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BACKGROUND: Invasive mechanical ventilation in critically ill adults involves adjusting the fraction of inspired oxygen to maintain arterial oxygen saturation. The oxygen-saturation target that will optimize clinical outcomes in this patient population remains unknown. METHODS: In a pragmatic, cluster-randomized, cluster-crossover trial conducted in the emergency department and medical intensive care unit at an academic center, we assigned adults who were receiving mechanical ventilation to a lower target for oxygen saturation as measured by pulse oximetry (Spo(2)) (90%; goal range, 88 to 92%), an intermediate target (94%; goal range, 92 to 96%), or a higher target (98%; goal range, 96 to 100%). The primary outcome was the number of days alive and free of mechanical ventilation (ventilator-free days) through day 28. The secondary outcome was death by day 28, with data censored at hospital discharge. RESULTS: A total of 2541 patients were included in the primary analysis. The median number of ventilator-free days was 20 (interquartile range, 0 to 25) in the lower-target group, 21 (interquartile range, 0 to 25) in the intermediate-target group, and 21 (interquartile range, 0 to 26) in the higher-target group (P = 0.81). In-hospital death by day 28 occurred in 281 of the 808 patients (34.8%) in the lower-target group, 292 of the 859 patients (34.0%) in the intermediate-target group, and 290 of the 874 patients (33.2%) in the higher-target group. The incidences of cardiac arrest, arrhythmia, myocardial infarction, stroke, and pneumothorax were similar in the three groups. CONCLUSIONS: Among critically ill adults receiving invasive mechanical ventilation, the number of ventilator-free days did not differ among groups in which a lower, intermediate, or higher Spo(2) target was used. (Supported by the National Heart, Lung, and Blood Institute and others; PILOT ClinicalTrials.gov number, NCT03537937.)

BACKGROUND AND AIMS: The number of procedures performed by internal medicine residents in the United States (US) is declining. An increasing proportion of residents do not feel confident performing essential invasive bedside procedures and, upon graduation, desire additional training. Several residency programs have utilized the medical procedure service (MPS) to address this issue. We aim to summarize the current state of evidence by systematically evaluating the effect of the MPS on resident education, comfort, and training, as well as patient safety and procedural outcomes in the US. METHODS: We conducted a systematic review of all studies reporting the use of an MPS with supervision from a board-certified physician in internal medicine residencies in the US. Database search was performed on PubMed, Embase, ERIC, and Cochrane Library from January 2000 to November 2020 for relevant studies. Quality of evidence assessment and random-effects proportion meta-analyses were performed. RESULTS: A total of nine studies reporting on 3879 procedures performed by MPS were identified. Procedures were safely performed, with a pooled complication rate of 2.1% (95% CI: 1.0–3.5) and generally successful, with a pooled success rate of 94.7% (95% CI: 90.8–97.7). The range of procedures performed by residents under MPS was 6.7–72.8 procedures per month (n = 9) compared to 4.3–64.4 procedures (n = 4) without MPS. MPS significantly increased confidence, comfort, and use of appropriate safety measures among residents. CONCLUSION: There are a limited number of published studies on MPS supervised by a board-certified physician in US internal medicine residencies. Procedures performed by MPS are generally successfully completed and safe. MPS benefits internal medicine residents training by improving competency, comfort, and confidence. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11606-020-06526-2.

We report a case of a 31-year-old immunocompetent male who presented with altered mental status and agitation requiring intubation. As sedation was weaned, he demonstrated choreiform movements with associated hemiballismus of the right upper and lower extremities, and he was ultimately diagnosed with cryptococcal meningitis. The patient’s chorea did not terminate after the completion of induction antifungal therapy and all pharmacologic options for the management of chorea were ineffective. He underwent a successful unilateral pallidotomy using standard stereotactic methodology targeting the posterior-ventral pallidum, and his choreiform movements dramatically improved post-operatively within 48 hours.

Objectives Neurocysticercosis (NCC) and human immunodeficiency virus (HIV) have high disease burden and are prevalent in overlapping low- and middle-income areas. Yet, treatment guidance for people living with HIV/AIDS (PLWH/A) co-infected with NCC is currently lacking. This study aims to scope the available literature on HIV/AIDS and NCC co-infection, focusing on epidemiology, clinical characteristics, diagnostics, and treatment outcomes. Methods The scoping literature review methodological framework, and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. A total of 16,969 records identified through database searching and 45 additional records from other sources were reduced to 52 included studies after a standardised selection process. Results Two experimental studies, ten observational studies, 23 case series/case reports and 17 reviews or letters were identified. Observational studies demonstrated similar NCC seroprevalence in PLWH/A and their HIV-negative counterparts. Of 29 PLWH/A and NCC co-infection, 17 (59%) suffered from epileptic seizures, 15 (52%) from headaches, and 15 (52%) had focal neurological deficits. Eighteen (62%) had viable vesicular cysts and six (21%) had calcified cysts. Fifteen (52%) were treated with albendazole, of which 11 (73%) responded well to treatment. Five individuals potentially demonstrated an immune-reconstitution inflammatory syndrome after commencing anti-retroviral therapy, although this was in the absence of immunological and neuroimaging confirmation. Conclusions There is a paucity of evidence to guide treatment of PLWH/A and NCC co-infection. There is a pressing need for high-quality studies in this patient group to appropriately inform diagnostic and management guidelines for HIV-positive patients with NCC.

Rationale: Respiratory support (noninvasive ventilation or high-flow nasal cannula) applied at the time of extubation has been reported to reduce reintubation rates, but concerns regarding effectiveness have limited uptake into practice. Objectives: To determine if providing postextubation respiratory support to all patients undergoing extubation in a medical ICU would decrease the incidence of reintubation. Methods: We conducted a pragmatic, two-armed, cluster–crossover trial of adults undergoing extubation from invasive mechanical ventilation between October 1, 2017, and March 31, 2019, in the medical ICU of an academic medical center. Patients were assigned to either protocolized postextubation respiratory support (a respiratory therapist–driven protocol in which patients with suspected hypercapnia received noninvasive ventilation and patients without suspected hypercapnia received high-flow nasal cannula) or usual care (postextubation management at the discretion of treating clinicians). The primary outcome was reintubation within 96 hours of extubation. Measurements and Main Results: A total of 751 patients were enrolled. Of the 359 patients assigned to protocolized support, 331 (92.2%) received postextubation respiratory support compared with 66 of 392 patients (16.8%) assigned to usual care, a difference driven by differential use of high-flow nasal cannula (74.7% vs. 2.8%). A total of 57 patients (15.9%) in the protocolized support group experienced reintubation compared with 52 patients (13.3%) in the usual care group (odds ratio, 1.23; 95% confidence interval, 0.82 to 1.84; P = 0.32). Conclusions: Among a broad population of critically ill adults undergoing extubation from invasive mechanical ventilation at an academic medical center, protocolized postextubation respiratory support, primarily characterized by an increase in the use of high-flow nasal cannula, did not prevent reintubation compared with usual care. Clinical trial registered with www.clinicaltrials.gov (NCT0328831).

“Smoothing” is the phenomenon by which manually recorded vital signs are less likely to document values at the extremes compared to automated systems [1]. Intermittent manual recording of the peripheral oxygen saturation (SpO2) has the potential to inaccurately reflect patients’ true physiological states. Taenzer et al. observed that manually charted SpO2 values were, on average, 6.5 percentage points greater than SpO2 values collected by automated sampling [2]. Automated high-frequency SpO2 monitoring may more reliably quantify the incidence and severity of hypoxemia and hyperoxemia. Intensive Care Units (ICUs) have traditionally been more vigilant in preventing hypoxemia than hyperoxemia, and ICU patients frequently receive more supplemental oxygen than required to maintain normal values for SpO2 [3]. Hyperoxemia has been associated with worse patient outcomes in observational studies [4, 5], but randomized trials evaluating oxygen saturation targets for mechanically ventilated adults have reported conflicting results [6–9]. These trials have used intermittent recording of SpO2 by study personnel every 4 to 24 h to assess oxygenation. Automating data collection for SpO2 has the potential to facilitate the design, conduct, and analysis of pragmatic clinical trials examining SpO2 targets in mechanically ventilated ICU patients [10]. We developed a technique for automated extraction of large-volume data on SpO2 values for use in a randomized clinical trial. We aimed to quantify the completeness and density of SpO2 data among mechanically ventilated patients. We evaluated the reliability of automated extraction of SpO2 data from pulse oximetry using physician manual review of photoplethysmographic waveforms.

Introduction Age-related immunosenescence influences the presentation of tuberculosis (TB) in older patients. Here, we explore the clinical and radiological presentation of TB in the elderly and the factors associated with time to treatment for TB. Methods This is a retrospective cohort study comparing the clinical, radiological and demographic characteristics of TB patients aged ≥65 years with TB patients aged 18–64 years in a large cohort of TB patients in the UK. Factors associated with the time to presentation and time to treatment were identified using a multivariable analysis model. Results 1023 patients were included in the analyses: 679 patients aged 18–64 years and 344 patients aged ≥65 years. “Classical” symptoms of TB (cough, haemoptysis, fever, nights sweats and weight loss) were less common among older patients with pulmonary TB (PTB) (p, A retrospective UK study in patients with TB shows only age remains an independent predictor of time to treatment in a multivariable model. Older patients have fewer of the “classical” clinical and radiological features of TB compared with younger patients. http://bit.ly/2yHc0OL

IntroductionFollowing extubation from invasive mechanical ventilation, nearly one in seven critically ill adults requires reintubation. Reintubation is independently associated with increased mortality. Postextubation respiratory support (non-invasive ventilation or high-flow nasal cannula applied at the time of extubation) has been reported in small-to-moderate-sized trials to reduce reintubation rates among hypercapnic patients, high-risk patients without hypercapnia and low-risk patients without hypercapnia. It is unknown whether protocolised provision of postextubation respiratory support to every patient undergoing extubation would reduce the overall reintubation rate, compared with usual care.Methods and analysisThe Protocolized Post-Extubation Respiratory Support (PROPER) trial is a pragmatic, cluster cross-over trial being conducted between 1 October 2017 and 31 March 2019 in the medical intensive care unit of Vanderbilt University Medical Center. PROPER compares usual care versus protocolized post-extubation respiratory support (a respiratory therapist-driven protocol that advises the provision of non-invasive ventilation or high-flow nasal cannula based on patient characteristics). For the duration of the trial, the unit is divided into two clusters. One cluster receives protocolised support and the other receives usual care. Each cluster crosses over between treatment group assignments every 3 months. All adults undergoing extubation from invasive mechanical ventilation are enrolled except those who received less than 12 hours of mechanical ventilation, have ‘Do Not Intubate’ orders, or have been previously reintubated during the hospitalisation. The anticipated enrolment is approximately 630 patients. The primary outcome is reintubation within 96 hours of extubation.Ethics and disseminationThe trial was approved by the Vanderbilt Institutional Review Board. The results will be submitted for publication in a peer-reviewed journal and presented at one or more scientific conferences.Trial registration numberNCT03288311.

ObjectiveThe aim of this study was to determine the occurrence of latent tuberculosis infections (LTBI) and active TB in a cohort of patients with inflammatory bowel disease (IBD) treated with biologics. We also examined the effects of immunosuppressive drugs on indeterminate interferon-gamma release assays (IGRA) in LTBI screening.DesignRetrospective study of patients treated with biologics between March 2007 and November 2015.SettingSt Mark’s Hospital, North West London, UK.Patients732 patients with IBD who were screened for LTBI using either tuberculin skin test or IGRA before starting a biologic treatment.MethodsRetrospective case note review of all patients with IBD who were screened for LTBI prior to initiating biologics. Patients who developed active TB were identified from the London TB register.ResultsOf 732 patients with IBD, 31 (4.2%) were diagnosed with and treated for LTBI with no significant side effects. Six of 596 patients (1.0%) who received biologic treatment developed active TB. There was a higher proportion of indeterminate IGRA in the immunosuppressive medication group compared with the non-immunosuppressive group (33% (59/181) compared with 9% (6/66), pConclusionsThis study highlights the challenges of LTBI screening prior to commencing biologic therapy and demonstrates the risk of TB in patients who have been screened and who are receiving prolonged and continuing doses of antitumour necrosis factor.

Background: Scientific literature can contain errors. Discrepancies, defined as two or more statements or results that cannot both be true, may be a signal of problems with a trial report. In this study, we report how many discrepancies are detected by a large panel of readers examining a trial report containing a large number of discrepancies. Methods: We approached a convenience sample of 343 journal readers in seven countries, and invited them in person to participate in a study. They were asked to examine the tables and figures of one published article for discrepancies. 260 participants agreed, ranging from medical students to professors. The discrepancies they identified were tabulated and counted. There were 39 different discrepancies identified. We evaluated the probability of discrepancy identification, and whether more time spent or greater participant experience as academic authors improved the ability to detect discrepancies. Results: Overall, 95.3% of discrepancies were missed. Most participants (62%) were unable to find any discrepancies. Only 11.5% noticed more than 10% of the discrepancies. More discrepancies were noted by participants who spent more time on the task (Spearman’s ρ = 0.22, P

Empyema necessitans is a complication of a pleural space infection that dissects through the pleura into the soft tissues of the chest and skin. Due to the widespread availability of antibiotics, empyema necessitans is rare in modern medicine and is most commonly caused by Mycobacterium tuberculosis. A 38-year-old immunocompetent male presented with left shoulder pain and his chest radiograph revealed a mass in the upper left lung and/or pleural space. He underwent multiple debridements of the chest wall due to a left chest wall abscess and empyema necessitans. All operative samples were positive for Streptococcus agalactiae (Group B Streptococcus). The patient's clinical course was complicated by septic arthritis of the left sternoclavicular joint and first rib, vertebral osteomyelitis/discitis, and pelvic abscesses. This case report illustrates the pathogenic process of empyema necessitans and summarizes the clinical management for practicing clinicians. It also documents the second case of Streptococcus agalactiae-associated empyema necessitans, to our knowledge, with significantly greater disease extension than the first.

IntroductionCauda equina syndrome (CES) is a neurosurgical emergency. Early diagnosis with MRI and subsequent surgical decompression surgery can prevent permanent neurological dysfunction. Charing Cross Hospital (CXH) is a tertiary neurosurgical referral centre where in the emergency department (ED), current practice mandated a neurosurgery review prior to requesting MRI.HypothesisIt was hypothesised that a new clinical pathway, with better coordination from the ED, radiology and neurosurgical teams could reduce the time of presentation to diagnosis or exclusion of CES.MethodRetrospective case-note analysis of patients presenting with back pain to CXH ED over a 3-month period was performed. The primary outcome was the time interval between the patient’s arrival to the ED and the MRI preliminary report.ResultsThe baseline primary outcome was recorded at 8 hours and 16 min (n=30). A new clinical pathway was designed empowering ED senior decision makers to order MRIs prior to neurosurgical review. Two Plan-Do-Study-Act (PDSA) cycles were performed, each measured over a 2-month period. The first PDSA cycle was performed after the pathway was initially launched (n=17), while the second PDSA cycle measured the effect of staff education and active promotion of the pathway (n=17). MRI was requested earlier, waiting and reporting time for MRI were reduced. The exclusion or diagnosis of CES was reduced to 5 hours and 54 min in PDSA 1 and 5 hours 17 min in PDSA 2, a 29% and 36% reduction (p=0.048 and p=0.012, respectively).ConclusionThe clinical protocol was a cost-neutral and sustainable intervention that effectively reduced the time taken to diagnose or exclude CES and ED waiting times.

ATP is a known mediator of inflammatory and neuropathic pain. However, the mechanisms by which specific purinergic receptors contribute to chronic pain states are still poorly characterized. Here, we demonstrate that in response to peripheral nerve injury, P2X4receptors (P2X4R) are expressedde novoby activated microglia in the spinal cord. Usingin vitroandin vivomodels, we provide direct evidence that P2X4R stimulation leads to the release of BDNF from activated microglia and, most likely phosphorylation of the NR1 subunit of NMDA receptors in dorsal horn neurons of the spinal cord. Consistent with these findings, P2X4-deficient mice lack mechanical hyperalgesia induced by peripheral nerve injury and display impaired BDNF signaling in the spinal cord. Furthermore, ATP stimulation is unable to stimulate BDNF release from P2X4-deficient mice microglia in primary cultures. These results indicate that P2X4R contribute to chronic pain through a central inflammatory pathway. P2X4R might thus represent a potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain injury and neurodegenerative disorders.

This is a study of the HTLV-1/HIV-1 co-infected cohort attending the National Centre for Human Retrovirology in London. In a retrospective analysis, demographic, clinical, immunological and virological data was described. A subgroup of patients on combination antiretroviral therapy (cART) for more than twelve months with an undetectable HIV viral load was identified and compared to a matched HTLV-1 mono-infected group. In an ongoing prospective study, HIV-1 mono-infected patients are recruited and virological and immunological parameters compared to the HTLV/HIV co-infected and HTLV mono-infected groups. 29 co-infected patients had a median age of 47 years, 55% were male and over 80% identified with the Black – African/Caribbean ethnic group. The median time on cART was 6 months. CD4/CD4% cell counts were lower in stable co-infected patients (508 cells/mm3, p=0.2 / 27.9%, p

Cell fusion is a central phenomenon during the immune response that leads to formation of large elements called multinucleated giant cells (MGCs) of common occurrence at sites of granulomatous inflammation. We have previously reported on the involvement in this event of a novel receptor expressed to high level by mononuclear phagocytes, the purinergic P2X7receptor. Herein, we show that blockade of this receptor by a specific monoclonal antibody prevents fusion in vitro. In contrast, cell fusion is stimulated by addition of enzymes that destroy extracellular ATP (i.e., apyrase or hexokinase). Experiments performed with phagocytes selected for high (P2X7hyper) or low (P2X7hypo) P2X7expression show that fusion only occurs between P2X7hyper/P2X7hyper and not between P2X7hyper/P2X7hypo or P2X7hypo/P2X7hypo. During MGCs formation we detected activation of caspase 3, an enzyme that is powerfully stimulated by P2X7. Finally, we observed that during MGCs formation, the P2X7receptor is preferentially localized at sites of cell-to-cell contact. These findings support the hypothesis originally put forward by our group that the P2X7receptor participates in multinucleated giant cell formation.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2′,3′-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X 1 (or P2X 3 ) receptors had strong desensitization, whereas currents in cells expressing P2X 2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X 1 receptor of either one of two 34-residue segments from the P2X 2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X 2 receptor only by providing both these segments of the P2X 1 (or P2X 3 ) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

In the immune system, apoptosis is involved in intrathymic elimination of self-reactive thymocytes and in peripheral T cell tolerance to exogenous antigens. Here, we describe the role in T cell apoptosis of P(2x1), a nonselective cation channel activated by ATP. P(2X1) molecules are up-regulated in thymocytes during dexamethasone-induced apoptosis, and antagonists to these receptors protect thymocytes from cell death. Moreover, P(2X1) mRNA and protein levels increase in thymocytes induced to die in vivo by the superantigen staphylococcal enterotoxin B. In contrast, T cells undergoing apoptosis in the periphery do not express P(2X1). The demonstration that P(2X1) ion channels play a role in the apoptosis of thymocytes but not peripheral T cells illustrates a novel mechanism contributing to thymocyte cell death and opens new possibilities for investigating clonal deletion in the thymus.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.

1. The binding of [3H]-alpha beta meATP, [35s]-ATP gamma S and [alpha 33P]-ATP to a human bladder P2X purinoceptor, transiently expressed in CHO-K1 cells using the Semliki Forest Virus (SFV) expression system, was examined. The characteristics of the binding sites were compared with results obtained in rat vas deferens, a tissue in which the radioligands are thought to label P2X purinoceptors and in which the endogenous P2X purinoceptor displays high homology with the human bladder P2X purinoceptor. 2. In non-infected CHO-K1 cells, 100 microM ATP evoked only small inward currents (40 pA) in approximately 30% of the cells when studied by the whole-cell voltage clamp technique. In membranes prepared from either these non-infected cells or cells infected with SFV containing the LacZ gene (SFV-LacZ), [3H]-alpha beta meATP bound with low affinity (pKd = 7.04; Bmax = 8.88 pmol ml-1 protein) and there was only a low density of [35S]-ATP gamma S binding sites (pKd = 8.74; Bmax = 358 fmol ml-1 protein). These binding sites differed from those present in rat vas deferens. Thus, pIC50 values for alpha beta meATP (6.5) and L-beta gamma meATP (4.0) at the [3H]-alpha beta meATP binding sites in non-infected CHO-K1 cells were much lower than the respective pIC50 values of 8.3 and 7.7, determined in rat vas deferens. Similarly, affinity estimates (pIC50 values) for ATP (6.82), 2-meS-ATP (5.43), ATP gamma S (7.06) and alpha beta meATP (4.84) at the [35S]-ATP gamma S binding sites in non-infected CHO-K1 cells were up to 2291 fold lower than the respective values of 9.01, 8.79, 8.73 and 7.57, determined in rat vas deferens. 3. In CHO-K1 cells infected using SFV containing the cDNA for the human bladder P2X purinoceptor (SFV-h.P2X), ATP, 2-meS-ATP and alpha beta meATP evoked large inward currents (2-7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV-h.P2X infected cells, [3H]-alpha beta meATP binding was increased, compared to that measured in the non infected or SFV-LacZ infected cells, with only high affinity [3H]-alpha beta meATP binding sites being detected (pKd = 9.21; Bmax = 3.54 pmol mg-1 protein). The pIC50 values for alpha beta meATP (8.2) and L-beta gamma meATP (7.2) in competing for these sites were the same or similar to the values determined in rat vas deferens. 4. A high density of [35H]-ATP gamma S binding sites (pKd = 9.09; Bmax = 6.82 pmol mg-1 protein) was also present in the membranes from CHO-K1 cells infected with SFV-h.P2X and affinity estimates (pIC50 values) for ATP (8.93), 2-meS-ATP (8.23), ATP gamma S (8.08), and alpha beta meATP (7.17) at competing for these sites were as much as 631 fold higher than the respective values determined in non-infected CHO-K1 cells but were close to the values determined in rat vas deferens. Similar data were obtained with [alpha 33P]-ATP as radioligand. 5. These data suggest that [3H]-alpha beta meATP, [35S]-ATP gamma S and [33P]-ATP label the human bladder recombinant P2X purinoceptor expressed in CHO-K1 cells following infection with SFV-h.P2X and provide further corroborative evidence to support the contention that the high affinity binding sites for these radioligands in rat vas deferens are P2X purinoceptors.

Cation-selective P2X receptor channels were first described in sensory neurons where they are important for primary afferent neurotransmission and nociception. Here we report the cloning of a complementary DNA (P2X3) from rat dorsal root ganglia that had properties dissimilar to those of sensory neurons. We also found RNA for (P2X1)(ref. 7), (P2X2)(ref. 8) and P2X4 (ref. 9) in sensory neurons; channels expressed from individual cDNAs did not reproduce those of sensory ganglia. Coexpression of P2X3 with P2X2, but not other combinations, yielded ATP-activated currents that closely resembled those in sensory neurons. These properties could not be accounted for by addition of the two sets of channels, indicating that a new channel had formed by subunit heteropolymerization. Although in some tissues responses to ATP can be accounted for by homomeric channels, our results indicate that ATP-gated channels of sensory neurons may form by a specific heteropolymerization of P2X receptor subunits.

Introduction and objectives In 2013, Public Health England (PHE) reported that 39% of pulmonary tuberculosis (TB) patients >65 years old started treatment >4 months after symptom onset compared to 25% of patients aged 15–44 years. A longer symptomatic period, particular if smear positive, may result in increased transmission and poorer treatment outcomes. We investigated age-related differences in symptom duration and time to starting TB treatment in a large UK cohort of TB patients. Methods The cohort comprised patients on the London TB register (LTBR) who were treated at Northwick Park Hospital between 2002–2015. Patients aged >65 years were compared with a random sample of patients aged 18–40 years, with respect to symptoms, symptom duration at presenting to secondary care and time to starting treatment after presenting to secondary care. Results 357 patients over 65 and 517 younger patients were identified. Demographics for the total number of patients are included in Table 1. Data were available for 489 of the younger group and 73 of the older group. 26 of the >65s (35.6%) and 9 (1.8%) of the 65s were diabetic. Conclusions This study has identified that a potential cause for a delay in diagnosis in the elderly may be related to the decreased frequency of ‘classical’ TB symptoms in those >65 years of age. Clinicians need to be vigilant despite the lack of these symptoms. Median duration of symptoms was twice that in older patients versus younger patients (90 days versus 45) with a longer time to starting treatment (7 days versus 2). The total duration (in months) from symptom onset was also higher at 3 months versus 2 months. A higher proportion of older patients (32%) started treatment greater than 4 months from symptom onset compared to 13.1% in younger patients. Biases in this work including recording bias due to the retrospective nature of symptom recording, the effect of selecting younger patients from more recent years (to improve data availability) and the low proportion of patients over 65 which had symptom and treatment data available.

Introduction and objectives It has been suggested that TB has a different phenotype in older patients with age-related changes to the cell-mediated immune response and co-existent organ dysfunction. Older patients with tuberculosis (TB) may have different radiographic features than younger patients; this may lead to less immediate suspicion of TB resulting in delays to diagnosis and starting treatment. We wanted to identify if there are differences in the most common radiological differences in older and younger patients with pulmonary TB (PTB). Methods Patients with PTB > 65 were noted from the London TB register between 2002 and 2015. A random selection of younger patients aged 18–40 with PTB were also identified. All available chest x-ray (CXR) reports were obtained from online radiology systems. CXR features were classified according to reported features with particular note of cavitation, nodules and miliary changes, consolidation, lymphadenopathy and effusions. Results The CXR reports of 239 patients with PTB 65 were collated. Demographic details as well as CXR changes are detailed in Table 1. Cavitation, lymphadenopathy and effusions were more common in younger patients whereas consolidation was more evident in older patients. Upper zone involvement was similar in both groups. Conclusions Studies by other groups have suggested a higher proportion of cavitation and upper zone changes in younger patients with TB with less specific changes in older patients. This may lead to less suspicion of TB and potentially a longer infective period; this is important given that 23% and 19% of younger and older patients have smear positive PTB. In our study, the proportion with upper zone changes are similar though cavitation is more frequent in younger patients. Of note, is the much higher presence of lymphadenopathy and effusions seen in younger patients. This may potentially be related to differences in the immune function of both groups or primary infection versus reactivation. These findings re-enforce the need for clinical suspicion for PTB in both older and younger patients with both specific and non-specific radiographic changes.

Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.

Human neurokinin-1 receptor cDNA was introduced into the pSFV1 Semliki Forest virus (SFV) vector and the in vitro transcribed RNA was electroporated into BHK cells with pSFV-Helper RNA. This procedure resulted in the packaging of a high-titer SFV-NK-1 virus stock containing approximately 5 x 10(9) infective units/ml. Infection of baby hamster kidney, COS-7, Chinese hamster ovary and human osteosarcoma cells yielded high levels of human neurokinin-1 receptor expression as assessed by [3H]substance P binding. The maximal receptor expression level obtained was 4 x 10(6) receptors/cell and studies of the post-infection time indicated that a high level of receptor expression was observed 10-24 h post-infection. The human neurokinin-1 receptor expressed in infected baby hamster kidney, COS-7 and Chinese hamster ovary cells was able to stimulate Ca2+ mobilization indicating functional coupling to guanine-nucleotide-binding proteins. The application of the Semliki Forest virus expression system will permit the rapid and efficient production of large quantities of receptor protein for both pharmacological and structural studies.

The complete amino acid sequence of the human neurokinin-3 receptor was deduced by DNA sequence analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobic regions with maximum divergence occuring at the amino- and carboxy-termini. The position of intron-exon junctions are identical to those in other reported neurokinin genes. Using a chimeric genomic-cDNA gene, the human NK-3 receptor was expressed in Xenopus laevis oocytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the gene in mammalian cells resulted in detection of receptor binding as well as neurokinin-stimulated calcium mobilization and arachidonic acid release, all displaying the pharmacological characteristics expected of a neurokinin-3 receptor, By using the polymerase chain reaction we have shown that mRNA for the human neurokinin-3 receptor is expressed predominantly in the central nervous system.

Introduction and objectives Tuberculosis (TB) in older persons presents challenges related to diagnosis, management, comorbidities and polypharmacy potentially contributing to increased morbidity and mortality. This retrospective cohort review compares the baseline characteristics, diagnosis, management and outcome between older patients (OPs) (over 65 years) and younger patients (YPs) (25–35 years.) Method All patients ≥65 years treated at Northwick Park Hospital during 2002–2014 were identified from London TB register; a comparison group of patients aged 25–35 years were randomly selected. Clinical, microbiological, radiological and biochemical parameters together with management and outcomes were obtained from electronic records. Characteristics of patients were compared between the two groups using Chi-squared and Kruskal-Wallis tests; analyses were performed using Stata (Stata Corp. 2013). Results The comparison groups comprised 313 patients aged ≥65 years and 339 patients aged 25–35. Demographics, site of disease, TB culture, treatment regimens and outcomes are recorded in Table 1. 35.6% of OPs and 29.6% of YPs were symptomatic for >2 months at review in secondary care. Median duration to starting treatment from review was 17 days (IQR: 4–57) in OPs compared to 2 (IQR: 1–19) in YPs (p = 0.001). 44.8% of OPs experienced drug toxicity compared to 27.3% of YPs (p = 10 days (p = Conclusion These data characterise the delays in presentation and treatment initiation in older patients who also experience a more complicated treatment course with an increased side effect profile, more variation from standard quadruple therapy, lower completion rates and poorer outcomes. This, together with longer inpatient stays impacts patients, but also has financial implications for services.

The efficacy of treatments used for patients with HTLV-1 associated myelopathy/tropical spastic paraperesis (HAM/TSP) is uncertain although corticosteroids are widely prescribed. The effect of pulsed IV methylprednisolone was retrospectively analysed in an open cohort of 26 patients. 1g IV methylprednisolone was infused on three consecutive days. The outcomes were pain, gait, urinary frequency and nocturia, a range of inflammatory markers and HTLV-1 proviral load. A plasma cytokine profile was conducted in nine patients and correlated to gait and pain. Significant improvements in pain and 10m timed walk were observed immediately after the 3rd infusion and maintained, for pain, for up to six months. A shorter duration of disease was strongly correlated with improvement in the 10m timed walk (p=0.05) but not with the reduction in pain. Although baseline cytokine concentrations did not correlate to baseline pain or gait impairment a decrease in tumour necrosis factor-alpha (TNF-α) concentration after pulsed methylprednisolone was associated with improvements in both. Pulsed IV methylprednisolone significantly reduced pain in patients with HAM/TSP and was associated with a transient improvement in gait that may be related to a reduction in TNF-α concentration.

P2X4purinergic receptors are calcium-permeable, ATP-activated ion channels. In the CA1 area of the hippocampus, they are located at the subsynaptic membrane somewhat peripherally to AMPA receptors. The possible role of P2X4receptors has been difficult to elucidate because of the lack of selective antagonists. Here we report the generation of a P2X4receptor knock-out mouse and show that long-term potentiation (LTP) at Schaffer collateral synapses is reduced relative to that in wild-type mice. Ivermectin, which selectively potentiates currents at P2X4, was found to increase LTP in wild-type mice but had no effect in P2X4knock-out mice. We suggest that calcium entry through subsynaptic P2X4receptors during high-frequency stimulation contributes to synaptic strengthening.

P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.

Human leukocytes can express the P2X(7) purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X(7) cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X(7) transfectants is abolished by the P2X(7) receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X(7)-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X -S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.

P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X2 receptor splice variant (P2X2-2) carrying a 207 bp deletion in the intracellular C-terminus and the analysis of the corresponding genomic structure of the P2X2 gene. P2X2-2 is as highly expressed as the original P2X2 sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X2 or P2X2-2 receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X2 isoforms. © 1997 Federation of European Biochemical Societies.