Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion

Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 microm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.     

PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia.     

Uptake of L-lysine by a double mutant ofSaccharomyces cerevisiae

A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport. Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system. The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol. The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate). The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine. The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule. The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre. It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached. Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue.     

Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer

Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio‐ and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre‐treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease‐free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence.     

Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.     

Resistance to glyphosate in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> as result of pre-selective mutations

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (G^s) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (G^r) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10⁻⁷ to 3.6 × 10⁻⁷ mutants per cell per generation in two strains of M. aeruginosa; the frequency of the G^r allele ranged from 6.14 × 10⁻⁴ to 6.54 × 10⁻⁴. The G^r mutants are slightly elliptical in outline, whereas the G^s cells are spherical. Since G^r mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via G^r clone selection.     

Demographic history has influenced nucleotide diversity in European Pinus sylvestris populations

To infer the role of natural selection in shaping standing genetic diversity, it is necessary to assess the genomewide impact of demographic history on nucleotide diversity. In this study we analyzed sequence diversity of 16 nuclear loci in eight Pinus sylvestris populations. Populations were divided into four geographical groups on the basis of their current location and the geographical history of the region: northern Europe, central Europe, Spain, and Turkey. There were no among-group differences in the level of silent nucleotide diversity, which was approximately 0.005/bp in all groups. There was some evidence that linkage disequilibrium extended further in northern Europe than in central Europe: the estimates of the population recombination rate parameter, rho, were 0.0064 and 0.0294, respectively. The summary statistics of nucleotide diversity in central and northern European populations were compatible with an ancient bottleneck rather than the standard neutral model.     

A novel mutation in the mitochondrial tRNAAla gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA^Ala in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.