8 possesses similar co-stimulatory properties on rat a/b and g/d T cells. Consequently a correlation between CD8ab expression and a possible MHC I restriction of rat g/d T cells was tested. Because no antigens have been found for rat g/d T cells so far, the antigen binding regions of the g/d TCR were investigated. It is known that the complementarity determining region 3 (CDR3) protein loops, which are encoded by the region of V, D, and J gene segment joining, show characteristic length differences: CDR3 lengths of mouse and human TCRd chains show the same variability and are as long as CDR3 of the B cell receptor heavy chain. In contrast, the CDR3 regions of TCRb chains are very short, what probably results from the necessity of interaction between MHC and all three CDR loops. In consequence, determining the CDR3d lengths should allow predictions about a possible MHC restriction of rat g/d T cells. For CDR3d length analysis a method called spectratyping was established. First of all, common TCRd V segments from rat spleen (DV105 and five members of the ADV7 gene family) were cloned and sequenced partially. Both gene families were expressed in about the same frequency by CD8 negative and CD8 positive g/d T cells, respectively. For both V segment families the spectratyping analyses revealed somewhat longer CDR3d loops in rat than in mouse g/d T cells. CD8ab or CD8aa positive and CD8 negative g/d T cells showed exactly the same CDR3d length spectra. Therefore the CD8ab expression of rat g/d T cells is no indication for a MHC restricted antigen recognition. As a specific property of rat g/d T cells an activation dependent down modulation of the CD8b chain could be demonstrated, that increased with the strength of the activating signal. Following activation in vitro, a major percentage of g/d T cells which previously expressed CD8ab, solely expressed the CD8aa homodimer. On MHC I restricted a/b T cells CD8aa is known to show poor co-stimulatory potential compared to CD8ab. The modulation was irreversible, occurred on a pre-translational level and did not influence g/d T cell effector functions like interferon-g production or cytotoxicity. A physiological role of the modulation is speculative so but the CD8b modulation might be a mechanism to increase the signalling threshold for a following T cell activation. Moreover, the modulation limits the use of CD8ab expression as a marker for the thymically derived g/d T cell lineage.

Offprints of author's four journal articles in pocket / Bibliography: leaves 131-147 / x, 148 leaves, [13] leaves of plates : ill. (1 col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986

In experimentally exposed mice, the environmental contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) produces significant suppression of adaptive immune responses at low doses. However, the underlying biochemical and cellular mechanisms of TCDD-induced immunotoxicity have remained elusive since the identification of these effects nearly 30 years ago. Antigen-presenting cells (APC) constitute various populations of cells essential for the initiation and maintenance of adaptive immune responses, and represent a potential target of TCDD toxicity. Thus, the studies presented here address the ability of TCDD to directly affect APC. The underlying objectives of these studies focus on the investigation of molecular signaling pathways and cellular processes potentially affected by TCDD. In order to eliminate conflicting variables found in vivo, we used ex vivo and in vitro models to address these objectives. Initial studies investigated the status and behavior of the aryl hydrocarbon receptor (AhR), a transcription factor recognized as the principal mediator of TCDD-induced immunotoxic effects, in the two main APC populations, macrophages and dendritic cells (DC). The results demonstrated that both APC populations expressed AhR. However, TCDD induced binding of AhR to dioxin response elements only in macrophages, and not DC. Because TCDD has been shown to alter DC function and survival in vivo, the possibility that TCDD altered other signaling pathways was addressed. Specifically, activation of the transcription factor NF-kB/Rel, integral in DC generation and function, was found to be suppressed by TCDD. This suppression was apparently mediated by a physical association between the AhR and proteins of NF-kB/Rel. Additional studies demonstrated that TCDD enhances the maturation of DC and appears to sensitize DC to apoptosis. These data establish that TCDD directly affects DC on the molecular and cellular levels and support several potential mechanisms of TCDD-induced immunotoxicity. / Graduation date: 2002

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 1999. / Includes bibliographical references. Also available on the Internet.

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

Naja naja atra Cardiotoxins (CTXs), basic polypeptides of 60 amino acid residues adopt a three-fingered loop-folding topology and show cytotoxicity for human tissues in targeting cell membrane. Despite having highly similar sequence, the six CTX isoforms also display different cytotoxic potencies and hemolytic activities. The goal of these studies is to explore the mechanical processes that involved in membrane-damaging activities of CTXs on vesicles composed of different cell membrane components, and to delineate the events that lead to different biological activities of CTXs. The studies were performed by estimating the color transformation of phospholipid/polydiacetylene vesicles and the fluorescence enhancement of fluorescein-labeled phospholipid/protein or fluorescein released from vesicles. It was found that vesicles consisted of unsaturated phospholipids improve membrane-damaging activity of CTXs and adopt a vital membrane-bound conformation of CTXs. In contract, the characteristic of vesicles consisted of saturated phospholipids was against CTXs adopting an essential membrane-damaging structure. It was also found that not only electrostatic force but also hydrophobic force were involved in the interaction between CTXs and membrane. Comparing with phosphatidylcholine-only vesicles, CTXs displayed higher membrane-damaging activity for the sphingomyelin-containing vesicles, and the loop2 region of CTXs play a crucial role for the membrane-damaging activity of sphingomyelin-containing vesicles. Besides, the CTX3 and CTX5 would interact with the H-antigen of blood group O red blood cells, but only the binding of CTX3 with H-antigen reduce its membrane-damaging activity for red blood cells membrane. Moreover, the fusogenicity of CTXs is responsible for the membrane-damaging activity of CTXs toward bacterial membrane-mimicking vesicles. The cardiolipin have the potency to improve the fusogenicity of CTX3, which induced the bactericidal activity toward the cardiolipin-containing bacterium.

No description available.

Mycoplasma hyopneumoniae causes swine enzootic pneumonia (SEP) and leads to economic loss worldwide. The mechanism of pathogenesis is still not clear. Since this pathogen remains extracellulary after infection, the surface proteins on M. hyopneumoniae should play very important roles in adhering and affecting tracheal mucosal cells. Therefore, the potential of using the surface proteins as the basic to develop molecular vaccine is currently being investigated. The recombinant clone expressing the 42 kDa protein was isolated from the

Investigations detailed in this thesis constitue a part of continuing programme of research work undertaken in this laboratory on vitamin binding proteins. Avidin from the chicken egg white, streptavidin &om the bacterium Streptromyces avidin and biotin binding proteins (BBP-I and BBP-11) from chicken egg yolk constitute a family of proteins that bind the vitamin biotin with extremely high affinities. The yolk BBPs are involved in the deposition of the vitamin in the developing oocyte in chicks whereas an antimicrobial function has been attributkl to avidin.. The fact that all these proteins bind the vitamin in the same manner, unlike biotin-dependent enzymes, indicates that the structural features involved in ligand binding could be similar, if not identical in these proteins. To delineate the basis of putative structural similarity among these proteins, studies were carried out using antibodies as the immunological probes. Avidin, a homotetremer glycoprotein, with a subunit Mr of 17,000 has been purified to homogeneity from chicken egg white using a novel procedure involving ammonium sulphate fractionation, ethanol precipitation and S-Sepharose column chromatography. Despite their lesser abundance in chicken egg yolk associated with a large amount of interfering lipids during the purification, both BBP-I (monomer and shown to be precursor for BBP-11) and BBP-I1 (tetramer) have been purified to homogeneity by employing a common method using butanol extraction to remove the lipids, DEAE-Sephacel column chromatography, biotin-AH-Sepharose affinity chromatography and fast performance liquid chrometography (FPLC) system. The purity of all these proteins was confirmed by SDS-PAGE analysis.