Imaging Inflammation - From Whole Body Imaging to Cellular Resolution

Imaging techniques have evolved impressively lately, allowing whole new concepts like multimodal imaging, personal medicine, theranostic therapies, and molecular imaging to increase general awareness of possiblities of imaging to medicine field. Here, we have collected the selected (3D) imaging modalities and evaluated the recent findings on preclinical and clinical inflammation imaging. The focus has been on the feasibility of imaging to aid in inflammation precision medicine, and the key challenges and opportunities of the imaging modalities are presented. Some examples of the current usage in clinics/close to clinics have been brought out as an example. This review evaluates the future prospects of the imaging technologies for clinical applications in precision medicine from the pre-clinical development point of view

Highly Pathogenic H5N1 Influenza Viruses Carry Virulence Determinants beyond the Polybasic Hemagglutinin Cleavage Site

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05poly). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HAR65) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HATG05poly). In vitro, TG05poly and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05poly, TG05-HAR65, and R65-HATG05poly are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05poly led to temporary non-lethal disease in all animals, the reassortant TG05-HAR65 caused death in 3 of 10 animals. Furthermore, the reassortant R65-HATG05poly displayed the highest lethality as 8 of 10 chickens died, resembling “natural” HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins

Molekulare Mechanismen der Adaptation sowie Impfstudien zur Bekämpfung von aviären Influenza-A-Viren

Wildvögel stellen die natürlichen Wirte und das Hauptreservoir für Influenza-A-Viren dar. Einige Influenza-Stämme konnten sich zudem an verschiedene Säugetierarten wie Mensch, Schwein oder Pferd anpassen. Die molekularen Mechanismen der Adaptation von Influenza-A-Viren an einen neuen Wirt sind komplex. Sie werden u. a. auf eine modifizierte Interaktion viraler Proteine mit Wirtszellproteinen zurückgeführt, die in Verbindung mit dem Auftreten von Punktmutationen in viralen Proteinen, vor allem den Polymeraseproteinen, steht und zu einer optimierten Replikation von Influenza-A-Viren im neuen Wirt führen kann. Im Rahmen dieser Doktorarbeit wurden molekulare Werkzeuge entwickelt, die der Identifizierung wirtsspezifischer Interaktionspartner der viralen Ribonukleoprotein-Komplexe (vRNP) dienen können. Dazu wurden mittels reverser Genetik rekombinante Influenza-A-Viren unterschiedlichen Wirtsspektrums mit einem Strep-tag als Markierung am C-Terminus der Polymerase-Untereinheit PB2 generiert. Zu den verwendeten Viren zählten das humane A/HongKong/1/68 (H3N2), die beiden speziesübergreifenden Viren A/swan/Germany/R65/06 (H5N1) (‚R65‘) sowie A/seal/Massachusetts/1/80 (H7N7) und das aviäre A/duck/Ukraine/1/63 (H3N8). Durch Immunfluoreszenz- und Western-Blot-Analysen wurde die stabile Expression des Strep-PB2-Fusionsproteins in infizierten Zellen bestätigt. Es wurde zudem gezeigt, dass die markierten Viren auf Säuger- und Vogelzellen vergleichbar mit den entsprechenden unmarkierten Viren replizieren. Anhand des Strep-getaggten PB2-Proteins des R65-Virus wurden erfolgreich virale RNP-Komple xe aus infizierten Säuger- und Vogel-Zellen mittels Affinitätschromatographie aufgereinigt und deren vier Protein-Bestandteile PB2, PB1, PA und NP durch MALDI-tof-Massenspektrometrie identifiziert. Die Palette getaggter Viren bildet die Grundlage für weiterführende Studien zur Untersuchung Virus-Wirt-spezifischer Wechselwirkungen, die für den Wirtswechsel und die Adaptation von Influenza-A-Viren entscheidend sein können. Die Entstehung des pandemischen Influenza-Virus A/HongKong/1/68 (H3N2) (‚Hk68‘) geht auf ein Reassortment zwischen dem zuvor zirkulierenden humanen H2N2-Virus und einem aviären H3-Stamm zurück. Hierbei wurden die Segmente des humanen Virus, die für das Rezeptor-bindende Protein Haemagglutinin (HA) und die Polymerase-Untereinheit PB1 kodieren, gegen die entsprechenden Segmente des aviären H3-Virus ausgetauscht. Bei einem Sequenzvergleich zwischen dem Hk68-PB1 und dem PB1 des dem unbekannten aviären Donor nahestehenden Isolates A/duck/Ukraine/1/63 (H3N8) (‚dUk‘) wurden lediglich sechs Unterschiede in der Aminosäuresequenz identifiziert, die möglicherweise Folge der Adaptation des Hk68-Virus an den humanen Wirt sind. Nach dem Einfügen der einzelnen Mutationen in das dUk-PB1 wurden homologe RNP-Komplexe (PB2, PB1 und PA sowie NP von Hk68) und heterologe RNP-Komplexe (PB2, PA, NP von Hk68 und PB1 bzw. PB1-Punktmutante von dUk) in transfizierten Säugerzellen rekonstituiert. Mit Hil fe eines Luciferase-Reportertests konnte gezeigt werden, dass der heterologe Hk68/dUk-PB1-Komplex im Vergleich zum homologen Hk68-Komplex eine um 50% erniedrigte Polymerase-Aktivität aufweist. Dieser negative Effekt konnte durch das Einfügen der Mutation PB1 I12V in das dUk-PB1, aber nicht durch eine der anderen fünf Punktmutationen, vollständig aufgehoben werden. Folglich könnte es sich bei dieser Mutation um eine adaptive Mutation handeln. Untersuchungen an in vitro rekonstituierten RNP-Komplexen anderer Viren konnten diese Theorie unterstützen. Wachstumskinetiken des homologen Hk68- und dUk-Virus sowie von ihnen abgeleiteter PB1-Reassortanten und PB1-Mutanten deuteten ebenfalls auf einen Einfluss von Aminosäureposition 12 im PB1-Protein auf die Virusreplikation hin. Mit Hilfe eines ELISAs durchgeführte Bindungsstudien zwischen den PA-Proteinen verschiedener Influenza-A-Viren und PB1-Peptiden mit Valin oder Isoleucin an Position 12 legten zudem eine Zu- bzw. Abnahme der Af finität zwischen PA und PB1 als Ursache für die veränderte Polymeraseaktivität nahe. Hochpathogene aviäre Influenza-A-Viren (HPAIV) vom Subtyp H5 oder H7 verursachen enorme wirtschaftliche Schäden und stellen eine potentielle Bedrohung für den Menschen dar. Die Entwicklung effektiver Impfstoffe ist deshalb in vielfacher Hinsicht sinnvoll. In der vorliegenden Arbeit wurde mittels reverser Genetik eine Elastase-abhängige Mutante des HPAIV A/swan/Germany/R65/06 (H5N1), genannt R65-E, als potentielle lebend-attenuierte Vakzine erzeugt. Dazu wurde die polybasische Spaltstelle im HA des hochpathogenen Virus gegen eine Spaltstelle für die in vivo kaum verfügbare Protease Elastase ersetzt. In vitro wurde mit Hilfe von Plaquetests, Wachstumskinetiken und Western-Blot-Analysen die strikte Abhängigkeit der R65-E-Replikation und der R65-E-HA-Spaltung von Elastase nachgewiesen. Im Gegensatz zum R65-Wildtyp war die R65-E-Mutante in vivo aufgrund der Abwesenheit von Elastase auf einen Replikationszyklus beschränkt und somit hochgradig attenuiert. Insgesamt erwies sich die R65-E-Mutante im Huhn jedoch als wenig immunogen. So kam es 7 Tage nach okulonasaler Infektion von Eintagsküken lediglich zu einer schwachen zellulären Immunantwort basierend auf CD8+ zytotoxischen T-Zellen in der Milz. Eine Antikörper-Antwort wurde nach o kulonasaler oder in ovo Infektion nur bei jeweils einem von zehn bzw. einem von sieben Tieren induziert. Das Vorhandensein H5-spezifischer Antikörper korrelierte hierbei mit einem Schutz der Tiere gegen eine Belastungsinfektion mit dem homologen HPAIV R65. Gleichermaßen ging die Abwesenheit H5-spezifischer Antikörper bei den übrigen Versuchstieren mit einem letalen Verlauf der homologen R65-Belastungsinfektion einher. Ein partieller Schutz gegen eine heterosubtypische Belastungsinfektion mit dem HPAIV R65-H9R66mutR65 sowie eine reduzierte Virusausscheidung bei einigen Tieren der Boostergruppe, die drei Wochen nach okulonasaler Infektion eine zweite Dosis R65-E erhalten hatten, deuteten auf eine R65-E-induzierte zellvermittelte Schutzwirkung hin. Es ist zu vermuten, dass die R65-E-Mutante in vivo überattenuiert war und aus diesem Grund keine protektive Immunabwehr induzieren konnte. R65-E eignet sich daher nicht als lebend-attenuierte Geflügelvakzine.Wild birds are the natural hosts and main reservoir for influenza A viruses of all known subtypes. However, some influenza viruses have established stable lineages in mammals. The molecular basis of host restriction and adaptation of influenza viruses is determined by the interaction of viral proteins with specific host cell factors and based on adaptive point mutations in the viral polymerase proteins enhancing viral replication in a species-specific manner. To identify host-specific interaction partners of viral ribonucleoprotein (vRNP) complexes, recombinant influenza A viruses with a Strep affinity tag at the C-terminus of the polymerase protein PB2 were generated by reverse genetics. For this, a human H3N2 strain, an avian H3N8 strain and two strains of subtype H5N1 and H7N7 infecting avian and mammalian hosts were used. The stable expression of Strep-tagged fusion protein in infected cells was confirmed by immunofluorescence and Western blot analysis. Replication of tagged mutants was shown to be comparable to that of the untagged recombinant parent viruses. Tagged vRNP complexes were purified from human and avian cell extracts by affinity chromatography. All vRNP protein components were successfully identified by MALDI-tof mass spectrometry. These experiments form the basis for ongoing studies to investigate virus-host-specific interactions that are important for host switch and adaptation of influenza A viruses. The pandemic virus A/Hong Kong/1/68 (H3N2) (Hk68) is a reassortant between the at that time circulating human H2N2 and an avian H3 strain which provided its HA and PB1 segments. The avian donor virus is supposed to have a common ancestor with reference strain A/duck/Ukraine/1/63 (H3N8) (dUk). The alignment of the PB1 segments revealed six amino acid substitutions suggesting relevance for adaptation of Hk68-PB1. To investigate their adaptive potential, these point mutations were introduced into dUk-PB1. Homologous (PB2, PB1, PA, NP from Hk68) and heterologous (PB2, PA, NP from Hk68 and PB1 or PB1 mutant from dUk) RNP complexes were reconstituted in vitro in transfected human cells and polymerase activity was determined by a luciferase reporter assay. dUk-PB1 reduced the polymerase activity of the heterologous Hk68 complex by 50%. This negative effect was abolished by insertion of PB1 I12V into dUk-PB1 but not by any of the other 5 point mutations. Therefore, PB1 I12V could be an adaptive mutation. This hypothesis was supported by comparing the polymerase activity of other influenza A viruses with either valin or isoleucin at PB1 position 12. Growth kinetics of homologous Hk68 and dUk virus, their reassortants and PB1 mutants also implied an influence of PB1 amino acid position 12 on viral replication. Binding studies of different PA proteins to PB1 peptides with either valin or isoleucin at position 12 also suggested an altered affinity between both proteins as a reason for the differing polymerase activity. Highly pathogenic avian influenza A viruses (HPAIV) of subtype H5 and H7 are a major economic burden and a threat to human health. The development of efficient vaccines against HPAIV is therefore highly desirable. In the present study, an elastase-dependent mutant of HPAIV A/swan/Germany/R65/06 (H5N1) was generated by reverse genetics (R65-E). The polybasic cleavage site of parental R65 was replaced by a cleavage site for elastase. The strict dependence of R65-E replication on the availability of elastase was confirmed in vitro by plaque assays, growth kinetics and western blot analysis. In contrast to HPAIV R65 and due to the absence of elastase, R65-E was highly attenuated in vivo. Unexpectedly, R65-E was not immunogenic in chicken. After oculonasally infecting 1-day-old chicks with R65-E only a slight cell-mediated immune response based on CD8+ cytotoxic T-lymphocytes was detected. Additionally, antibody was induced in only one of ten or seven chickens each after oculonasal or in ovo R65-E infection. Here, the presence of H5-specific antibodies correlated with protection against homologous HPAIV challenge. Partial protection against heterosubtypic challenge with HPAIV R65-H9R66mutR6 5 and reduced viral shedding in some animals immunized twice with R65-E was probably linked to a cell-mediated immune response. R65-E is very likely to be over-attenuated in vivo and thus did not induce a protective immune response. It is not a suitable live attenuated vaccine against HPAIV in chicken

The inhibitory effects of anacardic acid on hepatitis C virus life cycle.

Hepatitis C virus (HCV) is a small positive-sense single-stranded RNA virus that causes severe liver diseases. Current anti-HCV therapies involving direct-acting antivirals have significantly enhanced efficacy in comparison to traditional interferon and ribavirin combination. However, further improvement is needed to eradicate HCV. Anacardic acid (AnA) is a phytochemical compound that can inhibit the activity of various cellular enzymes including histone acetyltransferases (HATs). In this study, we investigated the effects of AnA on different phases of HCV life cycle. Our data showed that AnA can inhibit HCV entry, replication, translation, and virion secretion in a dose-dependent manner with no measurable effects on cell viability. In addition, we showed that two HAT inhibitors and knocking down HAT (PCAF) by RNAi can reduce HCV replication, suggesting a mechanism of AnA's inhibitory effects on HCV. Elucidation of the AnA-mediated inhibitory mechanism should facilitate the development of new drug candidates for HCV infection

Effect of injected dexamethasone on relative cytokine mRNA expression in bronchoalveolar lavage fluid in horses with mild asthma

Abstract Background Mild equine asthma is a common inflammatory airway disease of the horse. The primary treatment of mild equine asthma is corticosteroids. The purpose of this study was to investigate the effects of injected dexamethasone on relative IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-17, IL-23, IFN-γ, Eotaxin-2 and TNF-α mRNA expression in bronchoalveolar lavage (BAL) fluid in healthy Thoroughbred horses (n = 6), and those with mild equine asthma (n = 7). Results Horses with mild equine asthma had a significantly greater bronchoalveolar lavage mast cell percentage than healthy horses both before and after treatment. Mild equine asthma was associated with a 4.95-fold up-regulation of IL-17 (p = 0.026) and a 2.54-fold down-regulation of IL-10 (p = 0.049) compared to healthy horses. TNF-α was down-regulated in response to dexamethasone treatment in both healthy horses (3.03-fold, p = 0.023) and those with mild equine asthma (1.75-fold, p = 0.023). IL-5 was also down-regulated in horses with mild asthma (2.17-fold, p = 0.048). Conclusions Horses with mild equine asthma have a lower concentration of IL-10 in BAL fluid than healthy controls which concurs with human asthmatics. The marked up-regulation of IL-17 in horses with mild asthma suggests these horses had a true tendency of “allergic” airway inflammation in response to environmental allergens. Dexamethasone administration exerted anti-inflammatory effects associated with down-regulation of TNF-α in all horses, and decreased levels of IL-5 mRNA expression in horses with mild equine asthma. The inhibition of the Th-2 response, without any alterations to the airway cytology, indicates that maintained exposure to environmental allergens perpetuates airway inflammation

Pure Award Final Report

Epstein-Barr virus (EBV) has infected more 90% of the world’s population, and is the cause of 2% of all neoplasms globally. In pigs, a closely related gammaherpes virus was identified called Porcine Lymphotrophic Herpesvirus 3 (PLHV3), that also causes lymphoproliferative disorders that resemble those caused by EBV. The purpose of this research is to generate a high titer viral stock of PLHV3 by shifting the viral life cycle from latency to lytic replication using baculovirus expression systems (BEVS) in latently infected cell lines. Infected lymphoblastic cell lines (LCL) will be infected with baculovirus vector carrying two PLHV3 immediate early genes that are crucial for lytic reactivation, BZLF1 and BRLF1. These immediate early genes will become expressed after the infection to produce recombinant proteins in high quantity, thus allowing viral reactivation. BZLF1, BRLF1 and an appropriate mammalian promoter will be cloned into the vector using PCR cloning. Acquiring a high titer viral stock will allow the generation of a chimeric PLHV3-EBV virus that could be used to establish a porcine model for studying EBV

Histone acetyltransferase (HAT) inhibitors and knockdown suppress HCV replication.

<p>HuH-7-HCV-2a J6/JFH-1(p7-rLuc2A) replicon cells were incubated with DMSO or different concentrations of p300i (<b>A</b>) or HATIIi (<b>B</b>) for 48 h. <i>Renilla</i> luciferase activity was measured and normalized to total protein amount. Statistical differences are indicated as * if <i>p</i> ≤ 0.05, *** if <i>p</i> ≤ 0.001, or <i>NS</i> for not significant. <b>(C)</b>. HuH-7-HCV-2a J6/JFH-1(p7-rLuc2A) replicon cells were transfected with non-silencing control or PCAF RNAi. At 24 h after transfection, <i>renilla</i> luciferase activity was measured and normalized to total protein amount. Statistical differences are indicated as * if <i>p</i> ≤ 0.05. <b>(D)</b>. To determine the transcript level of PCAF, RNA was extracted from HuH-7-HCV-2a J6/JFH-1(p7-rLuc2A) replicon cells 16 h after transfecting with non-silencing control or PCAF RNAi. After reverse transcription, real-time PCR experiment was performed using PCAF-specific primers. The transcript level of β-glucuronidase (GUSB) was determined in parallel and used for normalization. Statistical difference is indicated as * if <i>p</i> ≤ 0.05.</p

AnA inhibits HCV entry and release.

<p>(<b>A)</b>. HuH-7.5 cells were pretreated with DMSO and different concentrations of AnA for 12 h and infected with HCVpp-Luc for 4 h. followed by replacement by fresh medium. Luciferase activity was measured 48 h after infection and normalized to total protein amount. Statistical differences between samples are indicated as * if <i>p</i> ≤ 0.05, and ** if <i>p</i> ≤ 0.01. (<b>B)</b>. Viability of cells treated as in (<b>A)</b> was determined by MTT. <i>NS</i> = not significant. <b>(C)</b>. HuH-7-HCV-2a J6/JFH-1(p7-rLuc2A) cells were treated with DMSO and different concentrations of AnA for 5 h. After removal of inhibitors in the supernatant, HuH-7.5 cells were infected with the supernatant for 4 h followed by replacement of fresh medium. <i>Renilla</i> luciferase activity was measured 72 h after infection and normalized to total protein amount. Statistical differences are indicated as ** if <i>p</i> ≤ 0.01, or <i>NS</i> for not significant. <b>(D)</b>. Viability of cells treated as in (<b>C)</b> was determined by MTT. <i>NS</i> = not significant.</p