Vor Einführung der direkt antiviralen Kombinationstherapien war die Kombination aus pegyliertem Interferon plus Ribavirin die Standardbehandlung für Patienten mit chronischer Hepatitis-C-Infektion. Bei 30% der Patienten zeigten sich neurokognitive sowie depressive Nebenwirkungen, die das dauerhafte Therapieansprechen negativ beeinflussen können. Vor diesem Hintergrund untersuchten wir in unserer Arbeit bei 93 Patienten mit chronischer Hepatitis-C-Infektion den Zusammenhang zwischen drei Single Nucleotide Polymorphismen im Bereich des IL28B-Gens und der Verträglichkeit sowie dem Therapieerfolg einer interferonbasierten Behandlung. Der Vergleich zwischen den Ergebnissen im HADS-(Hospital Anxiety and Depression Scale) sowie TAPS- (Testbatterie zur Aufmerksamkeitsprüfung) Testverfahren mit den Genotypen der drei SNPs zeigte im Studienkollektiv keinen signifikanten Zusammenhang. Hinsichtlich des Therapieerfolges konnten wir bei einem der drei SNPs das C-Allel als positiven Prognosefaktor für das dauerhafte Therapieansprechen nachweisen. / Prior to the introduction of direct antiviral combination therapies, the combination of pegylated interferon plus ribavirin was the standard treatment for patients with chronic hepatitis C infection. In 30% of the patients, neurocognitive and depressive side effects were observed, which could negatively influence the sustained virological response. Against this background, the central question that motivates our work was to investigate the association between three single nucleotide polymorphisms in the IL28B gene and the tolerability and therapeutic outcome of interferon-based treatment in 93 patients with chronic hepatitis C infection. The comparison between two test procedures, the HADS (hospital anxiety and depression scale) and TAPS (test battery for attention testing), with the genotypes of the three SNPs showed no significant association in the study population. In terms of therapeutic success, we were able to demonstrate the C allele in one of the three SNPs as a positive prognostic factor for the sustained virological response.

Techn. Universiẗat, Diss., 2005--München.

The complexity of melanoma is pronounced at many levels, whereby both environmental influences and genetic predisposition are involved and interact. Embedded within this complexity is heterogeneity, a defining characteristic of this malignancy. The rearrangement of genomic material on chromosomes 1p, 6q, 9p or 10q, 11q and 17q has been frequently reported during the development and progression of cutaneous malignant melanoma (CMM), suggesting several putative tumour suppressor genes and oncogenes in these regions. The genomic complexity of chromosome 9p21 in melanoma development is well documented. This region encodes a potent cyclin-dependent kinase inhibitor CDKN2/INK4A/p16 as a tumour suppressor gene (TSG) that is frequently inactivated in melanomas. Functional evidence suggested the presence of additional TSG loci in the 9p21-22 chromosome region (Parris et al., 1999). In pursuit of identifying novel TSG(s), our previous group’s collaborative research provided experimental evidence that suggests IFNA1 as a candidate TSG for melanoma development. Therefore, the aim of this work was to provide a further functional validation of such tumour-suppressive activity in CMM. Firstly, I have successfully subcloned IFNA1 cDNA into pcDNA3 expression vector and established a panel of stably IFNA1-expressing clones. Subsequently, I have assessed their tumourigenicity in soft agar by measuring the colony-forming ability of each transfected clone. Expression analyses of IFNA1, at both post-transcriptional and translational levels, were also carried out. I have also demonstrated a strong correlation between anchorage-independent growth in soft agar and IFNA1 expression in qRT-PCR. The antiproliferative and pro-apoptotic effects of IFNα have been widely documented, however, the precise mechanisms that trigger and potentiate this behaviour are not completely known. Based on previous findings, I have investigated whether IFNA1 exerts its antitumoural activity through apoptosis. I was able to demonstrate a moderate relationship between anchorage-independent growth in soft agar and the apoptotic levels in the transfected clones. Although unpersuasive and inconclusive, the results seemed encouraging since this study was carried out using only the highly tumourigenic malignant melanoma UACC903 cell line.

ABSTRACT In sub-Saharan Africa a causal relationship has been established between hepatitis B virus (HBV) infection and membranous nephropathy (MN), especially in Black children. The most common method of treatment is interferon therapy, which is however, only effective in 30-40% of patients. The reason for this is unclear. The objective of this pilot study was to determine whether mutations in the complete surface gene of HBV isolated from Black children with HBV-associated MN before, during and after treatment with interferon, had any effect on treatment response and vice versa. HBV DNA was extracted from the serum of a responder, reverter and non-responder patient before, during (4 and 16 weeks) and after (40 weeks) IFN treatment. The preS1/preS2/S region was amplified and cloned, and the clones sequenced. Sequence analyses revealed the preS2 region to be the most variable in the reverter and non-responder and HBsAg was the most variable in the non-responder. Phylogenetic analysis showed that the viral population dynamics between the responder strains and the reverter/non-responder strains differed as a result of various mutations found within the surface gene. Thus the presence of mutations in preS2 and HBsAg of the non-responding patients may carry predictive markers for nonresponse but further investigation would be needed to conclusively prove this.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Tsang Sai Leong. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 96-104). / Abstracts in English and Chinese. / Abstract (in Chinese) --- p.(i) / Abstract --- p.(iii) / Table of contents --- p.(v) / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The interferon --- p.1 / Chapter 1.1.1 --- About type I IFN --- p.1 / Chapter 1.1.2 --- IFN α/β receptor and signal transduction --- p.3 / Chapter 1.1.3 --- IFN induction --- p.3 / Chapter 1.1.4 --- Functions --- p.4 / Chapter 1.1.5 --- MuIFN α subtypes --- p.8 / Chapter 1.1.6 --- Gene expression --- p.9 / Chapter 1.2 --- Aim of study: Functions and gene expression --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.15 / Chapter 2.1.1 --- "Cell line, bacterial strain, virus strain and plasmid vector" --- p.15 / Chapter 2.1.2 --- Chemicals --- p.20 / Chapter 2.1.3 --- "Culture media, buffer and other solutions" --- p.20 / Chapter 2.1.4 --- Reagents and nucleic acids --- p.21 / Chapter 2.1.5 --- Reaction kits --- p.22 / Chapter 2.1.6 --- Solutions --- p.22 / Chapter 2.1.7 --- Major equipments --- p.24 / Chapter 2.1.8 --- Primers used --- p.24 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- "Cloning of MuIFN αl2, MuIFN αl and MuIFN α4 from L929 genomic DNA and their subcloning into pEGFP-Nl mammalian expression vector" --- p.26 / Chapter 2.2.1.1 --- PCR of MuIFN αl2 --- p.26 / Chapter 2.2.1.2 --- Gel purification of MuIFN αl2 PCR product --- p.26 / Chapter 2.2.1.3 --- Ligation of MuIFN αl2 PCR product into pGEM-T vector --- p.26 / Chapter 2.2.1.4 --- Sequencing of clones which were positive in PCR screening --- p.26 / Chapter 2.2.1.5 --- Subcloning of the gene from pGEM-T vector to pEGFP-Nl --- p.28 / Chapter 2.2.1.6 --- Construction of expression vectors for MuIFN αl and MuIFN a4 gene --- p.28 / Chapter 2.2.2 --- Preparation ofplasmid DNA --- p.29 / Chapter 2.2.3 --- Preparation of cell culture medium --- p.30 / Chapter 2.2.4 --- Production of recombinant MuIFN α (rMuIFN α) --- p.30 / Chapter 2.2.5 --- Production of native MuIFN α by polyI：polyC induction --- p.31 / Chapter 2.2.6 --- Influenza A virus strain A/NWS/33 preparation and titration --- p.31 / Chapter 2.2.7 --- Virus infection in Influenza A virus challenge assay --- p.32 / Chapter 2.2.8 --- Cell culture techniques --- p.32 / Chapter 2.2.9 --- "MTT cell proliferation assay of JCS cell line, for measuring MuIFN α anti-proliferation activity" --- p.33 / Chapter 2.2.10 --- Quantitative analysis of MuIFN α --- p.34 / Chapter 2.2.11 --- Flow cytofluorometric analysis of cell cycle of MuIFN α treated JCS cells by propidium iodide staining --- p.34 / Chapter 2.2.12 --- FACS study on the effect of MuIFN α on MHC-I up-regulation in JCS cells --- p.35 / Chapter 2.2.13 --- FACS study on the effect of MuIFN α on MHC-I up-regulation on primary macrophages from Balb/c mice --- p.35 / Chapter 2.2.14 --- Anti-viral activity by transfection of MuIFN α gene --- p.36 / Chapter 2.2.15 --- Sequencing of MuIFN al2 coding region from genomic DNA of L929 and JCS cell lines --- p.37 / Chapter 2.2.16 --- "RNA extraction from L929 cell lines, with or without Influenza A virus infection or polyI:polyC induction" --- p.37 / Chapter 2.2.17 --- RNA extraction from tissues of Balb/c mouse --- p.38 / Chapter 2.2.18 --- Reverse transcription --- p.39 / Chapter 2.2.19 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Overview --- p.40 / Chapter 3.2 --- "Subcloning of MuIFN α 12, MuIFN αl and MuIFN α4 coding sequences into the pEGFP-Nl vector" --- p.40 / Chapter 3.3 --- The growth inhibitory effect of different MuIFN α subtypes on murine myeloid leukemia cell line JCS --- p.41 / Chapter 3.4 --- Quantitation of MuIFN α subtype samples --- p.50 / Chapter 3.5 --- Cell cycle analysis of MuIFN α treated JCS cells --- p.50 / Chapter 3.6 --- FACS analysis of the effect of different MuIFN α subtypes on MHC-I expression in JCS cell line --- p.57 / Chapter 3.7 --- FACS analysis of the effect of different MuIFN α subtypes on MHC-I expression in primary macrophages in Balb/c mice --- p.65 / Chapter 3.8 --- Effect of MuIFN α subtype transgenes on L929 cells challenged with Influenza A virus --- p.72 / Chapter 3.9 --- Sequencing of MuIFN αl2 coding region from genomic DNA of L929 and JCS cell line --- p.78 / Chapter 3.10 --- "MuIFN αl2 expression in untreated, Influenza A virus infected or polyl:polyC induced L929 cells" --- p.78 / Chapter 3.11 --- Detection of MuIFN α12 transcripts in tissues of the 8-10 week untreated Balb/c mice --- p.85 / Chapter Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- Overview --- p.89 / Chapter 4.2 --- rMuIFN α 12 has anti-proliferative and apoptotic effects on JCS cell line --- p.89 / Chapter 4.3 --- "Up-regulation of MHC-I expression in JCS cells and primary macrophages by rMuIFN αl2, rMuIFN αl, rMuIFN α4 and mixed type I IFN" --- p.91 / Chapter 4.4. --- Transfection of MuIFN α12 gene could induce anti-viral state in L929 cell line --- p.91 / Chapter 4.5 --- Gene regulation of MuIFN al2 in L929 cells infected with Influenza A virus or induced by polyI:polyC --- p.92 / Chapter 4.6 --- Gene expression of MuIFN αl2 in different tissues of Balb/c mice --- p.94 / Conclusion --- p.95 / Reference List --- p.96 / List of figures: / Fig. 1.1 The 3D structure of recombinant human interferon alpha (HuIFN α) subtype 2B --- p.11 / Fig. 1.2 Current model of lFN induction --- p.12 / Fig. 1.3 Activation of RNase L --- p.13 / Fig. 2.1 Graphical map of plasmid vector pEGFP-Nl --- p.17

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors. One of the factors implicated in disease pathogenesis is early life viral infection. A typical immune response to viral infection includes production of type 1 interferons (IFN), such as IFN-α, which can induce stress in the pancreatic β-cells. Reactive oxygen species (ROS) accumulation occurs after exposure to other inflammatory cytokines, causing oxidative stress that may be linked to T1D pathogenesis. Therefore, we hypothesized that IFN-α may also elicit β-cell ROS accumulation. Our in vivo and in vitro experiments with human islets showed rapid and heterogenous ROS accumulation with IFN-α. Although T1D is characterized by autoimmune destruction of β-cells, some cells survive this persistent attack. We hypothesized that survival/ death of β-cells could be attributed to the ability to effectively mitigate ROS accumulation. One mechanism to mitigate ROS is autophagy, which degrades and recycles cellular components to promote cellular homeostasis. We observed an impairment in autophagy in β-cells of donors with T1D as well as in islets of diabetic non-obese diabetic (NOD) mouse model of autoimmune diabetes. Autophagic flux was also impaired in diabetic NOD mouse islets, further confirming impairment of autophagy. Interestingly, we observed an induction of autophagy after acute treatment with IFN-α both in vitro and in vivo, suggesting compensatory upregulation of autophagy to restore homeostasis. Similarly, we observed an increase in autophagosomes and telolysosomes in β-cells of normoglycemic autoantibody positive organ donors compared to nondiabetic organ donors. Together, these data implicate a defect in the final degradation step of autophagy involving lysosomes. Therefore, we analyzed the activity and expression of lysosomal cysteine protease Cathepsin H (CTSH, a T1D susceptibility locus), and found both to be increased in islets of pre-diabetic NOD mice. Together, these data support compensatory hyperactivation of lysosomal enzymes prior to overt diabetes, potentially to rid the cell of ROS and degradation-resistant oxidized proteins and lipids. We also observed that C57Bl/6J mice lacking a key autophagy enzyme, ATG7, in their β-cells, spontaneously developed hyperglycemia. Collectively, these data highlight the importance of -phagic degradation process in the pathogenesis of T1D. / 2022-12-28

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases.