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Pathologic data indicate that human cytomegalovirus (HCMV) infection might be associated with the pathogenesis of several human malignancies. However, no definitive evidence of a causal link between HCMV infection and cancer dissemination has been established to date. This study describes the modulation of the invasive behavior of NCAM-expressing tumor cell lines by HCMV. Neuroblastoma (NB) cells, persistently infected with the HCMV strain AD169 (UKF-NB-4AD169 and MHH-NB-11AD169), were added to endothelial cell monolayers and adhesion and penetration kinetics were measured. The 140- and 180-kDa isoforms of the adhesion receptor NCAM were evaluated by flow cytometry, Western blot, and reverse transcriptionpolymerase chain reaction (RT-PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides or NCAM transfection. HCMV infection profoundly increased the number of adherent and penetrated NB, compared to controls. Surface expression of NCAM was significantly lower on UKF-NB-4AD169 and MHH-NB-11AD169, compared to mock-infected cells. Western-blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoform. An inverse correlation between NCAM expression and adhesion capacity of NB has been shown by antisense and transfection experiments. We conclude that HCMV infection leads to downregulation of NCAM receptors, which is associated with enhanced tumor cell invasiveness.
Measles virus (MeV) is an aerosol-borne and one of the most contagious pathogenic viruses known. Almost every MeV infection becomes clinically manifest and can lead to serious and even fatal complications, especially under conditions of malnutrition in developing countries, where still 115,000 to 160,000 patients die from measles every year. There is no specific antiviral treatment. In addition, MeV infections cause long-lasting memory B and T cell impairment, predisposing people susceptible to opportunistic infections for years. A rare, but fatal long-term consequence of measles is subacute sclerosing panencephalitis. Fifteen years ago (2001), WHO has launched a programme to eliminate measles by a worldwide vaccination strategy. This is promising, because MeV is a human-specific morbillivirus (i.e. without relevant animal reservoir), safe and potent vaccine viruses are sufficiently produced since decades for common application, and millions of vaccine doses have been used globally without any indications of safety and efficacy issues. Though the prevalence of wild-type MeV infection has decreased by >90 % in Europe, measles is still not eliminated and has even re-emerged with recurrent outbreaks in developed countries, in which effective vaccination programmes had been installed for decades. Here, we discuss the crucial factors for a worldwide elimination of MeV: (1) efficacy of current vaccines, (2) the extremely high contagiosity of MeV demanding a >95 % vaccination rate based on two doses to avoid primary vaccine failure as well as the installation of catch-up vaccination programmes to fill immunity gaps and to achieve herd immunity, (3) the implications of sporadic cases of secondary vaccine failure, (4) organisation, acceptance and drawbacks of modern vaccination campaigns, (5) waning public attention to measles, but increasing concerns from vaccine-associated adverse reactions in societies with high socio-economic standards and (6) clinical, epidemiological and virological surveillance by the use of modern laboratory diagnostics and reporting systems. By consequent implementation of carefully designed epidemiologic and prophylactic measures, it should be possible to eradicate MeV globally out of mankind, as the closely related morbillivirus of rinderpest could be successfully eliminated out of the cattle on a global scale.
The human immunodeficiency virus (HIV) had spread unrecognized in the human population as sexually transmitted disease and was finally identified by its disease AIDS in 1981. Even after the isolation of the causative agent in 1983, the burden and death rate of AIDS accelerated worldwide especially in young people despite the confection of new drugs capable to inhibit virus replication since 1997. However, at least in industrialised countries, this trend could be reversed by the introduction of combination therapy strategies. The design of new drugs is on going; besides the inhibition of the three enzymes of HIV for replication and maturation (reverse transcriptase, integrase and protease), further drugs inhibits fusion of viral and cellular membranes and virus maturation. On the other hand, viral diagnostics had been considerably improved since the emergence of HIV. There was a need to identify infected people correctly, to follow up the course of immune reconstitution of patients by measuring viral load and CD4 cells, and to analyse drug escape mutations leading to drug resistance. Both the development of drugs and the refined diagnostics have been transferred to the treatment of patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). This progress is not completed; there are beneficial aspects in the response of the scientific community to the HIV burden for the management of other viral diseases. These aspects are described in this contribution. Further aspects as handling a stigmatising disease, education of self-responsiveness within sexual relationships, and ways for confection of a protective vaccine are not covered.
Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.
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.
The human cytomegalovirus (HCMV) is suspected to increase tumour malignancy by infection of cancer and/or stroma cells (oncomodulation). So far, oncomodulatory mechanisms have been attributed to the presence of HCMV and direct action of its gene products on cancer cells. Here, we investigated whether the prolonged presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. The neuroblastoma cell line UKF-NB-4 was long-term (200 passages) infected with the HCMV strain Hi91 (UKF-NB-4Hi) before virus eradication using ganciclovir (UKF-NB-4HiGCV). Global gene expression profiling of UKF-NB-4, UKF-NB-4Hi and UKF-NB-4HiGCV cells and subsequent bioinformatic signal transduction pathway analysis revealed clear differences between UKF-NB-4 and UKF-NB-4Hi, as well as between UKF-NB-4 and UKF-NB-4HiGCV cells, but only minor differences between UKF-NB-4Hi and UKF-NB-4HiGCV cells. Investigation of the expression of a subset of five genes in different chronically HCMV-infected cell lines before and after virus eradication suggested that long-term HCMV infection reproducibly causes specific changes. Array comparative genomic hybridisation showed virtually the same genomic differences for the comparisons UKF-NB-4Hi/UKF-NB-4 and UKF-NB-4HiGCV/UKF-NB-4. UKF-NB-4Hi cells are characterised by an increased invasive potential compared with UKF-NB-4 cells. This phenotype was completely retained in UKF-NB-4HiGCV cells. Moreover, there was a substantial overlap in the signal transduction pathways that differed significantly between UKF-NB-4Hi/UKF-NB-4HiGCV and UKF-NB-4 cells and those differentially regulated between tumour tissues from neuroblastoma patients with favourable or poor outcome. In conclusion, we present the first experimental evidence that long-term HCMV infection can result in the selection of tumour cell populations with enhanced malignancy.
Oncolytic effects of a novel Influenza A virus expressing Interleukin-15 from the NS reading frame
(2012)
Oncolytic influenza A viruses with deleted NS1 gene (delNS1) replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15) coding sequence into the viral NS gene segment (delNS1-IL-15). DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1) infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected) melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.
Background: Europe was certified to be polio-free in 2002 by the WHO. However, wild polioviruses remain endemic in India, Pakistan, Afghanistan, and Nigeria, occasionally causing polio outbreaks, as in Tajikistan in 2010. Therefore, effective surveillance measures and vaccination campaigns remain important. To determine the poliovirus immune status of a German study population, we retrospectively evaluated the seroprevalence of neutralizing antibodies (NA) to the poliovirus types 1, 2 and 3 (PV1, 2, 3) in serum samples collected from 1,632 patients admitted the University Hospital of Frankfurt am Main, Germany, in 2001, 2005 and 2010.
Methods: Testing was done by using a standardized microneutralization assay.
Results: Level of immunity to PV1 ranged between 84.2% (95%CI: 80.3-87.5), 90.4% (88.3-92.3) and 87.5% (85.4-88.8) in 2001, 2005 and 2010. For PV2, we found 90.8% (87.5-90.6), 91.3% (89.3-93.1) and 89.8% (88.7-90.9), in the same period. Seroprevalence to PV3 was 76.6% (72.2-80.6), 69.8% (66.6-72.8) and 72.9% (67.8-77.5) in 2001 and 2005 and 2010, respectively. In 2005 and 2010 significant lower levels of immunity to PV3 in comparison to PV1 and 2 were observed. Since 2001, immunity to PV3 is gradually, but not significantly decreasing.
Conclusion: Immunity to PV3 is insufficient in our cohort. Due to increasing globalization and worldwide tourism, the danger of polio-outbreaks is not averted - even not in developed countries, such as Germany. Therefore, vaccination remains necessary.
Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFKappaB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.
Background In October 2007, the working group CEN/TC 216 of the European Committee for standardisation suggested that the Sabin oral poliovirus vaccine type 1 strain (LSc-2ab) presently used for virucidal tests should be replaced by another attenuated vaccine poliovirus type 1 strain, CHAT. Both strains were historically used as oral vaccines, but the Sabin type 1 strain was acknowledged to be more attenuated. In Germany, vaccination against poliomyelitis was introduced in 1962 using the oral polio vaccine (OPV) containing Sabin strain LSc-2ab. The vaccination schedule was changed from OPV to an inactivated polio vaccine (IPV) containing wild polio virus type 1 strain Mahoney in 1998. In the present study, we assessed potential differences in neutralising antibody titres to Sabin and CHAT in persons with a history of either OPV, IPV, or OPV with IPV booster. Methods Neutralisation poliovirus antibodies against CHAT and Sabin 1 were measured in sera of 41 adults vaccinated with OPV. Additionally, sera from 28 children less than 10 years of age and immunised with IPV only were analysed. The neutralisation assay against poliovirus was performed according to WHO guidelines. Results The neutralisation activity against CHAT in adults with a complete OPV vaccination series was significantly lower than against Sabin poliovirus type 1 strains (Wilcoxon signed-rank test P < 0.025). In eight sera, the antibody titres measured against CHAT were less than 8, although the titre against Sabin 1 varied between 8 and 64. Following IPV booster, anti-CHAT antibodies increased rapidly in sera of CHAT-negative adults with OPV history. Sera from children with IPV history neutralised CHAT and Sabin 1 strains equally. Conclusion The lack of neutralising antibodies against the CHAT strain in persons vaccinated with OPV might be associated with an increased risk of reinfection with the CHAT polio virus type 1, and this implies a putative risk of transmission of the virus to polio-free communities. We strongly suggest that laboratory workers who were immunised with OPV receive a booster vaccination with IPV before handling CHAT in the laboratory.