Quantitative expression analysis of HHV-6 cell receptor CD46 on cells of human cord blood, peripheral blood and G-CSF mobilised leukapheresis cells

Human herpesvirus-6 (HHV-6) can infect blood cells and thereby may inhibit hematopoietic stem and progenitor cell expansion and differentiation. In this context, it has been discussed if early progenitor cells can be infected by HHV-6. CD46 was identified as one possible cellular surface receptor for HHV-6. The study presented here had been done to get insight into the susceptibility of various leukocyte subpopulations to HHV-6 (including early hematopoietic progenitors) by determining the amount of CD46 molecules expressed on their surfaces. Human cord blood cells, peripheral blood cells and G-CSF mobilised progenitor cells were analysed by flow cytometry. CD46 molecule number per cell was determined and compared to calibration beads conjugated with known ratio of PE per bead. Highest CD46 expression was detected on B- lymphocytes, whereas T-lymphocytes only showed about half of the amount found on B cells. Hematopoietic progenitors also carried CD46 at intermediate levels. Unexpectedly, CD46 expression on progenitors from G-CSF mobilised leukapheresis products was approximately 20% of that found on comparable cells from untreated cord blood. In conclusion, hematopoietic progenitor cells express CD46 on their surface, thereby fulfilling a basic requirement for the susceptibility of HHV-6 infection

Screening and characterisation of anti-β-herpesvirus activities in substances from phototrophic, aquatic microorganisms.

Infektionen mit den β-Herpesviren humanes Cytomegalievirus (HCMV) und humanes Herpesvirus-6 (HHV-6) sind mit bis zu 90 % Seroposititvität aller Erwachsenen sehr weit verbreitet. Sie verlaufen in der Regel asymptomatisch, können jedoch bei immunsupprimierten Patienten zum Teil lebensbedrohliche Erkrankungen hervorrufen, z.B. Entzündungen der Lunge, des Gehirns oder der Netzhaut. Gegenwärtig werden verschiedene antiherpale Medikamente verwendet, die partiell ernsthafte Nebenwirkungen hervorrufen. Außerdem ist die Verbreitung resistenter Stämme zu beobachten. Daher existiert ein verstärktes Interesse an der Entwicklung neuartiger antiviraler Substanzen. Naturstoffe spielen dabei wegen ihrer großen Strukturvielfalt eine zentrale Rolle, wobei Verbindungen aus aquatischen, phototrophen Mikroorganismen (pMO, Mikroalgen und Cyanobakterien), ein bisher unzureichend genutztes Potential darstellen. Die vorliegende Arbeit war Teil eines Projektes mit dem Ziel, antivirale Substanzen aus pMO zu identifizieren und zu isolieren. Die Substanzen wurden aus monoseptischen pMO-Kulturen in sterilisierbaren Photobioreaktoren aufgearbeitet, ihre chemische Struktur bestimmt und im Rahmen der vorliegenden Arbeit hinsichtlich einer antiviralen Aktivität gegenüber HCMV getestet. Antiviral aktive Substanzen wurden ferner hinsichtlich ihrer Wirkungsweise untersucht. Zunächst wurden die Screeningmethoden zur Bestimmung der Zellvitalität von MRC-5-Fibroblasten (WST-1-Test) und der anti-HCMV-Aktivität (Peroxidasefärbung viraler Antigene) etabliert und mit dem bei HCMV-Infektionen klinisch eingesetzten Basenanalogon Ganciclovir (GCV) als Referenzsubstanz getestet. Im entwickelten Screeningsystem inhibiert es bei einer Konzentration von etwa 6 mg/ml die Zellvitalität um 50 % (CC50) und bei einer Konzentration von 14 µg/ml die HCMV-Replikation um 50 % (IC50). Aus fast 500 unterschiedlich gewonnenen Extrakten wurden die folgenden wirksamen Substanzen erfolgreich identifiziert und charakterisiert: 1. Das anionische Exopolysaccharid (EPS) aus dem Rhodophyten Porphyridium purpureum weist im Screeningsystem eine anti-HCMV-Aktivität von IC50 = 90 µg/ml auf (CC50 > 5 mg/ml). Die Substanz ist außerdem wirksam gegen HHV-6 und Vacciniavirus (VV). Damit werden erstmals Aktivitäten aus P. purpureum gegen -Herpesviren und VV beschrieben. 2. Für das intrazelluläre aPS TK V2 aus der Biomasse des Cyanobakteriums Arthrospira platensis wurde eine Wirksamkeit von IC50 = 39 µg/ml nachgewiesen (CC50 > 5 mg/ml). In diesem Extrakt ist wahrscheinlich das bereits beschriebene Ca-Spirulan enthalten. 3. Das davon zu unterscheidende, bisher unbekannte anionische Exopolysaccharid TK V3 aus A. platensis zeigt sogar eine 10fach höhere antivirale Aktivität (IC50 = 4 µg/ml, CC50 > 5 mg/ml) als TK V2 und ist darüber hinaus etwa 3,5fach besser als das herkömmlich verwendete GCV. Hervorzuheben ist weiterhin die 100 %ige Inhibierung der HCMV-Replikation mit lediglich 20 µg/ml TK V3, was mit 250 µg/ml EPS, 40 µg/ml TK V2 und ebenso mit 40 µg/ml GCV nicht erzielt werden konnte. Sowohl TK V2 als auch TK V3 besitzen außerdem eine Wirksamkeit gegen HHV-6, VV, das Herpes Simplex-Virus-1 und das Humane Immundefizienz-Virus-1. Anhand von in vitro Infektionskinetiken wurde für EPS, TK V2 und TK V3 weiterhin veranschaulicht, dass sie zu einer signifikanten Reduktion der viralen DNA- und RNA-Replikation führen. Das herausragende Charakteristikum der aPS ist die Verminderung der Virusreplikation durch Inhibierung der Adsorption und/oder Fusion der Viruspartikel an die Zellmembran. Im Gegensatz dazu hemmen herkömmliche Virostatika direkt die DNA-Replikation, zum Beispiel Basenanaloga. Die mikrobiellen aPS stellen somit eine Erfolg versprechende Basis für die Entwicklung von Virostatika mit neuartigem Wirkmechanismus und darüber hinaus mit einem sehr breiten antiviralen Wirkungsspektrum dar.Infections with human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6), both belonging to the β-subfamily of herpesviruses, are very widespread with a rate up to 90 % of seropositive adults. Usually, no infection related symptoms can be detected. Nevertheless, these infections can cause life-threatening diseases in immunosuppressed patients, for instance, pneumonitis, encephalitis or retinitis. Nowadays, several different therapeutic agents are used against herpesvirus infections but still this way of treatment can come up with severe partial side effects. Furthermore, the spreading out of resistant strains can be watched, and so there is a great need for and an increasing interest in the development of novel substances. As natural products exhibit a great structural diversity, they play a central role in this process. In fact, concerning antiviral drug development, there should have been paid more attention to phototrophic microorganisms providing many different metabolic substances. All data presented here were achieved by a project work aiming at the isolation and chemical characterisation of anti-herpetic drugs produced by phototrophic microorganisms (pMO, micro algae and cyanobacteria). Therefore, metabolically synthesised sulphated polysaccharides and sulpholipides were selectively extracted from pMO-culture and their chemical structures were determined. Also screening and characterisation of their antiviral activity were performed. Substances positively tested for antiviral action were further analysed in view of their mode of action. Initially, screening methods to ensure cytotoxicity against MRC-5-fibroblasts (WST-1 test) and anti-HCMV activity (peroxidase staining of viral antigens) were established. The therapeutically exerted base analogue ganciclovir (GCV) was used as reference substance. It inhibits cellular vitality by 50 % (CC50) at a concentration of about 6 mg/ml and leads to 50 % inhibition of viral replication (IC50) at a concentration of 14 µg/ml. After having screened nearly 500 extracts, the following agents were successfully identified and characterised: 1. The anionic exopolysaccharide EPS of rhodophyte Porphyridium purpureum exhibits a low cytotoxicity (CC50 > 5 mg/ml) and a significant anti-HCMV activity (IC50 = 90 µg/ml). EPS is also active against HHV-6 and vaccinia virus (VV). So this is the first report about P. purpureum showing activities against -herpesviruses and VV. 2. Furthermore, the anionic polysaccharide TK V2 was isolated from the biomass of the cyanobacterium Arthrospira platensis exhibiting an anti-HCMV activity of IC50 = 39 µg/ml. TK V2 presumably contains the already known Ca-Spirulan. 3. In contrast to TK V2, the structurally differing anionic exopolysaccharide TK V3, also isolated from Arthrospira platensis, demonstrated a 10fold higher antiviral activity (IC50 = 4 µg/ml). Compared to clinically used GCV, TK V3 is 3.5fold more active. It is to emphasise, that using the screening system only 20 g/ml TK V3 completely inhibited HCMV-replication, whereas 250 µg/ml EPS, 40 µg/ml TK V2 or 40 µg/ml GCV did not achieve 100 % inhibition of HCMV-replication. TK V2 and TK V3 possess low cytotoxicities (CC50 > 5 mg/ml) and are also active against HHV-6, VV, HSV-1 and human immunodeficiency virus-1 (HIV-1). Performing in vitro infection kinetics, anionic polysaccharides EPS, TK V2 and TK V3 were shown to reduce viral DNA- and RNA-replication. Compared to antiherpal therapeutical agents, these agents were demonstrated to act by inhibiting the adsorption of viral particles to the cellular membrane. Because of the different mode of action of clinically used agents, which directly inhibit the viral DNA-replication, this has to be considered as an outstanding attribute. Due their broad antiviral activity and their novel mode of action, the identified anionic polysaccharides constitute a fundamental potential for the development of antiviral agents

Anionic Polysaccharides From Phototrophic Microorganisms Exhibit Antiviral Activities to Vaccinia Virus

The aim of the present study was to characterise anti-vaccinia virus activities of anionic exopolysaccharides TK V3 isolated from cyanobacterium Arthrospira platensis and an exopolysaccharide isolated from the rhodophyt Porphyridium purpureum, respectively. These substances have previously been shown to be active against other enveloped viruses. We determined the in vitro inhibition of GFP-expressing vaccinia virus replication of 50 % at a concentration of 0.65 µg/ ml for EPS and of 0.78 µg/ml for TK V3. Substances also had an antiviral effect against ectromelia virus which is a most distinct orthopoxvirus genetically and the causative agent of mousepox. Anti-vaccinia virus and anti-ectromelia virus activities were demonstrated to increase with decreasing multiplicity of infection. Furthermore, non-toxic reduction of vaccinia virus in ovo replication was shown. Using time-of-addition assays, inhibition of viral entry by polyanionic substances was verified. EPS and TK V3 derived from phototrophic microorganisms represent novel anti-orthopoxvirus substances

Common Protein Biomarkers Assessed by Reverse Phase Protein Arrays Show Considerable Intratumoral Heterogeneity in Breast Cancer Tissues

<div><p>Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2–5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22–43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range18–38%) or between different tumor zones (CV 24%, range 17–38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18–34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29–98%) and lymph node metastases (CV 65%, range 40–146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.</p> </div

Heterogeneity in the expression of 35 proteins assessed by reverse-phase-protein-arrays.

<p>There was considerable intratumoral heterogeneity in the expression of all 35 proteins analyzed, and even more pronounced variation amongst different patients.</p><p>Intratumoral, differences between individual samples of the same primary tumor or between individual lymph node metastases from the same patient.</p><p>CV, coefficient of variation given as percentage; Primary tumor, whole tumor including central, peripheral, and intermediate zone; LN, lymph node; Total, total case including all primary tumor and lymph node samples.</p><p>Phospho, all phosphorylated proteins combined; Non-phospho, all non-phosphorylated proteins combined;</p><p>Overall, all 35 proteins combined.</p