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The pandemic caused by novel coronavirus disease of 2019 (COVID-19) is a global threat. Wastewater surveillance in Japan and abroad has led to the detection of SARS-CoV-2, causing concern that SARS-CoV-2 in the feces of infected persons may contaminate the aquatic environment. Bivalves such as oysters cultivated in coastal areas are known to filter and concentrate viruses such as norovirus present in seawater in their bodies; however, whether they do so with SARS-CoV-2 is unknown. Therefore, we examined cultivated oysters sold in Japan for the presence of SARS-CoV-2 between October 2021 and April 2022 to clarify the extent of viral contamination and evaluate the risk of food-borne transmission of SARS-CoV-2. Porcine epidemic diarrhea virus (PEDV), known as pig coronavirus, was used to spike midgut-gland samples as a whole process control. The presence of SARS-CoV-2 and PEDV was investigated using a modified polyethylene glycol precipitation method and RT-qPCR. While all samples spiked with the whole process control were positive, no SARS-CoV-2 was detected in any of the 145 raw oyster samples surveyed, despite a marked increase in infections caused by the Omicron variant from January to April 2022 in Japan. Therefore, our results suggest that with well-developed sewage treatment facilities, consumption of oysters cultivated in coastal areas may not be a risk factor for SARS-CoV-2 outbreaks.

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persica L. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting 'Carmel' cultivar and early flowering peach ('40A17') produced an F1 where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as "crazy-top"), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1 progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitati

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persica L. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting 'Carmel' cultivar and early flowering peach ('40A17') produced an F1 where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as "crazy-top"), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1 progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitati

by Julia Hartl, Arbeit gesperrt, University of Innsbruck, Diplomarbeit, 2020, (VLID)4738921

Pituitary-dependent hypercortisolism (PDH) is a common endocrinopathy in dogs, but the promotors and initiators of the tumourigenesis of corticotroph pituitary adenomas remain unknown. Based on human data, we investigated mRNA expression of pituitary tumour transforming gene 1 (PTTG1) with quantitative RT-PCR in canine corticotroph pituitary adenomas. PTTG1 was overexpressed in adenomas approximately 3-fold. A strong association was observed between PTTG1 expression and disease-free interval; dogs with high PTTG1 expression had a significantly (4 times; P=0.02) shorter disease-free interval than dogs with low PTTG1 expression. This paper shows that PTTG1 expression is a negative prognosticator in relation to disease-free interval and recurrence in dogs undergoing transsphenoidal hypophysectomy as treatment for PDH.

BackgroundThe root extract of Rhodiola rosea has historically been used in Europe and Asia as an adaptogen, and similar to ginseng and Shisandra, shown to display numerous health benefits in humans, such as decreasing fatigue and anxiety while improving mood, memory, and stamina. A similar extract in the Rhodiola family, Rhodiola crenulata, has previously been shown to confer positive effects on the gut homeostasis of the fruit fly, Drosophila melanogaster. Although, R. rosea has been shown to extend lifespan of many organisms such as fruit flies, worms and yeast, its anti-aging mechanism remains uncertain. Using D. melanogaster as our model system, the purpose of this work was to examine whether the anti-aging properties of R. rosea are due to its impact on the microbial composition of the fly gut.ResultsRhodiola rosea treatment significantly increased the abundance of Acetobacter, while subsequently decreasing the abundance of Lactobacillales of the fly gut at 10 and 40 days of age. Additionally, supplementation of the extract decreased the total culturable bacterial load of the fly gut, while increasing the overall quantifiable bacterial load. The extract did not display any antimicrobial activity when disk diffusion tests were performed on bacteria belonging to Microbacterium, Bacillus, and Lactococcus.ConclusionsUnder standard and conventional rearing conditions, supplementation of R. rosea significantly alters the microbial community of the fly gut, but without any general antibacterial activity. Further studies should investigate whether R. rosea impacts the gut immunity across multiple animal models and ages.

BackgroundThe root extract of Rhodiola rosea has historically been used in Europe and Asia as an adaptogen, and similar to ginseng and Shisandra, shown to display numerous health benefits in humans, such as decreasing fatigue and anxiety while improving mood, memory, and stamina. A similar extract in the Rhodiola family, Rhodiola crenulata, has previously been shown to confer positive effects on the gut homeostasis of the fruit fly, Drosophila melanogaster. Although, R. rosea has been shown to extend lifespan of many organisms such as fruit flies, worms and yeast, its anti-aging mechanism remains uncertain. Using D. melanogaster as our model system, the purpose of this work was to examine whether the anti-aging properties of R. rosea are due to its impact on the microbial composition of the fly gut.ResultsRhodiola rosea treatment significantly increased the abundance of Acetobacter, while subsequently decreasing the abundance of Lactobacillales of the fly gut at 10 and 40 days of age. Additionally, supplementation of the extract decreased the total culturable bacterial load of the fly gut, while increasing the overall quantifiable bacterial load. The extract did not display any antimicrobial activity when disk diffusion tests were performed on bacteria belonging to Microbacterium, Bacillus, and Lactococcus.ConclusionsUnder standard and conventional rearing conditions, supplementation of R. rosea significantly alters the microbial community of the fly gut, but without any general antibacterial activity. Further studies should investigate whether R. rosea impacts the gut immunity across multiple animal models and ages.

Travellers' diarrhoea (TD) remains the most frequent health problem encountered by visitors to the (sub) tropics. Traditional stool culture identifies the pathogen in only 15% of cases. Exploiting PCR-based methods, we investigated TD pathogens with a focus on asymptomatic travellers and severity of symptoms. Pre- and post-travel stools of 382 travellers with no history of antibiotic use during travel were analysed with a multiplex quantitative PCR for Salmonella, Yersinia, Campylobacter, Shigella, Vibrio cholerae and five diarrhoeagenic Escherichia coli: enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) and enteroinvasive (EIEC). The participants were categorized by presence/absence of TD during travel and on return, and by severity of symptoms. A pathogen was indentified in 61% of the asymptomatic travellers, 83% of those with resolved TD, and 83% of those with ongoing TD; 25%, 43% and 53% had multiple pathogens, respectively. EPEC, EAEC, ETEC and Campylobacter associated especially with ongoing TD symptoms. EAEC and EPEC proved more common than ETEC. To conclude, modern methodology challenges our perception of stool pathogens: all pathogens were common both in asymptomatic and symptomatic travellers. TD has a multibacterial nature, but diarrhoeal symptoms mostly associate with EAEC, EPEC, ETEC and Campylobacter. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

[EN] Tomato torrado virus (ToTV) causes serious damage to the tomato industry and significant economic losses. A quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan (R) MGB probe for ToTV was developed for sensitive detection and quantitation of different ToTV isolates. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 10(4) to 10(10) ToTV RNA copies/ng of total RNA. The specificity of the RT-qPCR was tested with twenty-three ToTV isolates from tomato (Solanum lycopersicum L), and black nightshade (Solanum nigrum L) collected in Spain, Australia, Hungary and France, which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies. (C) 2015 Elsevier B.V. All rights reserved.

[EN] Tomato torrado virus (ToTV) causes serious damage to the tomato industry and significant economic losses. A quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan (R) MGB probe for ToTV was developed for sensitive detection and quantitation of different ToTV isolates. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 10(4) to 10(10) ToTV RNA copies/ng of total RNA. The specificity of the RT-qPCR was tested with twenty-three ToTV isolates from tomato (Solanum lycopersicum L), and black nightshade (Solanum nigrum L) collected in Spain, Australia, Hungary and France, which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies. (C) 2015 Elsevier B.V. All rights reserved.

Using drugs such as alcohol and morphine among others can be addictive in some individuals, and progress into a substance abuse disorder. The mesolimbic dopaminergic system (MD-system) is involved in the reward process during the development of drug addiction. The MD-system is critical for survival and affects different behaviors in both man and animal. Neurochemical pathways drive for instance physical activity, food intake, love and reproduction and are part of the natural reward process involved partly in the release of dopamine (DA) into frontal lobes. Within the MD-system opioid receptors throughout the brain are affected by drug intake, and activation of these receptors modulate DA-release in brain regions involved in reward-behavior. The aim of this study was to evaluate gene expression of MOR and DOR within the endogenous opioid system (EO-system) in relation to voluntary physical activity, a natural reinforcer. Further on investigations of the drug alcohol was compared to the natural reinforcer sucrose using voluntary consumption. For both experiments qRT-PCR was used to measure mRNA levels of MOR and DOR from brain areas of interest. We found a small significant up regulation in NAc, PFC and VTA but for DOR in VTA a down regulation in gene expression of physical exercising mice. Additionally these two different genes OPRM1- and the OPRD1- gene are down regulated in VTA and NAc due to alcohol- and sugar-intake. This implicate that the natural reward system and their ORs point in the direction of earlier findings; the opioid receptors have a key role in regulate alcohol intake and the natural rewarding stimuli as food intake.

Using drugs such as alcohol and morphine among others can be addictive in some individuals, and progress into a substance abuse disorder. The mesolimbic dopaminergic system (MD-system) is involved in the reward process during the development of drug addiction. The MD-system is critical for survival and affects different behaviors in both man and animal. Neurochemical pathways drive for instance physical activity, food intake, love and reproduction and are part of the natural reward process involved partly in the release of dopamine (DA) into frontal lobes. Within the MD-system opioid receptors throughout the brain are affected by drug intake, and activation of these receptors modulate DA-release in brain regions involved in reward-behavior. The aim of this study was to evaluate gene expression of MOR and DOR within the endogenous opioid system (EO-system) in relation to voluntary physical activity, a natural reinforcer. Further on investigations of the drug alcohol was compared to the natural reinforcer sucrose using voluntary consumption. For both experiments qRT-PCR was used to measure mRNA levels of MOR and DOR from brain areas of interest. We found a small significant up regulation in NAc, PFC and VTA but for DOR in VTA a down regulation in gene expression of physical exercising mice. Additionally these two different genes OPRM1- and the OPRD1- gene are down regulated in VTA and NAc due to alcohol- and sugar-intake. This implicate that the natural reward system and their ORs point in the direction of earlier findings; the opioid receptors have a key role in regulate alcohol intake and the natural rewarding stimuli as food intake.

Honey bee virus research is an enormously broad area, ranging from subcellular molecular biology through physiology and behaviour, to individual and colony-level symptoms, transmission and epidemiology. The research methods used in virology are therefore equally diverse. This article covers those methods that are very particular to virological research in bees, with numerous cross-referrals to other BEEBOOK papers on more general methods, used in virology as well as other research. At the root of these methods is the realization that viruses at their most primary level inhabit a molecular, subcellular world, which they manipulate and interact with, to produce all higher order phenomena associated with virus infection and disease. Secondly, that viruses operate in an exponential world, while the host operates in a linear world and that much of the understanding and management of viruses hinges on reconciling these fundamental mathematical differences between virus and host. The article concentrates heavily on virus propagation and methods for detection, with minor excursions into surveying, sampling management and background information on the many viruses found in bees.

Bm86 midgut protein has been used in order to control ticks of the Hyalomma genus. Previous studies demonstrated the inefficacity of this antigen in the control of Hyalomma scupense, whereas recombinant Hd86 antigen, the Bm86 ortholog in H. scupense produced in Pichia pastoris, was protective against larval H. scupense tick stage infestations but ineffective in the control of the adult stage. One possible explanation for this result is the variation in Hd86 expression levels between these two developmental stages. To test this hypothesis, Hd86 mRNA levels were characterized in H. scupense developmental stages. The expression profile of Hd86 demonstrated a significant variation between tick life stages and showed a significant reduction in the number of transcripts during feeding and, particularly after molting to adults. The most interesting result was noted after molting of engorged nymphs in unfed adults where the expression levels decreased significantly by 12.78 (10.77–17.39) (p < 0.001) and 9.25 (5.77–15.72)-fold (p < 0.001) in unfed males and unfed females, respectively. Comparing unfed nymphs to unfed adult ticks, the Hd86 expression levels decreased by 13.82 (5.39–24.45) (p = 0.035) and 9.93 (2.87–22.08)-fold (p = 0.038) in males and females respectively. Lower Hd86 mRNA levels in adult ticks should result in lower protein levels and thus less antibody–antigen interactions necessary for vaccine efficacy in ticks fed on vaccinated animals. Thus, the observed differences in Hd86 expression profile between immature and adult stages might explain, in part, the discrepancy of the Hd86 vaccine efficacy against these two life stages of H. scupense.

Background: In breast cancer, little is known about the consequences of co-expression of ER alpha with the second estrogen receptor, ER beta, and its isoforms in light of their joint prognostic value. Previously reported correlations have been based mostly on independent ER alpha and ER beta expression levels in breast tumors. Purpose: To address whether the expression ratio of ER alpha and ER beta and its isoforms may be a more important parameter than their absolute levels, we analyzed relative mRNA expression ratios of ER beta 1 to ER beta 2 and ER alpha in 74 clinical samples of invasive breast cancer including 39 early-onset and 35 late-onset breast cancers. Expression levels were correlated with clinical and histopathological parameters and disease-free interval. Results: A specific correlation of ER beta 1 expression levels with tumor size was detected in early-onset breast cancer patients and of ER beta 2 levels with tumor size in late-onset patients. Expression of both ER beta isoforms inversely correlated with expression of the two estrogen regulated genes, progesterone receptor and pS2 in both groups. Higher levels of ER beta 2 than ER beta 1 isoform were associated with a better outcome in late-onset patients. Conclusions: Our results suggest that different isoforms of ER beta may be involved in suppression of tumor growth in young and elder patients and may have different prognostic values. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Background:A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery. Methods:Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT–PCR. Results:Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8). Conclusion:In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence., Originally included in thesis in manuscript form with title: "Lymph node kallicrein 6 mRNA: a new progression marker for colorectal cancer".

Background:A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery. Methods:Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT–PCR. Results:Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8). Conclusion:In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence., Originally included in thesis in manuscript form with title: "Lymph node kallicrein 6 mRNA: a new progression marker for colorectal cancer".

Background: In breast cancer, little is known about the consequences of co-expression of ER alpha with the second estrogen receptor, ER beta, and its isoforms in light of their joint prognostic value. Previously reported correlations have been based mostly on independent ER alpha and ER beta expression levels in breast tumors. Purpose: To address whether the expression ratio of ER alpha and ER beta and its isoforms may be a more important parameter than their absolute levels, we analyzed relative mRNA expression ratios of ER beta 1 to ER beta 2 and ER alpha in 74 clinical samples of invasive breast cancer including 39 early-onset and 35 late-onset breast cancers. Expression levels were correlated with clinical and histopathological parameters and disease-free interval. Results: A specific correlation of ER beta 1 expression levels with tumor size was detected in early-onset breast cancer patients and of ER beta 2 levels with tumor size in late-onset patients. Expression of both ER beta isoforms inversely correlated with expression of the two estrogen regulated genes, progesterone receptor and pS2 in both groups. Higher levels of ER beta 2 than ER beta 1 isoform were associated with a better outcome in late-onset patients. Conclusions: Our results suggest that different isoforms of ER beta may be involved in suppression of tumor growth in young and elder patients and may have different prognostic values. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Background:A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery. Methods:Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT–PCR. Results:Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8). Conclusion:In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence., Originally included in thesis in manuscript form with title: "Lymph node kallicrein 6 mRNA: a new progression marker for colorectal cancer".

Background:A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery. Methods:Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT–PCR. Results:Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8). Conclusion:In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence., Originally included in thesis in manuscript form with title: "Lymph node kallicrein 6 mRNA: a new progression marker for colorectal cancer".

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan (R) MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidenialis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan (R) MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidenialis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

It is well known that breast tumors which are estrogen positive ER(+) are more likely to respond to hormone therapy. However, a certain percentage of ER(+)/PR(+) tumors do not respond to this therapy. Identification of the second estrogen receptor, named estrogen receptor beta (ER), as well as the existence of numerous isoforms/splice variants of both ER alpha and ER beta, suggests that a complex regulation of estrogen action exists. In this study, we analyzed the expression ratio of ER beta 1 isoform and ER beta Delta 5 splice variant mRNAs, and its correlation with ER/PR status by quantitative RT-PCR and clinical and histopathological parameters. We found that the relative proportion of ER beta Delta 5 in the total ER beta 1 transcript pool inversely correlates with the PR level (rho = -0,359, p LT 0,003, Spearman). It may be that the ER beta Delta 5 variant modulates the ERa activity of downstream targets. In addition, we suggest that the determination of the expression profiles of ER alpha and ER beta isoforms and splice variants in the defined groups of patients are necessary for elucidating their involvement in endocrine resistance.

It is well known that breast tumors which are estrogen positive ER(+) are more likely to respond to hormone therapy. However, a certain percentage of ER(+)/PR(+) tumors do not respond to this therapy. Identification of the second estrogen receptor, named estrogen receptor beta (ER), as well as the existence of numerous isoforms/splice variants of both ER alpha and ER beta, suggests that a complex regulation of estrogen action exists. In this study, we analyzed the expression ratio of ER beta 1 isoform and ER beta Delta 5 splice variant mRNAs, and its correlation with ER/PR status by quantitative RT-PCR and clinical and histopathological parameters. We found that the relative proportion of ER beta Delta 5 in the total ER beta 1 transcript pool inversely correlates with the PR level (rho = -0,359, p LT 0,003, Spearman). It may be that the ER beta Delta 5 variant modulates the ERa activity of downstream targets. In addition, we suggest that the determination of the expression profiles of ER alpha and ER beta isoforms and splice variants in the defined groups of patients are necessary for elucidating their involvement in endocrine resistance.

Cerebral gene expression changes in response to traumatic brain injury will provide useful information in the search for future trauma treatment. In order to characterize the outcome of mild brain injury, we studied C57BL/6J mice in a weight-drop (30 g), closed head injury model. At various times post-injury, mRNA was isolated from neocortex and hippocampus and transcriptional alterations were studied using quantitative reverse transcriptase PCR and gene array analysis. At three days post-injury, the results showed unilateral injury responses, both in neocortex and hippocampus, with the main effect seen on the side of the skull hit by the dropping weight. Upregulated transcripts encoded products characterizing reactive astrocytes, phagocytes, microglia and immune-reactive cells. Markers for oligodendrocytes and T-cells were not altered. Notably, strong differences in the responses among individual mice were seen, e.g. for the Gfap transcript expressed by reactive astrocytes and the chemokine Ccl3 transcript expressed by activated microglial cells. In conclusion, mild TBI chiefly activates transcripts leading to tissue remodeling, inflammatory processes and chemokine signalling, as in focal brain injury, suggesting putative targets for drug development.

Transcription factors are pivotal in regulating cell fate and development. We analyzed five transcription factors - FoxN4, Ptf1a, Prox1, Isl1 and Lim1 - with putative functions in the formation of early-generated retinal interneurons. A full-length chicken FoxN4 cDNA was characterized and in situ as well as RT-PCR showed that FoxN4 expression commenced already in the stage 12-14 optic vesicles. Ptf1a, Prox1, Isl1 and Lim1 expression appeared later by stage 20-24, concomitant with the first post-mitotic ganglion-, amacrine- and horizontal cells. The FoxN4 and Ptf1a expression was transient with peak levels by stage 32-35. Expression disappeared as the retinal progenitor cells differentiated. Prox1, Isl1 and Lim1 expression remained in several differentiated cells including the horizontal cells. The order of expression supports a scheme where Ptf1a and Prox1 is downstream of FoxN4 and that FoxN4 and Ptf1a have transient roles during fate specification while Prox1, Isl1 and Lim1 have roles that are important for the generation of the neuronal subtypes.

Background Type 1 diabetes (T1D) is characterised by loss of tolerance to beta-cell antigens, and the insulin-producing beta-cells in the pancreatic islets are destroyed by the host's own immune system. Immunological risk factors associated with T1D are related to the defects in the polarization of T-cells and in the function of regulatory T (Treg)-cells. We set out to study whether an impaired induction of regulatory mechanisms during the generation of T-cell responses upon stimulation is associated with T1D. Methods Naive T-cells were isolated from 18 children with recent T1D (0–14days from diagnosis; mean age 9.3 years), 11 children who had had T1D for at least 1 year (mean age 10.6) and 14 non-diabetic children (mean age 8.1). CD45RA+ T-cells were stimulated with PHA for 72 h in type 1 cytokine [interleukin (IL)-12 and anti-IL-4] or type 2 cytokine (IL-4 and anti-IL-12) environment. T-cell polarization and regulation related markers were analysed by quantitative reverse transcription polymerase chain reaction (QRT-PCR) (Th1 promoting T-bet, Th2 promoting GATA-3 and regulation related FOXP3, ICOS and NFATc2). Results Children with recently diagnosed T1D showed decreased induction of FOXP3, ICOS and NFATc2 in T-cells activated in type 1 cytokine milieu (p = 0.007, p = 0.001, and p = 0.02), whereas no differences between the diabetic and healthy children were seen in the up-regulation of activation markers, T-bet and GATA-3. Conclusions The poor induction of factors that mediate down-modulation of T-cell responses upon stimulation in type 1 cytokine environment may contribute to the development of autoreactive type 1 responses in T1D.

Purpose In addition to Estrogen Receptor alpha (ER alpha) and Progesterone Receptor (PR), the Second Estrogen Receptor (ER beta) appears to play an important role not only in estrogen signaling, but also in the pathogenesis of cancer in estrogen dependent tissues. The existence of various isoforms and splice variants of both ERs additionally complicates elucidation of their physiological role and involvement in the process of carcinogenesis. Methods In this study, the expression of ER beta 1 mRNA (wild type of beta receptor) and splice variant ER beta Delta 5 mRNA (which codes for truncated protein) was measured by the quantitative RT-PCR (q RT-PCR) in the 60 samples of Breast Cancer (BC) and correlated with ER alpha and PR protein levels and with clinical and histopathological parameters. Results We found the inverse correlation of ER beta Delta 5 mRNA expression with the levels of PR and ER alpha proteins in the group of postmenopausal patients; we also report the lower expression of ER beta 1 and ER beta Delta 5 mRNA in the larger tumors ( GT 20 mm, T2, and T3) than in smaller ones ( LT = 20 mm, T1). The decrease of ER beta Delta 5 mRNA expression in larger tumors is found to arise from ER-positive breast carcinomas. In addition, the portion of tumors with concomitant high expression of both transcripts matches up the known percentage of tumors resistant to endocrine therapy in patients with different ER/PR status. Conclusions As far as we know, this is the first study in which ER beta Delta 5 mRNA splice variant was quantified by realtime RT-PCR in the clinical samples of breast cancer tissue. Until now, the focus of clinical reports was the level of ER beta 1, ER beta 2, and ER beta 5 isoforms. The higher expression of ER beta Delta 5 mRNA is associated with the indicators of low biological aggressiveness of tumor (low tumor size within ER-positive status in our study) suggesting that the uncontrolled local tumor growth may occur as the expression of ER beta De

Purpose In addition to Estrogen Receptor alpha (ER alpha) and Progesterone Receptor (PR), the Second Estrogen Receptor (ER beta) appears to play an important role not only in estrogen signaling, but also in the pathogenesis of cancer in estrogen dependent tissues. The existence of various isoforms and splice variants of both ERs additionally complicates elucidation of their physiological role and involvement in the process of carcinogenesis. Methods In this study, the expression of ER beta 1 mRNA (wild type of beta receptor) and splice variant ER beta Delta 5 mRNA (which codes for truncated protein) was measured by the quantitative RT-PCR (q RT-PCR) in the 60 samples of Breast Cancer (BC) and correlated with ER alpha and PR protein levels and with clinical and histopathological parameters. Results We found the inverse correlation of ER beta Delta 5 mRNA expression with the levels of PR and ER alpha proteins in the group of postmenopausal patients; we also report the lower expression of ER beta 1 and ER beta Delta 5 mRNA in the larger tumors ( GT 20 mm, T2, and T3) than in smaller ones ( LT = 20 mm, T1). The decrease of ER beta Delta 5 mRNA expression in larger tumors is found to arise from ER-positive breast carcinomas. In addition, the portion of tumors with concomitant high expression of both transcripts matches up the known percentage of tumors resistant to endocrine therapy in patients with different ER/PR status. Conclusions As far as we know, this is the first study in which ER beta Delta 5 mRNA splice variant was quantified by realtime RT-PCR in the clinical samples of breast cancer tissue. Until now, the focus of clinical reports was the level of ER beta 1, ER beta 2, and ER beta 5 isoforms. The higher expression of ER beta Delta 5 mRNA is associated with the indicators of low biological aggressiveness of tumor (low tumor size within ER-positive status in our study) suggesting that the uncontrolled local tumor growth may occur as the expression of ER beta De

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels c

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels c

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels c

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels c

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels c

OBJECT: The cellular events leading to cerebral vasospasm after subarachnoid haemorrhages (SAH) involve a number of members of the protein kinase C (PKC) family. However, whereas calcium is thought to play a number of major roles in the pathophysiology of SAH, a number of PKCs function independently of calcium. We recently emphasized the potential role of the calcium-binding S100 proteins in a 'double haemorrhage' rat model of SAH-induced vasospasm. A number of S100 proteins are known to interfere directly with PKC, or indirectly with PKC substrates. We therefore investigated whether specific S100 proteins and PKCs are co-expressed/co-located in a rat model of SAH-induced vasospasm. METHODS AND RESULTS: SAH-induced vasospasm in rats (by means of a double cisternal injection of autologous blood from a rat femoral artery) distinctly modified the expression levels of calcium-dependent PKC-alpha and PKC-beta and calcium-independent PKC-eta and PKC-zeta in endothelial and smooth-muscle cells. The RNA levels of these four PKC isotypes were determined by quantitative RT-PCR. The present study reveals that, in endothelial cells, the S100B expression/location correlate well with those of PKC-eta, and those of S100A1 with PKC-beta. In smooth-muscle cells S100A2 expression/location correlate with those of PKC-eta, and those of S100B with PKC-zeta. CONCLUSION: The present data argue in favour of a joint action of the S100 protein network and the PKC signalling pathway during cerebral vasospasm., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

OBJECT: The cellular events leading to cerebral vasospasm after subarachnoid haemorrhages (SAH) involve a number of members of the protein kinase C (PKC) family. However, whereas calcium is thought to play a number of major roles in the pathophysiology of SAH, a number of PKCs function independently of calcium. We recently emphasized the potential role of the calcium-binding S100 proteins in a 'double haemorrhage' rat model of SAH-induced vasospasm. A number of S100 proteins are known to interfere directly with PKC, or indirectly with PKC substrates. We therefore investigated whether specific S100 proteins and PKCs are co-expressed/co-located in a rat model of SAH-induced vasospasm. METHODS AND RESULTS: SAH-induced vasospasm in rats (by means of a double cisternal injection of autologous blood from a rat femoral artery) distinctly modified the expression levels of calcium-dependent PKC-alpha and PKC-beta and calcium-independent PKC-eta and PKC-zeta in endothelial and smooth-muscle cells. The RNA levels of these four PKC isotypes were determined by quantitative RT-PCR. The present study reveals that, in endothelial cells, the S100B expression/location correlate well with those of PKC-eta, and those of S100A1 with PKC-beta. In smooth-muscle cells S100A2 expression/location correlate with those of PKC-eta, and those of S100B with PKC-zeta. CONCLUSION: The present data argue in favour of a joint action of the S100 protein network and the PKC signalling pathway during cerebral vasospasm., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.

Representational difference analysis, a cDNA subtraction approach, was used to identify genes that are expressed in acute leukemia but not in normal hematological tissues. We isolated a cDNA fragment from a cell line derived from a B cell acute lymphoblastic leukemia bearing two Philadelphia chromosomes. The cDNA derives from an orphan gene that was named BLACE. BLACE is located in region 7q36 and encodes a major 5.3-kb transcript and several alternatively spliced minor transcripts. Significant expression of BLACE was detected by RT-PCR and quantitative RT-PCR in bone marrow samples from B cell acute lymphoblastic leukemia patients. BLACE was not or was scarcely expressed in other types of hematological malignancies, in normal tissues, and in solid tumors.

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.

We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID50 produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.

Wie sich in den letzten Jahren gezeigt hat, ist Fettgewebe nicht nur ein inerter Fettspeicher, sondern produziert auch eine Vielzahl endokrin wirksamer Substanzen, die unter anderem auch an der Blutdruckregulation beteiligt sind. Da Adipositas ein wichtiger Risikofaktor für die Entwicklung der Hypertonie ist, sollte im Rahmen dieser Dissertation ein System zur quantitativen Untersuchung der Genexpression in Primärkulturen humaner Adipocyten entwickelt werden und dessen Funktionalität am Beispiel der hormonellen Regulation der Gene des Renin-Angiotensin-Systems demonstriert werden. Dies beinhaltete die Etablierung der Adipocytenisolierung und -kultivierung, eines Stimulationsassays, die Entwicklung einer der besonderen Größe und dem hohen Fettgehalt der Zellen angepaßten Zellzahl- und Vitalitätsbestimmungsmethode, die Untersuchung vier verschiedener RNA-Extraktionsmethoden auf ihre Eignung für Adipocyten und die Etablierung eines besonders sensitiven RT-PCR Systems zur Untersuchung der Genexpression mittels einer fluoreszenzmarkierten Sonde. Exemplarisch konnte anhand der Renin-Angiotensin-System-Gene die Funktionalität der Methoden demonstriert werden, indem nicht nur die Genexpression aller Komponenten des Renin-Angiotensin-Systems in humanen Adipocyten nachgewiesen wurden, sondern auch gezeigt werden konnte, dass Hydrocortison sowohl die Genexpression als auch die Dichte des Angiotensin II Typ 1-Rezeptors in der Adipocytenmembran stimuliert. Dieser Aspekt könnte möglicherweise nicht nur bei der besonderen Adipositasform des Cushing-Syndroms, sondern auch für die Entstehung der zentralen Adipositas von Bedeutung sein., Adipose tissue has functions above-and-beyond storing fat. It also produces a variety of different endocrine substances, some of which influence blood pressure regulation. Obesity is a well known risk factor for the development of hypertension Thus, the genes regulating expression of vasoactive molecules in adipose tissue, possibly contributing to an increase in blood pressure are of great interest. The aim of this work was to develope a system for quantitative gene expression analysis in primary cultured human adipocytes and to demonstrate its utility for studying the hormonal regulation of genes encoding the renin-angiotensin-system. We established procedures for the isolation and culture of human adipocytes, as well as a stimulatory assay. We also developed methods for the determination of cell number and vitality. Above this, four RNA extraction protocols were evaluated regarding their suitability for adipocytes, and a very sensitive RT-PCR system for gene expression analysis using fluorescent labeled probes was established. As an example for the functionality of these methods we showed that all genes of the renin-angiotensin-system are expressed in human adipocytes. We also demonstrated that hydrocortisone stimulates the gene expression as well as the density of the angiotensin II receptor type 1 on cultured human adipocytes. This finding may be of interest for the development of the obesity phenotype found in cushing syndrome, but could also contribute to the development of central obesity.

Wie sich in den letzten Jahren gezeigt hat, ist Fettgewebe nicht nur ein inerter Fettspeicher, sondern produziert auch eine Vielzahl endokrin wirksamer Substanzen, die unter anderem auch an der Blutdruckregulation beteiligt sind. Da Adipositas ein wichtiger Risikofaktor für die Entwicklung der Hypertonie ist, sollte im Rahmen dieser Dissertation ein System zur quantitativen Untersuchung der Genexpression in Primärkulturen humaner Adipocyten entwickelt werden und dessen Funktionalität am Beispiel der hormonellen Regulation der Gene des Renin-Angiotensin-Systems demonstriert werden. Dies beinhaltete die Etablierung der Adipocytenisolierung und -kultivierung, eines Stimulationsassays, die Entwicklung einer der besonderen Größe und dem hohen Fettgehalt der Zellen angepaßten Zellzahl- und Vitalitätsbestimmungsmethode, die Untersuchung vier verschiedener RNA-Extraktionsmethoden auf ihre Eignung für Adipocyten und die Etablierung eines besonders sensitiven RT-PCR Systems zur Untersuchung der Genexpression mittels einer fluoreszenzmarkierten Sonde. Exemplarisch konnte anhand der Renin-Angiotensin-System-Gene die Funktionalität der Methoden demonstriert werden, indem nicht nur die Genexpression aller Komponenten des Renin-Angiotensin-Systems in humanen Adipocyten nachgewiesen wurden, sondern auch gezeigt werden konnte, dass Hydrocortison sowohl die Genexpression als auch die Dichte des Angiotensin II Typ 1-Rezeptors in der Adipocytenmembran stimuliert. Dieser Aspekt könnte möglicherweise nicht nur bei der besonderen Adipositasform des Cushing-Syndroms, sondern auch für die Entstehung der zentralen Adipositas von Bedeutung sein., Adipose tissue has functions above-and-beyond storing fat. It also produces a variety of different endocrine substances, some of which influence blood pressure regulation. Obesity is a well known risk factor for the development of hypertension Thus, the genes regulating expression of vasoactive molecules in adipose tissue, possibly contributing to an increase in blood pressure are of great interest. The aim of this work was to develope a system for quantitative gene expression analysis in primary cultured human adipocytes and to demonstrate its utility for studying the hormonal regulation of genes encoding the renin-angiotensin-system. We established procedures for the isolation and culture of human adipocytes, as well as a stimulatory assay. We also developed methods for the determination of cell number and vitality. Above this, four RNA extraction protocols were evaluated regarding their suitability for adipocytes, and a very sensitive RT-PCR system for gene expression analysis using fluorescent labeled probes was established. As an example for the functionality of these methods we showed that all genes of the renin-angiotensin-system are expressed in human adipocytes. We also demonstrated that hydrocortisone stimulates the gene expression as well as the density of the angiotensin II receptor type 1 on cultured human adipocytes. This finding may be of interest for the development of the obesity phenotype found in cushing syndrome, but could also contribute to the development of central obesity.

In natural environments bacteria and filamentous fungi often compete for the same resources. Consequently, production of antibiotic secondary metabolites and defence mechanisms against these compounds have evolved in these organisms. An experimental model has been developed to study the response in fungi exposed to one such antibiotic. The filamentous fungus Aspergillus nidulans was treated with bafilomycin B-1, a Streptomyces-produced antibiotic which reduces radial growth rate and induces morphological changes in fungi. mRNA differential display was used to study changes in fungal gene expression. For five genes, changes in abundance of the corresponding mRNAs, directly or indirectly caused by bafilomycin, were observed. Of these, three were up-regulated and two repressed. With four of these the change in mRNA abundance measured ranged from 10- to 60-fold. However, for one gene the mRNA was only detected after bafilomycin treatment. One of the downregulated mRNAs encodes ASPND1, a glycoprotein that belongs to a known family of antigens identified in aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The remaining three genes were also cloned and sequenced; their sequences do not correspond to known genes in A. nidulans, and no similarities with published nucleotide or protein sequences in other organisms were found. These results indicate the feasibility of using mRNA differential display to study interactions between bacteria and filamentous fungi.