Asymmetric Synthesis and Biological Screening of Quinoxaline-Containing Synthetic Lipoxin A₄ Mimetics (QNX-sLXms)

Failure to resolve inflammation underlies many prevalent pathologies. Recent insights have identified lipid mediators, typified by lipoxins (LXs), as drivers of inflammation resolution, suggesting potential therapeutic benefit. We report the asymmetric preparation of novel quinoxaline-containing synthetic-LXA4-mimetics (QNX-sLXms). Eight novel compounds were screened for their impact on inflammatory responses. Structure–activity relationship (SAR) studies showed that (R)-6 (also referred to as AT-02-CT) was the most efficacious and potent anti-inflammatory compound of those tested. (R)-6 significantly attenuated lipopolysaccharide (LPS)- and tumor-necrosis-factor-α (TNF-α)-induced NF-κB activity in monocytes and vascular smooth muscle cells. The molecular target of (R)-6 was investigated. (R)-6 activated the endogenous LX receptor formyl peptide receptor 2 (ALX/FPR2). The anti-inflammatory properties of (R)-6 were further investigated in vivo in murine models of acute inflammation. Consistent with in vitro observations, (R)-6 attenuated inflammatory responses. These results support the therapeutic potential of the lead QNX-sLXm (R)-6 in the context of novel inflammatory regulators

Asymmetric Synthesis and Biological Screening of Quinoxaline-Containing Synthetic Lipoxin A4 Mimetics (QNX-sLXms)

Failure to resolve inflammation underlies many prevalent pathologies. Recent insights have identified lipid mediators, typified by lipoxins (LXs), as drivers of inflammation resolution, suggesting potential therapeutic benefit. We report the asymmetric preparation of novel quinoxaline-containing syntheticLXA4-mimetics (QNX-sLXms). Eight novel compounds were screened for their impact on inflammatory responses. Structure− activity relationship (SAR) studies showed that (R)-6 (also referred to as AT-02-CT) was the most efficacious and potent anti-inflammatory compound of those tested. (R)-6 significantly attenuated lipopolysaccharide (LPS)- and tumor-necrosis-factor-α (TNF-α)-induced NF-κB activity in monocytes and vascular smooth muscle cells. The molecular target of (R)-6 was investigated. (R)-6 activated the endogenous LX receptor formyl peptide receptor 2 (ALX/FPR2). The anti-inflammatory properties of (R)-6 were further investigated in vivo in murine models of acute inflammation. Consistent with in vitro observations, (R)-6 attenuated inflammatory responses. These results support the therapeutic potential of the lead QNX-sLXm (R)-6 in the context of novel inflammatory regulators

Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specifc antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria

Interaction of hnRNPA1/A2 and DAZAP1 with an Alu-Derived Intronic Splicing Enhancer Regulates ATM Aberrant Splicing

We have previously identified an Alu-derived Intronic Splicing enhancer (ISE) in the Ataxia Teleangectasia Mutated gene (ATM) that facilitates intron pre-mRNA processing and leads to the inclusion of a cryptic exon in the final mRNA transcript. By using an RNA pull-down assay, we show here that hnRNPA1/A2, HuR and DAZAP1 splicing factors and DHX36 RNA helicase bind to the ISE. By functional studies (overexpression and siRNA experiments), we demonstrate that hnRNPA1 and DAZAP1 are indeed involved in ISE-dependent ATM cryptic exon activation, with hnRNPA1 acting negatively and DAZAP1 positively on splicing selection. On the contrary, HuR and DHX36 have no effect on ATM splicing pattern. These data suggest that splicing factors with both negative and positive effect can assemble on the intronic Alu repeats and regulate pre-mRNA splicing

Evaluation of dipstick analysis among elderly residents to detect bacteriuria: a cross-sectional study in 32 nursing homes

Background: Up to half the residents of nursing homes for the elderly have asymptomatic bacteriuria (ABU), which should not be treated with antibiotics. Thus, it is difficult to know if new symptoms in residents with bacteriuria are caused by urinary tract infection (UTI), or if bacteriuria only represents an ABU. This is especially difficult in the presence of non-urinary tract specific symptoms. The diagnostic uncertainty is likely to generate significant overtreatment with UTI antibiotics. Aim: The general aim was to clarify the association between symptoms, bacteriuria, dipstick urinalysis and urine Interleukin-6 (IL-6) among nursing home residents to improve the diagnostic procedure of a suspected lower UTI. Methods: In 2003 a study protocol including newly onset symptoms was completed, and single voided urine specimens collected for dipstick urinalysis and cultures from 651 residents of 32 participating Swedish nursing homes for the elderly. This data was used for a study of dipstick urinalysis (Paper I) and for a study of nonspecific symptoms and bacteriuria (Paper II). In 2012, similar data was collected for 421 elderly residents of 22 nursing homes, which also included an analysis of IL-6 in urine and urine specimens from another 59 residents with urinary catheters. The association between bacteriuria, IL-6 in urine, dipstick urinalysis and newly onset symptoms was analysed (Paper III). Antimicrobial resistance rates were described among residents of nursing homes in 2012 and compared with those from 2003 (Paper IV). Results: Paper I: The negative predictive value for predicting absence of bacteriuria was 88 (84-92)% when dipstick urinalysis for nitrite and leukocyte esterase were simultaneously negative. A positive dipstick or any combination thereof could not sufficiently predict bacteriuria. Papers II-III: New or increased nonspecific symptoms were common among elderly residents of nursing homes. Residents without nonspecific symptoms had positive urine cultures as often as those with nonspecific symptoms with a duration of up to one month. Paper III: Residents with positive urine cultures had higher concentrations of IL-6 in the urine. However, among residents with positive urine cultures there were no differences in IL-6 concentrations or dipstick findings between those with or without nonspecific symptoms. Paper IV: The average rates of antimicrobial resistance were low and did not increase between 2003 and 2012 in Escherichia coli (E. coli) urinary isolates among Swedish nursing home residents. Any antibiotic treatment during the last month and hospitalization during the last six months predicted higher resistance rates among E. coli. Conclusions: Nonspecific symptoms among elderly residents of nursing homes are unlikely to be caused by bacteria in the urine. Therefore, dipstick urinalysis, IL-6 in the urine and urine cultures are of little or no value in clarifying the aetiology of nonspecific symptoms. If there is a reason for testing for bacteriuria, dipstick urinalysis for nitrite and leukocyte esterase can rule out but cannot reliably rule in bacteriuria. Antimicrobial resistance in urinary pathogens among Swedish nursing home residents remained low. It is important to use antibiotics rationally to preserve the effectiveness of antibiotics

Intronic Alus Influence Alternative Splicing

Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA) formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptom

HLA-DQA1*05 carriage associated with development of anti-drug antibodies to infliximab and adalimumab in patients with Crohn's Disease

Anti-tumor necrosis factor (anti-TNF) therapies are the most widely used biologic drugs for treating immune-mediated diseases, but repeated administration can induce the formation of anti-drug antibodies. The ability to identify patients at increased risk for development of anti-drug antibodies would facilitate selection of therapy and use of preventative strategies.This article is freely available via Open Access. Click on Publisher URL to access the full-text

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

BACKGROUND: The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. METHODS: We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). FINDINGS: Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca