Studien zur Funktion der Koaktivatoren ZIP-Kinase, AATF und TSG101 in der Androgenrezeptor-vermittelten Transkription

In unserer Arbeitsgruppe wurden mit der murinen ZIPK, dem Transkriptionsfaktor AATF und dem Ubiquitin-Bindeprotein TSG101 neue Koaktivatoren der AR-abhängigen Transkription identifiziert, deren funktionelle Kooperation in dieser Arbeit untersucht wurde. Dabei lag der Fokus auf der zeitlichen Abfolge und der gegenseitigen Abhängigkeit der Assemblierung des AR und der Koaktivatoren im Transkriptionsinitiationskomplex, sowie dem Mechanismus über den die ZIPK koaktivierend wirkt. Transiente Reportergenstudien bestätigten zunächst die humanen Orthologen als Koaktivatoren des AR. Die von der Kinaseaktivität abhängende koaktivierende Wirkung der ZIPK wurde für Enhancer- und Promotorbereiche von Zielgenen des AR und unter den Steroidhormonrezeptoren für die GR-, aber nicht die ER- und PR-vermittelte Transkription nachgewiesen. Auch die p53-abhängige Reportergenexpression wurde durch die ZIPK koaktiviert. Die Herunterregulation von ZIPK, AATF und TSG101 resultierte in verminderten mRNA-Leveln der endogenen AR-Zielgene PSA, KLK2 und TMPRSS2, was die endogenen Proteine als echte AR-Koaktivatoren klassifizierte. Die ZIPK zeigte dabei eine gewisse Zielgenspezifität, da das TMPRSS2-Gen von Überexpression und Reduktion der ZIPK unbeeinflusst blieb. Sequentielle Chromatin-Immunpräzipitations- (ChIP)-Analysen bestätigten, dass ZIPK, AATF und TSG101 in einem gemeinsamen Komplex mit dem AR an Enhancer- und Promotor-bereichen von AR-Zielgenen gebunden vorliegen. ChIP-Analysen nach siRNA-vermitteltem Knockdown von AATF zeigten, dass die Rekrutierung von TSG101 von AATF abhängt. Die Rekrutierung der ZIPK erfolgt dagegen direkt über den AR, wobei AATF stabilisierend wirkt. Die Assemblierung des AR und der Koaktivatoren an regulatorische Bereiche des PSA-, KLK2- und TMPRSS2-Gens erfolgte dynamisch in einer zyklischen Abfolge. Die Zykluslänge des AR betrug ca. 90 min, mit einem verkürzten ersten Zyklus von 30 min am PSA-Promotor. Die Koaktivatoren zeigten dabei ein deutlich dynamischeres Anlagerungs- und Dissoziations-verhalten. Innerhalb des AR-Zyklus fand eine mehrfache Assoziation und Dissoziation mit einem individuellen Rekrutierungsverhalten statt. Die in vitro von der ZIPK durchgeführte Phosphorylierung von Histon H3 an T11, welche ursprünglich als Mitose- und Centromerspezifisch beschrieben wurde (Preuss et al., 2003), konnte an den regulatorischen Regionen des PSA-, KLK2- und TMPRSS2-Gens als neue Histonmodifikation identifiziert, allerdings nicht der ZIPK zugeordnet werden. Die H3T11-Phosphorylierung erfolgte im Zuge der Genaktivierung noch vor der H3S10-Phosphorylierung und korrelierte mit der Demethylierung von H3K9. Um die Ursache für die zyklische Assoziation/Dissoziation des AR und der Koaktivatoren zu ermitteln wurden Proteasomen- oder Transkriptionsinhibitoren eingesetzt. Die Inhibition des Proteasoms bewirkte einerseits ein Ausbleiben der zyklischen Assoziation/Dissoziation und damit eine deutliche Akkumulation des AR an Response-Elementen, andererseits führte sie zur vollständigen Repression der AR-abhängigen Transkription von Reportergenen. Das zyklische Verhalten des AR kann somit auf den Abbau des AR zurückgeführt werden und ist für eine effiziente Transkription unerlässlich. Andererseits bewirkte die Inhibition der Transkription ebenso eine Akkumulation des AR. Daraus lässt sich schließen, dass die Initiation der Transkription eine Vorraussetzung für den Abbau darstellt und der Abbau wiederum die Vorraussetzung für eine erneute Transkriptionsinitiation. Interessanterweise führte die Herunterregulation der ZIPK ebenfalls zur Akkumulation des AR und von TSG101. Dieses Ergebnis wies auf eine Beteiligung der ZIPK an der Regulation des proteasomalen Abbaus des AR hin. Reportergenstudien zeigten einen funktionellen Zusammenhang zwischen der E3-Ligase Mdm2 und der ZIPK. In-vivo-Ubiquitinierungs-analysen nach Expression kinaseinaktiver ZIPK, sowie nach Herunterregulation endogener ZIPK führten zu einer verminderten Polyubiquitinierung des AR. Demgegenüber konnte nach ektopischer Expression der ZIPK eine Zunahme an polyubiquitiniertem AR detektiert werden. Der Nachweis einer schwachen Phosphorylierung von Mdm2 durch die ZIPK deutet eine Funktion der ZIPK in der Regulation der Mdm2-vermittelten Ubiquitinierung und damit dem Abbau des AR an, der vermutlich die Ursache des zyklischen Verhaltens des AR ist. Es lässt sich somit postulieren, dass das Zusammenspiel von AATF, TSG101 und ZIPK die Aktivität und den Abbau des AR reguliert. Nach Rekrutierung von ZIPK und AATF durch an den Enhancer/Promotor-gebundenen AR, rekrutiert AATF wiederum TSG101. Dieses wirkt koaktivierend, indem es die monoubiquitinierte, vermutlich aktive Form des AR stabilisiert. Nach erfolgter Transkriptionsinitiation ist die ZIPK an der Regulation des Ubiquitinierungs-zustandes des AR beteiligt, möglicherweise durch Aufhebung der Schutzwirkung von TSG101 und Aktivierung von Mdm2 durch Phosphorylierung, welches daraufhin die Polyubiquitinierung und damit den Abbau des AR einleitet. Dies bewirkt ein „Promotor-clearing“, welches dann die Bildung eines neuen Transkriptionsinitiationskomplexes ermöglicht

Diagnostic utility of cerebrospinal fluid immunocytochemistry for diagnosis of feline infectious peritonitis manifesting in the central nervous system

Objectives The aim of the study was to evaluate whether an ante-mortem diagnosis of central nervous system (CNS) feline infectious peritonitis (FIP) is possible via immunocytochemical staining (ICC) of feline coronavirus antigen (FCoV) within macrophages of cerebrospinal fluid (CSF). Methods Prospectively, CSF samples of 41 cats were investigated, including cats with histopathologically confirmed FIP and neurological signs (n = 10), cats with confirmed FIP without CNS involvement (n = 11), cats with neurological signs but another confirmed CNS disease (n = 17), and cats without neurological signs and a disease other than FIP (n = 3). ICC staining of CSF macrophages was performed in all cats. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of CSF ICC were calculated. Results Of 10 samples from cats with CNS FIP, eight had detectable CSF macrophages, seven of which were positive for FCoV. Ten of 11 samples from cats with confirmed FIP without neurological signs had macrophages in the CSF, with all 10 being ICC-positive. In cats with other CNS disorders, 11/17 had macrophages, two of which stained positively. In cats with diseases other than FIP and without neurological disorders, 2/3 revealed macrophages, with one cat showing positive ICC staining. Diagnosis of FIP via CSF ICC had a sensitivity of 85.0% and a specificity of 83.3%. PPV and NPV were 85.0% and 83.3%. Conclusions and relevance CSF ICC is a highly sensitive test for ante-mortem diagnosis of FIP manifesting in the CNS. However, CNS ICC specificity is too low to confirm FIP and the method should only be applied in conjunction with other features such as CSF cytology. CNS ICC could be helpful to discover pre-neurological stages of CNS FIP

Detection of feline coronavirus spike gene mutations as a tool to diagnose feline infectious peritonitis

Objectives Feline infectious peritonitis (FIP) is an important cause of death in the cat population worldwide. The ante-mortem diagnosis of FIP in clinical cases is still challenging. In cats without effusion, a definitive diagnosis can only be achieved post mortem or with invasive methods. The aim of this study was to evaluate the use of a combined reverse transcriptase nested polymerase chain reaction (RT-nPCR) and sequencing approach in the diagnosis of FIP, detecting mutations at two different nucleotide positions within the spike (S) gene. Methods The study population consisted of 64 cats with confirmed FIP and 63 cats in which FIP was initially suspected due to similar clinical or laboratory signs, but that were definitively diagnosed with another disease. Serum/plasma and/or effusion samples of these cats were examined for feline coronavirus (FCoV) RNA by RT-nPCR and, if positive, PCR products were sequenced for nucleotide transitions within the S gene. Results Specificity of RT-nPCR was 100% in all materials (95% confidence interval [CI] in serum/plasma 83.9-100.0;95% CI in effusion 93.0-100.0). The specificity of the sequencing step could not be determined as none of the cats of the control group tested positive for FCoV RNA. Sensitivity of the 'combined RT-nPCR and sequencing approach' was 6.5% (95% CI 0.8-21.4) in serum/plasma and 65.3% (95% CI 50.4-78.3) in effusion. Conclusions and relevance A positive result is highly indicative of the presence of FIP, but as none of the control cats tested positive by RT-nPCR, it was not possible to confirm that the FCoV mutant described can only be found in cats with FIP. Further studies are necessary to evaluate the usefulness of the sequencing step including FCoV-RNA-positive cats with and without FIP. A negative result cannot be used to exclude the disease, especially when only serum/plasma samples are available

Clinical Follow-Up and Postmortem Findings in a Cat That Was Cured of Feline Infectious Peritonitis with an Oral Antiviral Drug Containing GS-441524

This is the first report on a clinical follow-up and postmortem examination of a cat that had been cured of feline infectious peritonitis (FIP) with ocular manifestation by successful treatment with an oral multicomponent drug containing GS-441524. The cat was 6 months old when clinical signs (recurrent fever, lethargy, lack of appetite, and fulminant anterior uveitis) appeared. FIP was diagnosed by ocular tissue immunohistochemistry after enucleation of the affected eye. The cat was a participant in a FIP treatment study, which was published recently. However, 240 days after leaving the clinic healthy, and 164 days after the end of the 84 days of treatment, the cured cat died in a road traffic accident. Upon full postmortem examination, including histopathology and immunohistochemistry, there were no residual FIP lesions observed apart from a generalized lymphadenopathy due to massive lymphoid hyperplasia. Neither feline coronavirus (FCoV) RNA nor FCoV antigen were identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively, in any tissues or body fluids, including feces. These results prove that oral treatment with GS-441524 leads to the cure of FIP-associated changes and the elimination of FCoV from all tissues. Keywords: FCoV; FIP; Mutian; Xraphconn®; antiviral chemotherapy; feline coronavirus; necropsy; therapy; treatmen

Long-term impact of myocardial inflammation on quantitative myocardial perfusion-a descriptive PET/MR myocarditis study.

PURPOSE Whether myocardial inflammation causes long-term sequelae potentially affecting myocardial blood flow (MBF) is unknown. We aimed to assess the effect of myocardial inflammation on quantitative MBF parameters, as assessed by 13N-ammonia positron emission tomography myocardial perfusion imaging (PET-MPI) late after myocarditis. METHODS Fifty patients with a history of myocarditis underwent cardiac magnetic resonance (CMR) imaging at diagnosis and PET/MR imaging at follow-up at least 6 months later. Segmental MBF, myocardial flow reserve (MFR), and 13N-ammonia washout were obtained from PET, and segments with reduced 13N-ammonia retention, resembling scar, were recorded. Based on CMR, segments were classified as remote (n = 469), healed (inflammation at baseline but no late gadolinium enhancement [LGE] at follow-up, n = 118), and scarred (LGE at follow-up, n = 72). Additionally, apparently healed segments but with scar at PET were classified as PET discordant (n = 18). RESULTS Compared to remote segments, healed segments showed higher stress MBF (2.71 mL*min-1*g-1 [IQR 2.18-3.08] vs. 2.20 mL*min-1*g-1 [1.75-2.68], p < 0.0001), MFR (3.78 [2.83-4.79] vs. 3.36 [2.60-4.03], p < 0.0001), and washout (rest 0.24/min [0.18-0.31] and stress 0.53/min [0.40-0.67] vs. 0.22/min [0.16-0.27] and 0.46/min [0.32-0.63], p = 0.010 and p = 0.021, respectively). While PET discordant segments did not differ from healed segments regarding MBF and MFR, washout was higher by ~ 30% (p < 0.014). Finally, 10 (20%) patients were diagnosed by PET-MPI as presenting with a myocardial scar but without a corresponding LGE. CONCLUSION In patients with a history of myocarditis, quantitative measurements of myocardial perfusion as obtained from PET-MPI remain altered in areas initially affected by inflammation. CMR = cardiac magnetic resonance; PET = positron emission tomography; LGE = late gadolinium enhancement

Long-term impact of myocardial inflammation on quantitative myocardial perfusion-a descriptive PET/MR myocarditis study

PURPOSE Whether myocardial inflammation causes long-term sequelae potentially affecting myocardial blood flow (MBF) is unknown. We aimed to assess the effect of myocardial inflammation on quantitative MBF parameters, as assessed by 13N-ammonia positron emission tomography myocardial perfusion imaging (PET-MPI) late after myocarditis. METHODS Fifty patients with a history of myocarditis underwent cardiac magnetic resonance (CMR) imaging at diagnosis and PET/MR imaging at follow-up at least 6 months later. Segmental MBF, myocardial flow reserve (MFR), and 13N-ammonia washout were obtained from PET, and segments with reduced 13N-ammonia retention, resembling scar, were recorded. Based on CMR, segments were classified as remote (n = 469), healed (inflammation at baseline but no late gadolinium enhancement [LGE] at follow-up, n = 118), and scarred (LGE at follow-up, n = 72). Additionally, apparently healed segments but with scar at PET were classified as PET discordant (n = 18). RESULTS Compared to remote segments, healed segments showed higher stress MBF (2.71 mL*min$^{-1}$*g$^{-1}$ [IQR 2.18-3.08] vs. 2.20 mL*min$^{-1}$*g$^{-1}$ [1.75-2.68], p < 0.0001), MFR (3.78 [2.83-4.79] vs. 3.36 [2.60-4.03], p < 0.0001), and washout (rest 0.24/min [0.18-0.31] and stress 0.53/min [0.40-0.67] vs. 0.22/min [0.16-0.27] and 0.46/min [0.32-0.63], p = 0.010 and p = 0.021, respectively). While PET discordant segments did not differ from healed segments regarding MBF and MFR, washout was higher by ~ 30% (p < 0.014). Finally, 10 (20%) patients were diagnosed by PET-MPI as presenting with a myocardial scar but without a corresponding LGE. CONCLUSION In patients with a history of myocarditis, quantitative measurements of myocardial perfusion as obtained from PET-MPI remain altered in areas initially affected by inflammation. CMR = cardiac magnetic resonance; PET = positron emission tomography; LGE = late gadolinium enhancement

Curing cats with Feline Infectious Peritonitis with an oral multi-component drug containing GS-441524

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn$^{®}$ in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn$^{®}$. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn$^{®}$ displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn$^{®}$ containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species

Effect of Duration and Intermittency of Rifampin on Tuberculosis Treatment Outcomes: A Systematic Review and Meta-Analysis

In a systematic review of randomized controlled trials on tuberculosis treatment, Dick Menzies and colleagues find shorter courses of rifampin to be associated with poorer treatment outcomes