Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.     

The Evolution of Fungal Metabolic Pathways

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.     

A novel function of eIF2alpha kinases as inducers of the phosphoinositide-3 kinase signaling pathway

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.     

Interferons induce tyrosine phosphorylation of the eIF2alpha kinase PKR through activation of Jak1 and Tyk2

The interferon (IFN)-inducible, double-stranded RNA activated protein kinase (PKR) is a dual-specificity kinase, which has an essential role in the regulation of protein synthesis by phosphorylating the translation eukaryotic initiation factor 2 (eIF2). Here, we show the tyrosine (Tyr) phosphorylation of PKR in response to type I or type II IFNs. We show that PKR physically interacts with either Jak1 or Tyk2 in unstimulated cells and that these interactions are increased in IFN-treated cells. We also show that PKR acts as a substrate of activated Jaks, and is phosphorylated at Tyr 101 and Tyr 293 both in vitro and in vivo. Moreover, we provide strong evidence that both the induction of eIF2alpha phosphorylation and inhibition of protein synthesis by IFN are impaired in cells lacking Jak1 or Tyk2, which corresponds to a lack of induction of PKR tyrosine phosphorylation. We conclude that PKR tyrosine phosphorylation provides an important link between IFN signalling and translational control through the regulation of eIF2alpha phosphorylation.     

Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation

Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.     

Industry involvement and baseline assumptions of cost-effectiveness analyses: diagnostic accuracy of the Papanicolaou test

Background

Industry involvement has been associated with more favourable cost-effectiveness ratios in cost-effectiveness analyses, but the mechanisms for this association are unclear. We evaluated whether the assumed accuracy of the Papanicolaou (Pap) test was correlated with the features of cost-effectiveness analysis studies.

Methods

We searched PubMed (last updated April 2010) for cost-effectiveness analysis studies in which at least one strategy involved the Pap test for cervical cancer. We assessed the baseline assumed diagnostic sensitivity and specificity of the Pap test in each study and the association of these values with three levels of manufacturer involvement in the study.

Results

Among 88 analyzed cost-effectiveness analysis studies, the assumed sensitivity of the Pap test was lower in studies with manufacturer-affiliated authors, manufacturer funding or manufacturer-related competing interests versus studies without (mean sensitivity 60% v. 70%, p < 0.001). The assumed specificity of the Pap test was lower in cost-effectiveness analyses involving new screening tests (mean 93% v. 96%, p = 0.016). The assumed specificity did not differ between trials with manufacturer involvement versus those without (mean 95% v. 95%, p = 0.755).

Interpretation

The results of cost-effectiveness analyses may be affected by a downgrading of the assumed diagnostic accuracy of the standard Pap test against which newer tests or interventions are compared. New technology then seems to have more favourable results against a straw-man comparator.Given the substantial impact of cost-effectiveness analyses on public health policies, it is important to safeguard their robustness. However, most published cost-effectiveness analyses report favourable incremental cost-effectiveness ratios, and industry-funded analyses are more likely to report desirable ratios.¹ Some journals discourage cost-effectiveness analysis studies that have been conducted or funded by sponsors of the examined interventions.² Examples exist in which cost-effectiveness analysis studies on the same topic have reached different conclusions depending on who funded them.³ Yet the mechanism for such discrepancies in seemingly rigorous quantitative analyses is unclear.The process of cost-effectiveness analysis involves making assumptions about the main parameters of interest that enter calculations. Baseline values and plausible ranges are specified. When baseline assumptions vary across different cost-effectiveness analyses, results may diverge considerably. We evaluated the baseline assumptions of the diagnostic accuracy of a widely used and studied screening test,⁴ the Papanicolaou (Pap) test (i.e., conventional cervical cytology) in cost-effectiveness analysis studies. The diagnostic accuracy of the Pap test has been studied extensively,^5–7 and assumptions about its accuracy are pivotal for any cost-effectiveness analysis related to screening for and prevention of cervical cancer. Moreover, alternative approaches, such as DNA testing for human papilloma virus (HPV) or HPV vaccines, are quite expensive. New tests may get favourable cost-effectiveness ratios against the Pap test if the performance of the Pap test is assumed to be suboptimal.We therefore examined whether cost-effectiveness analysis studies involving screening for and prevention of cervical cancer assumed different diagnostic performance estimates for the Pap test and whether differences reflected sponsor-related biases.     

Role of guanylate binding protein‐1 in vascular defects associated with chronic inflammatory diseases

Rheumatic autoimmune disorders are characterized by a sustained pro‐inflammatory microenvironment associated with impaired function of endothelial progenitor cells (EPC) and concomitant vascular defects. Guanylate binding protein‐1 (GBP‐1) is a marker and intracellular regulator of the inhibition of proliferation, migration and invasion of endothelial cells induced by several pro‐inflammatory cytokines. In addition, GBP‐1 is actively secreted by endothelial cells. In this study, significantly increased levels of GBP‐1 were detected in the sera of patients with chronic inflammatory disorders. Accordingly we investigated the function of GBP‐1 in EPC. Interestingly, stable expression of GBP‐1 in T17b EPC induced premature differentiation of these cells, as indicated by a robust up‐regulation of both Flk‐1 and von Willebrand factor expression. In addition, GBP‐1 inhibited the proliferation and migration of EPC in vitro. We confirmed that GBP‐1 inhibited vessel‐directed migration of EPC at the tissue level using the rat arterio‐venous loop model as a novel quantitative in vivo migration assay. Overall, our findings indicate that GBP‐1 contributes to vascular dysfunction in chronic inflammatory diseases by inhibiting EPC angiogenic activity via the induction of premature EPC differentiation.     

Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities

Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3′-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.Fast detection, differentiation, and identification of bacteria are crucial tasks in clinical, food, and environmental microbiology. Cultivation-independent tools not only save time compared to cultivation-based techniques but also allow access to the difficult-to-cultivate part of a microbial community. Molecular detection methods are usually based on hybridization of oligonucleotide probes to signature sequences (phylogenetically informative regions) in the nucleic acids (RNA or DNA) of the target microorganisms. Verification of the hybridization event can be accomplished by detection of hybridized labeled probes in situ (e.g., fluorescence in situ hybridization [FISH]) or ex situ (dot blot hybridization). Combining two specific oligonucleotides in a PCR increases the sensitivity of specific detection, while real-time monitoring of the amplification product formed allows quantification of the original template (for a review, see reference 17). Multiple detection can be achieved by using more than one primer pair targeting several loci in multiplex PCR assays (for a review, see reference 32). However, the main disadvantages of FISH are its restricted capability for parallel analysis of several target groups in the same sample and limitations in probe design due to differences in accessibility of the probes to their target sites (3, 7). Moreover, detection of slowly growing bacteria with low ribosome contents requires labor-intensive techniques (30, 36). Multiplex PCR also has limitations for multiplexing and challenges for primer design (32).Recently, single-nucleotide primer extension (SNuPE) was proposed as a fast, semiquantitative multiplex detection tool for analyzing sequence variants. This method is frequently used for determination of single-nucleotide polymorphisms and benefits from the fidelity of dideoxynucleoside triphosphate (ddNTP) incorporation catalyzed by a DNA polymerase. When primer extension takes place on a solid support, the method is called minisequencing (35, 37), while a reaction in solution is referred to as SNuPE (34) or single-base extension (15). These methods were originally developed for routine medical diagnosis of genetic disorders (23, 35) or for use in forensic research (38). Different versions of the primer extension technique have also been used recently for fast identification and genotyping of microbial strains (9, 31). Recent studies showed that detection of a hybridization event via labeling of the hybridized primer in the extension reaction is possible. However, the use of this method as a detection tool in applied and environmental microbiology has not been fully exploited so far. Rudi and coworkers were the first workers who used a minisequencing approach with PCR products from environmental DNA to detect toxic cyanobacteria by labeling only one of the four ddNTPs used in the reaction (27). Multiplexing was accomplished by hybridizing the labeled products to complementary oligonucleotides in an array format. In combination with antibody-based chromogenic visualization, genetic profiles of cyanobacterial diversity (28), microbial communities in vegetable salads (25), and Listeria strains (26) were obtained. However, this approach is labor-intensive and time-consuming and requires specific equipment; furthermore, the primer is restricted to certain positions since only one terminator nucleotide is labeled.An alternative strategy for multiplexing in solution benefits from incorporation of four differently labeled ddNTPs and attachment of mobility modifiers to the different primers. Subsequent separation using capillary electrophoresis and laser-induced fluorescence detection results in a very fast assay that is easy to interpret. Determination of the incorporated nucleotide provides additional proof of the assay specificity or may even provide extra phylogenetic information. The first application of primer extension with four differently labeled ddNTPs in environmental microbiology was the use of this method by Wu and Liu (41) for multiplex detection of different Bacteroides spp. This study also addressed different methodological issues and aspects, such as the effects of noncomplementary tail length, annealing temperature, cycle number, and primer-to-template ratio on extension efficiency. In a previous study, Nikolausz et al. (19) reported development and application of a multiplex SNuPE assay for detection and typing of “Dehalococcoides” sp. sequences obtained from chloroethene-contaminated groundwater samples. However, there still has not been a systematic evaluation of factors that affect primer design and the discriminatory power of primer extension. Moreover, quantitative aspects of the method have not been thoroughly addressed so far.The present study focused on these crucial issues by investigating the effects of the type, number, and position of primer mismatches on the extension efficiency and hence the specificity. Furthermore, quantitative aspects of SNuPE were investigated in a model community experiment by using defined template mixtures of 16S rRNA gene PCR products or reverse-transcribed RNA.