BackgroundH5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals.Methodology/Principal FindingsWe demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH≤5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice.Conclusion/SignificanceOur finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

Recent reports indicate a broad spectrum of antileukemic activity for arsenic trioxide (As 2 O 3 ) due to its ability to induce apoptosis via intracellular production of reactive oxygen species (ROS). Despite its potent apoptotic mechanism, As 2 O 3 is not equally effective in all leukemic cells, which has prompted a search for agents enhancing As 2 O 3 efficacy. Recently, evidence has been gathered that the polyunsaturated fatty acid docosahexaenoic acid (DHA) may sensitize tumor cells to ROSinducing anticancer agents. The aim of our investigation was to evaluate whether DHA enhances As 2 O 3 -mediated apoptosis in As 2 O 3 -resistant HL-60 cells. While 1 M As 2 O 3 or 25 M DHA reduced cell viability to 85.8% ؎ 2.9% and 69.2% ؎ 3.6%, combined treatment with As 2 O 3 and DHA reduced viability to 13.0% ؎ 9.9% with a concomitant increase of apoptosis. Apoptotic cell death was preceded by collapse of the mitochondrial membrane potential, increased expression of proapoptotic B-cell lymphoma protein-2-associated X protein (Bax), and caspase-3 activation. Importantly, the combined effect of As 2 O 3 and DHA was associated with increased production of intracellular ROS and toxic lipid peroxidation products and was abolished by the antioxidant vitamin E or when oleic acid (a nonperoxidizable fatty acid) was used in place of DHA. Intracellular ROS and toxic lipid peroxidation products most likely constitute the key mediators contributing to the combined effect of As 2 O 3 and DHA. Our data provide the first evidence that DHA may help to extend the therapeutic spectrum of As 2 O 3 and suggest that the combination of As 2 O 3 and DHA could be more broadly applied in leukemia therapy.

Interleukin-10-deficient mice develop colitis and colorectal cancer similar to the inflammatory bowel disease associated cancer in humans. The aim of this study was to identify possible mutations of oncogenes and tumour suppressor genes involved in tumorigenesis in Interleukin-10 (IL-10)-deficient mice. Twenty colon carcinomas from IL-10-deficient mice were screened for mutations in the K-ras and p53 genes by 'cold' single-strand-conformation polymorphism. Immunohistochemical staining was performed to detect mutations in the proteins P53, APC and MSH2, and the transforming growth factor beta type II receptor. Microsatellite instability was analysed at eight chromosomal loci and plasma levels of transforming growth factor beta1 (TGF-beta1) were also measured. At 9 weeks, 14% of the animals developed colorectal cancer, and at 10-31 weeks the incidence of carcinoma was 65%. No mutations were detected in the analysed oncogene and tumour suppressor genes. Plasma TGF-beta1 levels in IL-10-deficient mice 10-31 weeks old were higher than in wild-type littermates e.g. 45.7 +/- 4.6 ng/ml versus 19.8 +/- 4.5 ng/ml (P<0.01). No alterations in K-ras, p53, APC: and Msh2 genes suggests that other genes are involved in the development of these tumours. Elevated TGF-beta1 plasma levels correspond to the high incidence of dysplasia and cancer. Normal expression of the TGF-beta II receptors hints at genetic alterations in other members of the TGF-beta receptor signal transduction pathway.

It has been shown that the polyunsaturated fatty acid docosahexaenoic acid (DHA) can sensitize various tumor cells to reactive oxygen species (ROS)-inducing anticancer agents. Recently, we demonstrated that DHA also enhances the apoptotic effect of clinically achievable concentrations (1-2 M) of arsenic trioxide (As 2 O 3 ) in several As 2 O 3 -resistant human leukemic cell lines via a ROS-dependent mechanism. The aim of the present study was to evaluate whether this combined effect of As 2 O 3 and DHA is also applicable to Several studies have shown that exogenous polyunsaturated fatty acids (PUFAs) may sensitize tumor cells to reactive oxygen species (ROS)-inducing anticancer drugs both in vitro and in vivo. [1][2][3][4][5][6][7] It has been shown that the susceptibility of PUFAs to lipid peroxidation is of pivotal importance for this drug-enhancing effect. 2,4,7 The ROS-generating anticancer drug arsenic trioxide (As 2 O 3 ) has been used for the treatment of several types of leukemia with remarkable clinical success. 8 -12 Although several mechanisms of action have been proposed for the antileukemic activity of As 2 O 3 , the generation and accumulation of ROS most likely constitutes the key mechanism responsible for its cytotoxic action. 13,14 However, clinically achievable concentrations (1-2 M) of As 2 O 3 are not effective in all tumor types. 15 Hence, investigations to enhance the effect of As 2 O 3 without increasing its concentration have been impelled. 16,17 Recently, we showed that the PUFA docosahexaenoic acid (DHA) enhances the apoptotic effect of clinically achievable concentrations (1 M) of As 2 O 3 in As 2 O 3 -resistant leukemia cells. 18 The combined effect of As 2 O 3 and DHA was due to the induction of apoptosis and associated with accumulation of intracellular ROS and increased production of toxic lipid peroxidation products. Importantly, the combined effect of As 2 O 3 and DHA was not restricted to one type of leukemia cells, but was effective in cells derived from several hematologic malignancies. As a consequence, we hypothesized that the combined effect of As 2 O 3 /DHA treatment may also be applicable to solid tumor cells that are known to be intrinsically less sensitive to As 2 O 3 . To elucidate whether a possible enhancing effect of DHA was PUFA-specific and to define the relationship between enhanced lipid peroxidation and increased As 2 O 3 toxicity, we also tested the effects of oleic acid (OA), a nonperoxidizable monounsaturated fatty acid, on As 2 O 3 -mediated apoptosis. Finally, we also determined the efficacy of As 2 O 3 and DHA in the presence of the lipophilic antioxidant vitamin E. MATERIAL AND METHODS ChemicalsNinety-nine percent pure cis-4,7,10,13,16,19-DHA and cis-9-OA, dl-␣-tocopherol (vitamin E) and As 2 O 3 (Sigma Chemical, St. Louis, MO) were prepared as stock solutions and dissolved to the respective working concentrations with complete culture medium before use as described previously. 18

Human endogenous retroviruses (HERVs) represent a cellular reservoir of potentially pathogenic retroviral genes. A growing body of evidence indicates that the activation of endogenous retroviral sequences might be involved in the transformation of melanocytes. In this study, we investigated the effects of ultraviolet radiation (UVR) on the expression of human endogenous retrovirus type K (HERV-K) in melanoma cells and non-melanoma cells in vitro. Solely in melanoma cell lines, irradiation with UVB (200 mJ/cm(2)) resulted in a significant transcriptional activation of the retroviral pol gene as well as in an enhanced expression of the retroviral envelope protein (env). In addition, UVB treatment induced the production of retroviral particles in the supernatants of melanoma cell lines. These data indicate that HERV-K expression can be activated by UVB irradiation and suggest an involvement of HERV-K in UVR-related melanoma pathogenesis.

Segment 8 of the influenza A virus codes for two proteins (NS1 and NS2/NEP) via splicing. Here, we developed a viral vector expressing a cytokine or chemokine instead of the interferon antagonist NS1. To achieve both the desired genetic stability and high transgene expression levels, NS2/NEP mRNA splicing efficacy had to be fine-tuned by modification of splicing elements. Expression levels of secreted foreign proteins could be further enhanced by fusing the N-terminal 13 amino acids of NS1 with an IgK-derived secretion signal peptide. Thus, the first start codon was used for translation initiation of both NS2/NEP and the foreign protein.Genetic engineering allowed the rational design of influenza A virus vaccine prototypes based on partial or complete deletions (4, 7, 30) of the viral interferon (IFN) antagonist nonstructural protein 1 (NS1) (7).A recently completed clinical phase I trial of a vaccine using the complete NS1 deletion (delNS1) virus confirmed its excellent safety profile for humans after a single intranasal application (31). These clinical data, as well as the fact that influenza viruses lack a DNA intermediate in their replication cycle, render the delNS1 virus attractive for further development as a viral expression vector.Several bicistronic strategies have been explored for the expression of foreign genes from the segmented genome of the influenza A virus. Those include internal promoters (13), minigenes (24), internal ribosomal entry sites (8), protease cleavage sites (11,14,22,28), and a stop-start translation reinitiation sequence (UAAUG) (10; for a review, see reference 16).Here, we developed a strategy to express cytokines or chemokines from the genome of the delNS1 virus. In our initial construct (pdelNS1-IL-2-spl-wt), the human interleukin-2 (IL-2) open reading frame (ORF) including the signal peptide preceded by the stop-start pentamer UAAUG was inserted between the splice donor and the splice acceptor site of the nonstructural protein (NS) segment (Fig. 1b). The NS1 protein is thereby truncated to the N-terminal 21 amino acids, rendering it nonfunctional. Mutations were introduced by standard molecular biology techniques (e.g., restriction enzyme digests, PCR mutagenesis, overlapping PCR) into a PolI promoter-driven plasmid coding for influenza virus A/PuertoRico/ 8/34 (PR8) segment 8 and confirmed by sequencing. We rescued the corresponding virus, delNS1-IL-2-spl-wt, by transfecting Vero cells with the plasmid pdelNS1-IL-2-spl-wt together with seven bidirectional plasmids (9) coding for the remaining segments of a Vero cell-adapted IVR-116 (20) strain.Surprisingly, reverse transcription-PCR (RT-PCR) (Fig. 2) and sequence analysis (data not shown) demonstrated that a major part of the introduced IL-2 sequence was already deleted from the virus after one passage in Vero cells, despite the fact that IL-2 had been stably expressed previously from the partial NS1 deletion mutant NS1-125 (10).We hypothesized that the introduction of a foreign sequence at this position of the NS segment mig...

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.

Recently we showed that the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) sensitizes arsenic trioxide (As2O3)-resistant tumor cells to a clinically achievable concentration (1 microM) of As2O3 via a reactive oxygen species (ROS)-dependent mechanism. The aim of the present study was to evaluate, whether this combined effect of As2O3 and DHA is also applicable to other PUFAs [i.e., eicospentaenoic acid (EPA), arachidonic acid (AA), and gamma-linolenic acid (GLA)]. Fourteen tumor cell lines were incubated with As2O3 (1 microM), PUFA (25-100 microM), or the combination thereof (+/- vitamin E). Cell viability (colorimetric), apoptosis (bivariate annexin V/propidium iodide staining, detection of hypodiploid DNA), and thiobarbituric acid reactive substances (TBARS) were evaluated. Twelve of 14 As2O3-resistant cell lines tested were resistant to PUFA monotherapy. However, combined treatment with As2O3 and either PUFA significantly reduced cell viability in a dose-dependent manner with AA being the most potent As2O3 enhancer. The combined cytotoxic effect of As2O3/AA treatment was due to induction of apoptosis, preceded by increased intracellular TBARS and was abolished by the antioxidant vitamin E. Importantly, the combined effect of As2O3 and AA was selectively toxic for malignant cells because no cytotoxic effect was observed in normal skin fibroblasts and human microvascular endothelial cells. In conclusion, our study shows that also other PUFAs than DHA-and in particular the omega-6-PUFA AA--can be used as effective modulators of tumor cell chemosensitivity to clinically achievable concentrations of As2O3. Enhanced lipid peroxidation most likely constitutes the key mechanism for the combined effect.