published_or_final_version / abstract / Zoology / Master / Master of Philosophy

The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

Certain strains of mice, e.g. C57BL/6, are highly resistant to serious infections with the protozoan pathogen, Leishmania major, whereas other strains, e.g. BALB/c, are not. It has beenproposed that interferon gamma (IFN-y) is one of the most critical lymphokines produced in a protective response to these intracellular pathogens. IFN-y has been classified as a Thi lymphokine produced by the Thl subset of T lymphocytes which not only activates macrophages to kill the protozoa but also helps regulates the immune system overall to form a lasting immunity to the microorganism (4,19). Mice susceptible to L. major arethought to produce inadequate amounts of IFN-y and instead produce an excessive amount of a Th2 lymphokine, IL-4, produced by Th2 T cells. (6) We have previously found that prophylactic treatment with cyclosporine A (CsA), a T cell specific immunosuppressant, reduces the susceptibility of the BALB/c to L. major by either preventing disease entirely or delaying itsdevelopment significantly (19). In this murine model, it may be that CsA causes a switch from the production of the less protective Th2 lymphokines to the more protective Thl lymphokines. In order to test this hypothesis we examined the IFN-y produced by lymph node and spleen cells over time after infection in three groups of mice: C57BL/6, BALB/c and cyclosporine- protected BALB/c. Interestingly, cells taken from all three groups of mice were able to secrete IFN-y in vitro in response to co-culture with Leishmania, antigens. The pattern of secretion over time, however, varied and may indicate a significant difference in the animals' response to the pathogen. In addition to this work, we also examined the ability of another immunosuppressant, FK506, which is very similar in action to but much less toxic than CsA, to induce enhanced resistence to L. major. FK506 also appears to be effective in reversing the susceptibility of the BALB/c mice towards this pathogen with much less apparent toxicity. / Department of Biology

Virus infection of mammalian cells induces several stress mechanisms, including autophagy and type-I interferon (IFN). Autophagy, a cellular homeostatic mechanism in which intracellular materials are sequestered into double-membrane vesicles and targeted to lysosomes for degradation, is also activated in response to virus infection. Most positive single-stranded RNA viruses studied to date utilise autophagy to increase virus replication. IFN is a potent anti-viral mechanism, which can be divided into two parts: (i) induction and secretion of IFN and (ii) IFN signalling and priming of uninfected cells for a rapid response upon infection and induction of an anti-viral state in infected cells. Alphaviruses are medically important RNA viruses. Semliki Forest virus (SFV) provides a well-characterised model for studying alphavirus infection. A number of strains have been identified, which differ in virulence in adult mice. In this thesis three hypotheses were investigated: (i) that SFV infection induces autophagy in cell culture and utilises this response to enhance virus replication, (ii) that the quality, quantity and/or protective efficacy of the IFN response differ between virus strains and between human and murine cells and (iii) that non-structural protein (nsP)-2 and/or nsP3 antagonise the IFN response. SFV4, SFV L10 and SFV A7(74) infection induced autophagy in Huh7 cells as early as one hour post-infection. Pharmacological induction or inhibition of autophagy had no affect on SFV4 replication, except at a very low multiplicity of infection. NsP3, capsid and dsRNA rarely colocalised with the autophagosome marker LC3. Taken together these results indicate that SFV does not use autophagosomes for replication and autophagy is not important in controlling SFV4 infection at a high MOI, at least in Huh7 cells. However, autophagy may be important in controlling SFV4 spread at a low MOI. An IFN bioassay was established. In fibroblasts, SFV4, SFV L10 and SFV A7(74) induced relatively little IFN in comparison to that induced by Sendai virus. In human fibroblasts, similar levels of IFN were induced by all three virus strains. In mouse fibroblasts, SFV4 induced more IFN than SFV L10. Treatment of fibroblasts with IFN prior to infection greatly reduced, but did not abolish, the replication and spread of all three strains. Therefore, SFV is sensitive to IFN. Analysis of IFN signalling demonstrated that all three strains of SFV inhibited STAT1 phosphorylation during infection of fibroblasts. The growth and viability of SFV infected cells varied between human and mouse cells. The complete genetic sequences of SFV L10 and SFV A7(74) were determined using Solexa (Illumina) sequencing and compared to the sequence of SFV4. The sequences of SFV L10 and SFV4 were extremely similar; only seven differences were identified. Multiple amino acid substitutions were identified in SFV A7(74) compared to SFV4, these mostly mapped to nsP3. To investigate the hypothesis that nsP2 and or nsP3 antagonise the IFN response, two virus mutants were studied: SFV4nsP2RDR and SFV4nsP3Δ50. SFV4nsP2RDR encodes a point mutation in the nuclear localisation signal of nsP2, which largely restricts nsP2 to the cell cytoplasm. SFV4nsP3Δ50 contains a deletion of 50 amino acids in the C-terminus hyperphosphorylated region of nsP3. Neither mutant inhibited STAT1 phosphorylation as efficiently as WT SFV4; SFV4nsP2RDR was particularly poor at inhibiting STAT1 phosphorylation. Both mutants induced more IFN in fibroblasts than SFV4. In summary, autophagy had a limited affect on SFV replication. In contrast, strains of SFV were highly sensitive to IFN, but antagonised this response through the nsP2 protein inhibiting STAT1 phosphorylation.

Memoria para optar al título de Químico Farmacéutico / En los últimos años se ha descrito y dado énfasis al rol determinante que tiene el fibroblasto cardiaco (FC) en el proceso inflamatorio, dónde cumple un rol clave a fin de mantener la homeostasis del órgano. En los procesos inflamatorios y en cualquier episodio de daño, los neutrófilos son la primera línea de defensa del organismo, los cuales son atraídos al sitio de daño por la quimioquina, interleuquina 8 (IL-8), quien además, provoca su activación. Posteriormente, el reclutamiento de neutrófilos en los sitios de daño es dependiente de las proteínas de adhesión E-selectina, ICAM-1 y VCAM-1. Por lo tanto, controlar la expresión de estas proteínas constituye un nuevo enfoque terapéutico en el tratamiento de los procesos inflamatorios. En ese sentido, el interferón beta (IFN-β), ha demostrado moderar la respuesta inflamatoria; pero hasta la fecha no se ha demostrado su utilidad como agente antiinflamatorio en el corazón y específicamente en el FC. Debido a estos antecedentes, el objetivo general fue estudiar el efecto preventivo del IFN-β sobre la expresión de IL-8, ICAM-1 y VCAM-1 bajo un contexto inflamatorio, inducido por estímulo con LPS. Consecuente con este hallazgo, se observó que el IFN-β disminuye la adhesión de neutrófilos sobre FC inducida por LPS. De esta manera, se demostró que el tratamiento con IFN-β puede modular la cascada inflamatoria, y así, transformarse en una potencial y valiosa herramienta terapéutica capaz de controlar la expresión de estas proteínas de vital importancia partícipes del proceso inflamatorio luego de algún evento de injuria cardiaca como lo puede ser una miocarditis, o bien, un infarto al miocardio / In recent years it is described the determining role that cardiac fibroblast (CF) has in the inflammatory process, where plays a major role to maintain homeostasis. In inflammatory process and any event of damage, neutrophils are the first line of defense, which are attracted to the site of damage through interleukin 8 (IL-8), who initially causes activation. Subsequently, neutrophil recruitment at sites of damage depends on Eselectin, ICAM-1 and VCAM-1 adhesion proteins. Therefore, controlling the expression of these proteins is a new therapeutic approach in the treatment of inflammatory process. In this sense, interferon beta (IFN-β) has shown be a inflammatory moderator; but to date it has not been proved useful as an anti-inflammatory agent in the heart, specifically on CF. On the basis of this background, the overall objective was to study the preventive effect of IFN-β on the expression of IL-8, ICAM-1 and VCAM-1 on CF under an inflammatory stimuli with LPS and evaluate neutrophil adhesion to CF. The results showed that IFN-β prevents the increased expression on IL-8, ICAM-1 and VCAM-1 induced by LPS. Consistent with this finding, we observed that IL-8, ICAM-1 and VCAM-1 decreases the neutrophil adhesion induced also by LPS on CF. Thus, it was shown that IL-8, ICAM-1 and VCAM-1 can modulate the inflammatory cascade, and thus become a potentially valuable therapeutic tool able to manage these important participants proteins of the inflammatory process after an event of cardiac injury as can be a myocarditis, or myocardial infarction

The human Class Ib gene, HLA-E inhibits both Natural Killer Cells and a subset of CD8+ cytotoxic T lymphocytes by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for the killing of target cells. Since HLA-E has negative effects on immune killing of target cells, we have sought to identify locus specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrate that tumor cells that express GATA factors as determined by western blot and quantitative PCR, mediate a 2.4 to 4.0 fold enhanced response to IFN-γ stimulation. Functional constructs containing mutations of the core nucleotides in the GATA binding site had a 4.8 fold decreased response to IFN-γ in A2780 cells and a 8.5 to 14.0 fold decreased response to IFN-γ in SKOV3 cells. Knockdown of GATA-6 using siRNA resulted in a 40% decrease in HLA-E induction in Seg1 cells and a 30% decrease in HLA-E induction in HCT116 cells. Tetracycline regulated shRNA knockdown of GATA-6 expression in the SKOV3 cell line revealed a 3 fold decrease in the IFN-γ response of HLA-E reporter driven constructs. Additionally we observed a decreased IFN-γ response in SKOV3 cells transfected with siRNA specific for CBP and IRF-9. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression and that IRF-9 may be a target for the differential manipulation of classical MHC and HLA-E.

The work of the previous chapters describes the role of Nipah virus (NiV) V and W proteins regarding their role in interferon antagonism and regulation of viral replication. Previous publications have shown that NiV encodes IFN antagonist activity in its V, W and C protein (Park et al., 2003b; Rodriguez et al., 2002). In order to study the effect of both NiV proteins in the context of a virus infection, recombinant Newcastle disease viruses (rNDVs) expressing NiV V or NiV W were constructed. As a control virus served rNDV expressing NDV V proteins, which behaved like wildtype NDV. Growth kinetic experiments demonstrated that rNDVs expressing NiV V or W grew to higher titers than rNDV expressing NDV V in human A549 cells. This result suggested that both NiV V and W were able to render the avian virus, which normally does not replicate well in human cells, into a better growing virus. This hypothesis was supported by the fact that all rNDVs grew similarly in avian DF1 or Vero cells. When rNDV-infected A549 cells were specifically stained for NiV V or W protein it was observed that V is localized in the cytoplasm whereas W could be predominantly found in the nucleus. This observation was in agreement with previous studies reporting a nucleus export signal (NES) for NiV V and a nuclear localization signal (NLS) for NiV W (Rodriguez et al., 2004; Shaw et al., 2005). The specific localization of each NiV protein has also been shown to contribute to different functions in terms of IFN antagonism (Shaw et al., 2005). Here, NiV V and W proteins caused a severe attenuation of the immune response in rNDV-infected human A549 and dendritic cells. The transcription of type I interferons and ISGs was significantly downregulated in the presence of NiV V and W proteins. As a consequence of the transcriptional block, there was also an inhibition at the level of translation (as seen for A549 cells) and the secretion of IFNs and cytokines/chemokines (as seen for DCs). In contrast, NDV V protein induced a host immune response. Both NiV V and W also displayed a strong inhibitory effect on the function DCs. DCs represent a very important cell class because they link the innate immune response to the adaptive immune response (Banchereau & Steinman, 1998). By downregulating the production and secretion of important cytokines/chemokines that are important for the activation of B and T lymphocytes, NiV V and W were able to disrupt that link. Interestingly, NiV W seemed to be a stronger inhibitor than NiV V in both A549 cells and DCs. Overall, it was demonstrated that NiV V and W were able to prevent the induction of the innate and adaptive host immune response cascade by inhibiting the transcription of immune genes in DCs and A549 cells. The second part of this work addressed the question whether NiV V and W proteins have a regulatory role in viral replication. This has been previously reported for Nipah virus itself (Sleeman et al., 2008) and other viruses (Atreya et al., 1998; Horikami et al., 1996; Witko et al., 2006). In order to study the ability of the V and W proteins of NiV to regulate viral transcription and/or replication, an existing NiV minireplicon assay was used (Halpin et al., 2004). Here, it was shown that NiV V and W (but not C) proteins significantly downregulated NiV minireplicon activity. The common N terminal region was shown to harbor the inhibitory activity. Co-immunoprecipitation experiments showed that both NiV V and W (but not C) were able to interact with NiV N, one component of the NiV polymerase. This result was supported by immunofluorescence experiments that revealed co-localization of NiV N with V and W. The binding of NiV V or W to NiV N occurred via their N terminus and more specifically amino acids 1-50. This suggested that V and W might inhibit viral replication by interacting with the viral polymerase resulting in a loss of function. Exact mechanisms still have to be elucidated. / Die Arbeit der vorangegangenen Kapitel geht auf die Rolle von Nipah Virus (NiV) V und W Proteinen bezueglich deren Rolle im Interferon-Antagonismus und Regulation der viralen Replikation. In vergangenen Veroeffentlichungen wurde gezeigt, dass NiV seine interferon-antagonistische Aktivitaet in dessen V, W und C Proteinen kodiert (Park et al., 2003b; Rodriguez et al., 2002). Um den Effekt beider NiV Proteine im Rahmen einer Virusinfektion zu untersuchen, wurden rekombinante Newcastle disease Viren (rNDV), welche entweder NiV V oder W exprimieren, konstruiert. Als Kontrollvirus diente ein rNDV, der das NDV V Protein exprimiert. Dabei verhaelt sich der rekombinante Virus wie NDV-Wildtyp. Kinetische Wachstumsexperimente in A549 Zellen zeigten, dass rNDVs, die NiV V oder W exprimieren, zu hoeheren Titern wuchsen als rNDV, der NDV V exprimiert. Dieses Ergebnis deutete darauf hin, dass sowohl NiV V als auch NiV W im Stande waren, den Vogelvirus, welcher normalerweise schlecht in humanen Zellen repliziert, zu einem besseren Wachstum anzuregen. Diese Hypothese wurde durch die Tatsache unterstuetzt, dass alle rNDVs ein recht recht aehnliches Wachstumsverhalten in Vogelzellen (DF1 Zellen) und Vero Zellen aufwiesen. Ausserdem wurden rNDV-infizierte A549 Zellen spezifisch gegen NiV V oder W Protein angefaerbt und es konnte beobachtet werden, dass V im Zytoplasma und W ueberwiegend im Nukleus der Zelle lokalisiert ist. Diese Beobachtung stimmte mit vorhergehenden Studien ueberein, die von ein Nukleus-Exportsignal (NES) fuer NiV V und ein Nukleus-Lokalisationsignal (NLS) fuer NiV W berichteten (Rodriguez et al., 2004; Shaw et al., 2005). Es wurde gezeigt, dass diese unterschiedliche, jedoch spezifische Lokalisation der NiV Proteine zu verschiedenen Funktionen bezueglich des Interferon-Antagonismus beitraegt (Shaw et al., 2005). In der vorliegenden Arbeit verursachten NiV V und W Proteine eine heftige Attenuation der Immunantwort in rNDV-infizierten humanen A549 und dendritischen Zellen (DC). In der Anwesenheit von NiV V und W Proteinen wurde die Transkription der Typ 1-Interferonen und ISGs signifikant herunterreguliert. Als Konsequenz des transkriptionellen Blocks konnte auch eine Inhibition auf translationalem Niveau (zu sehen in A459 Zellen) und eine Inhibition der Sekretion von IFNs und Zytokinen/ Chemokinen (zu sehen in DCs) verzeichnet werden. Im Gegensatz dazu induzierte NDV V Protein ein Immunantwort im Wirt. Sowohl NiV V als auch W zeigten auch einen starken inhibitorischen Wirkung auf die Funktion von DCs. DCs repraesentieren einen sehr wichtigen Zelltyp, da sie eine Verbindung zwischen der angeborenen und erworbenen Immunantwort herstellen (Banchereau & Steinman, 1998). Durch die Herunterregulierung der Produktion und Sekretion wichtiger Zytokine/ Chemokine, die fuer die Aktivierung von B- und T-Lymphozyten wichtig sind, waren NiV V und W faehig, diese Verbindung zu zerstoeren. Interessanterweise schien NiV W in A549 Zellen ein viel staerkerer Inhibitor zu sein als NiV V. Insgesamt wurde demonstriert, dass NiV V und W durch Inhibierung der Transkription von Immungenen im Stande waren, die Induktion der angeborenen und erworbenen Immunantwort-Kaskade zu unterbinden. Der zweite Teil dieser Arbeit beschaeftigt sich mit der Frage, ob NiV V oder W Proteine eine regulatorische Aufgabe in der viralen Replikation uebernehmen. Vorherige Berichte hatten dies fuer Nipah Virus selbst (Sleeman et al., 2008) und andere Viren demonstriert (Atreya et al., 1998; Horikami et al., 1996; Witko et al., 2006). Um die Faehigkeit von V und W Proteinen bezueglich der Regulation der viralen Transkription und/ oder Replikation zu bestimmen, wurde Gebrauch von einem bereits existierenden NiV Minireplikon-Assay gemacht (Halpin et al., 2004). In der vorliegenden Studie wurde gezeigt, dass NiV V und W (jedoch nicht C) Proteine die NiV Minireplikon-Aktivitaet in signifikanter Weise reduzierten. Es wurde gezeigt, dass die gemeinsame N-terminale Region die inhibitorische Aktivitaet besitzt. Ko-Immunopraezipitationsexperimente demonstrierten weitehin, dass sowohl NiV V als auch W (jedoch nicht C) im Stande waren, mit NiV N, einer Komponente der NiV Polymerase, zu interagieren. Dieses Ergebnis wurde von Immunfluoreszenzexperimenten untermauert, die eine Kolokalisation zwischen NiV N und V und W demonstrierten. Der N-Terminus von NiV V und W, und hierbei speziell die Aminosaeuren 1-50, waren fuer die Bindung von NiV V und W an NiV N verantwortlich. Dies deutete darauf hin, dass V und W durch das Binden der viralen Polymerase die virale Replikation inhibiert.

In der vorliegenden Arbeit wurden die Behandlungsabläufe von Patienten mit chronischer Hepatitis C unter Therapie mit Standard-Interferon (Kollektiv 1) bzw. mit pegyliertem Interferon (Kollektiv 2) ausgewertet. Die meisten Patienten erhielten eine Kombinationstherapie mit Ribavirin. Es bestand Strukturgleichheit für die Kollektive hinsichtlich Alter, Geschlecht, BMI vor Therapie, Übertragungsweg der Hepatitis, Hepatitis B-Infektion, HAI-Grading-Score und HAI-Staging-Score. Ein signifikanter Unterschied bestand für das Merkmal HIV-Koinfektion. Nach Therapiebeginn zeigte sich ein schnell einsetzendes serologisches und virologisches Ansprechen. Patienten unter Therapie mit pegyliertem Interferon und Ribavirin hatten die besten Chancen auf ein anhaltendes Therapieansprechen. Eine early virological Response war ein guter Prädiktor für das Erreichen einer sustained virological Response. Die meisten Patienten berichteten über Nebenwirkungen unter Therapie. Die häufigsten Nebenwirkungen waren Müdigkeit und Schmerzen, v.a. in Form von Kopfschmerzen. Diese kamen jeweils bei ca. 70% der Patienten vor. Eine Anämie trat bei ca. 9% der Patienten auf. Hämatokrit, Hämoglobin und Erythrozyten sanken im Kollektiv 2 stärker ab als im Kollektiv 1. Unter Kombinationstherapie mit Ribavirin sank das Hämoglobin zudem mehr ab als unter Interferon- Monotherapie, was auf den hämolytischen Effekt des Ribavirins zurückzuführen ist. Thrombozyten fielen unter Kombinationstherapie im Kollektiv 1 deutlich geringer ab als im Kollektiv 2, was durch einen stärkeren myelosuppressiven Effekt des pegylierten Interferons bedingt sein könnte. Im Kollektiv 1 sanken die Thrombozyten unter Monotherapie stärker ab als unter Kombinationstherapie. Leukopenien traten häufiger unter Therapie mit pegyliertem Interferon auf. Insgesamt zeigte sich im hier analysierten Kollektiv ein geringes Risiko für eine Neutropenie oder Lymphopenie. Vor allem ältere Patienten mit niedrigen neutrophilen Granulozyten bzw. Lymphozyten vor Therapie schienen ein erhöhtes Risiko für eine Neutropenie bzw. Lymphopenie zu haben. Das Therapieansprechen und die Therapiedauer waren für Patienten mit bzw. ohne Leukopenie, Neutropenie oder Lymphopenie ähnlich. Für Infektionen fand sich ebenfalls kein signifikant erhöhtes Risiko bei Patienten mit Leukopenie, Neutropenie oder Lymphopenie. 16% der Patienten im Gesamtkollektiv hatten eine Infektion unter Therapie. Es zeigte sich kein Unterschied zwischen Kollektiv 1 und 2 für Infektionen unter Therapie. Patienten mit bzw. ohne Infektion wurden hinsichtlich der Merkmale Alter, Geschlecht, BMI, Hepatitis B-Infektion, Hepatitis G/GB-Infektion und HIV-Infektion verglichen. Zudem wurden die prätherapeutischen Laborwerte Ferritin, Viruslast, Eisen, TSH, GPT, GOT, Hämoglobin, Hämatokrit, Leukozyten, neutrophile Granulozyten, Lymphozyten, Thrombozyten und Erythrozyten gegenübergestellt und Therapiedauer und Therapieansprechen für Patienten mit bzw. ohne Infektion erhoben. Für keines dieser Kriterien lag ein signifikanter Unterschied zwischen Patienten mit Infektion und Patienten ohne Infektion vor. Die meisten Infektionen waren unkomplizierte, respiratorische Infektionen. Diese traten für Patienten mit Neutropenie und Patienten ohne Neutropenie gleich häufig auf. HIV-Patienten hatten ein höheres Infektionsrisiko. Jedoch war der Unterschied nicht signifikant. Beim Vergleich des prozentualen Absinkens der Lymphozyten vom Ausgangswert zeigte sich ein schwach signifikanter Unterschied zwischen den Werten zu Infektionszeitpunkten und den Werte für Patienten ohne Infektion. Für die absoluten Werte war der Unterschied nicht signifikant. Für neutrophile Granulozyten und Leukozyten fanden sich keine Unterschiede zwischen den Werten für Infekt-Patienten zum Infektionszeitpunkt, den Werten zu infektfreien Zeitpunkten und den Werten für Patienten ohne Infektion. Insgesamt fand sich im hier untersuchten Kollektiv keine Assoziation von Infektionen unter Interferontherapie mit Leukopenien oder Neutropenien. Ein Absinken der neutrophilen Granulozyten scheint daher in größerem Maße ohne Dosisreduktion tolerierbar zu sein als bisher empfohlen. Ein relativer Lymphozytenmangel könnte mit dem Auftreten von Infektionen assoziiert sein. Für den absoluten Lymphozytenmangel fand sich diese Assoziation jedoch nicht. / In the present study, the treatment procedures for patients with chronic hepatitis C during therapy with standard interferon (group 1) or with pegylated interferon (group 2) were evaluated. Most patients received combination therapy with ribavirin. It was par for the collective structure in terms of age, gender, BMI before therapy, transmission of hepatitis, hepatitis B infection, HAI grading and staging score. A significant difference was found for the characteristic HIV co-infection. After starting therapy a rapid onset of serological and virological responses was seen. Patients treated with pegylated interferon and ribavirin had the best chances of sustained response to therapy. An early virological response was a good predictor of sustained virological response. Most patients reported side effects during treatment. The most common side effects were fatigue and pain (both for about 70% of patients), especially in the form of headache. Anaemia occurred in approximately 9 % of patients. The decreases of hematocrit, hemoglobin and erythrocytes was stronger in group 2 than in the first group Under combination therapy with ribavirin hemoglobin decreased also stronger than under interferon monotherapy, which is due to the haemolytic effect of ribavirin. On combination therapy in group 1 platelets were significantly lower than in group 2, which could be due to a greater myelosuppressive effect of pegylated interferon. In group 1, the platelets decreased stronger under monotherapy compared with combination therapy. Leukopenia occurred more frequently during treatment with pegylated interferon. Overall, the analyzed risk of neutropenia and lymphopenia in this collective was small. Especially elderly patients with low neutrophil granulocytes or lymphocytes before starting therapy seemed to have an increased risk for neutropenia and lymphopenia. The treatment response and duration of therapy were similar for patients with or without leukopenia, neutropenia and lymphopenia. There was also no significant increased risk for infections in patients with leucopenia, neutropenia and lymphopenia. 16 % of patients in the total group had an infection during treatment. There was no difference between the collective 1 and 2 for infections during treatment. Patients with and without infection were compared for age, gender, BMI, hepatitis B infection, hepatitis G / GB-infected and HIV-infection. In addition, the pretherapeutic laboratory values ferritin, viral load, iron, TSH, ALT, AST, hemoglobin, hematocrit, leukocytes, neutrophils, lymphocytes, platelets and red blood cells, treatment duration and response rate were compared for patients with and without infection. For none of these criteria a significant difference between patients with infection and patients without infection was found. Most infections were uncomplicated respiratory infections. These occurred equally in patients with neutropenia and patients without neutropenia. HIV patients had a higher risk of infection. However, the difference was not significant. Comparing the percentage drop in the lymphocytes from baseline a weak significant difference was shown between the values at infection times and the avarage values for patients without infection. For the absolute values the difference was not significant. For neutrophils and leukocytes, there were no differences between the values for patients at times of infection, the values at infection-free times and the values for patients without infections. In total no association of infection with interferon therapy with leukopenia or neutropenia was found. A decrease in neutrophils seems therefore to be more tolerable without dose reduction than recommended till now. A relative lack of lymphocytes could be associated with the occurrence of infections. For the absolute lymphocyte deficiency this association was not found.

Foi produzida a proteína recombinante do domínio extracelular da subunidade 2 do receptor do IFN-alfa em forma de proteína de fusão com a glutatione-S-transferase (GST-IFNAR2cEC) em E. coli, com o objetivo de avaliar seu efeito bloqueador sobre a ação do IFN-alfa. A GST-IFNAR2cEC de forma solúvel, com peso molecular 54 kDa, foi obtida quando a proteína recombinante foi expressa a 25 graus C por indução de IPTG e foi lisada por French Press. A avaliação do efeito bloqueador da GST-IFNAR2cEC foi feita utilizando-se 3 métodos: 1) Ensaio antiproliferativo com células Daudi, 2) ensaio antiviral com células Hep 2/c e vírus encefalomiocarditis, e 3) expressão gênica semi-quantitativa do STAT 1 (transdutor de sinal e ativador de transcrição) e da 2',5'-oligoadenilato sintetase (OAS) pela RT-PCR duplex nas células Daudi e Hep 2/c. A GST-IFNAR2cEC, mesmo a 420 nM, não inibiu as atividades antiproliferativa e antiviral de 24,5 pM e 49 pM de rIFN-alfa2 provavelmente devido à sua ineficiência na competição com o IFNAR da superfície celular durante o longo período de incubação nos ensaios realizados. A expressão gênica do STAT 1 não foi inibida pela GST-IFNAR2cEC nas células Daudi e Hep 2/c, porém inibiu-se a da 2’, 5’-OAS. Estes resultados sugerem que a expressão gênica do STAT 1 é independente da ligação do IFN-alfa com a IFNAR 2c, e que a da 2’, 5’-OAS é dependente dessa ligação nessas células

by Kin-wai Lee. / Includes bibliographies / Thesis (M.Phil.)--Chinese University of Hong Kong, 1982