Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent Apoptosis, but are insensitive to direct activation with exogenous fas ligand

Introduction Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma. Aims To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines. Results Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both. Conclusions 1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes

The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication

The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Adenovirus-mediated CD40 Ligand Immunotherapy of Prostate and Bladder Cancer

Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses. We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy. In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models

Bakomliggande nätverksfaktorer till en utlandsetablering : en fallstudie av Scanias etableringsprocess i Polen

Syftet med denna utredning är att ur ett nätverksperspektiv undersöka ett antal bakomliggande faktorer till Scanias påverkat etableringsprocessen. För att kunna utföra detta har vi använt oss av en modell av Blankenburg (1989) som beskriver utlandsetableringen ur ett nätverksperspektiv. Modellen har ytterligare anpassats till den aktuella marknaden och tidpunkten för inträdet. De nätverksfaktorer som undersöks i uppsatsen är: grad av motstridande intressen, företagets rykte, det polska nätverkets internationaliseringsgrad, företagets nätverkskunskap och nätverkskontextens internationaliseringsgrad. Undersökningen är en fallstudie där personliga intervjuer genomförts för insamlig av data. Blankenburgs modell har använts vid utformningen av en intervjumall. I analysen har det funnits att de undersökta nätverksfaktorerna hade olika grad av betydelse för Scanias etablering i Polen. Mycket betydelsefull var graden av motstridande intressen bland de polska nätverksaktörerna, där enskilda aktörers engagemang ökade företagets marknadskunskap och var en länk till polska myndigheter. Likaså oförutsedda händelser, som en tull på lastbilar, styrde och påverkade etableringsförloppet. Faktorerna företagets rykte och nätverkskontextens internationaliseringsgrad var också av betydelse, då Scanias kundkrets snabbt ökade och företagets stora erfarenhet av tidigare etableringar ökade sannolikheten till ytterligare en lyckad etablering. Däremot har det utländska nätverkets internationaliseringsgrad och Scanias nätverkskunskap inte visat sig haft en avgörande roll vid marknadsinträdet. Polens fordonsindustri bedrev vid tidpunkten inte internationella affärer och nätverkskunskapen införskaffade företaget genom marknadsaktivitet

Bakomliggande nätverksfaktorer till en utlandsetablering : en fallstudie av Scanias etableringsprocess i Polen

Syftet med denna utredning är att ur ett nätverksperspektiv undersöka ett antal bakomliggande faktorer till Scanias påverkat etableringsprocessen. För att kunna utföra detta har vi använt oss av en modell av Blankenburg (1989) som beskriver utlandsetableringen ur ett nätverksperspektiv. Modellen har ytterligare anpassats till den aktuella marknaden och tidpunkten för inträdet. De nätverksfaktorer som undersöks i uppsatsen är: grad av motstridande intressen, företagets rykte, det polska nätverkets internationaliseringsgrad, företagets nätverkskunskap och nätverkskontextens internationaliseringsgrad. Undersökningen är en fallstudie där personliga intervjuer genomförts för insamlig av data. Blankenburgs modell har använts vid utformningen av en intervjumall. I analysen har det funnits att de undersökta nätverksfaktorerna hade olika grad av betydelse för Scanias etablering i Polen. Mycket betydelsefull var graden av motstridande intressen bland de polska nätverksaktörerna, där enskilda aktörers engagemang ökade företagets marknadskunskap och var en länk till polska myndigheter. Likaså oförutsedda händelser, som en tull på lastbilar, styrde och påverkade etableringsförloppet. Faktorerna företagets rykte och nätverkskontextens internationaliseringsgrad var också av betydelse, då Scanias kundkrets snabbt ökade och företagets stora erfarenhet av tidigare etableringar ökade sannolikheten till ytterligare en lyckad etablering. Däremot har det utländska nätverkets internationaliseringsgrad och Scanias nätverkskunskap inte visat sig haft en avgörande roll vid marknadsinträdet. Polens fordonsindustri bedrev vid tidpunkten inte internationella affärer och nätverkskunskapen införskaffade företaget genom marknadsaktivitet

Bakomliggande nätverksfaktorer till en utlandsetablering : en fallstudie av Scanias etableringsprocess i Polen

Syftet med denna utredning är att ur ett nätverksperspektiv undersöka ett antal bakomliggande faktorer till Scanias påverkat etableringsprocessen. För att kunna utföra detta har vi använt oss av en modell av Blankenburg (1989) som beskriver utlandsetableringen ur ett nätverksperspektiv. Modellen har ytterligare anpassats till den aktuella marknaden och tidpunkten för inträdet. De nätverksfaktorer som undersöks i uppsatsen är: grad av motstridande intressen, företagets rykte, det polska nätverkets internationaliseringsgrad, företagets nätverkskunskap och nätverkskontextens internationaliseringsgrad. Undersökningen är en fallstudie där personliga intervjuer genomförts för insamlig av data. Blankenburgs modell har använts vid utformningen av en intervjumall. I analysen har det funnits att de undersökta nätverksfaktorerna hade olika grad av betydelse för Scanias etablering i Polen. Mycket betydelsefull var graden av motstridande intressen bland de polska nätverksaktörerna, där enskilda aktörers engagemang ökade företagets marknadskunskap och var en länk till polska myndigheter. Likaså oförutsedda händelser, som en tull på lastbilar, styrde och påverkade etableringsförloppet. Faktorerna företagets rykte och nätverkskontextens internationaliseringsgrad var också av betydelse, då Scanias kundkrets snabbt ökade och företagets stora erfarenhet av tidigare etableringar ökade sannolikheten till ytterligare en lyckad etablering. Däremot har det utländska nätverkets internationaliseringsgrad och Scanias nätverkskunskap inte visat sig haft en avgörande roll vid marknadsinträdet. Polens fordonsindustri bedrev vid tidpunkten inte internationella affärer och nätverkskunskapen införskaffade företaget genom marknadsaktivitet

Will–Skill–Tool Components as Key Factors for Digital Media Implementation in Education: Austrian Teachers’ Experiences with Digital Forms of Instruction during the COVID-19 Pandemic

Although comprehensive digitalization (e.g., the provision of skills and resources) had already been placed on Austria’s education policy agenda prior to the emergence of COVID-19, there is evidence that educators had some difficulty ensuring digital learning opportunities for their students when schools closed in early 2020. Against this backdrop, the present study, which drew on qualitative data from the large-scale INCL-LEA (Inclusive Home Learning) study, aimed to determine whether secondary school teachers (n = 17) from Viennese schools met the prerequisites for successfully implementing digital instruction, formulated in the Will–Skill–Tool model developed by Christensen and Kzenek (2008). Findings reveal that teachers primarily associated their sufficient digital skills with three factors: (1) basic interest and competence, (2) recently attended training, and/or (3) a positive attitude toward changing teaching practices. Interestingly, some educators recognized that digitization offers great potential for implementing individualized teaching approaches. However, the findings point to the didactic necessity of digital socialization in terms of social communication and inclusion when establishing emergency digital education