Effect of Inhaled Xenon on Cerebral White Matter Damage in Comatose Survivors of Out-of-Hospital Cardiac Arrest: A Randomized Clinical Trial

IMPORTANCE: Evidence from preclinical models indicates that xenon gas can prevent the development of cerebral damage after acute global hypoxic-ischemic brain injury but, thus far, these putative neuroprotective properties have not been reported in human studies. OBJECTIVE: To determine the effect of inhaled xenon on ischemic white matter damage assessed with magnetic resonance imaging (MRI). DESIGN, SETTING, AND PARTICIPANTS: A randomized single-blind phase 2 clinical drug trial conducted between August 2009 and March 2015 at 2 multipurpose intensive care units in Finland. One hundred ten comatose patients (aged 24-76 years) who had experienced out-of-hospital cardiac arrest were randomized. INTERVENTIONS: Patients were randomly assigned to receive either inhaled xenon combined with hypothermia (33°C) for 24 hours (n = 55 in the xenon group) or hypothermia treatment alone (n = 55 in the control group). MAIN OUTCOMES AND MEASURES: The primary end point was cerebral white matter damage as evaluated by fractional anisotropy from diffusion tensor MRI scheduled to be performed between 36 and 52 hours after cardiac arrest. Secondary end points included neurological outcome assessed using the modified Rankin Scale (score 0 [no symptoms] through 6 [death]) and mortality at 6 months. RESULTS: Among the 110 randomized patients (mean age, 61.5 years; 80 men [72.7%]), all completed the study. There were MRI data from 97 patients (88.2%) a median of 53 hours (interquartile range [IQR], 47-64 hours) after cardiac arrest. The mean global fractional anisotropy values were 0.433 (SD, 0.028) in the xenon group and 0.419 (SD, 0.033) in the control group. The age-, sex-, and site-adjusted mean global fractional anisotropy value was 3.8% higher (95% CI, 1.1%-6.4%) in the xenon group (adjusted mean difference, 0.016 [95% CI, 0.005-0.027], P = .006). At 6 months, 75 patients (68.2%) were alive. Secondary end points at 6 months did not reveal statistically significant differences between the groups. In ordinal analysis of the modified Rankin Scale, the median (IQR) value was 1 (1-6) in the xenon group and 1 (0-6) in the control group (median difference, 0 [95% CI, 0-0]; P = .68). The 6-month mortality rate was 27.3% (15/55) in the xenon group and 34.5% (19/55) in the control group (adjusted hazard ratio, 0.49 [95% CI, 0.23-1.01]; P = .053). CONCLUSIONS AND RELEVANCE: Among comatose survivors of out-of-hospital cardiac arrest, inhaled xenon combined with hypothermia compared with hypothermia alone resulted in less white matter damage as measured by fractional anisotropy of diffusion tensor MRI. However, there was no statistically significant difference in neurological outcomes or mortality at 6 months. These preliminary findings require further evaluation in an adequately powered clinical trial designed to assess clinical outcomes associated with inhaled xenon among survivors of out-of-hospital cardiac arrest. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00879892

Detection of Pathogenic Mycobacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 104 bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples

Lanthanide-based time-resolved luminescence immunoassays

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized

An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1

A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117–121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1−/− animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context

Re‐weighing the 5% tagging recommendation: assessing the potential impacts of tags on the behaviour and body condition of bats

Considerable advances and breakthroughs in wildlife tracking technology have occurred in recent years, allowing researchers to gain insights into the movements and behaviours of a broad range of animals. Considering the accessibility and increase in use of tracking devices in wildlife studies, it is important to better understand the effects on these on animals. Our endeavour revisits a guideline established in 1988, which proposes that bats may encounter body condition or health problems and alter their behaviour when carrying tags weighing more than 5% of their body mass. Through a systematic literature review, we conducted a meta‐analysis to identify the impacts of tags on bats, including 367 papers from 1976 to 2023 that discussed, mentioned, employed, or quantified tagging of bats. We noted that the proportion of studies exceeding the 5% rule has not changed in recent years. However, the impact of tags was quantified in few studies for behaviour (n = 7) and body condition (n = 10) of bats. We were unable to assess whether tags weighing less or more than 5% of the bat's body mass impacted bats, but our meta‐analysis did identify that tags, irrespective of mass, affect the behaviour and body condition of bats. Although the overall magnitude of measured effects of tags on bats was small, progress has been made to advance our understanding of tag mass on bats. Naturally, there is a bias in reporting of significant results, illustrating the need of reporting results when there is no apparent effect of tags on bats. Our findings highlight the need for rigorous reporting of behaviour and body condition data associated with tagging of animals and illustrate the importance for studies comparing how tracking devices of different dimensions and masses may impact bat species to ensure research meets rigorous ethical standards

Protease-activated receptor-2 mediates the expression of inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in keratinocytes in response to Propionibacterium acnes

Propionibacterium acnes (P. acnes) has been known to produce various exogenous proteases, however, their role in acne pathogenesis remains largely unknown. Proteases elicit cellular responses, at least in part, via proteinase-activated receptor-2 (PAR-2), which is known to mediate inflammation and immune response. In this study, we investigated whether proteases from P. acnes could activate PAR-2 on keratinocytes and induce pro-inflammatory cytokines, antimicrobial peptides (AMPs), and matrix metalloproteinases (MMPs) via PAR-2 signaling. We examined PAR-2 expression and protease activity in acne lesions using immunofluorescence staining and in situ zymography. The effect of the culture supernatant of P. acnes on Ca2+ signaling in immortalized keratinocytes (HaCaT) was measured using a fluorescence method. HaCaT cells were treated with P. acnes strain ATCC 6919 culture supernatant, with or without pretreatment with serine protease inhibitor or selective PAR-2 antagonist and the gene expression of pro-inflammatory cytokines, AMPs, and MMPs was detected using real-time reverse transcription-polymerase chain reaction. We found that the protease activity and PAR-2 expression were increased in acne lesions. The P. acnes culture supernatant induced calcium signaling in keratinocytes via PAR-2 and stimulated the mRNA expression of interleukin (IL)-1α, -8, tumor necrosis factor (TNF)-α, human beta defensin (hBD)-2, LL-37, MMP-1, -2, -3, -9, and -13 in keratinocytes, which was significantly inhibited by serine protease inhibitor as well as selective PAR-2 specific antagonist. These results indicate that PAR-2 plays an important role in the pathogenesis of acne by inducing inflammatory mediators in response to proteases secreted from P. acnes

The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST4) that correctly predicted the phylogroup (IA1, IA2, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http:// pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST4 method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium’s evolution. The secreted/cell-associated ‘virulence’ factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages

A Spaetzle-like role for nerve growth factor beta in vertebrate immunity to Staphylococcus aureus

Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity

Comparative Genomics and Transcriptomics of Propionibacterium acnes

The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with maintenance of skin homeostasis. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits differ between phylotypes, as judged from PCR analysis of a collection of P. acnes strains. Comparative transcriptome analysis of strains KPA171202 (type I-2) and 266 during exponential growth revealed inter-strain differences in gene expression of transport systems and metabolic pathways. In addition, transcript levels of genes encoding possible virulence factors such as dermatan-sulphate adhesin, polyunsaturated fatty acid isomerase, iron acquisition protein HtaA and lipase GehA were upregulated in strain 266. We investigated differential gene expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by upregulation of genes involved in stress responses and amino acid metabolism. Our data highlight the genomic basis for strain diversity and identify, for the first time, the actively transcribed part of the genome, underlining the important role growth status plays in the inflammation-inducing activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by the phylotype-specific genome content but also by variable gene expression