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Psychische Belastungsfaktoren bei Patienten mit chronischer Hepatitis-C-Infektion während und außerhalb einer antiviralen InterferontherapieSchäfer, Arne 31 January 2008 (has links)
I) Hintergrund Die chronische Hepatitis-C-Infektion stellt global ein wesentliches Gesundheitsproblem dar. Diese Virusinfektion kann bei unbehandelten Patienten zur Leberzirrhose und im weiteren Verlauf bis hin zur Entwicklung eines hepatozellulären Karzinoms führen. Die einzige Behandlungsoption mit der Aussicht auf dauerhafte Viruselimination besteht in modernen Kombinationstherapien, die das Zytokin Interferon alfa enthalten. Wesentliche Merkmale sind – neben inzwischen sehr hohen Ansprechraten – eine Behandlungsdauer zwischen 24 und 48 Wochen, hohe Therapiekosten und ein Nebenwirkungsprofil, das sowohl somatische als auch psychopathologische Symptome umfassen kann. II) Untersuchungsgegenstand und Fragestellungen Sowohl die chronische Virusinfektion an sich als auch die aktuell verfügbaren Therapieverfahren bergen ein erhebliches psychisches Belastungspotential. Hauptgegenstand dieser Dissertation ist die Erfassung der psychologischen Aspekte der Erkrankung und der psychischen und psychopathologischen Nebenwirkungen einer Interferonbehandlung. Wesentliche bearbeitete Fragestellungen sind: - Welchen Belastungsfaktoren sind Hepatitis-C-Patienten bereits ohne aktuelle antivirale Interferontherapie ausgesetzt bzw. welche psychopathologischen Symptome zeigen diese Patienten? - Wie ist der zeitliche Verlauf psychopathologischer Symptome bei Hepatitis-C-Patienten vor, während und nach einer antiviralen Therapie? - Wie wirksam und wie sicher ist eine medikamentöse Behandlung der Interferon-induzierten Depression mit selektiven Serotonin-Wiederaufnahmehemmern (SSRI) unter Fortführung der antiviralen Therapie? III) Patienten und Methoden Studienteilnehmer waren Hepatitis-C-Patienten, die sich ambulant vorstellten bzw. in unsere Ambulanz überwiesen wurden und die jeweiligen Einschlusskriterien erfüllten. Zu den wichtigsten verwendeten psychometrischen Selbstbeurteilungsskalen zählen: HADS (Depressivität, Angst), SCL-90-R (psychopathologische Symptome), SF-36 (Lebensqualität) und FKV (Krankheitsverarbeitung). IV) Wesentliche Forschungsergebnisse Bereits ohne Einfluss des Zytokins Interferon bestehen starke Krankheits-assoziierte psychische bzw. psychosoziale Belastungen der Patienten, die sich in einem erhöhten Depressionsrisiko ausdrücken. Die erhobenen Depressionsscores stehen in signifikantem Zusammenhang mit der Erkrankungsdauer und den individuell bestehenden Optionen und Erfolgsaussichten einer antiviralen Interferontherapie. Prospektive Erfassungen der Auftretenshäufigkeit klinisch relevanter Interferon-assoziierter Depressionen ergeben Raten von ca. 30 %. Diese Größenordnung wurde sowohl in einer eigenen prospektiven Studie als auch im Rahmen einer vorgestellten Übersichtsarbeit bestätigt. Die Umstellung der verwendeten Formulierung des Medikaments von herkömmlichem Interferon alfa auf die pegylierte Variante brachte keine Verbesserung der Verträglichkeit z.B. im Hinblick auf die interferonassoziierte Depression. Ein rechtzeitiges Erkennen der entsprechenden Symptome vorausgesetzt, ist die antidepressive Behandlung der Interferon-assoziierten Depression mit Hilfe von selektiven Serotonin-Reuptake-Inhibitoren auch ohne generelle Prophylaxe sehr effektiv und sicher möglich. V) Diskussion Empfohlen wird ein engmaschiges psychometrisches Monitoring aller Hepatitis-C-Patienten im Therapieverlauf. Ausführliche Aufklärung, enger Arzt-Patienten-Kontakt während der Therapie, sowie die Betreuung durch einen festen Ansprechpartner während der bis zu einem Jahr dauernden Therapie sind wichtige Rahmenbedingungen für eine solche Behandlung. Für die medikamentöse Behandlung der Interferon-induzierten Depression gilt: Bei besonderer Indikation (z.B. Interferon-assoziierte Depression bei früheren Therapieversuchen) sollte eine SSRI-Sekundärprophylaxe in Betracht gezogen werden. Ansonsten ist eine entsprechende SSRI-Intervention beginnend mit dem Einsetzen einer klinisch relevanten Depression ausreichend.
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Dynamik histomorphologischer Veränderungen in der Leber von Milchkühen im peripartalen ZeitraumPietsch, Fabian 09 November 2022 (has links)
Dynamik histomorphologischer Veränderungen in der Leber von Milchkühen im peripartalen Zeitraum
Einleitung
Das Lipomobilisationssydrom verursacht bei Milchkühen vielfältige Produktionskrankheiten und hohe ökonomische Verluste. Aus der Humanmedizin ist bekannt, dass chronische Leberverfettungen zu Steatohepatitiden und im Weiteren zu Leberzirrhosen führen können.
Ziel der Untersuchung
Ziel der Untersuchung war es, Schädigungen des Lebergewebes unter Betrachtung der Dynamik verschiedener Einzelmerkmale (Fett- und Glykogeneinlagerung, Hepatitis, Fibrose) histopathologisch vergleichend darzustellen und dabei auf Parallelen zur nichtalkoholischen Steatohepatitis (NASH) des Menschen zu untersuchen. Weiterhin sollen Einflüsse des Temperatur-Feuchtigkeits-Indexes (THI) auf die Dynamik histomorphologischer Leberveränderungen untersucht werden.
Material und Methoden
In die Studie wurden 80 Deutsch Holstein Kühe einbezogen. Zwei Gruppen (je n = 20) erhielten (5 oder 10 ml / 100 kg Körpergewicht) Butaphosphan und Cyanocobalamin (BCC) und eine Gruppe (n = 40) ein Placebo. Leberbioptate wurden 14 Tage (d) ante partum (a.p.) sowie 7, 28, 42 d post partum (p.p.) entnommen. Sie wurden mittels vier verschiedener Verfahren (Hämalaun-Eosin, Sudan III, Periodsäure-Schiff-Reaktion und Pikrosiriusrot) aufgearbeitet und auf Fett- und Glykogeneinlagerungen sowie degenerative, entzündliche, fibrotische und proliferative Lebergewebsveränderungen semiquantitativ untersucht. In der statistischen Auswertung wurden Effekte aus den Leberbiopsiezeitpunkten, der Laktationsanzahl, der metaphylaktischen BCC Behandlung, des THIs und der Metabotypen (Massenspektrometrie von Leberproben) ausgewertet. Mittels einer Metabolomanalyse wurden drei verschiedene Metabotypen identifiziert.
Ergebnisse
Bereits in den ersten Wochen vor der Abkalbung wiesen die Kühe 37 % ggr. bis mgr. Fetteinlagerungen in der Leber auf. Bis zur ersten Woche p.p. konnte ein deutlicher Anstieg des Leberfettgehaltes beobachtet werden (66 % mgr. bis hgr.), der bis einschließlich der sechsten Woche p.p. wieder abfiel (ca. 25 % mgr. bis hgr.). Der Grad der Leberverfettung korrelierte positiv mit dem Anteil an Leberzelldegenerationen und negativ mit den Glykogeneinlagerungen. Trotz hgr. Leberverfettungen kam es zu keinem Zeitpunkt zu einer vollständigen Glykogendepletion in den Leberzellen. Bei 39 % der Tiere wurde während der gesamten Transitperiode eine mgr. bis hgr. lymphozytäre Hepatitis nachgewiesen. Kühe ab der fünften Laktation wiesen signifikant häufiger perisinusoidale Fibrosen auf. In keinem Fall wurden hgr. Fibrosen diagnostiziert. Kühe einer der drei Metabotypengruppen wiesen eine höhere Chance für vermehrte Fettinfiltrationen, geringere Glykogeneinlagerungen und vermehrte degenerative, entzündlich-fibrotische sowie proliferative Leberparenchymveränderungen auf. Die Auswertung der Fütterungsdaten lässt einen Zusammenhang zwischen der Fütterung von Grassilage mit einer verminderten Qualität (hoher Rohascheanteil, erhebliche Schwankungen in Trockensubstanz- und Nährstoffgehalten), dem Lebermetabolom und histomorphologischen Veränderungen der Kühe dieser Metabotypengruppe vermuten. Für die Behandlung mit BCC konnten keine signifikanten Effekte festgestellt werden. Der THI hatte einen positiven Einfluss auf den Grad der Fetteinlagerung und die Zelldegenerationen sowie einen negativen Einfluss auf den Grad der Glykogeneinlagerung.
Schlussfolgerungen
Während des peripartalen Zeitraums kam es in der Leber von Milchkühen zur Fettakkumulationen und Glykogendepletionen sowie zu Hepatitiden und Leberzelldegenerationen. Letztere sind Hinweise einer Steatohepatitis, ähnlich einer NASH des Menschen. Vor allem bei älteren Kühen waren perisinusoidale Fibrosen nachweisbar, die eine Vorstufe von Leberfibrosen sein können und mit höheren Milchleistungen sowie dem damit verbundenen erhöhten Blutfluss in der Leber zusammenhängen könnten. Die Ergebnisse weisen Zusammenhänge zwischen einem ausgeprägteren Körperkonditionsverlust, vermehrten Fettinfiltrationen, geringeren Glykogeneinlagerungen sowie wechselseitige Beziehungen zwischen degenerativen, entzündlichen, fibrotischen und proliferativen Leberveränderungen auf. Als ein ursächlicher Faktor wird eine verminderte Grassilagequalität vermutet. Durch den THI werden Fett- und Glykogeneinlagerungen sowie Leberzelldegenerationen beeinflusst.:1. Einleitung
2. Literaturübersicht
2.1 Die Funktionen der Leber bei Rindern
2.1.1 Allgemein
2.1.2 „First pass effect“
2.1.3 Entgiftung
2.1.4 Leber als Stoffwechselorgan
2.1.4.1 Allgemein
2.1.4.2 Aminosäuren- und Proteinstoffwechsel
2.1.4.2.1 Bedeutung
2.1.4.2.2 Proteinsynthese
2.1.4.2.3 Aminosäuresynthese und -abbau
2.1.4.2.4 Harnstoffsynthese
2.1.4.3 Fettstoffwechsel
2.1.4.3.1 Bedeutung
2.1.4.3.2 Cholesterolbiosynthese
2.1.4.3.3 Fettsäuresynthese
2.1.4.3.4 Fettsäureoxidation
2.1.4.3.5 Ketogenese
2.1.4.4 Kohlenhydratstoffwechsel
2.1.4.4.1 Bedeutung
2.1.4.4.2 Gluconeogenese
2.1.4.4.3 Glykogensynthese
2.1.4.4.4 Glykogenolyse
2.1.4.4.5 Glykolyse
2.1.5 Leber als Speicherorgan
2.1.5.1 Allgemein
2.1.5.2 Glykogen
2.1.5.2.1 Allgemein
2.1.5.2.2 Bedeutung
2.1.5.2.3 Einflussfaktoren
2.1.5.3 Fett
2.1.5.3.1 Allgemein
2.1.5.3.2 Bedeutung
2.1.5.3.3 Einflussfaktoren
2.2 Das Lipomobilisationssyndrom der Milchkuh und die nichtalkoholische
Leberverfettung des Menschen
2.2.1 Lipomobilisationssyndrom der Milchkuh
2.2.1.1 Allgemein
2.2.1.2 Pathogenese
2.2.1.3 Bedeutung und Epidemiologie
2.2.2 Nichtalkoholische Leberverfettung des Menschen
2.2.2.1 Allgemein
2.2.2.2 Pathogenese
2.2.2.3 Bedeutung und Epidemiologie
2.2.3 Hinweise einer nichtalkoholischen Leberverfettung bei Milchkühen
2.3 Die Lichtmikroskopie zur histologischen Beurteilung von
Lebergewebsveränderungen
2.3.1 Besonderheiten
2.3.2 Diagnostische Beurteilung von Lebergewebe
2.3.3 Bisherige histologische Untersuchungen der Leber von Rindern und
Forschungsbedarf
2.4 Zusammenfassende Schlussfolgerungen aus dem Literaturstudium und
Arbeitshypothesen
3. Material und Methoden
3.1 Studienprotokoll
3.2 Klimadaten
3.3 Entnahme der Leberbioptate
3.4 Aufbereitung der Leberbioptate für die histologische Untersuchung
3.5 Histopathologische Befundung
3.5.1 Durchführung der histopathologischen Untersuchung
3.5.2 Beurteilung der Fetteinlagerung
3.5.3 Beurteilung der Glykogeneinlagerung
3.5.4 Beurteilung der degenerativen, entzündlichen, fibrotischen und
proliferativen Veränderungen
3.6 Ergänzende Statistik (Temperatur-Feuchtigkeits-Index)
4. Publikation
5. Weitere Ergebnisse
5.1 Vorkommen von Glykogeneinlagerungen bei gleichzeitiger
Leberverfettung
5.2 Dynamik der Fett- und Glykogeneinlagerung im Leberläppchen
5.3 Dynamik der Hepatitis im Untersuchungszeitraum
5.4 Dynamik der Fibrose im Untersuchungszeitraum
5.5 Fallbeschreibungen von Einzeltieren mit entzündlichen, fibrotischen und
proliferativen Besonderheiten
5.6 Temperatur-Feuchtigkeits-Index Einflüsse
6. Diskussion
6.1 Der Temperatur-Feuchtigkeits-Index und sein Einfluss auf die
Histomorphologie
6.2 Periportale Fettakkumulation und Glykogendepletion
6.3 Periportales Auftreten von Hepatitis und Fibrose
6.4 Einzelfälle mit entzündlichen, fibrotischen und proliferativen
Veränderungen im Lebergewebe und deren mögliche Ursachen
6.5 Vergleich zwischen Lipomobilisationssyndrom der Milchkuh und der
nichtalkoholischen Steatohepatitis des Menschen
6.5.1 Vorkommen von Leberverfettung
6.5.2 Vorkommen von Hepatitis
6.5.3 Vorkommen von Leberzelldegeneration
6.5.4 Vorkommen von Leberfibrose
6.5.5 Unterscheidung der Ursachen und Folgen einer Leberverfettung bei
Mensch und Rind
6.6 Klinische Relevanz der histopathologischen Leberveränderungen
7. Zusammenfassung
8. Summary
9. Literaturverzeichnis
10. Danksagung
11. Anhang
11.1 Übersicht histopathologischer Untersuchungen der Leber von Rindern
11.2 Färbeanleitungen
11.2.1 Hämalaun-Eosin-Färbung
11.2.1.1 Verfahrensschritte
11.2.1.2 Benötigte Reagenzien
11.2.1.3 Ergebnis
11.2.2 Sudan-III-Hämalaun-Färbung
11.2.2.1 Verfahrensschritte
11.2.2.2 Benötigte Reagenzien
11.2.2.3 Ergebnis
11.2.3 Periodsäure-Schiff-Reaktion und Amylase Reaktion
11.2.3.1 Verfahrensschritte
11.2.3.2 Benötigte Reagenzien
11.2.3.3 Ergebnis
11.2.3.4 Amylase-Kontrollreaktion
11.2.3.4.1 Methode
11.2.3.4.2 Ergebnis
11.2.4 Pikrosiriusrot-Färbung
11.2.4.1 Verfahrensschritte
11.2.4.2 Benötigte Reagenzien
11.2.4.3 Ergebnis
11.3 Untersuchungsprotokolle / Dynamic histomorphological changes of the liver parenchyma during the transition period in dairy cows
Introduction
Lipomobilisation sydrome is associated with multiple production diseases in dairy cows and causes substantial economic losses. In people, chronic hepatic lipidosis can lead to steatohepatitis and subsequently liver cirrhosis.
Objectives
The main goal of this study was to investigate the histopathological changes in the liver parenchyma in terms of hepatitis, fibrosis and fat and glycogen deposits in dairy cows. Additionally, the histomorphological lesions in dairy cows with lipomobilisation syndrome were compared with those seen in people with non-alcoholic steatohepatitis (NASH). The effects of the temperature-humidity index (THI) on the dynamics of histomorphological hepatic changes were also evaluated.
Material and Methods
A total of 80 German Holstein cows were divided into three groups: the first group consisted of 20 cows that received 5 ml / 100 kg of butaphosphan and cyanocobalamin (BCC); the second group consisted of 20 cows that received 10 ml / 100 kg of BCC; and the third group consisted of 40 that cows received a placebo. Liver biopsy was carried out 14 days antepartum (a.p.) and 7, 28 and 42 days postpartum (p.p.). The liver tissue was processed routinely and stained with haematoxylin and eosin, sudan III, periodic acid-Schiff and picrosirius red stains. The histological sections were assessed semiquantitatively for fat and glycogen content and for degenerative, inflammatory, fibrotic and proliferative changes. The effects of time of biopsy, lactation number, metaphylactic BCC treatment, THI and metabotype (mass spectrometry of liver tissue) were examined statistically. A metabolom analysis was used to identify three metabotypes.
Results
Mild to moderate fat infiltration was seen in the liver of 37 % of cows in the last two weeks a.p. The degree of fat infiltration increased considerably from two weeks a.p. until the end of the first week when it was moderate to severe in 66 % of the cows. It then decreased again until the end of the study period when it was moderate to severe in 25 % of the cows. Lipidosis was significantly and positively correlated with the severity of hepatocyte degeneration and negatively correlated with the degree of glycogen deposition. Complete glycogen depletion of hepatocytes was not observed in cows even in the presence of severe hepatic lipidosis. Moderate to severe lymphocytic hepatitis was seen in 39 % of cows throughout the study period, and cows with lactation numbers five or greater had perisinusoidal fibrosis significantly more often than younger cows. Severe fibrosis of the liver did not occur. Cows of one of the three metabotypes had an increased risk of fatty infiltration of the liver, lower glycogen storage and degenerative, inflammatory, fibrotic, and proliferative changes of the liver parenchyma. Based on ration analysis, feeding of poor-quality grass silage (high ash content, fluctuations in dry matter and nutrient content) appeared to be associated with the liver metabolom and histomorphological liver changes. Treatment with BCC had no significant effects on any of the variables measured. The THI had a positive effect on the degree of lipid deposition and hepatocyte degeneration and a negative effect on the degree of glycogen deposition.
Conclusion
During the transition period, the liver of dairy cows is characterised by fat accumulation and glycogen depletion and histological signs of hepatitis and hepatocyte degeneration. The latter suggests steatohepatitis analogous to NASH in people. Perisinusoidal fibrosis was seen particularly in older cows and may represent an early stage of fibrosis of the liver. It is conceivable that these changes are associated with increased perfusion of the liver in association with increased milk production. There were correlations between pronounced loss of body condition, fatty infiltration of the liver and lower glycogen storage and degenerative, inflammatory, fibrotic, and proliferative changes of the liver parenchyma. These findings suggest that poor-quality grass silage was causative factor. Changes in the THI may also affect fat and glycogen deposition and liver cell degeneration in the liver.:1. Einleitung
2. Literaturübersicht
2.1 Die Funktionen der Leber bei Rindern
2.1.1 Allgemein
2.1.2 „First pass effect“
2.1.3 Entgiftung
2.1.4 Leber als Stoffwechselorgan
2.1.4.1 Allgemein
2.1.4.2 Aminosäuren- und Proteinstoffwechsel
2.1.4.2.1 Bedeutung
2.1.4.2.2 Proteinsynthese
2.1.4.2.3 Aminosäuresynthese und -abbau
2.1.4.2.4 Harnstoffsynthese
2.1.4.3 Fettstoffwechsel
2.1.4.3.1 Bedeutung
2.1.4.3.2 Cholesterolbiosynthese
2.1.4.3.3 Fettsäuresynthese
2.1.4.3.4 Fettsäureoxidation
2.1.4.3.5 Ketogenese
2.1.4.4 Kohlenhydratstoffwechsel
2.1.4.4.1 Bedeutung
2.1.4.4.2 Gluconeogenese
2.1.4.4.3 Glykogensynthese
2.1.4.4.4 Glykogenolyse
2.1.4.4.5 Glykolyse
2.1.5 Leber als Speicherorgan
2.1.5.1 Allgemein
2.1.5.2 Glykogen
2.1.5.2.1 Allgemein
2.1.5.2.2 Bedeutung
2.1.5.2.3 Einflussfaktoren
2.1.5.3 Fett
2.1.5.3.1 Allgemein
2.1.5.3.2 Bedeutung
2.1.5.3.3 Einflussfaktoren
2.2 Das Lipomobilisationssyndrom der Milchkuh und die nichtalkoholische
Leberverfettung des Menschen
2.2.1 Lipomobilisationssyndrom der Milchkuh
2.2.1.1 Allgemein
2.2.1.2 Pathogenese
2.2.1.3 Bedeutung und Epidemiologie
2.2.2 Nichtalkoholische Leberverfettung des Menschen
2.2.2.1 Allgemein
2.2.2.2 Pathogenese
2.2.2.3 Bedeutung und Epidemiologie
2.2.3 Hinweise einer nichtalkoholischen Leberverfettung bei Milchkühen
2.3 Die Lichtmikroskopie zur histologischen Beurteilung von
Lebergewebsveränderungen
2.3.1 Besonderheiten
2.3.2 Diagnostische Beurteilung von Lebergewebe
2.3.3 Bisherige histologische Untersuchungen der Leber von Rindern und
Forschungsbedarf
2.4 Zusammenfassende Schlussfolgerungen aus dem Literaturstudium und
Arbeitshypothesen
3. Material und Methoden
3.1 Studienprotokoll
3.2 Klimadaten
3.3 Entnahme der Leberbioptate
3.4 Aufbereitung der Leberbioptate für die histologische Untersuchung
3.5 Histopathologische Befundung
3.5.1 Durchführung der histopathologischen Untersuchung
3.5.2 Beurteilung der Fetteinlagerung
3.5.3 Beurteilung der Glykogeneinlagerung
3.5.4 Beurteilung der degenerativen, entzündlichen, fibrotischen und
proliferativen Veränderungen
3.6 Ergänzende Statistik (Temperatur-Feuchtigkeits-Index)
4. Publikation
5. Weitere Ergebnisse
5.1 Vorkommen von Glykogeneinlagerungen bei gleichzeitiger
Leberverfettung
5.2 Dynamik der Fett- und Glykogeneinlagerung im Leberläppchen
5.3 Dynamik der Hepatitis im Untersuchungszeitraum
5.4 Dynamik der Fibrose im Untersuchungszeitraum
5.5 Fallbeschreibungen von Einzeltieren mit entzündlichen, fibrotischen und
proliferativen Besonderheiten
5.6 Temperatur-Feuchtigkeits-Index Einflüsse
6. Diskussion
6.1 Der Temperatur-Feuchtigkeits-Index und sein Einfluss auf die
Histomorphologie
6.2 Periportale Fettakkumulation und Glykogendepletion
6.3 Periportales Auftreten von Hepatitis und Fibrose
6.4 Einzelfälle mit entzündlichen, fibrotischen und proliferativen
Veränderungen im Lebergewebe und deren mögliche Ursachen
6.5 Vergleich zwischen Lipomobilisationssyndrom der Milchkuh und der
nichtalkoholischen Steatohepatitis des Menschen
6.5.1 Vorkommen von Leberverfettung
6.5.2 Vorkommen von Hepatitis
6.5.3 Vorkommen von Leberzelldegeneration
6.5.4 Vorkommen von Leberfibrose
6.5.5 Unterscheidung der Ursachen und Folgen einer Leberverfettung bei
Mensch und Rind
6.6 Klinische Relevanz der histopathologischen Leberveränderungen
7. Zusammenfassung
8. Summary
9. Literaturverzeichnis
10. Danksagung
11. Anhang
11.1 Übersicht histopathologischer Untersuchungen der Leber von Rindern
11.2 Färbeanleitungen
11.2.1 Hämalaun-Eosin-Färbung
11.2.1.1 Verfahrensschritte
11.2.1.2 Benötigte Reagenzien
11.2.1.3 Ergebnis
11.2.2 Sudan-III-Hämalaun-Färbung
11.2.2.1 Verfahrensschritte
11.2.2.2 Benötigte Reagenzien
11.2.2.3 Ergebnis
11.2.3 Periodsäure-Schiff-Reaktion und Amylase Reaktion
11.2.3.1 Verfahrensschritte
11.2.3.2 Benötigte Reagenzien
11.2.3.3 Ergebnis
11.2.3.4 Amylase-Kontrollreaktion
11.2.3.4.1 Methode
11.2.3.4.2 Ergebnis
11.2.4 Pikrosiriusrot-Färbung
11.2.4.1 Verfahrensschritte
11.2.4.2 Benötigte Reagenzien
11.2.4.3 Ergebnis
11.3 Untersuchungsprotokolle
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Understanding the Role of the Hypervariable Region in the Open Reading Frame 1 of the Hepatitis E virus in Viral ReplicationPudupakam, Raghavendra Sumanth Kumar 15 March 2011 (has links)
Hepatitis E virus (HEV) is a major cause of enterically transmitted acute viral hepatitis in developing countries that lack proper hygienic infrastructure. Hepatitis E is globally distributed and has emerged as an important public health disease in both developing and industrialized countries. HEV is a non-enveloped virus carrying a single-stranded positive-sense RNA genome of approximately 7.200 bp in length. The life cycle of HEV is poorly understood due to the lack of an efficient cell culture system. Animal model systems, including non-human primates, swine, and chickens are being used to study some fundamental aspects of the HEV biology. Recently, novel animal strains of rat and rabbit HEV have been discovered, and whose usage as animal model systems needs to be established. HEV infections in pigs and chickens provide excellent model systems to study the replication and pathogenesis aspects of HEV. Recently, we identified a hypervariable region (HVR) in the open reading frame 1 (ORF1) of HEV. The objectives of this dissertation were to utilize chicken and swine model systems to study the role of HVR in HEV infection in vivo, to determine the effects of HVR on replication of HEV in vitro, and to analyze the effect of exchange of HVR among different genotypes on the replication-competency and virion production in vitro.
Extensive sequence variability in the HVR among HEV strains of different genotypes prompted us to study the dispensability of this region. Initially we constructed two partial deletion mutants of genotype 1 human HEV, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a sub-genomic GFP HEV replicon. Expression of enhanced green fluorescent protein by the mutant hHVRd2 confirmed the dispensability of amino acid residues 747-761 of the HVR. To confirm our in vitro results, specific-pathogen-free (SPF) chickens were intra-hepatically inoculated with capped RNA transcripts from three avian HEV HVR-deletion mutants: mutants aHVRd1 (Δ557-585), aHVRd2 (Δ612-641), and aHVRd3 (Δ557-641). Chickens intra-hepatically inoculated with the mutants, aHVRd1 and aHVRd2, developed active viral infection as evidenced by seroconversion, viremia, and fecal virus shedding. Mutant aHVRd3, with a larger HVR deletion, was apparently attenuated in chickens. Additionally, we used the swine model system to further verify our results from the chicken study. The infectivity of four genotype 3 swine HEV HVR-deletion mutants, sHVRd1 (Δ712-790), sHVRd2 (Δ722-781), sHVRd3 (Δ735-765), and sHVRd4 (Δ712-765) constructed using the genotype 3 swine HEV as the backbone was determined in SPF pigs. Pigs intra-hepatically inoculated with capped RNA transcripts from the mutants sHVRd2, sHVRd3, and sHVRd4 developed active viral infection, whereas mutant sHVRd1 (Δ712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.
To further elucidate the role of HVR in HEV replication, we investigated the effects of serial amino acid deletions in HVR on the replication of HEV. We first constructed a genotype 1 human HEV luciferase replicon by replacing the ORF2 gene that encodes for the capsid protein with the fire fly luciferase reporter gene. Using the backbone of human HEV genotype 1 luciferase replicon, we constructed a series of HVR-deletion mutants with deletions of variable lengths in the HVR. Amino acid deletions Δ711-725, 711-740 and Δ711-750 were engineered at the N-terminus, deletions Δ729-754, Δ721-766, and Δ716-771 were engineered in the central region, and deletions Δ761-775, Δ746-775, and Δ736-775 were engineered at C-terminus of the HVR. The effects of these serial deletions on HEV RNA replication in the human liver carcinoma cell line, Huh7, were examined. Replication levels of mutants carrying these deletions were compared with that of the wild-type HEV in Huh7 cells. We observed that deletions in the HVR did not abolish viral RNA synthesis but substantially reduced the replication levels of viral RNA, as measured by the reporter luciferase activity. To further verify the effects of HVR deletions on viral RNA replication as observed with the genotype 1 human HEV replicon, we subsequently used a genetically-distinct strain of HEV, avian HEV, and constructed an avian HEV sub-genomic luciferase replicon by substituting the ORF2 gene of avian HEV with the fire fly luciferase gene. Avian HEV HVR-deletion mutants Δ557-603, Δ566-595, and Δ573-587 were then engineered using the backbone of avian HEV luciferase replicon. The replication efficiency of the three deletion mutants of avian HEV in chicken liver hepatoma cell line, LMH, was evaluated. Compared with the wild-type avian HEV, the viral RNA synthesis of the avian HEV HVR-deletion mutants was considerably reduced by the HVR deletions. To analyze the impact of the complete HVR deletion on avian HEV infectivity, we constructed an avian HEV mutant with a deletion of the entire HVR region (aaΔ557-603) using the avian HEV infectious cDNA clone as the backbone. After confirming the viability of the complete HVR-deletion mutant in LMH cells, SPF chickens were intrahepatically inoculated with capped RNA transcripts generated from the mutant. None of the chickens inoculated with the complete HVR-deletion mutant showed evidence of HEV infection, indicating that drastic reduction in replication levels due to complete HVR deletion has resulted in the loss of virus infectivity. The results indicated that HVR may have critical residues that may interact with viral/and or host factors and modulate the replication efficiency of HEV.
In the final part of the dissertation research, we sought to determine if the variable sequences of HVR are genotype-specific for in vitro virus replication. By using the genotype 1 human HEV as the backbone, we swapped the HVR of genotype 1 human HEV with the HVRs of the genotype 3 swine HEV and the distantly-related avian HEV to construct two inter-genotypic chimeras, pSKHEV2-Sw and pSKHEV2-Av. Similarly, by using the genotype 3 swine HEV as the backbone, the HVR of genotype 3 swine HEV was swapped with the HVR of genotype 1 human HEV to construct the chimera, pSHEV3-Hu. The viability of these chimeras was tested in Huh7 cells that are permissive for HEV replication. Immunofluorescence assay (IFA) with anti-HEV antibodies revealed that all the three chimeras were replication-competent in Huh7 cells. The infectivity of these chimeras was subsequently evaluated in HepG2 cells. The results showed that exchange of the HVR between different genotypes of mammalian HEVs does not abolish the replication competency and infectivity of HEV. This finding suggests that HVR is not genotype-specific with respect to viral replication and infectivity. The absence of detectable viral antigen in HepG2 cells infected with chimera pSKHEV2-Av suggested a functional incompatibility of the HVR of avian HEV in the mammalian HEV genome.
In summary, we identified a highly variable sequence, HVR, in the ORF1 of the HEV genome, and the sequences of the HVR vary significantly among HEV strains of different genotypes. We found that the HVR contain sequences that are dispensable for virus infectivity both in vitro and in vivo. Deletion analysis of HVR revealed that the region may play a role in modulating the replication efficiency of HEV RNA by interacting with viral and/or host factors. Finally, we demonstrated that HVR is not genotype-specific for virus replication and the region can be functionally replaced between mammalian HEV genotypes for virus replication and virion production in vitro. The results from this dissertation research have important implications for better understanding the biology and mechanism of HEV replication and may aid in our efforts to eventually develop a modified live-attenuated vaccine against HEV. / Ph. D.
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Studies on host factors that regulate the replication of positive strand RNA virusesPatton, John B. January 1900 (has links)
Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include a diverse group of pathogens that cause a wide array of diseases that can range from sub-clinical to lethal. These viruses infect humans and mammals as well as a variety of other hosts. For their successful replication, viruses interact closely with host cells from the binding to the receptor to the exit as complete viral progenies. During the events, viruses are dependent on host factors for receptor bindings, genome synthesis, and trafficking of viral genome and proteins. Thus there have been major efforts on the studies of understanding the virus-host interactions in the field of virology. In my PhD program, I have studied the host factors that regulate the replication of viruses using porcine reproductive and respiratory syndrome virus (PRRSV) and hepatitis C virus (HCV). I found that modulation of either the viral receptor or cellular signaling pathways had pronounced effects in the replication of PRRSV or HCV respectively. Using PRRSV, I found that the modulation of the level of the putative receptor CD163 on cells with cytokines significantly influence virus replication, suggesting the importance of cytokine presence in environments to determine the replication and pathogenicity of PRRSV via receptor expression in vivo. With HCV, I found that the enhancement of the virus replication occurs through the activation of the epidermal growth factor receptor/extracellular signal-regulated kinase pathway by bile acids which are abundant in the liver where the virus targets in vivo. Furthermore, I found that the bile acid-mediated signaling pathway significantly inhibited the antiviral activities against HCV. These results indicate the importance of environmental factors such as bile acids and signaling pathways in the replication and pathogenicity of HCV in vivo.
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Recombinant Hepatitis B surface antigen production in Aspergillus nigerJames, Emmanuel Robin 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: See item for full text / AFRIKAANSE OPSOMMING: Sien item vir volteks
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Hepatocellular lipid metabolism in Hepatitis C Virus infectionBeer, Melanie January 2014 (has links)
The work described in this thesis investigates the lipid metabolism of human hepatocytes in the context of Hepatitis C Virus (HCV) infection. This includes lipoprotein signalling and cholesterol metabolism targeted analysis of gene expression as well as the influence of polyunsaturated ER targeting liposomes (PERLs) on infection. These analyses indicate that HCV suppresses the expression of key regulators throughout the cholesterol biosynthesis pathway. This effect was quantified and the influence of liposome treatment evaluated. The latter resulted in the formulation of the hypothesis that PERL treatment interfers with virus-induced abberations of the cholesterol biosynthesis pathway and normalises the expression of four genes directly involved in cholesterol regulation. In addition, the lipidome of isolated lipid droplet was analysed by mass spectrometry. These data, combined with microscopy data suggest that PERLs interfere with S-palmitoylation of the HCV core protein resulting in dissociation of core from lipid droplets. This is likely to interrupt the viral assembly process, leading to inhibition of the production of infectious viral particles. Further described here are two different yet unsuccessful approaches to fluorescently label HCV RNA for live cell microscopy studies, namely an MS2 coat protein mediated approach, and Alexa®UTP labelling.
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Hepatitis-B-associated glomerular disease : a clinicopathological study of Hepatitis B virus associated Membranous Glomerulonephritis in Namibian and South African children 1974 – 2005 and a comparison with hepatitis B associated Membranous Glomerulonephritis as well as Idiopathic Membranous Glomerulonephritis in adultsBates, William D. 12 1900 (has links)
Thesis (PhD (Med))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe
proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease
(MCD). This is also the pattern observed in white and Indian children in South Africa
(SA). By contrast, black and mixed race/coloured children of Southern Africa in the
1960s to 1990s were shown to have a different pattern of NS. One of the main
differences was the frequency of hepatitis B virus (HBV) associated
glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of
this project was a clinicopathological study of this subgroup of nephrotic children to
document the disease further and in particular to seek correlations between
pathological and clinical features including prognosis. A central focus was to
document the detailed ultrastructural examination of the renal biopsies of these
children and to correlate the spectrum of pathological features with demographic,
clinical, laboratory and prognostic features.
The hypothesis was that the clinicopathological features of HBV MGN in
children differed substantially from idiopathic MGN in general (children and
adults) and also from HBV MGN in adults and that HBV MGN in children should
be viewed as a distinct disease.
Patients and methods: The childhood (12 years and younger) patient cohort was
309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg
Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from
Namibia. The study group was 71 children with HBV MGN who were followed up to
2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN
in childhood as this centre only had 2 such patients during the study period.)
Demographic, clinical, laboratory and renal pathology data were collected, compared
and correlated.
Results: HBV associated MGN was the most frequent cause of NS in the Namibian
subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood
cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood
nephrotic cohort, by far the dominant subgroup.
The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years)
at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen
(HBeAg) was identified in the serum of 87% of children tested. Laboratory features
different from idiopathic MGN included more prominent haematuria, mildly raised
serum transaminases and more frequently lowered serum C3 and C4 levels. Light
microscopic examination of renal biopsies showed mesangial proliferation in all
patients but with minimal glomerular sclerosis and interstitial disease. On
ultrastructural examination mesangial and subendothelial deposits were common and
prominent as was mesangial interposition. The MGN of HBV in children therefore
frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition
to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies
displayed severe mesangial interposition in addition to the subepithelial deposits of
MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like
bodies and tubuloreticular inclusion bodies were both found in more than 80% of
biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits
in the glomeruli. This was the first time this feature was demonstrated in Africa. The
46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort
developed chronic renal failure (CRF). Age of 6 years and above at presentation and
severe mesangial deposits on biopsy correlated with fewer remissions and poorer
outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset
of CRF was more than 19 years with the longest being 23 years. The 358 cases of
childhood HBV MGN from Southern Africa constitute 37% of the reported childhood
patients.
Comparative data
A comparison was made between the 71 children with HBV MGN, 12 adults with
HBV MGN and 33 adults with idiopathic MGN. The main differences were that both
HBV MGN groups included only coloured and black patients and were more
predominantly male while the idiopathic MGN group included all races. In the HBV
patients, haematuria was more frequent and severe, liver enzymes were frequently
raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of
adult MGN patients had normal C4 levels while the childhood HBV MGN group had
reduced C4 levels.
The immune complex pattern in both of the HBV MGN adult and childhood groups on
biopsy was similar with more mesangial and subendothelial deposits as well as
mesangial interposition than the idiopathic group. Despite this similarity between the
two HBV groups, both adult groups showed more glomerular sclerosis and interstitial
disease than the childhood group. The clinical outcome of the children’s cohort was
better than the other 2 groups with remission (52%) more frequent at 4 years (p<
0.01) and better renal and patient survival.
Including the 83 cases from this series, at least 1243 renal biopsy proven cases of
HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar
(children 79%; adults 84%) and significantly greater than for idiopathic MGN.
Conclusions: The findings confirm that HBV MGN in children is a distinct form of
GN which broadens the classical morphologic description of MGN by often including
a number of mesangiocapillary GN features. The subgroup of renal biopsies with the
most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary
GN group. The MGN spectrum as a whole comprised 86% of the
HBV positive childhood group. HBV MGN was the most frequent association with
NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group
(19%) in the SA children. It showed a relatively high spontaneous remission rate but
at least 10% of the children developed renal failure. Age of 6 years and above at
presentation and severe mesangial deposits on biopsy correlated with fewer
remissions and poorer outcome. Extended follow up (more than 15 years) was
required to demonstrate renal failure in some patients in the poor outcome group.
Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated
routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this
disease in SA. HBV MGN has been a valuable and possibly unique model of human
GN and MGN in particular in that the HBeAg has been identified in both the serum
and glomeruli enabling confirmation of the aetiological role of HBeAg. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese
sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon
kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is
aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die
jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van
die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV)
geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN).
Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep
van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies
te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die
gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die
korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese,
laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea.
Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in
kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en
volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders
as ’n afsonderlike siekte beskou behoort te word.
Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met
erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel
was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië.
Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12
HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN
in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte
tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en
nierpatologie inligting is versamel, vergelyk en gekorreleer.
Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die
Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die
kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder
nefrotiese kohort en verreweg die oorheersende subgroep.
Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was
6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B
omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87%
van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese
MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum
transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese
ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon,
maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele
ondersoek was mesangiale en subendoteliële neerslae asook mesangiale
interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van
mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae
van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale
interposisie aangetoon het asook die subepiteliale neerslae van MGN is die
gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies
en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies
bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste
keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar
getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking
(KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer.
Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV
ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare
HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder
pasiënte.
Vergelykende data
’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met
HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide
HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik
was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was
hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer
dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN
pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN
verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die
HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en
subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese
groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee
volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep
vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee
groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met
beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde
gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse
literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV
MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir
idiopatiese MGN.
Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike
vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene
insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier
biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre
GN groep geklassifiseer. Die MGN spektrum in geheel het 86%
van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene
assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde
grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane
remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking
ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer.
Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van
die swak uitkomsgroep aan te toon.
Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met
algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp
daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n
waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die
HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol
van HBeAg te bevestig.
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Predicting Treatment Response and the Role of the ISG15/USP18 Ubiquitin-like Signaling Pathway in Hepatitis C Viral InfectionChen, Limin 14 February 2011 (has links)
Hepatitis C Virus (HCV) infects 170 million people worldwide. The current treatment regimen, which is combination therapy with pegylated interferon (PegIFN) and Ribavirin (Rib), cures only 50% of the patients infected with the most prevalent HCV genotype. Therefore, there is a pressing need to understand the molecular mechanism of interferon resistance and to develop a prognostic tool to predict who will respond to treatment before initiation of therapy. It has been firmly established that the virus-host interaction plays an important role in determining treatment outcomes. My thesis investigated the host factors that are involved in interferon resistance with an aim to provide insights into the molecular mechanism of IFN resistance.
cDNA microarray analysis identified 18 differentially expressed hepatic genes from pretreatment liver tissues of responders (Rs) and non-responders (NRs). Based on the differential expression levels of these 18 genes, a prognostic tool was developed to predict who will respond to therapy, with a positive predicting value (PPV) of 96%. Most of these 18 genes are interferon stimulated genes (ISGs) and they are more highly expressed in NR livers, indicating that preactivation of interferon signaling in the pre-treatment liver tissues contributes to NR. 3 out of the 18 genes are involved in an ubiquitin-like ISG15/USP18 signaling pathway that plays an important role in interferon response. Over-expression of USP18 and ISG15 in the pretreatment liver tissues of NR promotes HCV production and blunts interferon anti-HCV activity. There exists a distinct cell-type specific ISG activation in the pretreatment liver tissues of Rs and NRs. Up-regulation of the two ISGs that I tested (ISG15 and MxA) was found mainly in hepatocytes in NRs while ISG activation was preferentially observed in macrophages in Rs.
Taking all these data together, pre-activation of interferon signaling and cell-type specific gene activation in the pretreatment liver tissues of patients infected with HCV are associated with treatment non-response. HCV exploits the host interferon system to favour its persistence by enhanced replication /secretion stimulated by a few ISGs (ISG15, USP18) in response to IFN. The developed prognostic tool can be used to stratify patients for treatment and the novel insights of the molecular mechanism of IFN resistance in HCV patients offer potential drug targets for future development.
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Virusinių hepatitų A, B, ir C serologinių žymenų paplitimas neatlygintinų kraujo donorų populiacijoje 2010-2011 m / Prevalence of viral hepatitis a, b and c serological markers in non-remunerated blood donors population in 2010-2011Valentienė, Jolanta 30 June 2014 (has links)
Tyrimo tikslas – aprašyti bendruosius virusinių hepatitų, ŽIV ir kitų lytiškai plintančių infekcijų dėsningumus ir įvertinti HAV, HBV, HCV infekcijos paplitimą neatlygintinų kraujo donorų, duodančių pirmą kartą kraują, populiacijoje. Metodika. Tyrimui vykdyti gautas Vilniaus regioninio biomedicininių tyrimų etikos komiteto leidimas. Atlikta pirmą kartą neatlygintinai duodančių kraujo donorų anoniminė anketinė apklausa VŠĮ Nacionaliniame kraujo centre. Imunofermentiniu metodu buvo nustatomi: anti-HAV ir anti-HCV, anti-HBcor, anti-HBs, HBsAg. Nukleino rūgščių amplifikacijos testas taikytas HBV DNR ir HCV RNR nustatymui. Įvertinant ŽIV ir kitų lytiškai plintančių infekcijų ir virusinių hepatitų tendencijas buvo taikytas Mantel‘io testas ir paprastoji tiesinė regresija. Virusinių hepatitų serologinių žymenų paplitimas išreiškiamas procentais, įverčio tikslumui įvertinti apskaičiuotas pasikliautinis intervalas (PI) 95%, kategorinių duomenų analizei panaudotas χ² testas ir Fišerio tikslusis testas. Rizikos veiksnių įtaką vertinta taikant binarinę logistinę regresiją. Duomenų suderinamumui vertinti pasirinktas Hosmer‘io-Lemeshow‘o χ2 suderinamumo kriterijus. Vertinant, kaip modelio teoriniai dydžiai atitinka realiuosius, naudotas Cox’o ir Snell’o kriterijus, klasifikacinė analizuojamų požymių lentelė. Veiksnių įtaka įvertinta panaudojant šansų santykį su 95 % PI. Skirtumas statistiškai reikšmingas, kai p ≤ 0,05. Rezultatai. Tyrime dalyvavo 200 kraujo donorų. Analizei... [toliau žr. visą tekstą] / Aim: to describe epidemiology of the viral hepatitis A, B, C, HIV and other sexually transmitted infections (STD) and to estimate prevalence of HAV, HBV, HCV infections among first time non-remunerated blood donors population. Methodology: The study received approval from Vilnius Regional Biomedical Research Ethics Committee. First time non-remunerated blood donors participated in anonymous questionnaire survey in NGO National Blood Center. For anti-HAV, anti-HCV, anti-HBs, anti-Hbcor and HBsAg detection was used immunoenzyme method and for HBV DNR ir HCV RNR - nucleic acid amplification test. The Mantel trend test and linear regression method was used to evaluate the trend of viral hepatitis, HIV and other sexually transmitted infections. The prevalence of viral hepatitis serological markers was expressed in percentage points, the precision was evaluated at the confidence intervals (CI) of 95%, the comparison of categorical data was made using χ2 test and Fisher‘s exact test. For data analysis the following tests were used: for the risk factors – binary logistic regression; goodness of fit – Hosmer-Lemeshow χ2 test; Cox and Snell R Square, Classification Table. The statistical significance level p ≤ 0.05. Results: A total of 200 respondents haven been interviewed. Only 188 first time non-remunerated blood donors were selected for further analysis. Respondents minimum of age was 18 and maximum - 52 (Mean=22,6; Med=20,0), 47,9 % (n=90) of them were males, 52,1... [to full text]
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Hepatitis Delta Virus: Identification of Host Factors Involved in the Viral Life Cycle, and the Investigation of the Evolutionary Relationship Between HDV and Plant ViroidsSikora, Dorota 19 June 2012 (has links)
Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.
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