1 |
De novo synthesis of high purity CD3 epsilon peptides utilizing SUMO expression system in bacteriaKim, Albert 10 February 2022 (has links)
Cancer is one of the leading causes of death in the United States. Monoclonal antibody drugs became one of the commercially and clinically successful drugs for many diseases. Among the monoclonal antibodies, T cell-dependent bispecific antibodies directly target tumor cells for cancer treatment, they kill tumor cells by activating T cells through binding to the CD3 (cluster of differentiation 3) receptor on T cells. Many anti-CD3 antibodies bind to the surface exposed CD3 γ, δ, ε subunits for T cell activation. SP34, an anti-CD3ε antibody, specifically binds to the first 27 amino acids of CD3ε. Synthesis of CD3ε peptides proofed to be difficult due to its hydrophobic nature and presence of an N-terminal glutamine that caused many side reactions resulting in very poor peptide quality and purity. For some commercial full-length CD3ε proteins it is unclear whether N-terminal glutamine is present or absent. In cases where N-terminal glutamine is present it is modified to pyroglutamic acid. To study the SP34-CD3ε interaction a reliable and defined source of CD3ε peptide and peptide variants is required. By utilizing the SUMO (Small Ubiquitin-related Modifier) system from yeast, CD3ε1-27 amino acid and a truncated version 2-27 amino acid peptide are expressed in E. coli cells with an SMT3 (Mitotic Fidelity Gene 3) tag. Subsequently, SMT3 tag is removed with SUMO protease and the resulting peptide is further purified. This novel in vitro approach results in high yields of non-modified peptides with great purity (>95%).
|
2 |
Ligonių, kuriems atlikta širdies persodinimo operacija, kraujo T limfocitų membranos žymenų CD16/56, CD103, CD134 ekspresijos pokyčiai / Variation of peripheral blood t lymphocyte cd16/56, cd103, cd134 membrane markers expression in patients after heart transplantationBumblauskaitė, Jurga 09 July 2011 (has links)
Po pirmosios širdies transplantacijos 1967 metais, ši operacija iš eksperimentinio gydymo tapo šiuolaikiniu, pažangiu sunkių širdies ligų gydymo būdu. Širdis šiuo metu yra trečias pagal dažnumą transplantuojamas organas JAV. UNOS (angl. Unaited Network for Organs Sharing) duomenimis JAV kasmet atliekama 2700 širdies transplantacijos operacijų. Tyrimai rodo, kad pirmuosius metus po operacijos išgyvena apie 85% pacientų, pirmus trejus metus – 75% pacientų. ISHLT (angl. International Society for Heart and Lung Transplantation) registravimo duomenys rodo, kad šiuo metu pasaulyje gyvena apie 50 000 žmonių, turinčių širdies transplantatą [20]. Nepakankamas donorų skaičius skatina mokslininkus ieškoti alternatyvių širdies ligomis sergančių pacientų gydymo būdų. Šiuo metu daug dėmesio skiriama kamieninių ląstelių tyrimams ir galimam jų pritaikymui įvairiom ligom gydyti. Su eksperimentiniais pelių modeliais parodyta, kad kamieninės ląstelės, implantuotos į širdies raumenį, geba diferencijuotis į kardiomiocitus ir kraujagyslių endotelio ląsteles ir atkurti pažeistą raumenį. Taučiau publikuota ir nemažai nesėkmių, susijusių su kamieninių ląstelių terapija, pvz.: neprigyjimas implantuotoje vietoje, nepilna diferencijacija [59]. Žmogaus mezenchiminių kamieninių ląstelių auginimas užtrunka, o gydymo dažnai prireikia tuoj pat [50]. Šios ir daug kitų problemų reikalauja daugybės tyrimų ir eksperimentų, todėl transplantacija kol kas išlieka pagrindiniu gydymo būdu, prailginančiu pacientų... [toliau žr. visą tekstą] / The ame of this work was to evaluate the changes of the expresion of T lymphocyte CD16/56, CD103, CD134 surface markers in the blood of the patients who underwent heart transplantation. For this purpose we collected heparinized blood samples of 21 patients with cardiac transplant and 29 healthy volunteers. 9 patients out of 21 underwent acute cardiac transplant rejection. Blood samples of these patients were tested using flow cytometry 10 days before heart rejection and on the day when rejection was uphold. Our results showed that the expresion of T lymphocyte CD16/56 surface molecule is higher on the rejection day then in controle group. Exspresion of CD103 marker is higher 10 days before rjection than on the rejection day and is lower then in healthy volunteers. Expresion of CD134 marker is higher before transplant rejection and higher then in healthy controles. Monitoring of CD3+CD103+ and CD+CD134+ cells in the blood after heart transplantation could be helpful in prediction of heart transplant rejection.
|
3 |
Caractérisation de l'infiltrat inflammatoire de l'hépatite chronique du chienBoisclair, Julie January 2004 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
|
4 |
Förekomsten av Propionibacterium acnes är låg hos patienter med Rosacea : En studie av sambandet mellan Propionibacterium acnes och Rosacea med immunofluorescens / The Prevalence of Propionibacterium acnes is Low in Patients with Rosacea. A Study of the Connection Between Rosacea and Propionibacterium acnes with Immunofluorescence.Dahlberg, Ida January 2011 (has links)
No description available.
|
5 |
Analyse du rôle des PIP2 dans l'initiation de la signalisation TCR et l'activation lymphocytaire / Regulation of the T cell receptor membrane dynamics and triggering mechanism by phosphatidylinositol 4,5-bisphosphateChouaki-Benmansour, Nassima 31 October 2014 (has links)
L'activation des lymphocytes T est un événement fondamental de la réponse immunitaire adaptative. Elle est déclenchée par la transduction du signal médiée par le complexe TCR/CD3.Le mécanisme de déclenchement du signal via le TCR reste, mal compris. Mon projet de thèse vise à examiner la contribution des PI(4,5)P2 dans le mécanisme du déclenchement du signal TCR. L'expression ectopique d'une phosphatase spécifique de PIP2 a permis de réduire de 50% les PI(4,5)P2 membranaires. L'analyse du profil de phosphorylations spécifiques des tyrosines montre que l'expression ectopique de cette 5-phosphatase augmente les événements de phosphorylation à l'état basal comparés à des conditions analogues pour des cellules contrôles. En revanche, alors que suite à l'engagement du TCR par le complexe MHC-peptide indépendamment du co-récepteur CD4 nous observons une augmentation des phosphorylations, l'activation par le complexe MHC-peptide CD4 dépendant semble affectée. Nous avons analysé la contribution des PI(4,5)P2 dans la dynamique membranaire du TCR grâce à la technique svFCS. PIP2 peuvent jouer un rôle essentiel dans la régulation de la dynamique latérale du TCR à la membrane plasmique. Enfin, nous avons observé que l'inhibition par la néomycine (aminoglycoside qui en tant que polycation peut se lier et neutraliser le groupement anionique de PI(4,5)P2), aboutit à des changements similaires dans la dynamique membranaire du TCR et la régulation proximale dans des cellules T primaires murines CD4+. Ensemble, nos données révèlent le rôle régulateur fondamental de PI(4,5)P2 dans la dynamique membranaire du TCR et de CD4, pour le contrôle de l'initiation des voies de signalisation du TCR. / PI(4,5)P2 plays important roles in a large spectrum of membrane-based cellular activities . It is therefore surprising that it is currently not known if PI(4,5)P2 is also involved in the T cell receptor (TCR) signal transduction mechanism. We investigate here the role of PI(4,5)P2 in the regulation of the TCR membrane dynamics and signaling initiation using a combination of biophysical, biochemistry and cell biology approaches. Ectopic expression of the Inp54p, a 5-phophatase that hydrolyzes PI(4,5)P2 into PI(4)P, with a membrane targeting signal specifically decreased by 50% of the PI(4,5)P2 in a CD4+ T cell hybridoma. Interestingly, we observed that this decrease caused modified TCR (and CD4 co-receptor) dynamics in the plasma membrane. The lateral diffusion switched from a regime dominated by dynamic partitioning in the cholesterol- and sphingolipid-dependent nanodomains into one dominated by dynamic partitioning in the actin cytoskeleton-assisted nanodomains. This switch was associated with a change in activation of the TCR and proximal signaling pathways both at the basal level and upon stimulation. Upon pMHC engagement, the CD4-independent activation of the TCR signaling pathways was found significantly augmented while that of CD4-dependent was affected. We further provided evidence for the involvement of PI(4,5)P2 in the Finally, we found that inhibition of interactions between PI(4,5)P2 and endogenous proteins with neomycin resulted in the modified TCR membrane dynamics and proximal signaling in primary murine CD4+ T cells. Altogether, our data reveal that PI(4,5)P2 is crucially involved in the control of the activation of TCR early signaling pathways.
|
6 |
Understanding the rapid expression of lymphocyte activation gene 3 (LAG-3) on healthy human T cellsStalker, Andrew 01 September 2015 (has links)
LAG-3 is an immune inhibitory marker. These immune inhibitory markers function to reduce the capability of immune cells to elicit a proper immune response and to help maintain tolerance. During an acute infection, these markers assist in controlling the immune system, however, during a chronic infection these markers prevent the immune response from persisting to effectively fight the disease. Contrary to other immune inhibitory markers, LAG-3 is not highly expressed on T cells during chronic viral infections, such as HIV.
The majority of information available on LAG-3 has been gained from murine models and cell lines. This thesis uses primary human T cells in order to observe rapid expression of surface LAG-3 from a pre-formed intracellular store and cleavage of these surface molecules into soluble variants by matrix metalloprotease cleavage. LAG-3 and sLAG-3 expression is compared with CD69 and cytokine expression to help understand the early immune response. / October 2015
|
7 |
The mechanism and functional consequences of passive acquistion of membrane and integral membrane protein by bovine polymorphonuclear neutrophilsWhale, Tyler 04 November 2005
<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>
|
8 |
The mechanism and functional consequences of passive acquistion of membrane and integral membrane protein by bovine polymorphonuclear neutrophilsWhale, Tyler 04 November 2005 (has links)
<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>
|
9 |
Zytokinexpression der CD3+ T-Zellen bei Divertikulitis und Peritonitis /Russ, Martin Adam. January 2002 (has links)
Würzburg, Universität, Thesis (doctoral), 2001.
|
10 |
Characterization of a Novel Signaling Motif in the CD3epsilon Subunit of the T Cell ReceptorWatts, Laura January 2008 (has links)
Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.138-150
|
Page generated in 0.0378 seconds