Effects on cerebral blood flow after single doses of the ß2 agonist, clenbuterol, in healthy volunteers and patients with mild cognitive impairment or Parkinson's disease.

AIMS: Cerebral hypometabolism occurs years prior to a diagnosis of neurodegenerative diseases and coincides with reduced cerebral perfusion and declining noradrenergic transmission from the locus coeruleus. In pre-clinical models, ß-adrenoceptor (ß-AR) agonists increase cerebrocortical glucose metabolism, and may have therapeutic potential for neurodegenerative diseases. This study investigated the safety and effects on regional cerebral blood flow (rCBF) of the oral, brain-penetrant ß2-AR agonist, clenbuterol, in healthy volunteers (HV) and patients with mild cognitive impairment (MCI) or Parkinson's disease (PD). METHODS: This study evaluated the safety and effects on cerebral activity of the oral, brain-penetrant, ß2-AR agonist clenbuterol (20-160 µg) in healthy volunteers and patients with MCI or PD. Regional CBF, which is tightly coupled to glucose metabolism, was measured by arterial spin labelling MRI in 32 subjects (25 HV and 8 MCI or PD) across five cohorts. In some cohorts, low doses of nadolol (1-5 mg), a ß-AR antagonist with minimal brain penetration, were administered with clenbuterol to control peripheral ß2-AR responses. RESULTS: Significant, dose-dependent increases in rCBF were seen in multiple brain regions, including hippocampus, amygdala and thalamus, following the administration of clenbuterol to HVs (mean changes from baseline in hippocampal rCBF of -1.7%, 7.3%, 22.9%, 28.4% 3 h after 20, 40, 80 and 160 µg clenbuterol, respectively). In patients with MCI or PD, increases in rCBF following 80 µg clenbuterol were observed both without and with 5 mg nadolol (in hippocampus, 18.6%/13.7% without/with nadolol). Clenbuterol was safe and well-tolerated in all subjects; known side effects of ß2-agonists, including increased heart rate and tremor, were mild in intensity and were blocked by low-dose nadolol. CONCLUSIONS: The effects of clenbuterol on rCBF were evident both in the absence and presence of low-dose nadolol, suggesting central nervous system (CNS) involvement. Concomitant inhibition of the peripheral effects of clenbuterol by nadolol confirms that meaningful ß2-AR antagonism in the periphery was achieved without interrupting the central effects of clenbuterol on rCBF.

Single- and Multiple-Dose Pharmacokinetics of Gefapixant (MK-7264), a P2X3 Receptor Antagonist, in Healthy Adults.

Gefapixant (MK-7264, RO4926219, AF-219) is a first-in-class P2X3 antagonists being developed to treat refractory or unexplained chronic cough. The initial single- and multiple-dose safety, tolerability, and pharmacokinetics of gefapixant at doses ranging from 7.5 to 1800 mg were assessed in four clinical trials. Following single-dose administration of 10-450 mg, the pharmacokinetic (PK) profile of gefapixant in plasma and urine demonstrated low inter-subject variability and a dose-proportional exposure. Following administration of multiple doses twice daily, the plasma exposures were dose-proportional at doses ranging from 7.5 to 50 mg and less than dose-proportional at doses ranging from 100 to 1800 mg. The time to mean peak drug concentration ranged from 2 to 3 h post-dose, and steady state was achieved by 7 days after dosing, with an accumulation ratio of approximately 2, comparing data from day 1 to steady state. The mean apparent terminal half-life ranged from 8.2 to 9.6 h. Gefapixant was primarily excreted unmodified in urine. Gefapixant was well tolerated following single-dose administration up to 1800 mg and multiple doses up to 1800 mg twice daily; there were no serious adverse events (AEs) reported. The most common AE reported was dysgeusia. The PK profile supports a twice-daily dosing regimen.

Fingerprinting heterocellular ß-adrenoceptor functional expression in the brain using agonist activity profiles.

Noradrenergic projections from the brainstem locus coeruleus drive arousal, attentiveness, mood, and memory, but specific adrenoceptor (AR) function across the varied brain cell types has not been extensively characterized, especially with agonists. This study reports a pharmacological analysis of brain AR function, offering insights for innovative therapeutic interventions that might serve to compensate for locus coeruleus decline, known to develop in the earliest phases of neurodegenerative diseases. First, ß-AR agonist activities were measured in recombinant cell systems and compared with those of isoprenaline to generate Δlog(Emax/EC50) values, system-independent metrics of agonist activity, that, in turn, provide receptor subtype fingerprints. These fingerprints were then used to assess receptor subtype expression across human brain cell systems and compared with Δlog(Emax/EC50) values arising from ß-arrestin activation or measurements of cAMP response desensitization to assess the possibility of ligand bias among ß-AR agonists. Agonist activity profiles were confirmed to be system-independent and, in particular, revealed ß2-AR functional expression across several human brain cell types. Broad ß2-AR function observed is consistent with noradrenergic tone arising from the locus coeruleus exerting heterocellular neuroexcitatory and homeostatic influence. Notably, Δlog(Emax/EC50) measurements suggest that tested ß-AR agonists do not show ligand bias as it pertains to homologous receptor desensitization in the system examined. Δlog(Emax/EC50) agonist fingerprinting is a powerful means of assessing receptor subtype expression regardless of receptor expression levels or assay readout, and the method may be applicable to future use for novel ligands and tissues expressing any receptor with available reference agonists.

Prevalence and distribution of Plasmodium vivax Duffy Binding Protein gene duplications in Sudan.

Plasmodium vivax Duffy Binding Protein (PvDBP) is essential for interacting with Duffy antigen receptor for chemokines (DARC) on the surface of red blood cells to allow invasion. Earlier whole genome sequence analyses provided evidence for the duplications of PvDBP. It is unclear whether PvDBP duplications play a role in recent increase of P. vivax in Sudan and in Duffy-negative individuals. In this study, the prevalence and type of PvDBP duplications, and its relationship to demographic and clinical features were investigated. A total of 200 malaria-suspected blood samples were collected from health facilities in Khartoum, River Nile, and Al-Obied. Among them, 145 were confirmed to be P. vivax, and 43 (29.7%) had more than one PvDBP copies with up to four copies being detected. Both the Malagasy and Cambodian types of PvDBP duplication were detected. No significant difference was observed between the two types of duplications between Duffy groups. Parasitemia was significantly higher in samples with the Malagasy-type than those without duplications. No significant difference was observed in PvDBP duplication prevalence and copy number among study sites. The functional significance of PvDBP duplications, especially those Malagasy-type that associated with higher parasitemia, merit further investigations.

Locus Coeruleus and Noradrenergic Pharmacology in Neurodegenerative Disease.

Adrenoceptors (ARs) throughout the brain are stimulated by noradrenaline originating mostly from neurons of the locus coeruleus, a brainstem nucleus that is ostensibly the earliest to show detectable pathology in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. The α1-AR, α2-AR, and ß-AR subtypes expressed in target brain regions and on a range of cell populations define the physiological responses to noradrenaline, which includes activation of cognitive function in addition to modulation of neurometabolism, cerebral blood flow, and neuroinflammation. As these heterocellular functions are critical for maintaining brain homeostasis and neuronal health, combating the loss of noradrenergic tone from locus coeruleus degeneration may therefore be an effective treatment for both cognitive symptoms and disease modification in neurodegenerative indications. Two pharmacologic approaches are receiving attention in recent clinical studies: preserving noradrenaline levels (e.g., via reuptake inhibition) and direct activation of target adrenoceptors. Here, we review the expression and role of adrenoceptors in the brain, the preclinical studies which demonstrate that adrenergic stimulation can support cognitive function and cerebral health by reversing the effects of noradrenaline depletion, and the human data provided by pharmacoepidemiologic analyses and clinical trials which together identify adrenoceptors as promising targets for the treatment of neurodegenerative disease.

Gut microbiome immaturity and childhood acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Here, we map emerging evidence suggesting that children with ALL at the time of diagnosis may have a delayed maturation of the gut microbiome compared with healthy children. This finding may be associated with early-life epidemiological factors previously identified as risk indicators for childhood ALL, including caesarean section birth, diminished breast feeding and paucity of social contacts. The consistently observed deficiency in short-chain fatty-acid-producing bacterial taxa in children with ALL has the potential to promote dysregulated immune responses and to, ultimately, increase the risk of transformation of preleukaemic clones in response to common infectious triggers. These data endorse the concept that a microbiome deficit in early life may contribute to the development of the major subtypes of childhood ALL and encourage the notion of risk-reducing microbiome-targeted intervention in the future.

P2X3 receptor antagonism attenuates the progression of heart failure.

Despite advances in the treatment of heart failure, prognosis is poor, mortality high and there remains no cure. Heart failure is associated with reduced cardiac pump function, autonomic dysregulation, systemic inflammation and sleep-disordered breathing; these morbidities are exacerbated by peripheral chemoreceptor dysfunction. We reveal that in heart failure the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats. Purinergic (P2X3) receptors were upregulated two-fold in peripheral chemosensory afferents in heart failure, and when antagonized abolished these episodic discharges, normalized both peripheral chemoreceptor sensitivity and the breathing pattern, reinstated autonomic balance, improved cardiac function, and reduced both inflammation and biomarkers of cardiac failure. Aberrant ATP transmission in the carotid body triggers episodic discharges that via P2X3 receptors play a crucial role in the progression of heart failure and as such offer a distinct therapeutic angle to reverse multiple components of its pathogenesis.

A Randomized, Placebo-Controlled Study to Investigate the Safety, Tolerability, and Pharmacokinetics of 3 Weeks of Orally Administered Gefapixant in Healthy Younger and Older Adults.

PURPOSE: Patients with chronic cough are typically female and have a mean age of ~ 60 years. However, initial pharmacokinetic (PK) characterization of the P2X3-receptor antagonist gefapixant, developed to treat refractory or unexplained chronic cough, was performed in healthy participants who were predominantly younger adult males. The objective of this Phase 1 study was to assess the safety, tolerability, and PK of gefapixant in younger (18-55 years) and older (65-80 years) males and females. METHODS: A randomized, double-blind, placebo-controlled study was conducted. Healthy adult participants were stratified into 4 cohorts by age and sex (younger males/females and older males/females) and randomized 4:1 (younger adults) or 3:1 (older adults) to receive gefapixant 300 mg twice daily (BID) for 1 week, followed by gefapixant 600 mg BID for 2 weeks or placebo. Safety, tolerability, and PK were assessed. RESULTS: Of 36 randomized and treated participants, 28 (100%) receiving gefapixant and 6 (75%) receiving placebo reported ≥ 1 adverse event (AE). The most common treatment-related AEs in the gefapixant group were taste related. Predefined renal/urologic AEs were reported by 7 (25%) participants receiving gefapixant (all mild to moderate in severity). Gefapixant exposure was generally lower in younger males compared with younger females and older adults; however, differences may have been due to estimated glomerular filtration rate. CONCLUSION: The safety profile of gefapixant 300-600 mg BID was generally consistent with previous studies. Additional characterization of gefapixant PK as a function of age and sex using population PK modeling is warranted.

Germline allelic expression of genes at 17q22 locus associates with risk of breast cancer.

INTRODUCTION: Translation of genome-wide association study (GWAS) findings into preventive approaches is challenged by the identification of the causal risk variants and the understanding of the biological mechanisms by which they act. We present using allelic expression (AE) ratios to perform quantitative case-control analysis as a novel approach to identify risk associations, causal regulatory variants, and target genes. METHODS: Using the breast cancer (BC) risk locus 17q22 to validate this approach, we measured AE ratios in normal breast tissue samples from controls and cases, as well as from unmatched blood samples. Then we used in-silico and in-vitro analysis to map and functionally characterised candidate causal variants. RESULTS: We found a significant shift in the AE patterns of STXBP4 (rs2628315) and COX11 (rs17817901) in the normal breast tissue of cases and healthy controls. Preferential expression of the G-rs2628315 and A-rs17817901 alleles, more often observed in cases, was associated with an increased risk for BC. Analysis of blood samples from cases and controls found a similar association. Furthermore, we identified two putative cis-regulatory variants - rs17817901 and rs8066588 - that affect a miRNA and a transcription factor binding site, respectively. CONCLUSION: We propose causal variants and target genes for the 17q22 BC risk locus and show that using AE ratios in case-control association studies is helpful in identifying risk and mapping causal variants.

Clinical Formulation Bridging of Gefapixant, a P2X3-Receptor Antagonist, for the Treatment of Chronic Cough.

Gefapixant is a P2X3-receptor antagonist being developed for treatment of refractory or unexplained chronic cough. Four phase 1 studies were conducted in healthy participants that bridged the early-phase gefapixant formulation (F01) to the phase 3 (F04A) and intended commercial (F04B) formulations. In addition, food and proton pump inhibitor (PPI) coadministration effects on gefapixant exposure were assessed. The gefapixant free base formulation (F01) was used in the initial early-phase clinical studies. Adding citric acid to the F01 formulation (to generate F02) enhanced drug solubilization, resulting in similar bioavailability and mitigating food and gastric pH effects. The subsequently developed gefapixant citrate salt formulation (F04) achieved exposures that were comparable to F02 in the fed state (90%CIs of geometric mean ratios for area under the plasma concentration-time curve from time 0 extrapolated to infinity and maximum observed concentration were within 0.80 and 1.25) and were not meaningfully affected by food or PPIs (90%CIs of geometric mean ratios for area under the plasma concentration-time curve from time 0 extrapolated to infinity and maximum observed concentration were within 0.80 and 1.25). Minor compositional changes were made to generate the F04A and F04B formulations. In vitro dissolution studies were used to bridge F04 to F04A, and clinical bioequivalence was then established between F04A and F04B. These data support use of the proposed commercial gefapixant formulation without significant food and PPI effects.

The discovery and development of gefapixant.

Gefapixant is the approved generic name for a compound also known as MK-7264, and prior to that AF-219 and RO-4926219. It is the first-in-class clinically developed antagonist for the P2X3 subtype of trimeric ionotropic purinergic receptors, a family of ATP-gated excitatory ion channels, showing nanomolar potency for the human P2X3 homotrimeric channel and essentially no activity at related channels devoid of P2X3 subunits. As the first P2X3 antagonist to have progressed into clinical studies it has now progressed to the point of successful completion of Phase 3 investigations for the treatment of cough, and the NDA application is under review with US FDA for treatment of refractory chronic cough or unexplained chronic cough. The molecule was discovered in the laboratories of Roche Pharmaceuticals in Palo Alto, California, but clinical development then continued with the formation of Afferent Pharmaceuticals for the purpose of identifying the optimal therapeutic indication for this novel mechanism and establishing a clinical plan for development in the optimal patient populations selected. Geoff Burnstock was a close collaborator and advisor to the P2X3 program for close to two decades of discovery and development. Progression of gefapixant through later stage clinical studies has been conducted by the research laboratories of Merck & Co., Inc., Kenilworth, NJ, USA (MRL; following acquisition of Afferent in 2016), who may commercialize the product once authorization has been granted by regulatory authorities.

Treatment of Persistent Cough in Subjects with Idiopathic Pulmonary Fibrosis (IPF) with Gefapixant, a P2X3 Antagonist, in a Randomized, Placebo-Controlled Clinical Trial.

INTRODUCTION: Chronic cough is a highly problematic symptom for patients with idiopathic pulmonary fibrosis (IPF); limited therapeutic options are available. We evaluated gefapixant, a P2X3 receptor antagonist, for the treatment of chronic cough in IPF. METHODS: This randomized, double-blind, placebo-controlled, crossover study included subjects with IPF. Sequence A included gefapixant 50 mg BID (period 1; 14 days) followed by placebo (period 2; 14 days); sequence B had the opposite sequence of treatments. This regimen was specified in a protocol amendment that modified the original active treatment regimen of gefapixant 50 mg BID for 10 days and 150 mg BID for 4 days. Patients randomized to the original treatment regimen were excluded from efficacy analyses but included in safety assessments. The primary efficacy endpoint was change from baseline in awake cough frequency (coughs/hour) from periods 1 and 2 combined. Adverse events (AEs) were monitored throughout the study. RESULTS: A total of 51 subjects were randomized, 44 of whom were randomized to treatment sequences evaluated in the primary efficacy analysis (i.e., 22 subjects in sequence A and 22 subjects in sequence B); seven subjects received the treatment assigned before the protocol amendment and were excluded from efficacy analyses. The change from baseline in awake cough frequency from periods 1 and 2 combined (mixed model for repeated measures analysis) did not demonstrate a significant reduction versus placebo in cough at day 14 (p = 0.90); in a post hoc analysis of log-transformed data p value for reduction versus placebo at day 14 was 0.07. The most common AEs were related to taste (dysgeusia and ageusia). CONCLUSIONS: Gefapixant was generally well tolerated but was not associated with a significant improvement in chronic cough in subjects with IPF as defined by the primary endpoint in this study. TRIAL REGISTRATION: NCT02502097.

The inevitability of ATP as a transmitter in the carotid body.

Atmospheric oxygen concentrations rose markedly at several points in evolutionary history. Each of these increases was followed by an evolutionary leap in organismal complexity, and thus the cellular adaptions we see today have been shaped by the levels of oxygen within our atmosphere. In eukaryotic cells, oxygen is essential for the production of adenosine 5'-triphosphate (ATP) which is the 'Universal Energy Currency' of life. Aerobic organisms survived by evolving precise mechanisms for converting oxygen within the environment into energy. Higher mammals developed specialised organs for detecting and responding to changes in oxygen content to maintain gaseous homeostasis for survival. Hypoxia is sensed by the carotid bodies, the primary chemoreceptor organs which utilise multiple neurotransmitters one of which is ATP to evoke compensatory reflexes. Yet, a paradox is presented in oxygen sensing cells of the carotid body when during periods of low oxygen, ATP is seemingly released in abundance to transmit this signal although the synthesis of ATP is theoretically halted because of its dependence on oxygen. We propose potential mechanisms to maintain ATP production in hypoxia and summarise recent data revealing elevated sensitivity of purinergic signalling within the carotid body during conditions of sympathetic overactivity and hypertension. We propose the carotid body is hypoxic in numerous chronic cardiovascular and respiratory diseases and highlight the therapeutic potential for modulating purinergic transmission.

Gene Polymorphisms Among Plasmodium vivax Geographical Isolates and the Potential as New Biomarkers for Gametocyte Detection.

The unique biological features of Plasmodium vivax not only make it difficult to control but also to eliminate. For the transmission of the malaria parasite from infected human to the vector, gametocytes play a major role. The transmission potential of a malarial infection is inferred based on microscopic detection of gametocytes and molecular screening of genes in the female gametocytes. Microscopy-based detection methods could grossly underestimate the reservoirs of infection as gametocytes may occur as submicroscopic or as micro- or macro-gametocytes. The identification of genes that are highly expressed and polymorphic in male and female gametocytes is critical for monitoring changes not only in their relative proportions but also the composition of gametocyte clones contributing to transmission over time. Recent transcriptomic study revealed two distinct clusters of highly correlated genes expressed in the P. vivax gametocytes, indicating that the male and female terminal gametocytogeneses are independently regulated. However, the detective power of these genes is unclear. In this study, we compared genetic variations of 15 and 11 genes expressed, respectively, in the female and male gametocytes among P. vivax isolates from Southeast Asia, Africa, and South America. Further, we constructed phylogenetic trees to determine the resolution power and clustering patterns of gametocyte clones. As expected, Pvs25 (PVP01_0616100) and Pvs16 (PVP01_0305600) expressed in the female gametocytes were highly conserved in all geographical isolates. In contrast, genes including 6-cysteine protein Pvs230 (PVP01_0415800) and upregulated in late gametocytes ULG8 (PVP01_1452800) expressed in the female gametocytes, as well as two CPW-WPC family proteins (PVP01_1215900 and PVP01_1320100) expressed in the male gametocytes indicated considerably high nucleotide and haplotype diversity among isolates. Parasite samples expressed in male and female gametocyte genes were observed in separate phylogenetic clusters and likely represented distinct gametocyte clones. Compared to Pvs25, Pvs230 (PVP01_0415800) and a CPW-WPC family protein (PVP01_0904300) showed higher expression in a subset of Ethiopian P. vivax samples. Thus, Pvs230, ULG8, and CPW-WPC family proteins including PVP01_0904300, PVP01_1215900, and PVP01_1320100 could potentially be used as novel biomarkers for detecting both sexes of P. vivax gametocytes in low-density infections and estimating transmission reservoirs.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

Whole genome sequencing of Plasmodium vivax isolates reveals frequent sequence and structural polymorphisms in erythrocyte binding genes.

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale.

P2X3 receptor antagonism reduces the occurrence of apnoeas in newborn rats.

Hyperreflexia of the peripheral chemoreceptors is a potential contributor of apnoeas of prematurity (AoP). Recently, it was shown that elevated P2X3 receptor expression was associated with elevated carotid body afferent sensitivity. Therefore, we tested whether P2X3 receptor antagonism would reduce AoP known to occur in newborn rats. Unrestrained whole-body plethysmography was used to record breathing and from this the frequency of apnoeas at baseline and following administration of either a P2X3 receptor antagonist - AF-454 (5 mg/kg or 10 mg/kg s.c.) or vehicle was derived. In a separate group, we tested the effects of AF-454 (10 mg/kg) on the hypoxic ventilatory response (10 % FiO2). Ten but not 5 mg/kg AF-454 reduced the frequency of AoP and improved breathing regularity significantly compared to vehicle. Neither AF-454 (both 5 and 10 mg/kg) nor vehicle affected baseline respiration. However, P2X3 receptor antagonism (10 mg/kg) powerfully blunted hypoxic ventilatory response to 10 % FiO2. These data suggest that P2X3 receptors contribute to AoP and the hypoxic ventilatory response in newborn rats but play no role in the drive to breathe at rest.

Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche.

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILß pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILß-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILß compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.