J. Cinatl

An influenza A H1N1 virus revival – pandemic H1N1/09 virus

In April 2009, a novel H1N1 influenza A virus, the so-called pandemic H1N1/09 virus (former designations include swine influenza, novel influenza, swine-origin influenza A [H1N1] virus [S-OIV], Mexican flu, North American Flu) was identified in Mexico. The virus has since spread throughout the world and caused an influenza pandemic as defined by the criteria of the World Health Organization. This represents the first influenza A virus pandemic since the emergence of H3N2 (‘‘Hong Kong’’ Flu) in 1968. Vaccine production has started, and vaccines are expected to become available during the course of 2009. Although the pandemic H1N1/09 virus originates from the triple-reassortant swine influenza (H1) virus circulating in North American pigs, it is not epidemic in pigs. Although the H1N1/09 virus pandemic is currently mild, concerns remain that it may become more aggressive during spreading. The distribution of proper information to the public on the status of the H1N1/09 virus pandemic will be important to achieve a broad awareness of the potential risks and the optimum code of behavior during the pandemic. Here, the features of pandemic H1N1/09 virus are discussed within the framework of knowledge gained from previous influenza A virus pandemics.

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.

Preparation, characterization and cytotoxicity of methylmethacrylate copolymer nanoparticles with a permanent positive surface charge

Methylmethacrylate copolymer nanoparticles containing different cationic comonomers such as N-trimethylammoniumethylmethacrylate (TMAEMC), N-dimethylammoniumethylmethacrylate (DMAEMC), N-trimethylammoniumpropylmethylacrylamide (MAPTAC) or the anionic comonomer sulfopropylmethacrylate (SPM), respectively, were prepared by free radical polymerization. Particle size was determined by photon correlation spectroscopy (PCS), transmission and scanning electron microscopy (TEM, SEM), and surface charge by microelectrophoresis. Pure poly(methylmethacrylate) nanoparticles served as control. Depending on the method, mean diameters of permanently positively-charged nanoparticles MMA-TMAEMC and MMA-MAPTAC were 243 or 207 nm (PCS), 161 or 201 nm (TEM), and 158 or 197 nm (SEM), respectively. Zeta potential examined in demineralized water or NaCl solution was +63.4 or +32.1 mV for MMA-TMAEMC nanoparticles and +49.2 or +32.0 mV for MMA-MAPTAC nanoparticles, respectively. Cytotoxicity of nanoparticles was determined by MTT assay in three different cell cultures including human foreskin fibroblasts (HFF) and two monkey kidney cell lines MA-104 and Vero. Cell viability profiles of TMAEMC and MAPTAC containing nanoparticles were different, showing IC(50) values for MMA-TMAEMC nanoparticles of 189.6+/-11.4 µg/ml (MA-104), 110.9+/-3.1 µg/ml (Vero) and 27.2+/-4.0 µg/ml (HFF). Cell viability at maximum concentration of 500 µg/ml MMA-MAPTAC nanoparticles was 98.3% (Vero), 85.7% (MA-104), or 94.0% (HFF), respectively.

Prions and Orthopedic Surgery

Abstract.Prions are a novel class of infectious agents that cause subacute encephalopathy in man and animals as human Creutzfeldt-Jakob disease (CJD), sheep scrapie and bovine spongiform encephalopathy (BSE). Previously, prions were shown to be transmitted by neuro- and ophthalmosurgical measures and by application of brain-derived therapeutic hormones. Recently, prions have been detected in blood specimens of experimentally infected monkeys indicating a principal threat to transfusion medicine, furthermore in human or bovine materials used in reconstitutive surgery. In this article the risk of prion transmission from the surgeon to the patient or vice versa during (orthopedic) surgery is reevaluated including the issues of blood transfusion. This is accomplished based on recent epidemiologic findings and biometric calculations on the spread of prions in animals and humans as well as in terms of experimental data on artificially contaminated medical materials and devices. The overall risk of prion transmission in orthopedic surgery is considered very low if adequately prepared and sterilized materials and devices are used.

Efficacy of various disinfectants against SARS coronavirus.

Summary The recent severe acute respiratory syndrome (SARS) epidemic in Asia and Northern America led to broad use of various types of disinfectant in order to control the public spread of the highly contagious virus. However, only limited data were available to demonstrate their efficacy against SARS coronavirus (SARS-CoV). We therefore investigated eight disinfectants for their activity against SARS-CoV according to prEN 14476. Four hand rubs were tested at 30s (Sterillium, based on 45% iso-propanol, 30% n-propanol and 0.2% mecetronium etilsulphate; Sterillium Rub, based on 80% ethanol; Sterillium Gel, based on 85% ethanol; Sterillium Virugard, based on 95% ethanol). Three surface disinfectants were investigated at 0.5% for 30min and 60min (Mikrobac forte, based on benzalkonium chloride and laurylamine; Kohrsolin FF, based on benzalkonium chloride, glutaraldehyde and didecyldimonium chloride; Dismozon pur, based on magnesium monoperphthalate), and one instrument disinfectant was investigated at 4% for 15min, 3% for 30min and 2% for 60min [Korsolex basic, based on glutaraldehyde and (ethylenedioxy)dimethanol]. Three types of organic load were used: 0.3% albumin, 10% fetal calf serum, and 0.3% albumin with 0.3% sheep erythrocytes. Virus titres were determined by a quantitative test (endpoint titration) in 96-well microtitre plates. With all tested preparations, SARS-CoV was inactivated to below the limit of detection (reduction factor mostly ≥4), regardless of the type of organic load. In summary, SARS-CoV can be inactivated quite easily with many commonly used disinfectants.

In vitro inhibition of human cytomegalovirus replication by desferrioxamine

Desferrioxamine (DFO) is commonly used in therapy as a chelator of ferric ion in disorders of iron overload. We found that DFO inhibits human cytomegalovirus (HCMV) replication in infected cultures of human foreskin fibroblasts (HFF) at concentrations that have been achieved in humans with no significant adverse effects. The concentrations of DFO required for 50 and 90% reduction in the production of a HCMV-late antigen ranged for several HCMV strains from 3.1 to 4.9 microM and from 14.2 to 17.3 microM, respectively. DFO concentration of 60 microM had no significant effect on the viability of HFF cells. Inhibitory effects of DFO on HCMV replication were completely prevented by co-incubation with stoichiometric amounts of Fe3+.

Valproic acid inhibits adhesion of vincristine- and cisplatin-resistant neuroblastoma tumour cells to endothelium

Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). In pursuit of alternative treatments for chemoresistant tumour cells, we tested the response of multidrug-resistant SKNSH and of vincristine (VCR)-, doxorubicin (DOX)-, or cisplatin (CDDP)-resistant UKF-NB-2, UKF-NB-3 or UKF-NB-6 NB tumour cell lines to valproic acid (VPA), a differentiation inducer currently in clinical trials. Drug resistance caused elevated NB adhesion (UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-2CDDP, UKF-NB-3VCR, UKF-NB-3CDDP, UKF-NB-6VCR, UKF-NB-6CDDP) to an endothelial cell monolayer, accompanied by downregulation of the adhesion receptor neural cell adhesion molecule (NCAM). Based on the UKF-NB-3 model, N-myc proteins were enhanced in UKF-NB-3VCR and UKF-NB-3CDDP, compared to the drug naïve controls. p73 was diminished, whereas the p73 isoform deltaNp73 was upregulated in UKF-NB-3VCR and UKF-NB-3CDDP. Valproic acid blocked adhesion of UKF-NB-3VCR and UKF-NB-3CDDP, but not of UKF-NB-3DOX, and induced the upregulation of NCAM surface expression, NCAM protein content and NCAM coding mRNA. Valproic acid diminished N-myc and enhanced p73 protein level, coupled with downregulation of deltaNp73 in UKF-NB-3VCR and UKF-NB-3CDDP. Valproic acid also reverted enhanced adhesion properties of drug-resistant UKF-NB-2, UKF-NB-6 and SKNSH cells, and therefore may provide an alternative approach to the treatment of drug-resistant NB by blocking invasive processes.

Cytomegalovirus infection blocks apoptosis in cancer cells.

Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death is a major reason for failure of anticancer chemotherapy. HCMV infection interferes with both the intrinsic and extrinsic cellular apoptosis pathways. HCMV promotes cell survival signaling influencing the tumor suppressor p53 and its relative p73, and stimulates the antiapoptotic Ras/Raf/MEK/Erk- and PI-3K-signaling pathways. Antiapoptotic effects mediated by HCMV are inhibited by antiviral treatment in cell culture. Therefore, a better understanding of the influence of HCMV infection on tumor cell apoptosis might translate into improved anti-cancer therapy.

Proinflammatory Potential of Cytomegalovirus Infection

We observed the effects of antiviral therapy on CMV and/or oxidative-stress-induced stimulation of proinflammatory molecules including interleukin-8 (IL-8), melanoma growth stimulatory activity-α (GRO-α) and intercellular adhesion molecule 1 (ICAM-1) using human foreskin fibroblasts. Ganciclovir, foscarnet or cidofovir completely suppressed virus replication, as demonstrated by CMV late (L) antigen production. These drugs did not influence CMV immediate-early (IE) antigen expression and had no effects on CMV-induced cellular changes in IL-8, GRO-α and ICAM-1 levels. Phosphorothioate oligonucleotide (ISIS 2922) suppressed both CMV IE and L antigen by 99%. ISIS 2922 completely suppressed CMV-induced upregulation of both chemokines and ICAM-1. Induction of oxidative stress by H2O2 upregulated IL-8 expression. Oxidative stress and CMV infection showed synergistic effects on IL-8 expression. ISIS 2922 only partially inhibited the upregulation of IL-8 in infected cells treated with H2O2, whereas cotreatment with ISIS 2922 and antioxidants inhibited the upregulation almost completely. The results showed that inhibition of CMV IE expression alone or in combination with antioxidants is promising for the treatment of CMV disease.

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents.

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.