KO cocultures, with neither an intrinsic nor an extrinsic source of amphiregulin in the cultures, showed potent suppression (Fig 2, B and C). ...recombinant amphiregulin added to cultures of T cells ...in vitro impacted neither their proliferation nor their apoptosis, showing no evidence of an inhibitory role (data not shown).
Interleukin-2 inducible tyrosine kinase (ITK) is expressed in T cells and plays a critical role in signalling through the T cell receptor. Evidence, mainly from knockout mice, has suggested that ITK ...plays a particularly important function in Th2 cells and this has prompted significant efforts to discover ITK inhibitors for the treatment of allergic disease. However, ITK is known to have functions outside of its kinase domain and in general kinase knockouts are often not good models for the behaviour of small molecule inhibitors. Consequently we have developed a transgenic mouse where the wild type Itk allele has been replaced by a kinase dead Itk allele containing an inactivating K390R point mutation (Itk-KD mice). We have characterised the immune phenotype of these naive mice and their responses to airway inflammation. Unlike Itk knockout (Itk-/-) mice, T-cells from Itk-KD mice can polymerise actin in response to CD3 activation. The lymph nodes from Itk-KD mice showed more prominent germinal centres than wild type mice and serum antibody levels were significantly abnormal. Unlike the Itk-/-, γδ T cells in the spleens of the Itk-KD mice had an impaired ability to secrete Th2 cytokines in response to anti-CD3 stimulation whilst the expression of ICOS was not significantly different to wild type. However ICOS expression is markedly increased on αβCD3+ cells from the spleens of naïve Itk-KD compared to WT mice. The Itk-KD mice were largely protected from inflammatory symptoms in an Ovalbumin model of airway inflammation. Consequently, our studies have revealed many similarities but some differences between Itk-/-and Itk-KD transgenic mice. The abnormal antibody response and enhanced ICOS expression on CD3+ cells has implications for the consideration of ITK as a therapeutic target.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
IMPORTANCE: Whether β-lactam antibiotics administered by continuous compared with intermittent infusion reduces the risk of death in patients with sepsis is uncertain. OBJECTIVE: To evaluate whether ...continuous vs intermittent infusion of a β-lactam antibiotic (piperacillin-tazobactam or meropenem) results in decreased all-cause mortality at 90 days in critically ill patients with sepsis. DESIGN, SETTING, AND PARTICIPANTS: An international, open-label, randomized clinical trial conducted in 104 intensive care units (ICUs) in Australia, Belgium, France, Malaysia, New Zealand, Sweden, and the United Kingdom. Recruitment occurred from March 26, 2018, to January 11, 2023, with follow-up completed on April 12, 2023. Participants were critically ill adults (≥18 years) treated with piperacillin-tazobactam or meropenem for sepsis. INTERVENTION: Eligible patients were randomized to receive an equivalent 24-hour dose of a β-lactam antibiotic by either continuous (n = 3498) or intermittent (n = 3533) infusion for a clinician-determined duration of treatment or until ICU discharge, whichever occurred first. MAIN OUTCOMES AND MEASURES: The primary outcome was all-cause mortality within 90 days after randomization. Secondary outcomes were clinical cure up to 14 days after randomization; new acquisition, colonization, or infection with a multiresistant organism or Clostridioides difficile infection up to 14 days after randomization; ICU mortality; and in-hospital mortality. RESULTS: Among 7202 randomized participants, 7031 (mean SD age, 59 16 years; 2423 women 35%) met consent requirements for inclusion in the primary analysis (97.6%). Within 90 days, 864 of 3474 patients (24.9%) assigned to receive continuous infusion had died compared with 939 of 3507 (26.8%) assigned intermittent infusion (absolute difference, −1.9% 95% CI, −4.9% to 1.1%; odds ratio, 0.91 95% CI, 0.81 to 1.01; P = .08). Clinical cure was higher in the continuous vs intermittent infusion group (1930/3467 55.7% and 1744/3491 50.0%, respectively; absolute difference, 5.7% 95% CI, 2.4% to 9.1%). Other secondary outcomes were not statistically different. CONCLUSIONS AND RELEVANCE: The observed difference in 90-day mortality between continuous vs intermittent infusions of β-lactam antibiotics did not meet statistical significance in the primary analysis. However, the confidence interval around the effect estimate includes the possibility of both no important effect and a clinically important benefit in the use of continuous infusions in this group of patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03213990
B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization ...and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.
The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and ...there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.
People with Learning Disability (LD) often receive inequitable care within the NHS, something Mencap has described as ‘institutional discrimination’ 1. The NHS Long Term Plan states the need to ...improve the care of patients with LD 2. Simulation with debriefing is a useful approach for improving patient care with Human Factors teaching. To improve education around LD, we created a de novo course with concurrent LD theme for nursing educators to become trained in debriefing and simulation, to allow them to become champions in facilitating learning, especially in relation to LD.
To establish the current educational needs of our organisation around LD, we conducted a staff survey to further understand the educational needs of our colleagues. 108 professionals from a variety of disciplinary backgrounds (including nurses, dietitians, and doctors) across 4 sites within our Trust responded. Thematic analysis highlighted the need for further education, with anxiety about inequitable treatment of LD patients, and staff and patient physical safety when looking after a patient with LD. We subsequently ran a 2-day ‘Train the Trainers’ course for nursing educators, which used communication scenarios (online videos and actors) and games to develop generic debriefing skills. Day 2 focused on simulation design and incorporating LD into simulation design, and at the end of the course participants facilitated a high-fidelity simulation to the rest of the group. Throughout the course, patient feedback, serious incidents, staff survey, and our Trust LD specialist nurse’s expertise were incorporated.
8 nurses attended our course, taught by a diverse multidisciplinary faculty. Before the course, confidence in debriefing was on average 2.8/5 (5 being most confident). Afterwards they rated their confidence 3.8/5. Learners were asked about their confidence raising awareness of LD within their department. The rating was improved from the initial 2.6/5 to 3.6/5 after the course. To date, 1 participant has delivered LD-related teaching to their department using debriefing skills following an online LD video used on the course.
Our staff survey highlighted the need for further education within our organisation. The course was successful in increasing nursing educators’ confidence in debriefing and their confidence in raising awareness of LD during teaching sessions. We are currently creating further resources to aid teaching, including videos with service users. We will further signpost to existing resources and request delayed feedback to assess if our nursing educators have become LD Champions.
1. Mencap. Mencap.org.uk. 2022
2. NHS England. NHS Long Term Plan. NHS Long Term Plan. 2022.
Methylxanthines, such as theophylline, exhibit cyclic nucleotide phosphodiesterase inhibitory activity in high concentrations ($>$10$\sp{-4}$ M) and adenosine receptor antagonist activity in low ...concentrations (10$\sp{-5}$ M to 10$\sp{-4}$ M). Based on evidence that endogenously-produced adenosine inhibited neutrophil responses, the influence of methylxanthine bronchodilators on neutrophil responses stimulated in vitro by n-formyl-methionyl-leucyl-phenylalanine (fMLP) was examined. At concentrations between 10$\sp{-5}$ M and 10$\sp{-4}$ M, theophylline potentiated lysosomal enzyme release by 30 to 50% (p $<$ 0.005), superoxide anion formation by 30 to 60% (p $<$ 0.005), and neutrophil aggregation. Theophylline at concentrations $>$10$\sp{-4}$ M inhibited the same responses by $>$90%. Adenosine deaminase mimicked, whereas adenosine (3 $\times$ 10$\sp{-8}$ to 3 $\times$ 10$\sp{-7}$M) reversed the theophylline potentiation. A potential role for calcium in the modulation of the neutrophil responses by theophylline and adenosine was explored. Theophylline (3 $\times$ 10$\sp{-5}$ M) enhanced by $>$150% the fMLP-stimulated increase in cytoplasmic calcium concentration ((Ca$\sp{2+}$) $\sb{\rm i}$) at time points between 5 and 90 sec (p $<$ 0.01) as measured by Fura-2. Adenosine deaminase induced a comparable enhancement, whereas 3 $\times$ 10$\sp{-7}$ M adenosine and 10$\sp{-7}$ M N-ethylcarboxamideadenosine decreased the (Ca$\sp{2+}$) $\sb{\rm i}$ in fMLP-stimulated neutrophils. Extracellular calcium was not required for the opposing influences of theophylline and adenosine and neither compound altered fMLP-stimulated $\sp{45}$Ca uptake at the early time points. Inositol trisphosphate mediates the fMLP-stimulated increase in (Ca$\sp{2+}$) $\sb{\rm i}$ observed at 5 sec. Theophylline and adenosine deaminase each enhanced inositol tris phosphate production by $>$140% (p $<$ 0.002) within 5 sec of activation. At 5 sec, adenosine reduced the intracellular levels of inositol phosphate (p $<$ 0.05). These results indicate that theophylline and adenosine may modulate neutrophil inflammatory function by regulating the inositol trisphosphate-mediated mobilization of intracellular calcium.
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