Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.     

A critical evaluation of network and pathway-based classifiers for outcome prediction in breast cancer

Recently, several classifiers that combine primary tumor data, like gene expression data, and secondary data sources, such as protein-protein interaction networks, have been proposed for predicting outcome in breast cancer. In these approaches, new composite features are typically constructed by aggregating the expression levels of several genes. The secondary data sources are employed to guide this aggregation. Although many studies claim that these approaches improve classification performance over single genes classifiers, the gain in performance is difficult to assess. This stems mainly from the fact that different breast cancer data sets and validation procedures are employed to assess the performance. Here we address these issues by employing a large cohort of six breast cancer data sets as benchmark set and by performing an unbiased evaluation of the classification accuracies of the different approaches. Contrary to previous claims, we find that composite feature classifiers do not outperform simple single genes classifiers. We investigate the effect of (1) the number of selected features; (2) the specific gene set from which features are selected; (3) the size of the training set and (4) the heterogeneity of the data set on the performance of composite feature and single genes classifiers. Strikingly, we find that randomization of secondary data sources, which destroys all biological information in these sources, does not result in a deterioration in performance of composite feature classifiers. Finally, we show that when a proper correction for gene set size is performed, the stability of single genes sets is similar to the stability of composite feature sets. Based on these results there is currently no reason to prefer prognostic classifiers based on composite features over single genes classifiers for predicting outcome in breast cancer.     

Toxoplasma gondii actively inhibits neuronal function in chronically infected mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii-infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca(2+)) imaging studies revealed that tachyzoites actively manipulated Ca(2+) signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca(2+) uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca(2+) stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host.     

A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

Background

Trypanosoma cruzi is the causative agent of Chagas'' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.

Methodology/Principal Findings

We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.

Conclusions/Significance

Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.     

Central B-cell tolerance: where selection begins

The development of an adaptive immune system based on the random generation of antigen receptors requires a stringent selection process that sifts through receptor specificities to remove those reacting with self-antigens. In the B-cell lineage, this selection process is first applied to IgM⁺ immature B cells. By using increasingly sophisticated mouse models, investigators have identified the central tolerance mechanisms that negatively select autoreactive immature B cells and prevent inclusion of their antigen receptors into the peripheral B-cell pool. Additional studies have uncovered mechanisms that promote the differentiation of nonautoreactive immature B cells and their positive selection into the peripheral B-cell population. These mechanisms of central selection are fundamental to the generation of a naïve B-cell repertoire that is largely devoid of self-reactivity while capable of reacting with any foreign insult.B-cell generation in the bone marrow of adult mammals occurs through a tightly controlled developmental process (Fig. 1). Productive rearrangement of immunoglobulin heavy (IgH) and light (IgL) chain gene segments in B lymphocyte precursor cells, in addition to the expression of Ig-α (CD79a) and Ig-β (CD79b), result in the generation and expression on the cell surface of a mature B-cell antigen receptor (BCR). Whereas the combination of Ig H and L chains determines the antigenic specificity of the newly formed BCR, their association with Ig-α and Ig-β allows transduction of a signal inside the cell that directs cell fate. Developing B cells first express a mature BCR on the cell surface in the form of IgM and as such are classified as immature B cells (Fig. 1) (Hardy et al. 1991; Pelanda et al. 1996). It is at the immature B-cell stage that the BCR is tested for the first time for reactivity against autoantigens. This test determines whether the immature B cell and the antibody it expresses on the surface will be selected into the peripheral B-cell repertoire. Central B-cell tolerance, in fact, refers to the process that negatively selects newly generated immature B cells that react with a self-antigen in the bone marrow environment. This is considered the first checkpoint of B-cell tolerance, and the results of this checkpoint are fundamental to the generation of a naïve repertoire that contains foreign reactive antibodies and is largely devoid of self-reactive specificities.Open in a separate windowFigure 1.Schematic representation of B-cell development and Ig loci in mice. Large pro-B cells initiate Ig gene rearrangement at the IgH locus. Expression of a H chain following a productive V_HD_HJ_H recombination event promotes the differentiation of large pre-B cells in which the expression of pre-BCR (H chain pairing with surrogate light chains) results in the clonal expansion of H chain-positive pre-B cells and the development of small pre-B cells. Expression of conventional L chains following productive rearrangements at the IgL chain loci in small pre-B cells promotes the development of a diverse population of IgM⁺ immature B cells, which then differentiate into IgM⁺IgD⁺ transitional B cells. The scheme of mouse Ig H, κ, and λ loci (not to scale) indicate the presence of V (white rectangles), D (black vertical lines), J (brown vertical lines; a dashed line indicates a nonfunctional element), and C (black rectangles; a gray rectangle indicates a nonfunctional element) gene segments. The scheme does not represent the number of V_H, D_H, and Vκ gene segments in the actual Ig loci.On passing this central checkpoint, immature B cells continue to differentiate into transitional and mature B cells before and after they travel to the spleen (Loder et al. 1999; Allman et al. 2001; Su and Rawlings 2002; Tarlinton et al. 2003). Analysis of the bone marrow early immature B-cell repertoire indicates that a staggering 50%–75% of these cells express BCRs that are specific for self-antigens, both in mice and humans (Grandien et al. 1994; Wardemann et al. 2003). Similar studies performed on cell populations at the other end of this central checkpoint, namely, transitional and naïve mature B cells in spleen and blood, show a much lower frequency (20%–40%) of cells expressing autoreactive antibodies (Grandien et al. 1994; Wardemann et al. 2003), demonstrating the stringency and limitation of this initial selection step. Moreover, individuals affected by autoimmune disease such as lupus erythematosus or rheumatoid arthritis bear many more autoreactive cells in their new emigrant and naïve B-cell populations (Samuels et al. 2005; Yurasov et al. 2005), indicating a defect in central (and/or peripheral) B-cell selection. Thus, it seems important that the development of autoreactive immature B cells be constrained to prevent the potential occurrence of autoimmunity. However, there are also reasons to believe that the high frequency of autoreactive specificities generated during primary Ig gene rearrangements may be necessary for the generation of the peripheral B-cell repertoire (Pelanda et al. 1997; Kohler et al. 2008). Indeed, a fraction of autoreactive immature B cells, those manifesting a low level of self-reactivity, do bypass the central checkpoint of tolerance and differentiate into mature B cells (Hayakawa et al. 2003; Wardemann et al. 2003; Wen et al. 2005). The inclusion of these weakly self-reactive B cells in the peripheral B-cell repertoire may allow recognition of a broader spectrum of foreign molecules, potentially decreasing the negative impact of infections, especially at early stages (Mouquet et al. 2010).What are the rules that govern the selection of immature B cells? Most studies of central tolerance have been conducted by following the selection of B cells expressing BCRs displaying well-defined reactivity for natural or synthetic self-antigens. This has been accomplished through the use of Ig transgenic mice in which developing B cells have been altered to carry prerearranged Ig H and L chain genes encoding antibodies of defined antigen specificity and reactivity. Here we review some of these studies, what we have learned from them, and open questions that still await answers.     

Identification of pluripotent and adult stem cell genes unrelated to cell cycle and associated with poor prognosis in multiple myeloma

Gene expression-based scores used to predict risk in cancer frequently include genes coding for DNA replication, repair or recombination. Using two independent cohorts of 206 and 345 previously-untreated patients with Multiple Myeloma (MM), we identified 50 cell cycle-unrelated genes overexpressed in multiple myeloma cells (MMCs) compared to normal human proliferating plasmablasts and non-proliferating bone marrow plasma cells and which have prognostic value for overall survival. Thirty-seven of these 50 myeloma genes (74%) were enriched in genes overexpressed in one of 3 normal human stem cell populations - pluripotent (18), hematopoietic (10) or mesenchymal stem cells (9) - and only three genes were enriched in one of 5 populations of differentiated cells (memory B lymphocytes, T lymphocytes, polymorphonuclear cells, monocytes, osteoclasts). These 37 genes shared by MMCs and adult or pluripotent stem cells were used to build a stem cell score ((SC)score), which proved to be strongly prognostic in the 2 independent cohorts of patients compared to other gene expression-based risk scores or usual clinical scores using multivariate Cox analysis. This finding highlights cell cycle-unrelated prognostic genes shared by myeloma cells and normal stem cells, whose products might be important for normal and malignant stem cell biology.     

An acenocoumarol dosing algorithm using clinical and pharmacogenetic data in spanish patients with thromboembolic disease

Appropriate dosing of coumarins is difficult to establish, due to significant inter-individual variability in the dose required to obtain stable anticoagulation. Several genetic and other clinical factors have been associated with the coumarins dose, and some pharmacogenetic-guided dosing algorithms for warfarin and acenocoumarol have been developed for mixed populations. We recruited 147 patients with thromboembolic disease who were on stable doses and with an international normalized ratio (INR) between 2 and 3. We ascertained the influence of clinical and genetic variables on the stable acenocoumarol dose by multiple linear regression analysis in a derivation cohort (DC; n = 117) and developed an algorithm for dosing that included clinical factors (age, body mass index and concomitant drugs) and genetic variations of VKORC1, CYP2C9, CYP4F2 and APOE. For purposes of comparison, a model including only clinical data was created. The clinical factors explained 22% of the dose variability, which increased to 60.6% when pharmacogenetic information was included (p<0.001); CYP4F2 and APOE variants explained 4.9% of this variability. The mean absolute error of the predicted acenocoumarol dose (mg/week) obtained with the pharmacogenetic algorithm was 3.63 vs. 5.08 mg/week with the clinical algorithm (95% CI: 0.88 to 2.04). In the testing cohort (n = 30), clinical factors explained a mere 7% of the dose variability, compared to 39% explained by the pharmacogenetic algorithm. Considering a more clinically relevant parameter, the pharmacogenetic algorithm correctly predicted the real stable dose in 59.8% of the cases (DC) vs. only 37.6% predicted by the clinical algorithm (95% CI: 10 to 35). Therefore the number of patients needed to genotype to avoid one over- or under-dosing was estimated to be 5.     

Insights from Characterizing Extinct Human Gut Microbiomes

In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (∼8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.     

Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.