A magnetic nanoparticle-based microfluidic device fabricated using a 3D-printed mould for separation of Escherichia coli from blood.

Herein, A microfluidic device is described, produced with a 3D-printed master mould that rapidly separates and concentrates Escherichia coli directly from whole blood samples, enabling a reduction in the turnaround time of bloodstream infections (BSIs) diagnosis. Moreover, it promotes the cleansing of the blood samples whose complexity frequently hampers bacterial detection. The device comprises a serpentine mixing channel with two inlets, one for blood samples (spiked with bacteria) and the other for magnetic nanoparticles (MNPs) functionalized with a (bacterio)phage receptor-binding protein (RBP) with high specificity for E. coli. After the magnetic labelling of bacteria throughout the serpentine, the microchannel ends with a trapping reservoir where bacteria-MNPs conjugates are concentrated using a permanent magnet. The optimized sample preparation device successfully recovered E. coli (on average, 66%) from tenfold diluted blood spiked within a wide range of bacterial load (102 CFU to 107 CFU mL-1). The non-specific trapping, tested with Staphylococcus aureus, was at a negligible level of 12%. The assay was performed in 30 min directly from diluted blood thus presenting an advantage over the conventional enrichment in blood cultures (BCs). The device is simple and cheap to fabricate and can be tailored for multiple bacterial separation from complex clinical samples by using RBPs targeting different species. Moreover, the possibility to integrate a biosensing element to detect bacteria on-site can provide a reliable, fast, and cost-effective point-of-care device.

His bundle pacing and left bundle branch area pacing: Feasibility and safety.

INTRODUCTION AND OBJECTIVES: There has been increasing interest in pacing methods that provide physiological stimulation, such as His bundle pacing (HBP) or left bundle branch area pacing (LBBAP). Our goal was to assess the feasibility and safety of these techniques. METHODS: Prospective observational single-center study evaluating 46 patients with indication for a pacemaker that attempted HBP or LBBAP from July 2020 to November 2021. Procedural endpoints and pacing parameters were assessed and compared at implantation and three-month follow-up. RESULTS: Overall acute procedural success was achieved in 96% of the cases. Successful HBP was achieved in 91% of the patients and all patients for LBBAP. During implantation, HBP patients presented a higher capture threshold (0.80 [0.55-1.53] V vs. 0.70 [0.40-0.90] V, p=0.08) and lower R-wave amplitude (4.0 [2.9-6.2] mV vs. 7.8 [5.5-10.5] mV, p=0.001) compared to LBBAP patients. There was no difference between groups, either acutely or at 3-months, regarding paced QRS duration (125±22 ms vs. 133±16 ms, p=0.08; 118±16 ms vs. 124±14 ms, p=0.19). Although procedural time was similar with both techniques (95 [75-139] min vs. 95 [74-116] min, p=0.79), fluoroscopy time was significantly reduced during LBBAP (8.1 [5.3-13.4] min vs. 4.1 [3.1-11.3] min, p=0.05). At 3 months of follow-up, the pacing threshold remained with a stable profile in HBP as in LBBAP (1.25 [0.75-2.00] V, p=0.09 and 0.60 [0.50-0.80] V, p=0.78), respectively. The need for re-intervention occurred in 3 (6.5%) HBP cases during follow-up. CONCLUSION: This first national study demonstrates the feasibility and safety of the HBP and LBBAP in patients with pacemaker indication.

Potential of bacteriophage proteins as recognition molecules for pathogen detection.

Bacterial pathogens are leading causes of infections with high mortality worldwide having a great impact on healthcare systems and the food industry. Gold standard methods for bacterial detection mainly rely on culture-based technologies and biochemical tests which are laborious and time-consuming. Regardless of several developments in existing methods, the goal of achieving high sensitivity and specificity, as well as a low detection limit, remains unaccomplished. In past years, various biorecognition elements, such as antibodies, enzymes, aptamers, or nucleic acids, have been widely used, being crucial for the pathogens detection in different complex matrices. However, these molecules are usually associated with high detection limits, demand laborious and costly production, and usually present cross-reactivity. (Bacterio)phage-encoded proteins, especially the receptor binding proteins (RBPs) and cell-wall binding domains (CBDs) of endolysins, are responsible for the phage binding to the bacterial surface receptors in different stages of the phage lytic cycle. Due to their remarkable properties, such as high specificity, sensitivity, stability, and ability to be easily engineered, they are appointed as excellent candidates to replace conventional recognition molecules, thereby contributing to the improvement of the detection methods. Moreover, they offer several possibilities of application in a variety of detection systems, such as magnetic, optical, and electrochemical. Herein we provide a review of phage-derived bacterial binding proteins, namely the RBPs and CBDs, with the prospect to be employed as recognition elements for bacteria. Moreover, we summarize and discuss the various existing methods based on these proteins for the detection of nosocomial and foodborne pathogens.

Exploitation of a Klebsiella Bacteriophage Receptor-Binding Protein as a Superior Biorecognition Molecule.

Klebsiella pneumoniae is a Gram-negative bacterium that has become one of the leading causes of life-threatening healthcare-associated infections (HAIs), including pneumonia and sepsis. Moreover, due to its increasingly antibiotic resistance, K. pneumoniae has been declared a global top priority concern. The problem of K. pneumoniae infections is due, in part, to the inability to detect this pathogen rapidly and accurately and thus to treat patients within the early stages of infections. The success in bacterial detection is greatly dictated by the biorecognition molecule used, with the current diagnostic tools relying on expensive probes often lacking specificity and/or sensitivity. (Bacterio)phage receptor-binding proteins (RBPs) are responsible for the recognition and adsorption of phages to specific bacterial host receptors and thus present high potential as biorecognition molecules. In this study, we report the identification and characterization of a novel RBP from the K. pneumoniae phage KpnM6E1 that presents high specificity against the target bacteria and high sensitivity (80%) to recognize K. pneumoniae strains. Moreover, adsorption studies validated the role of gp86 in the attachment to bacterial receptors, as it highly inhibits (86%) phage adsorption to its Klebsiella host. Overall, in this study, we unravel the role and potential of a novel Klebsiella phage RBP as a powerful tool to be used coupled with analytical techniques or biosensing platforms for the diagnosis of K. pneumoniae infections.

Sticholysins, pore-forming proteins from a marine anemone can induce maturation of dendritic cells through a TLR4 dependent-pathway.

Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.

Bacteriophage-receptor binding proteins for multiplex detection of Staphylococcus and Enterococcus in blood.

Healthcare-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however, are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hr after an enrichment step.

The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes.

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.

Improving Cancer Immunotherapies through Empirical Neoantigen Selection.

Targeting neoantigens has become an attractive strategy for cancer immunotherapy. Epitope prediction algorithms facilitate rapid selection of potential neoantigens, but are plagued with high false-positive and false-negative rates. Here we review ex vivo technologies for biological identification of neoantigens to improve empirical prioritization for immunotherapy.

Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA.

We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

Protective immunity against Chlamydia trachomatis can engage both CD4+ and CD8+ T cells and bridge the respiratory and genital mucosae.

Understanding the cellular populations and mechanisms responsible for overcoming immune compartmentalization is valuable for designing vaccination strategies targeting distal mucosae. In this study, we show that the human pathogen Chlamydia trachomatis infects the murine respiratory and genital mucosae and that T cells, but not Abs, elicited through intranasal immunization can protect against a subsequent transcervical challenge. Unlike the genital infection where CD8(+) T cells are primed, yet fail to confer protection, we found that intranasal priming engages both CD4(+) and CD8(+) T cells, allowing for protection against genital infection with C. trachomatis. The protection is largely dependent on IFN-γ secretion by T cells. Moreover, different chemokine receptors are critical for C. trachomatis-specific CD4(+) T cells to home to the lung, rather than the CXCR3- and CCR5-dependent migration observed during genital infection. Overall, this study demonstrates that the cross-mucosa protective immunity against genital C. trachomatis infection following intranasal immunization is not dependent on Ab response but is mediated by not only CD4(+) T cells but also by CD8(+) T cells. This study provides insights for the development of vaccines against mucosal pathogens that threaten reproductive health worldwide.

Infectious disease is the most common cause of death among stroke patients: two-years of follow-up.

Stroke represents the second common cause of death in adults. Objective It was to evaluate the incidence and causes of mortality after 30 days in a group of patients with an atherothrombotic ischemic stroke who were followed-up for a period of up to two years. Methods We analyzed retrospectively the medical records of patients with ischemic stroke, who did not undergo thrombolysis, of the Santa Marcelina Hospital. We applied a research protocol to obtain information about risk factors and the etiology of death. Results They were followed 337: mean age was 66.6 years (SD 9.05) and 43.9% were females. The mortality rate was 11.9% with most deaths (37.5%) occurring due to infectious causes. The age was correlated with the risk of death, which was five times higher in patients older than 80. Conclusion The most important cause of death after 30 days was infectious disease, and advanced age was predictor of mortality among patients with an atherothrombotic stroke etiology.

IFNγ inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I.

The activation of host cells by interferon gamma (IFNγ) is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs) and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1), an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR) retanoic acid-inducible gene I (RIG-I), a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS)--but not cytosolic Nod-like receptors (NLRs)--are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5'-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing this bacterium. Additionally, these findings expand our understanding of how IFNγ recognizes, and ultimately restricts, bacterial pathogens within host cells.

Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein.

Coxiella burnetii and Legionella pneumophila are evolutionarily related pathogens with different intracellular infection strategies. C. burnetii persists within and is transmitted by mammalian hosts, whereas, L. pneumophila is found primarily in the environment associated with protozoan hosts. Although a type IV secretion system encoded by the defect in organelle trafficking (dot) and intracellular multiplication (icm) genes is a virulence determinant that remains highly conserved in both bacteria, the two pathogens encode a different array of effector proteins that are delivered into host cells by the Dot/Icm machinery. This difference suggests that adaptations to evolutionarily distinct hosts may be reflected in the effector protein repertoires displayed by these two pathogens. Here we provide evidence in support of this hypothesis. We show that a unique C. burnetii effector from the ankyrin repeat (Ank) family called AnkG interferes with the mammalian apoptosis pathway. AnkG was found to interact with the host protein gC1qR (p32). Either the addition of AnkG to the repertoire of L. pneumophila effector proteins or the silencing of p32 in mouse dendritic cells resulted in a gain of function that allowed intracellular replication of L. pneumophila in these normally restrictive mammalian host cells by preventing rapid pathogen-induced apoptosis. These data indicate that p32 regulates pathogen-induced apoptosis and that AnkG functions to block this pathway. Thus, emergence of an effector protein that interferes with a proapoptotic signaling pathway directed against intracellular bacteria correlates with adaptation of a pathogen to mammalian hosts.

Rapid pathogen-induced apoptosis: a mechanism used by dendritic cells to limit intracellular replication of Legionella pneumophila.

Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.

Type IV secretion-dependent activation of host MAP kinases induces an increased proinflammatory cytokine response to Legionella pneumophila.

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-kappaB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.