Evaluation of the QIAstat-Dx Meningitis/Encephalitis Panel, a multiplex PCR platform for the detection of community-acquired meningoencephalitis.

Rapid identification of the causative pathogens of central nervous system infections is essential for providing appropriate management and improving patient outcomes. The performance of QIAstat-Dx Meningitis/Encephalitis (ME) Panel-a multiplex PCR testing platform-in detecting pathogens implicated in meningitis and/or encephalitis was evaluated using BioFire FilmArray ME Panel as a comparator method. This multicenter study analyzed 585 retrospective residual cerebrospinal fluid specimens and 367 contrived specimens. The QIAstat-Dx ME Panel showed positive percent agreement (PPA) values of 100% for Neisseria meningitidis, Streptococcus agalactiae, Escherichia coli K1, Listeria monocytogenes, and Cryptococcus gattii/neoformans on clinical samples compared to the BioFire FilmArray ME Panel. The PPA values observed for Haemophilus influenzae and Streptococcus pneumoniae were 80% and 88.24%, respectively. Negative percent agreement (NPA) values were >99.0% for each of the six bacterial targets and one fungal target tested with clinical samples. One viral target, herpes simplex virus 1, exhibited a PPA value of 100.0%, while the remaining viral targets-human parechovirus, herpes simplex virus 2, human herpes virus 6, and varicella zoster virus-were >90.0%, with the exception of enterovirus, which had a PPA value of 77.8%. The QIAstat-Dx ME Panel detected five true-positive and four true-negative cases compared to BioFire FilmArray ME Panel. The NPA values for all viral pathogens were >99.0%. Overall, the QIAstat-Dx ME Panel showed comparable performance to the BioFire FilmArray ME Panel as a rapid diagnostic tool for community-acquired meningitis and encephalitis.

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation.

The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near-surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC-flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC-expressing bacteria outcompeted FljB-expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1ß by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

In vitro evaluation of the antielastase activity of polycyclic ß-lactams.

A series of bi- and tricyclic ß-lactam compounds was synthesized and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leukocyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive effect that the styryl group exerts on the HLE inhibitory activity in polycyclic ß-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.

Modeling the Arabidopsis seed shape by a cardioid: efficacy of the adjustment with a scale change with factor equal to the Golden Ratio and analysis of seed shape in ethylene mutants.

A new model for the description of Arabidopsis seed shape based on the comparison of the outline of its longitudinal section with a transformed cardioid is presented. The transformation consists of scaling the horizontal axis by a factor equal to the Golden Ratio. The elongated cardioid approximates the shape of the Arabidopsis seed with more accuracy than other figures. The length to width ratio in wild-type Columbia Arabidopsis dry seeds is close to the Golden Ratio and decreases over the course of imbibition. Dry seeds of etr1-1 mutants presented a reduced length to width ratio. Application of the new model based on the cardioid allows for comparison of shape between wild-type and mutant genotypes, revealing other general alterations in the seeds in ethylene signaling pathway mutants (etr1-1).

A Toll-like receptor 2-integrin beta3 complex senses bacterial lipopeptides via vitronectin.

Toll-like receptor 2 (TLR2) initiates inflammation in response to bacterial lipopeptide (BLP). However, the molecular mechanisms enabling the detection of BLP by TLR2 are unknown. Here we investigated the interaction of BLP with human serum proteins and identified vitronectin as a BLP-recognition molecule. Vitronectin and its receptor, integrin beta(3), were required for BLP-induced TLR2-mediated activation of human monocytes. Furthermore, monocytes from patients with Glanzmann thrombasthenia, which lack integrin beta(3), were completely unresponsive to BLP. In addition, integrin beta(3) formed a complex with TLR2 and this complex dissociated after BLP stimulation. Notably, vitronectin and integrin beta(3) coordinated responses to other TLR2 agonists such as lipoteichoic acid and zymosan. Our findings show that vitronectin and integrin beta(3) contribute to the initiation of TLR2 responses.

What is the role of Toll-like receptors in bacterial infections?

Innate immunity relies on signalling by Toll-like receptors (TLRs) to alert the immune system of the presence of invading bacteria. TLR activation leads to the release of cytokines that allow for effective innate and adaptive immune responses. However, the contribution of different TLRs depends on the site of the infection and the pathogen. This review will describe the involvement of TLRs in the development of three different bacterial infections as well as our current understanding of the role of TLRs during microbial pathogenesis.

The prohibitin genes in Arabidopsis thaliana: expression in seeds, hormonal regulation and possible role in cell cycle control during seed germination.

A fragment encoding a partial sequence of a prohibitin (Phb) gene was isolated. The expression of Phb mRNA and protein in seeds of wild type and mutant Arabidopsis thaliana is presented. Phb mRNA is abundant in wild-type seeds; thus, it may have sequence or structural characteristics responsible for this stability. The 3' untranslated region sequence of a Phb gene has interesting features. We found that Arabidopsis Phb does not interact with a retinoblastoma-related protein or E2F in a yeast two-hybrid system, thus suggesting that the plant protein may have not conserved such interaction, described for mammalian Phb. The possible role of Phb in cell cycle regulation during germination is discussed.

cDNA-AFLP analysis of seed germination in Arabidopsis thaliana identifies transposons and new genomic sequences.

A cDNA-AFLP experiment was designed to identify and clone nucleotide sequences induced during seed germination in Arabidopsis thaliana. Sequences corresponding to known genes involved in processes important for germination, such as mitochondrial biogenesis, protein synthesis and cell cycle progression, were isolated. Other sequences correspond to Arabidopsis BAC clones in regions where genes have not been annotated. Notably, a number of the sequences cloned did not correspond to available sequences in the databases from the Arabidopsis genome, but instead present significant similarity with DNA from other organisms, for example fish species; among them, some may encode transposons. A number of the sequences isolated showed no significant similarity with any sequences in the public databases. Oligonucleotides derived from these new sequences were used to amplify genomic DNA of Arabidopsis. Expression analysis of representative sequences is presented. This work suggests that, during germination, there may be a massive transposon mobilization that may be useful in the annotation of new genome sequences and identification of regulatory mechanisms.

Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis.

CD8 T lymphocytes are important effectors in protective immunity against Mycobacterium tuberculosis. We recently characterized the detour pathway of CD8 T cell activation in tuberculosis mediated by apoptotic vesicles from infected cells that transport mycobacterial antigens to dendritic cells (DCs). Here we demonstrate that apoptotic vesicles from mycobacteria-infected macrophages stimulate CD8 T cells in vivo. Homing of DCs to draining lymph nodes was critically required for effective crosspriming. Subsequent fate of vesicle-associated antigens in recipient DCs was characterized by endosomal mechanisms predominating over proteasomal processing. In addition, vesicle processing depended on the presence of saposins to disintegrate apoptotic membranes. Apoptotic vesicles displayed potent adjuvant activity by stimulating through Toll-like receptors (TLR). Ultimately, vaccination with vesicles from infected cells induced protection against M. tuberculosis infection. Taken together, we propose the detour pathway to represent a genuine immunological mechanism mediating crosspriming of CD8 T cells in vivo and protection against tuberculosis.

Proinflammatory effects of bacterial recombinant human C-reactive protein are caused by contamination with bacterial products, not by C-reactive protein itself.

Intravenous administration to human volunteers of a commercial preparation of recombinant human C-reactive protein (CRP) produced in Escherichia coli was recently reported in this journal to induce an acute phase response of serum amyloid A protein (SAA) and of CRP itself, and to activate the coagulation system. The authors concluded that CRP is probably a mediator of atherothrombotic disease. Here we confirm that this recombinant CRP preparation was proinflammatory both for mouse macrophages in vitro and for mice in vivo, but show that pure natural human CRP had no such activity. Furthermore mice transgenic for human CRP, and expressing it throughout their lives, maintained normal concentrations of their most sensitive endogenous acute phase reactants, SAA and serum amyloid P component (SAP). The patterns of in vitro cytokine induction and of in vivo acute phase stimulation by the recombinant CRP preparation were consistent with contamination by bacterial products, and there was 46.6 EU of apparent endotoxin activity per mg of CRP in the bacterial product, compared with 0.9 EU per mg of our isolated natural human CRP preparation. The absence of any proinflammatory activity in natural CRP for macrophages or healthy mice strongly suggests that the in vivo effects of the recombinant preparation observed in humans were attributable to proinflammatory bacterial products and not human CRP.