TY - JOUR A1 - Weigand, Isabel A1 - Ronchi, Cristina L. A1 - Vanselow, Jens T. A1 - Bathon, Kerstin A1 - Lenz, Kerstin A1 - Herterich, Sabine A1 - Schlosser, Andreas A1 - Kroiss, Matthias A1 - Fassnacht, Martin A1 - Calebiro, Davide A1 - Sbiera, Silviu T1 - PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation JF - Science Advances N2 - Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome. KW - mutation triggers KW - phosphorylation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270445 VL - 7 IS - 8 ER - TY - JOUR A1 - Weigand, Isabel A1 - Ronchi, Cristina L. A1 - Rizk-Rabin, Marthe A1 - Dalmazi, Guido Di A1 - Wild, Vanessa A1 - Bathon, Kerstin A1 - Rubin, Beatrice A1 - Calebiro, Davide A1 - Beuschlein, Felix A1 - Bertherat, Jérôme A1 - Fassnacht, Martin A1 - Sbiera, Silviu T1 - Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas JF - Scientific Reports N2 - Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion. KW - kinases KW - immunohistochemistry Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157952 VL - 7 IS - 49 ER - TY - JOUR A1 - Vetrivel, Sharmilee A1 - Zhang, Ru A1 - Engel, Mareen A1 - Oßwald, Andrea A1 - Watts, Deepika A1 - Chen, Alon A1 - Wielockx, Ben A1 - Sbiera, Silviu A1 - Reincke, Martin A1 - Riester, Anna T1 - Characterization of adrenal miRNA-based dysregulations in Cushing's syndrome JF - International Journal of Molecular Sciences N2 - MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing's syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing's disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes. KW - cortisol KW - ACTH KW - miRNA KW - Cushing's KW - hypercortisolism KW - pituitary Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284394 SN - 1422-0067 VL - 23 IS - 14 ER - TY - JOUR A1 - Vetrivel, Sharmilee A1 - Zhang, Ru A1 - Engel, Mareen A1 - Altieri, Barbara A1 - Braun, Leah A1 - Osswald, Andrea A1 - Bidlingmaier, Martin A1 - Fassnacht, Martin A1 - Beuschlein, Felix A1 - Reincke, Martin A1 - Chen, Alon A1 - Sbiera, Silviu A1 - Riester, Anna T1 - Circulating microRNA Expression in Cushing’s Syndrome JF - Frontiers in Endocrinology N2 - Context Cushing’s syndrome (CS) is a rare disease of endogenous hypercortisolism associated with high morbidity and mortality. Diagnosis and classification of CS is still challenging. Objective Circulating microRNAs (miRNAs) are minimally invasive diagnostic markers. Our aim was to characterize the circulating miRNA profiles of CS patients and to identify distinct profiles between the two major CS subtypes. Methods We included three groups of patients from the German Cushing’s registry: ACTH-independent CS (Cortisol-Producing-Adenoma; CPA), ACTH-dependent pituitary CS (Cushing’s Disease; CD), and patients in whom CS had been ruled out (controls). Profiling of miRNAs was performed by next-generation-sequencing (NGS) in serum samples of 15 CS patients (each before and after curative surgery) and 10 controls. Significant miRNAs were first validated by qPCR in the discovery cohort and then in an independent validation cohort of 20 CS patients and 11 controls. Results NGS identified 411 circulating miRNAs. Differential expression of 14 miRNAs were found in the pre- and postoperative groups. qPCR in the discovery cohort validated 5 of the significant miRNAs from the preoperative group analyses. Only, miR-182-5p was found to be significantly upregulated in the CD group of the validation cohort. Comparing all CS samples as a group with the controls did not reveal any significant differences in expression. Outcome In conclusion, our study identified miR-182-5p as a possible biomarker for CD, which has to be validated in a prospective cohort. Furthermore, our results suggest that presence or absence of ACTH might be at least as relevant for miRNA expression as hypercortisolism itself. KW - cortisol KW - ACTH KW - miRNA KW - biomarker KW - cortisol-producing adenoma KW - miR-182-5p KW - hypercortisolism KW - miR-183 cluster Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229761 SN - 1664-2392 VL - 12 ER - TY - JOUR A1 - Tamburello, Mariangela A1 - Altieri, Barbara A1 - Sbiera, Iuliu A1 - Sigala, Sandra A1 - Berruti, Alfredo A1 - Fassnacht, Martin A1 - Sbiera, Silviu T1 - FGF/FGFR signaling in adrenocortical development and tumorigenesis: novel potential therapeutic targets in adrenocortical carcinoma JF - Endocrine N2 - FGF/FGFR signaling regulates embryogenesis, angiogenesis, tissue homeostasis and wound repair by modulating proliferation, differentiation, survival, migration and metabolism of target cells. Understandably, compelling evidence for deregulated FGF signaling in the development and progression of different types of tumors continue to emerge and FGFR inhibitors arise as potential targeted therapeutic agents, particularly in tumors harboring aberrant FGFR signaling. There is first evidence of a dual role of the FGF/FGFR system in both organogenesis and tumorigenesis, of which this review aims to provide an overview. FGF-1 and FGF-2 are expressed in the adrenal cortex and are the most powerful mitogens for adrenocortical cells. Physiologically, they are involved in development and maintenance of the adrenal gland and bind to a family of four tyrosine kinase receptors, among which FGFR1 and FGFR4 are the most strongly expressed in the adrenal cortex. The repeatedly proven overexpression of these two FGFRs also in adrenocortical cancer is thus likely a sign of their participation in proliferation and vascularization, though the exact downstream mechanisms are not yet elucidated. Thus, FGFRs potentially offer novel therapeutic targets also for adrenocortical carcinoma, a type of cancer resistant to conventional antimitotic agents. KW - FGF-pathway KW - FGFR KW - FGFR-inhibitors KW - adrenocortical development KW - adrenocortical tumors Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324420 VL - 77 IS - 3 ER - TY - JOUR A1 - Sbiera, Silviu A1 - Tryfonidou, Marianna A. A1 - Weigand, Isabel A1 - Grinwis, Guy C. M. A1 - Broeckx, Bart A1 - Herterich, Sabine A1 - Allolio, Bruno A1 - Deutschbein, Timo A1 - Fassnacht, Martin A1 - Meij, Björn P. T1 - Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas JF - Plos One N2 - Purpose Cushing’s disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs. Methods Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas. Results None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours. Conclusions Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed. KW - cytoplasmic staining KW - dogs KW - adenomas KW - pituitary gland KW - pituitary adenomas KW - nuclear staining KW - mutation KW - protein expression Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148020 VL - 11 IS - 12 ER - TY - JOUR A1 - Sbiera, Silviu A1 - Ronchi, Cristina L. A1 - Leich, Ellen A1 - Henzel, Katharina A1 - Rosenwald, Andreas A1 - Allolio, Bruno A1 - Fassnacht, Martin T1 - Single Nucleotide Polymorphism Array Profiling of Adrenocortical Tumors - Evidence for an Adenoma Carcinoma Sequence? JF - PLoS ONE N2 - Adrenocortical tumors consist of benign adenomas and highly malignant carcinomas with a still incompletely understood pathogenesis. A total of 46 adrenocortical tumors (24 adenomas and 22 carcinomas) were investigated aiming to identify novel genes involved in adrenocortical tumorigenesis. High-resolution single nucleotide polymorphism arrays (Affymetrix) were used to detect copy number alterations (CNAs) and copy neutral losses of heterozygosity (cnLOH). Genomic clustering showed good separation between adenomas and carcinomas, with best partition including only chromosome 5, which was highly amplified in 17/22 malignant tumors. The malignant tumors had more relevant genomic aberrations than benign tumors, such as a higher median number of recurrent CNA (2631 vs 94), CNAs >100 Kb (62.5 vs 7) and CN losses (72.5 vs 5.5), and a higher percentage of samples with cnLOH (91% vs 29%). Within the carcinoma cohort, a precise genetic pattern (i.e. large gains at chr 5, 7, 12, and 19, and losses at chr 1, 2, 13, 17, and 22) was associated with a better prognosis (overall survival: 72.2 vs 35.4 months, P=0.063). Interestingly, >70% of gains frequent in beningn were also present in malignant tumors. Notch signaling was the most frequently involved pathway in both tumor entities. Finally, a CN gain at imprinted “IGF2” locus chr 11p15.5 appeared to be an early alteration in a multi-step tumor progression, followed by the loss of one or two alleles, associated with increased IGF2 expression, only in carcinomas. Our study serves as database for the identification of genes and pathways, such as Notch signaling, which could be involved in the pathogenesis of adrenocortical tumors. Using these data, we postulate an adenoma-carcinoma sequence for these tumors. KW - adenomas KW - cancer diagnosis KW - cancer detection KW - carcinogenesis KW - carcinomas KW - chromosomes KW - genetic loci KW - malignant tumors KW - notch signaling Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97218 ER - TY - JOUR A1 - Sbiera, Silviu A1 - Kunz, Meik A1 - Weigand, Isabel A1 - Deutschbein, Timo A1 - Dandekar, Thomas A1 - Fassnacht, Martin T1 - The new genetic landscape of Cushing’s disease: deubiquitinases in the spotlight JF - Cancers N2 - Cushing’s disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD’s genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways. KW - Cushing’s disease KW - pathogenesis KW - somatic mutations KW - deubiquitinases Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193194 SN - 2072-6694 VL - 11 IS - 11 ER - TY - JOUR A1 - Sbiera, Silviu A1 - Dexneit, Thomas A1 - Reichardt, Sybille D. A1 - Michel, Kai D. A1 - van den Brandt, Jens A1 - Schmull, Sebastian A1 - Kraus, Luitgard A1 - Beyer, Melanie A1 - Mlynski, Robert A1 - Wortmann, Sebastian A1 - Allolio, Bruno A1 - Reichardt, Holger M. A1 - Fassnacht, Martin T1 - Influence of Short-Term Glucocorticoid Therapy on Regulatory T Cells \(In\) \(Vivo\) JF - PLoS One N2 - Background: Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T(reg)) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs). Objective: We readdressed the influence of GC therapy on T(reg) cells in immunocompetent human subjects and naive mice. Methods: Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T(reg) cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T(reg) cells were analyzed prior and after a 14 day GC therapy based on different markers. Results: Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T(reg) cells in blood (100 mg dexamethasone/kg body weight: 2.8 +/- 1.8 x 10(4) cells/ml vs. 33 +/- 11 x 10(4) in control mice) and spleen (dexamethasone: 2.8 +/- 1.9 x 10(5)/spleen vs. 95 +/- 22 x 10(5)/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3(+) T(reg) cells amongst the CD4(+) T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T(reg) cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T(reg) cells in a relevant manner, although there was some variation depending on the definition of the T(reg) cells (FOXP3(+): 4.0 +/- 1.5% vs 3.4 +/- 1.5%*; AITR(+): 0.660.4 vs 0.5 +/- 0.3%, CD127(low): 4.0 +/- 1.3 vs 5.0 +/- 3.0%* and CTLA4+: 13.8 +/- 11.5 vs 15.6 +/- 12.5%; * p < 0.05). Conclusion: Short-term GC therapy does not induce the hitherto supposed increase in circulating T(reg) cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T(reg) cell numbers. KW - Systemic-Lupus-Erythematosus KW - Immunological Self-Tolerance KW - Multiple-Sclerosis KW - Suppressive Function KW - Autoimmune-Diseases KW - FoxP3 Expression KW - Dendritic Cells KW - Immune-System KW - Sex-Hormones KW - Antigen 4 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140822 VL - 6 IS - 9 ER - TY - THES A1 - Sbiera, Silviu T1 - Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery T1 - Interaktion zwischen Human Polyomavirus JC mit Zellen des haematopoietischen Systems in der Peripherie N2 - Primary contact with human polyomaviruses is followed by lifelong asymptomatic persistence of viral DNA. Under severe immunosuppression JCV activation may lead to unrestricted virus growth in the CNS followed by development of progressive multifocal leukoencephalopathy (PML). Besides the kidney and the brain, target cells of persistent infection were also found in the hematopoietic system. This included the presence of JCV genomes in peripheral blood cells (PBCs). In the attempt to understand the role of PBCs for the JCV infection in humans, we asked for the type of cells affected as well as for virus interaction with PBCs. Analysis of separated subpopulations by highly sensitive and specific polymerase chain reaction and Southern blot hybridization revealed the presence of JCV DNA mostly in circulating granulocytes. These cells have important functions in innate immunity and are professional phagocytes. This suggested that PCR amplified DNA might be the result of an extranuclear association of the virus due to membrane attachment or phagocytosis rather than JCV infection with presence of viral DNA in the nucleus. In the attempt to answer this question JCV DNA was subcellularly localized in the blood of 22 healthy donors by JCV specific fluorescence in situ hybridization (FISH). Granulocytes and peripheral blood mononuclear cells (PBMCs) were separated by Percoll gradient centrifugation. Intracellular JCV DNA was hybridized with Digoxigenin-labeled JCV specific DNA probes covering half of the viral genome. As the sensitivity of the anti-digoxigenin antibody system was lower than the PCR detection level, a chemical amplification step was included consisting of peroxidase labeled secondary antibody precipitating biotinylated tyramide followed by detection with streptavidin-Texas-Red and fluorescence microscopy. Comparison of the number of cells affected in healthy individuals with 15 HIV-1 infected patients with and without PML revealed that the rate of affected PBMCs was comparable in both groups (2.5±0.4 and 14.5±0.9 per 1000). In contrast, the rate of JCV positive granulocytes in the immunosuppressed group was 92.6±1.7% compared to 4±1.4% in healthy donors thus confirming that granulocytes are the major group of circulating cells affected by JCV and that HIV-1 associated immune impairment has an important effect on the virus-cell association. Localization revealed that JCV DNA was predominantly located within the cytoplasm, although hybridizing signals occasionally covered the nuclear compartment. The fluorescent glow of chemical amplification combined with classical fluorescence microscopy did not allow an unequivocal localization of viral DNA. However, confocal microscopy of 24 sections through single cells combined with FISH without chemical amplification confirmed cytoplasmic localization of JCV DNA in a large number of cells. Additionally, it clearly demonstrated that JCV DNA was also located in the nucleus and nuclear localization directly correlated with the number of cells affected. Calculation of the virus load in subcellular compartments revealed that up to 50% of the JCV genomes were located in the nucleus thus pointing to viral infection at least in the granulocytes of HIV-1 infected patients. This may contribute to the distribution of the virus from sites of peripheral infection to the CNS and may promote the development of active PML in the severely immune impaired patients. N2 - Primärer Kontakt mit dem humanen Polyomavirus JC führt zu lebenslanger asymptomatischer Persistenz der viralen DNA in den Zielorganen der Infektion insbesondere der Niere und dem ZNS. Unter schwerer Immunsuppression kann die Aktivierung des JCV zu uneingeschränkter Vermehrung des Virus im ZNS und zur Entwicklung einer zentralnervösen Erkrankung, der progressiven multifokalen Leukoenzephalopathie (PML) führen Neuerdings wurde JCV DNA auch in Zellen des blutbildenden Systems insbesondere in peripheren Blutzellen (PBCs) beschrieben. Um die Rolle der PBCs für die JCV-Infektion beim Menschen besser zu verstehen, sollte der virus-assoziierte Zelltyp bestimmt und die Virus-Zell Interaktion näher untersucht werden. Die Analyse von isolierten Blutzellsubpopulationen durch eine sensitive und spezifische Polymerase-Kettenreaktion mit folgender Southern Blot-Hybridisierung ergab die Präsenz von JCV-DNA zumeist in zirkulierenden Granulozyten. Diese Zellen haben eine wichtige Funktion in der angeborenen Immunität und sind professionelle Phagozyten. Dies legte nahe, dass die PCR-amplifizierte DNA eher das Ergebnis einer extranukleären Assoziation des Virus durch Membranassoziation oder Phagozytose als einer JCV-Infektion ist, die durch Virus-DNA im Kern charakterisiert ist. Bei dem Versuch, diese Frage zu klären, wurde JCV-DNA in Blutzellen von gesunden Spendern mittels JCV-spezifischer Fluoreszenz in situ Hybridisierung (FISH) subzellulär lokalisiert. Granulozyten und periphere mononukleäre Blutzellen (PBMCs) wurden isoliert und intrazelluläre JCV-DNA mit Digoxigenin-markierten JCV DNA-Sonden, die die Hälfte des viralen Genoms representierten, hybridisiert. Da die Empfindlichkeit des Anti-Digoxigenin-Antikörper-Systems niedriger war als die PCR-Nachweisgrenze, wurde ein chemischer Amplifikationsschritt benutzt, das sogenannte Tyramidsystem, um die Sensitivität der FISH in Kombination mit der klassischen Fluoreszenzmikroskopie zu erhöhen. Der Vergleich der Anzahl von JCV betroffenen Zellen in gesunden Individuen mit Zellen von HIV-1-infizierten Patienten mit und ohne PML zeigte, dass die Rate der betroffenen PBMCs in beiden Gruppen (2,5 ± 0,4 und 14,5 ± 0,9 pro 1000) vergleichbar war. Im Gegensatz dazu war die Rate der JCV positiven Granulozyten in der immunsupprimierten Gruppe, 92,6 ± 1,7%, im Vergleich zu denen bei gesunden Spendern 4 ± 1,4% deutlich höher. Dies bestätigte, dass mit den Granulozyten die größte Gruppe von zirkulierenden Zellen von JCV betroffen sind und dass die schwere Beeinträchtigung der immunologischen Kompetenz durch die HIV-1 Infektion einen bedeutenden Einfluss auf auf die Virus-Zell Interaktion hat. Die intrazelluläre Lokalisation der viralen DNA ergab, dass die Signale überwiegend im Zytoplasma lokalisiert waren, wenngleich gelegentlich auch nukleäre Kompartimente betroffen waren. Durch die chemischen Verstärkung der Fluoreszenzsignale in Kombination mit klassischer Fluoreszenzmikroskopie war es jedoch nicht möglich eine eindeutige Lokalisierung der viraler DNA zu erreichen. Erst die Anwendung der konfokalen Mikroskopie bestätigte die predominant zytoplasmatische Lokalisierung von JCV-DNA in einer großen Anzahl von Zellen und hat eindeutig gezeigt, dass JCV-DNA zusätzlich im Kern lokalisiert ist. Die Kern Lokalisation korreliert direkt mit der Anzahl der betroffenen Zellen. Berechnung der Viruslast in subzellulären Kompartimenten hat gezeigt, dass bis zu 50% der JCV Genome im Kern von Granulozyten von HIV-1 Patienten lokalisiert waren. Dies deutet auf eine virale Infektion der Granulozyten hin und lässt vermuten, dass sie unter der HIV-1 Infektion an der Disseminierung des JC Virus aus den Organen der peripheren Infektion in das ZNS beteiligt sind und in der Konsequenz auch bei der Entwicklung der PML eine wesentliche Rolle spielen könnten. KW - Polyomaviren KW - Persistenz KW - Blutbildendes Gewebe KW - persistierende Infektion KW - Polyomavirus JC Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-74183 ER -