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.

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.

Chemie Ingenieur TechnikVolume 78, Issue 9 p. 1382-1383 VortragFree Access Komponenten aus Mikroalgen mit Aktivitäten gegen humanpathogene Viren T. König, T. König tanja.koenig@bvt.cbi.uni-erlangen.de Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorS. Thulke, S. Thulke Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorI. Naumann, I. Naumann Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorW. Siegert Prof., W. Siegert Prof. Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorR. Buchholz Prof., R. Buchholz Prof. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorC. Walter Dr., C. Walter Dr. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this author T. König, T. König tanja.koenig@bvt.cbi.uni-erlangen.de Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorS. Thulke, S. Thulke Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorI. Naumann, I. Naumann Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorW. Siegert Prof., W. Siegert Prof. Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorR. Buchholz Prof., R. Buchholz Prof. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorC. Walter Dr., C. Walter Dr. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this author First published: 06 September 2006 https://doi.org/10.1002/cite.200650194Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 C. Walter, T. Steinau, N. Gerbsch, R. Buchholz, Biomol. Eng. 2003, 20 (4–6), 261. 2 S. Rechter et al., Jahrestagung der Gesellschaft für Virologie, München 2006. Citing Literature Volume78, Issue9Special Issue: GVC/DECHEMA-Jahrestagungen 2006 mit 24. DECHEMA-Jahrestagung der BiotechnologenSeptember, 2006Pages 1382-1383 ReferencesRelatedInformation

Natural substances offer interesting pharmacological perspectives for antiviral drug development in regard to broad-spectrum antiviral properties and novel modes of action. In this study we analyzed polysaccharide fractions isolated from Arthrospira platensis. Fractions containing intracellular or extracellular spirulan-like molecules showed a pronounced antiviral activity in the absence of cytotoxic effects. Using specific assays for the quantification of viral replication in vitro, these substances exhibited strong inhibition of human cytomegalovirus, herpes simplex virus type 1, human herpesvirus type 6 and human immunodeficiency virus type 1, while only weak or no inhibition was noted for Epstein-Barr virus and influenza A virus. Considering herpesviruses, antiviral effects were most pronounced when the cells were preincubated with the substances prior to the addition of virus, indicating that antiviral action may be primarily targeted to virus entry. However, an inspection of the inhibition of human cytomegalovirus protein synthesis clearly demonstrated that intracellular steps also contributed to the antiviral effect. In the case of human immunodeficiency virus, inhibition occurred at a stage later than viral entry. Thus, spirulan-like substances possess a marked antiherpesviral and anti-HIVactivity based on different modes of action. Further development of these substances might yield novel candidates of broad-spectrum antiviral drugs.

The clinical significance of human herpesvirus (HHV-6) infections after allogeneic stem cell transplantation (SCT) remains controversial. We analysed cryoconserved plasma samples from 82 patients after allogeneic SCT by quantitative polymerase chain reaction for HHV-6 variants A and B. Platelet engraftment was delayed in patients with HHV-6B infections but not with HHV-6A infections detected before day +28. In multivariate analysis early HHV-6B infections and the type of conditioning were associated with platelet engraftment. In conclusion, the two variants of HHV-6 should be studied separately; early infections with HHV-6B may contribute to delayed platelet engraftment after allogeneic SCT.

Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

Objective: quantitative evaluation of viruria (DNA-uria) and hematuria in patients with SCT and analyze both relationship clinically.

To elucidate the role of human herpesvirus 6 (HHV-6) in hematopoiesis, the influence of HHV-6A and HHV-6B on the in vitro expansion and differentiation of cord blood (CB) progenitor cells was investigated in liquid culture. Nonfractionated CB mononuclear cells (CB-MNC) or MACS-enriched CD34+ CB cells were seeded in liquid culture under conditions allowing maximal expansion of nucleated cells. Cells were either incubated with HHV-6A- or HHV-6B-containing cell culture supernatants or a virus-free control. After 7, 14, and 21 days, cells were analyzed for growth by cell count, for differentiation by flow cytometry, and for HHV-6 infection by antigen detection or PCR. Expansion of CB-MNC was significantly inhibited by HHV-6A and HHV-6B for a period of 3 weeks, including reduced proportions of CD34+ and CD33+ cells in HHV-6-treated cultures on day 7. In contrast, when starting with enriched CD34+ cells, the expansion was only affected by HHV-6A. Inhibition of CD34 and CD33 cell development was less pronounced in these cultures compared to CB-MNC cultures. However, HHV-6 antigen and DNA was detectable in these cultures. In conclusion, although HHV-6A inhibited expansion of CD34 progenitor cells, HHV-6B inhibited growth of immature CB cells only in interaction with nonfractionated CB-MNC.

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and β-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that “Classical” reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.

We recently presented a real-time duplex PCR method to determine the amount of human cells in chimeric mice. Although this assay was suitable for the quantitative detection of 1% of human cells in the background of murine cells in tissue, blood and bone marrow of chimeric mice, there is legitimate interest in detecting even lower amounts of human cells in these animals reliably. Therefore, we developed a new assay that reliably detects as few as 5 human cells in 100,000 mouse cells (0.005%). The assay specifically amplifies a human and a murine endogeneous retroviral sequence in two separate reactions. Based on the same primers, the assay can be performed either with 5' nuclease probes (TaqMan assay) or with a pair of hybridization probes (LightCycler assay), enabling the use of various real-time PCR instruments. The use of immunodeficient mice in hematological studies has dramatically increased during the last years. One possible approach to quantify chimerism is based on specific detection of human DNA in chimeric tissue. Published PCR methods are either highly sensitive or allow reliable real-time quantification; however, sensitivity is usually reduced at the expense of quantification. For the investigation of putative bone marrow engrafting cell types, kinetic studies of early engraftment events or the colonization of individual mouse organs with human cells, the combination of both, high sensitivity and reliable quantification, is required. Therefore, we established a new real-time PCR based assay, which combines i) all advantages of real-time PCR, including quantification, reduced contamination risk and specificity, with ii) an extremely high sensitivity. The assay is based on separate amplification of the human specific endogenous retroviral sequence ERV-3 and the mouse specific endogenous retroviral sequence ERV-L pol. For the detection of human ERV-3 a recently published TaqMan assay was modified and suitable hybridization probes were designed, whereas the mouse ERV-L assay was a completely new development. Both assays were optimized to be run under identical reaction conditions, permitting a simultaneous analysis of human and murine cells. TaqMan PCR was performed in a Perkin Elmer 7700 Sequence Detection System in 96-well microtiter plates in a final volume of 25 μL including 2.5 μL 10x PCR reaction buffer, 4.5 mM MgCl2, 1.0 mM dNTP, 1 U Platinum Taq DNA polymerase (all Invitrogen, Karlsruhe, Germany), 5 pmol of each primer, 3 pmol 5’ nuclease probe and 1 μM ROX (6-carboxy-X-rhodamine). 2 μL template DNA were amplified starting with 3 min at 94°C, followed by 45 cycles of 94°C for 30 s and at 58°C for 30 s. LightCycler PCR (Roche Molecular Diagnostics, Mannheim) was performed in a final reaction volume of 20 μL including 2 μL LightCycler-Fast Start DNA Master Hybridisation Probes mix (Roche, Mannheim, Germany), 5 mM Mg, 6 pmol of each primer, 2 pmol of each hybridization probe and 2 μL template DNA. Cycling conditions were 10 min at 95°C followed by 45 cycles of 8 s at 95°C, 8 s at 51°C and 8 s at 72°C. Table 1. shows primers and detection probes used for TaqMan and LightCycler PCR. To prove the sensitivity of the assay, human MCF-7 cells were diluted stepwise in murine P388 cells from 100% human cells to 0.005% human cells. DNA preparation was performed as described previously. Both assays were specific for each species. As shown

Since the incidence of cytomegalovirus (CMV) infections after hematopoietic stem cell transplantation (HSCT) may depend on the intensity of the pretreatment, we studied the incidence of CMV infections after reduced-intensity compared to myeloablative conditioning. A total of 82 patients with matched related or unrelated donors were prospectively monitored for CMV infections after HSCT by CMV-PCR techniques, CMV-antigenemia and clinical observation. A total of 45 patients received reduced-intensity conditioning consisting of fludarabine, busulfan and ATG and 37 patients received myeloablative conditioning. Leukocyte engraftment occurred after a median of 15 vs 18 days (P = 0.012) and platelet engraftment after 12 days vs 20 days (P = 0.001), respectively. Acute graft-versus-host disease (GVHD) grade II-IV was observed in 58 vs 54% patients (P = 0.737), respectively. The onset and peak values of CMV-antigenemia and DNAemia and the incidence of CMV infections did not differ statistically significantly between the two treatment groups. Multivariate analysis confirmed CMV seropositivity of the recipient (P = 0.035), acute GVHD II-IV (P = 0.001) but not the type of conditioning as significant risk factors for CMV-antigenemia. In conclusion, the kinetics of CMV-antigenemia and DNAemia and the incidence of CMV infections were not statistically different in patients who received HSCT after reduced-intensity conditioning with fludarabine, busulfan and ATG compared to myeloablative conditioning.

Cytomegalovirus (CMV) can compromise the life of patients who are immunosuppressed after organ transplantation or who have acquired immunodeficiency syndrome. CMV infections or reactivations can be successfully treated, provided treatment is instituted early in the course of developing disease. Current assays to detect the CMV pp65 antigen in peripheral blood leukocytes or CMV DNA by qualitative PCR of leukocytes or plasma can be slow and tedious (1)(2)(3). Antigen detection is limited by the availability of sufficient white blood cell numbers and is impossible to perform on leukopenic patients. In addition, blood for antigen detection cannot be stored for long periods because white blood cells need to be intact for staining procedures (4)(5). PCR techniques detect relatively stable double-stranded DNA present in the plasma or whole blood regardless of storage conditions or cell integrity, but qualitative PCR assays are not useful for follow-up studies of patients, and they do not allow the determination of viral load, which is useful for prognosis. In this study we compared quantitative real-time PCR (6)(7) with conventional pp65 antigen staining among 77 patients (1122 blood samples) surviving more than 30 days after stem cell transplantation (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue10/). In a retrospective study, results of the two assays were compared, and their sensitivity and specificity for predicting CMV disease were calculated. All patients were transplanted in the Virchow Klinikum (Berlin, Germany) between January 1998 and March 2001 (see Table 1 in the online Data supplement). Five patients developed CMV organ disease, which included pneumonitis and hepatitis (n = 1), hepatitis (n = 1), enteritis (n = 2), and retinitis (n = 1). Seven additional patients developed thrombo- and leukopenia not explained by other conditions. …