Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae

A lethal synergism exists between influenza virus and Streptococcus pneumoniae, accounting for excess mortality during influenza epidemics. Using a model of viral-bacterial synergism, we assessed the role that the influenza virus neuraminidase (NA) has in priming mice for pneumococcal infection. Administration of the selective NA inhibitor oseltamivir improved survival, independent of viral replication and morbidity from influenza. Both pathologic examination of the lungs and live imaging of pneumonic lesions, using a bioluminescent pneumococcus, suggested that the effect of NA inhibition was to limit the extent of pneumococcal pneumonia during early infection. Adherence assays and immunohistochemical staining for sialic acids in lungs from infected mice demonstrated that the influenza virus NA potentiates development of pneumonia by stripping sialic acid from the lung, thus exposing receptors for pneumococcal adherence. Selective NA inhibitors may be useful clinically to interrupt this novel mechanism of synergism and to prevent excess mortality from secondary bacterial pneumonia.     

Seroprevalence of seasonal and pandemic influenza A viruses in domestic cats

Infection of domestic cats with pandemic H1N1 influenza virus has recently been documented. We conducted a seroprevalence survey and found that 17 of 78 (21.8%) cats sampled during the 2009–2010 influenza season had antibody titers ≥40 against the novel H1N1 strain by hemagglutinin-inhibition assay, compared to only 1 of 39 (2.6%) sampled in 2008 prior to emergence of the pandemic (p = 0.006). Seroprevalance of seasonal H1N1 (41.9%) and H3N2 (25.6%) viruses was similarly high. These data reflecting past infection of household cats raise the possibility that they may act as a vector of influenza transmission within households.     

FluBlok, a recombinant influenza vaccine

Protein Sciences Corp and UMN Pharma Inc are developing FluBlok, an injectable trivalent influenza vaccine formulation composed of recombinant influenza hemagglutinin (HA) proteins that match the HA from the three influenza isolates that currently circulate in humans (H1N1, H3N2 and B), for the potential prevention of influenza virus infection. Phase III clinical trials to compare FluBlok and a licensed trivalent influenza vaccine for immunogenicity, safety and efficacy in preventing naturally occurring influenza are ongoing.     

Pneumococcal meningitis in children: relationship of antibiotic resistance to clinical characteristics and outcomes.

BACKGROUND: The relationship of antibiotic susceptibility to clinical outcome in children with pneumococcal meningitis is uncertain. Previous studies have been limited by inclusion of relatively few patients infected with nonsusceptible pneumococci and inconsistent use of empiric vancomycin. METHODS: Medical records of 86 children with culture-confirmed pneumococcal meningitis at a single institution from October, 1991, to October, 1999, were retrospectively reviewed, and differences in presentation and outcome based on antibiotic susceptibility of pneumococcal isolates were assessed. RESULTS: Of 86 isolates 34 were nonsusceptible to penicillin (12 resistant). Of 60 isolates for which cefotaxime susceptibility data were available, 17 were nonsusceptible (12 resistant). Antibiotic susceptibility was not significantly associated with death, intensive care unit admission, mechanical ventilation, focal neurologic deficits, seizures, secondary fever, abnormal neuroimaging studies or hospital days. Children with penicillin-resistant isolates had significantly higher median blood leukocyte counts (24,100/microliter vs. 15,700/microliter, P = 0.03) and lower median CSF protein concentrations (85 mg/dl vs. 219 mg/dl, P = 0.04), were more likely to have a CSF glucose concentration of > or = 50 mg/dl (7 of 11 vs. 15 of 68, P = 0.009) and had lower rates of sensorineural hearing loss (1 of 8 vs. 25 of 40, P = 0.02) than children with isolates that were not resistant to penicillin. Children with cefotaxime-nonsusceptible isolates had an increased median duration of primary fever compared with those with nonsusceptible strains (6 days vs. 3.5 days, P = 0.02). CONCLUSIONS: In children with pneumococcal meningitis, penicillin resistance was associated with a reduced risk of hearing loss, while cefotaxime resistance was associated with a longer duration of fever. Other outcome measures were not significantly influenced by the antibiotic susceptibility of pneumococcal isolates.     

Isolation and characterization of vancomycin-tolerant Streptococcus pneumoniae from the cerebrospinal fluid of a patient who developed recrudescent meningitis

The emergence of tolerance to vancomycin has recently been reported in Streptococcus pneumoniae, the most common cause of bacterial meningitis. A vancomycin- and cephalosporin-tolerant strain of S. pneumoniae, the Tupelo strain, was isolated from the cerebrospinal fluid of a patient who then developed recrudescence of meningitis despite treatment with vancomycin and a third-generation cephalosporin. The Tupelo strain evidenced no lysis in the exponential or stationary phase of growth when exposed to vancomycin and only minimal loss of viability. Further characterization revealed normal autolysin expression, localization, and triggering by detergents, indicating that the defect leading to tolerance in the Tupelo strain is in the control pathway for triggering of autolysis. Because tolerance is a precursor phenotype to resistance and may lead to clinical failure of antibiotic therapy, these observations may have important implications for vancomycin use in infections caused by S. pneumoniae.     

Effect of antiviral treatment on the outcome of secondary bacterial pneumonia after influenza

Secondary bacterial pneumonia is an important cause of influenza-associated death. Although antibacterial therapy is standard, antiviral therapy has been ignored because viral infections usually resolve by the time bacterial pneumonia presents. In the present study, antiviral compounds were tested in a mouse model of secondary pneumococcal pneumonia after influenza. Treatment with oseltamivir improved survival in mice from 0% to 75%, even when therapy was delayed for up to 5 days after infection with influenza virus. In mice, treatment with rimantadine had no effect on survival. Treatment with ampicillin cleared infection but, in the absence of treatment with oseltamivir, did not improve survival. Pneumonia developed in only 7 of the 22 mice receiving oseltamivir, and subsequent treatment with ampicillin resulted in cure (100% survival). Treatment of the predisposing influenza-virus infection with inhibitors specific for the viral neuraminidase may improve the efficacy of antibiotics and increase survival in persons who are at high risk for complications and mortality during influenza.     

Insights into the interaction between influenza virus and pneumococcus

Bacterial infections following influenza are an important cause of morbidity and mortality worldwide. Based on the historical importance of pneumonia as a cause of death during pandemic influenza, the increasingly likely possibility that highly pathogenic avian influenza viruses will trigger the next worldwide pandemic underscores the need to understand the multiple mechanisms underlying the interaction between influenza virus and bacterial pathogens such as Streptococcus pneumoniae. There is ample evidence to support the historical view that influenza virus alters the lungs in a way that predisposes to adherence, invasion, and induction of disease by pneumococcus. Access to receptors is a key factor and may be facilitated by the virus through epithelial damage, by exposure or up-regulation of receptors, or by provoking the epithelial regeneration response to cytotoxic damage. More recent data indicate that alteration of the immune response by diminishing the ability of the host to clear pneumococcus or by amplification of the inflammatory cascade is another key factor. Identification and exploration of the underlying mechanisms responsible for this synergism will provide targets for prevention and treatment using drugs and vaccines.     

Live attenuated influenza vaccine is safe and immunogenic in immunocompromised ferrets

Patients undergoing chemotherapy for cancer are highly susceptible to influenza virus infection. Prevention of influenza virus infection is complicated in the immunocompromised host because of suboptimal responses to the trivalent inactivated influenza vaccine (TIV). A new, live attenuated influenza vaccine (LAIV; FluMist) may offer a more effective alternative to TIV, but the safety of this LAIV in immunocompromised patients must first be established. In the present study, FluMist was administered to ferrets immunocompromised by treatment with dexamethasone and cytarabine. Ferrets exhibited no signs or symptoms attributable to FluMist, and nasal clearance of LAIV strains from immunocompromised ferrets was similar to that from control ferrets. Serum antibody responses against the vaccinating strains were analyzed as a measure of vaccine efficacy. Antibody titers to all 3 vaccine strains in immunocompromised ferrets were similar to those seen in mock-treated control ferrets, as assessed by microneutralization assay. These findings support the potential use of this vaccine in immunocompromised humans.     

The age distribution of mortality due to influenza: pandemic and peri-pandemic

Background

The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab.

Methods

NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index.

Results

EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA.

Conclusions

EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.