Dynamics of Influenza Virus Infection and Pathology

A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon (“cytokine storms”), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.Influenza A virus causes an acute respiratory disease in humans and other mammals; in humans, it is particularly important because of the rapidity with which epidemics develop, its widespread morbidity, and the seriousness of complications. Every year, an estimated 500,000 deaths worldwide, primarily of young children and the elderly, are attributed to seasonal influenza virus infections (49). Influenza pandemics may occur when an influenza virus with new surface proteins emerges, against which the majority of the population has no preexisting immunity. Both the emergence of H5N1 virus (34) and the current H1N1 virus pandemic (43) underline the importance of understanding the dynamics of infection and disease. A key question is, what regulates virus abundance in an individual host, causing the characteristic rapid decline in virus shedding following its initial peak? The main contenders in primary influenza virus infection are depletion of susceptible target cells and the impact of the host''s innate immune response (2, 20).On infection, the influenza virus elicits an immune response, including a rapid innate response that is correlated with the observed decline in the virus load after the first 2 days of infection (1). The slower adaptive response, including both humoral and cell-mediated components, takes several days to consolidate but is important for complete virus clearance and establishment of protective immunity. During infection of an immunologically naïve host, the innate immune response is particularly important as the first line of defense against infection. The innate immune response is regulated by chemokines and cytokines, chemical messengers produced by virus-infected epithelial cells and leukocytes (23), and natural interferon-producing cells, such as plasmacytoid dendritic cells (13). Among the key cytokines induced by epithelial cells infected with influenza A virus are type I interferons (IFNs) (IFN-α/β) (23), which directly contribute to the antiviral effect on infected and neighboring cells (38).Like other viruses, influenza A viruses have evolved strategies to limit the induction of innate immune responses (38). The NS1 protein plays a dominant role, and without it, the virus is unable to grow well or to cause pathology in an immunocompetent host (14). NS1 is multifunctional and counteracts both the induction of IFN expression and the function of IFN-activated antiviral effectors via multiple mechanisms (12, 17). Individual strains of influenza A virus possess these activities to various degrees (15, 21, 22, 26), and accordingly, NS1 has been implicated as a virulence factor (3, 17). A striking effect of the failure to control the innate response to virus infection is seen as a “cytokine storm,” which causes severe pathology (8).While there is an extensive literature on modeling influenza virus spread at the population level, the individual-host scale has received much less attention (2, 4, 5, 18, 19, 20, 27, 28). In a recent important paper, Baccam et al. modeled the kinetics of influenza A virus (2). The innate dynamics were included in the form of an IFN response that delayed and reduced virus production but did not prevent it; thus, the infection was resolved primarily through near-total depletion of epithelial cells. Their model was fitted to virus titers from human volunteers exposed to H1N1 influenza virus, but no data were available on the innate immune response or epithelial cell pathology. This has been a general difficulty in developing and validating more refined within-host models; there is a lack of detailed biological data from natural host systems, in particular, measures of immune kinetics and patterns of cellular depletion.The model presented here explicitly includes the ability of IFN to induce a fully antiviral state in order to explore the relative regulatory role of innate immunity and target cell depletion. Data from experimental infections of immunologically naïve horses with an equine influenza virus (36) allowed us to calibrate our model, not only to viral kinetics, but also to IFN dynamics and cell depletion in the context of infection of a naïve natural mammalian host. With our fitted model, we then investigate modulation of the immune response.     

Mechanism of the antimicrobial drug trimethoprim revisited.

We tested the hypothesis that the mechanism of action of the antifolate drug trimethoprim is through accumulation of bacterial dihydrofolate resulting in depletion of tetrahydrofolate coenzymes required for purine and pyrimidine biosynthesis. The folate pool of a strain of Escherichia coli (NCIMB 8879) was prelabeled with the folate biosynthetic precursor [(3)H]-p-aminobenzoic acid before treatment with trimethoprim. Folates in untreated E. coli were present as tetrahydrofolate coenzymes. In trimethoprim-treated cells, however, a rapid transient accumulation of dihydrofolate occurred, followed by complete conversion of all forms of folate to cleaved catabolites (pteridines and para-aminobenzoylglutamate) and the stable nonreduced form of the vitamin, folic acid. Both para-aminobenzoylglutamate and folic acid were present in the cell in the form of polyglutamates. Removal of trimethoprim resulted in the reconversion of the accumulated folic acid to tetrahydrofolate cofactors for subsequent participation in the one-carbon cycle. Whereas irreversible catabolism is probably bactericidal, conversion to folic acid may constitute a bacteriostatic mechanism since, as we show, folic acid can be used by the bacteria and proliferation is resumed once trimethoprim is removed. Thus, the clinical effectiveness of this important drug may depend on the extent to which the processes of either catabolism or folic acid production occur in different bacteria or during different therapeutic regimes.     

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.     

Genome-wide methylation and expression differences in HPV(+) and HPV(?) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(−) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(−) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(−) cell lines. Differentially methylated loci between HPV(+) and HPV(−) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(−) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(−) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(−) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.Key words: epigenetics, human papillomavirus, HNSCC, DNA methylation, squamous cell carcinoma, gene expression, microarrays, illumina infinium humanmethylation27 beadarray     

A novel system to quantify intestinal lipid digestion and transport

The zebrafish larva is a powerful tool for the study of dietary triglyceride (TG) digestion and how fatty acids (FA) derived from dietary lipids are absorbed, metabolized and distributed to the body. While fluorescent FA analogues have enabled visualization of FA metabolism, methods for specifically assaying TG digestion are badly needed. Here we present a novel High Performance Liquid Chromatography (HPLC) method that quantitatively differentiates TG and phospholipid (PL) molecules with one or two fluorescent FA analogues. We show how this tool may be used to discriminate between undigested and digested TG or phosphatidylcholine (PC), and also the products of TG or PC that have been digested, absorbed and re-synthesized into new lipid molecules. Using this approach, we explored the dietary requirement of zebrafish larvae for phospholipids. Here we demonstrate that dietary TG is digested and absorbed in the intestinal epithelium, but without dietary PC, TG accumulates and is not transported out of the enterocytes. Consequently, intestinal ER stress increases and the ingested lipid is not available support the energy and metabolic needs of other tissues. In TG diets with PC, TG is readily transported from the intestine and subsequently metabolized.     

Anticipating endoplasmic reticulum stress. A novel early response before pathogenesis-related gene induction

When it is attacked by a pathogen, a plant produces a range of defense-related proteins. Many of these are synthesized by the rough endoplasmic reticulum (RER) to be secreted from the cell or deposited in vacuoles. Genes encoding endoplasmic reticulum (ER)-resident chaperones, such as the lumenal binding protein (BiP), are also induced under these conditions. Here, we show that BiP induction occurs systemically throughout the plant. Furthermore, this induction occurs rapidly and precedes expression of genes encoding pathogenesis-related (PR) proteins. The underlying signal transduction pathway was shown to be independent of the signaling molecule salicylic acid and the unfolded protein response pathway. In addition, BiP induction was independent of PR gene induction. Overproduction of BiP alone was not sufficient to cause induction of PR gene expression; however, limiting the amount of BiP in the ER lumen via superimposed ER stress inhibited the induction of PR gene expression. We propose that the induction of BiP expression during plant-pathogen interactions is required as an early response to support PR protein synthesis on the RER and that a novel signal transduction pathway exists to trigger this rapid response.