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VGLUT and GAD65 Expression in Physiologically Characterized Ia AfferentsUkpabi, Ivonne Nkoli January 2007 (has links)
No description available.
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Vliv diabetogenních autoantigenů na cytokinovou odpověď mononukleárních buněk periferní krve pacientů s diabetem 1. typu / Effect of diabetogenic autoantigens on the cytokine production of peripheral blood mononuclear cells from type 1 diabetic patientsLabiková, Jana January 2012 (has links)
5 Abstract Type 1 diabetes (T1D) is a serious organ-specific autoimmune disease characterised by irreversible destruction of pancreatic β-cells by immune system. This process results in an absolute insulin deficiency. Both genetical predisposition and environmental factors influence the development of the disease. β-cell destruction is mediated by cellular components of an immune system. Proinflammatory Th1 response is considered as most pathological. Autoimmune destruction of β-cells can be identified by the detection of specific serum autoantibodies a long time before the T1D clinical onset. Currently, there is no efficient cure available to prevent or at least to delay the destructive insulitis. This diploma thesis describes the influence of synthetic diabetogenic autoantigens GAD65 and IA2 on the cytokine response of peripheral blood mononuclear cells (PBMC) obtained from T1D patients with regards to their antibody profile. The study has been carried out on patients with confirmed T1D diagnosis who tested positive for anti-GAD65 and/or anti-IA2 autoantibodies. By using flow cytometry we measured the cell type ratio in PBMC samples. The cells have been stimulated by three different concentrations of antigens and their IFNγ and IL-17 production has been detected by ELISPOT assay. In the case of both...
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The production, expression, and characterisation of insulin and GAD65 recombinant FAB for use inPadoa, Carolyn Jane 14 October 2009 (has links)
Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2006. / Objectives. Autoantibodies to the 65kDa isoform of glutamic acid decarboxylase
(GAD65Abs) are accepted markers for type 1 diabetes and, together with
autoantibodies to insulin (IAA) and a protein tyrosine phosphatase-like islet cell
antigen (IA-2), predict the disease. IAA are often the first autoantibodies detected
in type 1 diabetics and can be present before the onset of clinical diabetes. These
autoantibodies and their epitopes are however not well characterized. We
explored the use of monoclonal antibodies and their recombinant Fab (rFab) as
reagents for epitope analysis.
Methods and Results. Four rFab specific for insulin were cloned from murine
monoclonal antibodies (mAbs) 1E2, HB-126, HB-123, HB-127, and one rFab
specific for GAD65 was cloned from human mAb IgG antibody DP-D (derived from
autoimmune disease patients), to characterise insulin and GAD65 autoantibodies
present in the sera of patients with type 1 diabetes. Only rFab 126 and DP-D
showed insulin and GAD65 specific binding, respectively in radiobinding assays.
In competition experiments with sera positive for autoantibodies to insulin the rFab
126 significantly reduced the binding to 125I-insulin by sera of type 1 (n=35) and
type 1.5 diabetes (or LADA) (n=14) patients (p<0.0001). There was no difference
in the competition pattern in IAA positive type 1 diabetes patients (n=35) and IAA
positive type 1.5 diabetes patients (n=14). The insulin epitope that the rFab binds
to was mapped using competitive radiobinding assays with two monoclonal
antibodies (mAb 1 and mAb 125) whose epitopes are on the B chain and A chain
loop of insulin, respectively. We found the epitope of this recombinant antibody to
be located on the A chain loop of the insulin molecule. The 3-dimensional
structure of rFab 123, 126 and DP-D were determined using an automated
homology modelling programme. Using the computer programme ‘PatchDock’ we
attempted to further map the epitope that rFab 126 binds to on insulin. Of the
three models generated, only one supported our findings that rFab 126 binds to
the A chain loop of insulin.
The binding of GAD65Ab in 61 type 1 diabetes patients to GAD65 was analyzed
by competitive radioimmunoassays with rFab DP-D to ascertain disease-specific
GAD65Ab binding specificities. The median binding was reduced significantly by
rFab DP-D (80%) (p<0.0001). The competition pattern in type 1 diabetes patients
was different from that in GAD65Ab-positive type 1.5 diabetes patients (n=44), first
degree relatives (n=38), and healthy individuals (n=14) (Padoa et al., 2003).
Conclusions. We have shown that rFab specific for insulin and GAD65 can be
generated using PCR technology and that such agents can be used to determine
the insulin/GAD65 epitopes recognized by autoantibodies from type 1 and 1.5
diabetics. These novel findings with GAD65- and insulin-specific rFab support the
view that type 1 diabetes is associated with disease- and epitope-specific GAD65-
and insulin-autoantibodies and supports the notion that the middle epitope of
GAD65 is disease-specific. These GAD65-specific rFab should prove useful in
predicting type 1 diabetes. Furthermore, rFabs may be a novel method for
blocking autoimmune responses against β cell autoantigens in type 1 diabetics.
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Study of inhibitory neurons in Broca's area in autismHouse, Elva Lucille 20 June 2020 (has links)
Individuals with Autism Spectrum Disorder (ASD) experience a variety of symptoms that vary dramatically across individuals and can range from severe impairments to minor issues with social interactions and communication. The underlying cause of ASD is still unknown, and the level of influence that genetic and environmental factors have on the severity and occurrence of ASD is still a topic of great debate. Since the theories concerning cause or causes of ASD are multifactorial, the treatment options available are extremely limited and are based on behavioral testing. Alternatively, genetic testing might be considered in a diagnosis protocol. This study is designed to investigate ASD by assessing the variability of three genes associated with neuronal inhibition. Based on previous studies this experiment hypothesized that GAD1, GAD2, and PAVLB expression is decreased in Broca’s area in individuals with ASD when compared to controls, with the premise that this alteration could contribute to the symptoms involving language and communication. In situ hybridization was used to quantify the expression of the GAD1, GAD2, and PVALB genes in Broca’s area in postmortem human tissue. The variability of these three genes was quantified by measuring the amount of radioactively tagged mRNA in fifty cell bodies in each brain sample. This study used twenty-two brains of individuals with ASD and twenty-one control brains, including age matched males and females. The variables of age and sex are analyzed and discussed as well as the emulsion and film analyses. A decrease in parvalbumin expression was found between the ASD and control groups in Broca’s area. These finding were discussed in the context of symptoms and neuropathological features of ASD.
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Processing and Presentation of Glutamic Acid Decarboxylase 65 T cell-Inducing Epitopes: Implications in the Non-Obese Diabetic Mouse Model of Type 1 DiabetesRasche, Sarah S. January 2010 (has links)
No description available.
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Effectivization of GAD ELISA : Method-transfer from Dynex DSX to Tecan Freedom EVOlyzerBernal Salazar, Juan Manuel January 2022 (has links)
Due to a growing demand and need for faster results, effectivization as a concept has been pushed to the forefront of medical research. Here, a method transfer between 2 fully automized ELISA instruments is highlighted. The instruments were the Tecan Freedom EVOlyzer and the Dynex DSX. The Tecan instrument is a more modern instrument with a more extensive LIMS integration, better automatization and increased capacity for samples and assays. The method being transferred was an Anti-GAD ELISA for the diagnosis of Type 1 Diabetes Mellitus. The study design was a result comparison of 20 samples with varying Anti-GAD concentration and 15 replicates of a single sample for a precision study. To successfully transfer the method, acceptance criteria concerning percentual difference in result, intermediate precision, and repeatability had to fall below 20 %. The assay was set up as a sandwich ELISA in accordance with the kit used at the laboratory (Euroimmun), where each sample was set as duplicates. Preliminary programming and testing was performed to ensure proper function. An average bias of +33 % was reported, as was an intra-run variation and an inter-run variation of ca 11 %. Additionally, a recurring issue with samples and calibrators in specific locations on the plate was reported. The issue was deemed systemic and possible solutions include changing programming parameters, or instrument part replacement. Ultimately the method transfer was left incomplete, and several technical details must be overcome before taking Anti-GAD analysis into routine use on the instrument.
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Molekularbiologische Untersuchungen zu zentralnervösen Alterungsprozessen der Reproduktionsfunktion in der weiblichen Ratte / Molecular Biological Analysis of Central Nervous Age-Related Processes of the Reproduction Functions in the Female RatMakhouly, Bassel 03 October 2002 (has links)
Die GABA-ergen Neurone als Teil des GnRH-Netzwerkes spielen eine Rolle bei den Veränderungen der altersabhängigen Prozesse der Reproduktionsfunktion. Um die Regulation der gonadalen Steroide auf die Expression von GAD in reproduktionsabhängigen Regionen zu untersuchen, wurde im ersten Teil der vorliegenden Arbeit das männliche Rattenmodell gewählt. Nach der Untersuchung des endokrinen Zustands der Tiere anhand der Radioimmunoassay-Methode (RIA) wurden die zellulären Gentranskripte der beiden Isoformen von GAD, GAD65 und GAD67, mittels der Methode der in situ Hybridisierung in der POA, im Nukleus suprachiasmaticus (SCN), im mediobasalen Hypothalamus (MBH) und im Gyrus dentatus bestimmt. In allen untersuchten Regionen konnte nach der Kastration und einer anschließenden dreiwöchigen Erholungszeit kein Effekt beobachtet werden. Die Administration von Estradiol bewirkt in der POA eine signifikante Erhöhung der Expression von GAD65 und GAD67 um nahezu 40%. In den restlichen Regionen konnte dagegen kein Effekt gemessen werden. Die Testosteronbehandlung zeigte eine negative Wirkung auf die Regulation nur von GAD67: Eine 30%-ige Abnahme in der POA und eine 15%-ige im SCN. Im Gegensatz dazu trat im MBH und im Gyrus dentatus eine Verminderung der Expression nur bei GAD65 auf. Aus den hier vorgestellten Ergebnissen kann folgendes abgeleitet werden: Testosteron und Estradiol regulieren in unterschiedlicher Weise die Expression von GAD und so wiederum die inhibitorische Funktion von GABA. Da in der SCN, im MBH und im Gyrus dentatus im Gegensatz zu Estradiol eine Testosteron-Wirkung gemessen wurde, existiert eine eigene androgene Regulation von GAD. Weil die Estradiol-Zugabe eine Zunahme der Expression von GAD bewirkte und dieser Effekt von einer Abnahme der LH-Konzentration im Serum der betroffenen Tiergruppe begleitet wurde, ist die These bestätigt, dass GABA mit ihren inhibitorischen Funktionen zur Übermittlung der positiven Rückkopplung von Estradiol auf die LH-Freisetzung auf der Ebene der POA und nicht auf der Ebene der Axone agiert. Im Gegensatz zu Estradiol kann eine Progesteronbehandlung bei persistent östrischen Ratten einen LH-Peak auslösen und somit den Östrus-Zyklus wieder in Gang bringen. Aufgrund dieser Tatsache wurde im zweiten Teil der vorliegenden Arbeit ein Tiermodell zur Untersuchung der molekularbiologischen altersabhängigen Veränderungen entwickelt. Dabei wurden drei Monate alte proöstrische Ratten (Y) und 12 Monate alte persistent östrische Ratten (MA) benutzt. Die MA-Ratten wurden mit Progesteron behandelt. Sowohl die MA-Ratten als auch die Y-Ratten wurden um 13 Uhr und um 17 Uhr getötet. Eine unbehandelte MA-Gruppe, deren Tiere um 10 Uhr getötet wurden, diente hier als Kontrollgruppe. Anhand der LH-Messung der untersuchten Gruppen wurde ein Kontrollwert (5 ng/ml LH) für die positive Reaktion der Tiere auf Progesteron (responding animals) festgestellt. Es konnte bei 44% der persistent östrischen Ratten ein erhöhter LH-Spiegel erfolgreich wieder erreicht werden. In den Gruppen dieses Modells entstand eine Analogie zwischen den Gruppen der behandelten MA-13-Uhr und Y-13-Uhr Tiere sowie zwischen den responding animals und den Y-17 Uhr-Tieren. Um aussagekräftige statistische Veränderungen entlang der hypothalamo-hypophysio-ovariellen Achse in individuellen Tieren zu erhalten, wurde die Taqman®-PCR und die quantitative, kompetitive RT-PCR eingesetzt. Dabei wurden die folgenden Gene untersucht: ER α und ER β, GnRH, GnRH-R, GAD65 und GAD67, sowie FSH-β. In der POA, Hypophyse und im Ovar wurde altersabhängigen Genexpression beobachtet: Eine signifikante Abnahme der Expression von ER β sowohl in der Gruppe responding animals als auch in deren analoger Gruppe wurde in der POA (34 %), Hypophyse (44 %) und im Ovar (um die 30 %) gemessen. In der Hypophyse verzeichneten die mRNA-Transkripte von ER α bei der Gruppe der behandelten mittelalten Ratten der 13 Uhr-Gruppe eine Zunahme von 55% und bei der 13-Uhr-Gruppe der jungen Ratten einen Anstieg von 153 %. Ebenso nehmen die mRNA-Konzentrationen von FSH-β sowohl bei den responding animals als auch bei deren analoger Gruppe in gleichem Masse (ungefähr 300 %) zu. Da die Veränderungen der Expression von ER β, ER α und FSH-β bei den zwei analogen Gruppen auftritt, ist zu vermuten, dass diese Gene altersabhängig expremiert und an der Zyklusregulation ursächlich beteiligt sind. Die restlichen Gene zeigten entlang der Achse keine altersrelevanten Veränderungen. Da ER β-Expressionsveränderungen in der POA, in der Hypophyse und im Ovar gemessen wurden, konnte der wichtigste Schluss der hier vorgestellten Untersuchungen gezogen werden, dass nämlich ER β für den Erhalt des Zyklus essentiell sein kann. In diesem Teil der vorliegenden Arbeit wurde ein Tiermodell zur molekular biologischen Untersuchung der altersabhängigen Veränderungen mit sehr zufriedenstellender Ausbeute zur Wiederherstellung des Östrus-Zyklus (44%) erfolgreich entwickelt. Dieses Modell ermöglichte darüber hinaus die Untersuchung einer relativ hohen Anzahl an Genen entlang der hypothalamo-hypophysio-ovariellen Achse.
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Evaluation of the feasibility of intralymphatic injection of Diamyd®Fessehaye, Selam January 2019 (has links)
Type 1 diabetes affects a person’s life on many levels in terms of quality of life, health, and socioeconomic costs both for the patients but also their families. As of now there is no therapy that targets the underlying mechanism of the disease. Intralymphatic administration of Diamyd® is being evaluated in a phase IIb clinical trial, DIAGNODE-2. The aim was to examine if the intralymphatic administration is feasible for both patients and medical professionals, and to identify any aspects of the procedure that can be improved. This feasibility study is based on interviews and answers received from questionnaires. The medical professionals that were selected were radiologists and study nurses that are involved in the DIAGNODE-2 trial. The radiologists were the prime focus and were thus interviewed through face-to-face/skype or phone and answered a questionnaire. Study nurses, having more contact with the patient, answered a survey in order to gain additional insights into the patient perspective. The results show that the radiologists has a positive view towards the administration procedure, which was described as easy and safe. According to the study nurses the patients accept the procedure and they agreed that the patients understand the injection procedure once they received the information. In terms of the emotional state of the patients they were a bit nervous, but they became calmer after receiving the first injection. Based on the above-mentioned findings the intralymphatic injection procedure is described as feasible and has the potential to become a part of the standard clinical routine.
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