Roberto A. Diez

Cytokines in adenoviral disease in children: Association of interleukin-6, interleukin-8, and tumor necrosis factor alpha levels with clinical outcome ☆ ☆☆ ★ ★★

To explore the pathogenic mechanisms involved in adenovirus infection, we evaluated total levels of immunoglobulins, antiadenovirus antibodies, adenovirus-specific circulating immune complexes, and cytokines in serum samples obtained from 38 hospitalized children with adenovirus infection. According to their clinical findings and outcome, the infections were classified as follows: (1) moderate (group I, n = 10), (2) severe (group II, n = 12), and (3) fatal (group III, n = 16). About 60% of the children had elevated IgM levels. IgG-containing adenovirus-specific circulating immune complexes were initially detected in 7 of 16 group III patients, 4 of whom had low serum levels of the third component of complement. A decrease in initial antiadenovirus IgG antibodies was observed in 3 of 10 patients in group III. Serum interleukin-6 was not detected in group I (none of 10), but was present in group II (7 of 12, p = 0.016) and group III (13 of 16, p < 0.001). Interleukin-8 was detected in all groups; values in fatal cases were significantly higher than in surviving children. Tumor necrosis factor alpha was not observed in group I (none of 10) and was uncommon in group II (2 of 12) but was frequently detected in group III (9 of 15, p = 0.01). Interleukin-1 and interleukin-4 were rarely detected in serum samples. Increased concentrations of interleukin-6, interleukin-8, and tumor necrosis factor alpha were associated with hypoperfusion, febrile peaks, tonic-clonic seizures, and septic shock. In 5 of 10 patients in groups II and III, autoantibodies specific for smooth muscle were found. Our findings indicate that high serum values for interleukin-6, interleukin-8, and tumor necrosis factor alpha are associated with severity of adenovirus infection.

Emergence of intratreatment resistance to oseltamivir in pandemic influenza A H1N1 2009 virus

BACKGROUND Pandemic influenza A H1N1 2009 virus presents a new challenge to health authorities and communities worldwide. In Argentina, the outbreak was at its peak by the end of June 2009, during the southern winter. A systematic analysis of samples from patients with pandemic H1N1 2009 studied in our laboratory (Virology Laboratory, Hospital de Niños R Gutiérrez, Buenos Aires, Argentina) detected two patients presenting intratreatment emergence of the H275Y neuraminidase mutation, which confers resistance to oseltamivir. METHODS Complementary DNAs, including the 275 codon, were obtained by reverse transcriptase PCR using viral RNAs extracted from nasopharyngeal or tracheal aspirates. Conventional sequencing and pyrosequencing were performed on each sample. In order to measure the virus susceptibility to oseltamivir, 50% inhibitory concentration determinations were performed by chemiluminescence. RESULTS Sequential samples of two paediatric patients under oseltamivir treatment were analysed. Pretreatment samples were composed of 100% oseltamivir-sensitive variants. In case 1, the oseltamivir-resistant variant was found 8 days after the beginning of treatment. In case 2, the viral population became resistant on the second day of treatment, with 83% of the viral population bearing the mutation and this reached 100% on the seventh day. CONCLUSIONS We describe the intratreatment emergence of oseltamivir resistance in two paediatric patients. Pyrosequencing allowed us to detect variant mixtures, showing the transition of the viral population from sensitive to resistant.

Recombinant human interferon-gamma inhibits adenovirus multiplication without modifying viral penetration.

We have recently reported that adenovirus replication is inhibited by human recombinant interferon-gamma, but not by recombinant interferon-alpha, in a dose-dependent manner. The aim of this study was to determine whether the antiviral effect of recombinant interferon-gamma could be linked to interferon-induced alteration at the membrane level, inhibiting either adenovirus penetration of or release from WISH cells. Adsorption and penetration were investigated with an 125I-labelled adenovirus binding assay. To test defective virus release, the presence of newly synthesized virus proteins in the cytoplasmic and nuclear compartments was investigated. Binding studies showed that interferons-gamma and -alpha did not modify adenovirus attachment and penetration. Interferon-gamma but not interferon-alpha inhibited hexon protein synthesis in the cytosol as well as its accumulation in the nuclear compartment. The synthesis of polypeptides III, IV and VI was also inhibited. In cells infected before interferon-gamma treatment, its addition could be delayed up to 2 h after the infection to produce an inhibition of virus yield greater than 1 log10 unit (90% inhibition). We conclude that interferon-gamma acts on an intracellular step before or at adenovirus protein synthesis, probably through a mechanism not shared with interferon-alpha.

Inhibitory effect of interferon-γ on adenovirus replication and late transcription

Abstract We have previously shown that human interferon-γ inhibited adenovirus multiplication in vitro in a dose-dependent fashion. This action was previous to capsid proteins synthesis and did not involve virus adsortion nor penetration. In this report we have analysed viral mRNA levels at early (7 hr post infection (p.i.)) or late (20 hr p.i.) times, as well as DNA replication in Wish cells pretreated with interferon-γ and infected with adenovirus 5. Controls included untreated cells as well as cells treated with interferon-α, to which adenovirus are reported to be resistant. Transcription of adenovirus regions E1, E4, L1 and L2 has been analysed by Northern blot. Adenovirus DNA replication was determined by DNA-DNA hybridization with total adenovirus 2 DNA. We have also searched for adenovirus E1A proteins by immunoblot with a specific monoclonal antibody. Although pretreatment of cells with either interferon-α or interferon-γ resulted in reduced amounts of E1 and E4 mRNA in the early phase of infection (7 hr p.i.), the near complete inhibition of viral DNA and late transcription was only achieved by interferon-γ. Immunoblot has shown the absence of the 48-kD E1A protein in cells pretreated with interferon-γ. The lack of this regulatory adenovirus protein may be involved in the inhibitory mechanism of interferon-γ on adenovirus.

Corticosteroids Modulate the Binding of Recombinant Interferons Alpha and Gamma in Namalva Cells

To investigate possible mechanisms of interaction between corticosteroids and interferons (IFNs), the specific binding of recombinant human IFNs alpha 2 and alpha in Namalva cells after 72 h culture with dexamethasone (10(-8) M to 10(-6) M) was evaluated. Exponentially growing cells were incubated with different concentrations of the radiolabelled IFNs, with or without an excess of unlabelled IFN. The parameters of the interaction between each IFN and its specific receptor were analyzed by the Scatchard method. In the dose range tested, dexamethasone induced a dose-dependent inhibition of Namalva cells growth, which reached about 35% at 10(-6) M. The specific binding of IFN-alpha 2 was decreased to a maximum of 40%, for dexamethasone concentrations greater than or equal to 10(-7) M. The decrease in binding induced by the corticoid was additive with the down-regulation induced by IFN-alpha 2 itself. On the contrary, the specific binding of IFN-alpha was increased by dexamethasone in a dose-dependent fashion within the tested range. The maximal increase in the number of sites per cell was about 60%, with a slight decrease in affinity. These results suggest that complex interactions might arise between corticosteroids and IFNs in the course of their clinical use.