Inducible epithelial resistance protects mice against leukemia-associated pneumonia.

Despite widespread infection prevention efforts, pneumonia remains the leading cause of death among patients with acute leukemia, due to complex disease- and treatment-dependent immune defects. We have reported that a single inhaled treatment with a synergistic combination of Toll-like receptor 2/6 (TLR 2/6) and TLR9 agonists (Pam2-ODN) induces protective mucosal defenses in mice against a broad range of pathogens. As Pam2-ODN-induced protection persists despite depletion of several leukocyte populations, we tested whether it could prevent pneumonia in a mouse model of acute myeloid leukemia (AML) remission induction therapy. Pam2-ODN prevented death due to pneumonia caused by Pseudomonas aeruginosa, Streptococcus pneumoniae, and Aspergillus fumigatus when mice were heavily engrafted with leukemia cells, had severe chemotherapy-induced neutropenia or both. Pam2-ODN also extended survival of pneumonia in NSG mice engrafted with primary human AML cells. Protection was associated with rapid pathogen killing in the lungs at the time of infection and with reduced pathogen burdens at distant sites at the end of observation. Pathogen killing was inducible directly from isolated lung epithelial cells and was not abrogated by the presence of leukemia cells or cytotoxic agents. Pam2-ODN had no discernible effect on replication rate, total tumor population, or killing by chemotherapy of mouse or human leukemia cells, either in vitro or in vivo. Taken together, we report that therapeutic stimulation of lung epithelial defenses robustly protects against otherwise lethal pneumonias despite the profound immune dysfunction associated with acute leukemia and its treatment. These findings may suggest an opportunity to protect this population during periods of peak vulnerability.

Effects of quitting cannabis on respiratory symptoms.

Smoking cannabis is associated with symptoms of bronchitis. Little is known about the persistence of symptoms after stopping cannabis use. We assessed associations between changes in cannabis use and respiratory symptoms in a population-based cohort of 1037 young adults. Participants were asked about cannabis and tobacco use at ages 18, 21, 26, 32 and 38 years. Symptoms of morning cough, sputum production, wheeze, dyspnoea on exertion and asthma diagnoses were ascertained at the same ages. Frequent cannabis use was defined as ≥52 occasions over the previous year. Associations between frequent cannabis use and respiratory symptoms were analysed using generalised estimating equations with adjustments for tobacco smoking, asthma, sex and age. Frequent cannabis use was associated with morning cough (OR 1.97, p<0.001), sputum production (OR 2.31, p<0.001) and wheeze (OR 1.55, p<0.001). Reducing or quitting cannabis use was associated with reductions in the prevalence of cough, sputum and wheeze to levels similar to nonusers.Frequent cannabis use is associated with symptoms of bronchitis in young adults. Reducing cannabis use often leads to a resolution of these symptoms.

Prevalence and correlates of a 'knee' pattern on the maximal expiratory flow-volume loop in young adults.

BACKGROUND AND OBJECTIVE: Visual inspection of the maximal flow-volume curve is an important step in interpreting spirometry. Many young people have a convex inflection or 'knee' on the expiratory part of the loop. This is thought to be a normal variant, but this view is based on theoretical grounds, and the epidemiology of the knee pattern has never been reported. METHODS: Flow-volume loops from an unselected birth cohort of 1037 individuals at ages 18, 26, 32 and 38 years were visually inspected for a knee pattern. Associations with asthma diagnoses, smoking history, body mass index (BMI) and spirometry were assessed. RESULTS: The knee pattern was found in approximately two thirds of men and women at age 18. The prevalence decreased with age, but it was more likely to persist in women. The knee was more common after bronchodilator and was associated with higher forced expiratory volume in 1 s/forced vital capacity ratios and mid-expiratory flow rates. There was no association with smoking, except for an inverse correlation in men at age 18. No association was found with BMI. Women with asthma were less likely to have a knee at both ages 18 and 38, whereas men with asthma showed an inverse association at age 18. CONCLUSIONS: A knee is a very common pattern on flow-volume loop in young adults. In accordance with theoretical predictions, the prevalence of the knee declines with age, but it is more likely to persist in women. It is associated with less airflow obstruction and is less common in people with asthma.

Piezo1 opposes age-associated cortical bone loss.

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

Pancreatic islet cell type-specific transcriptomic changes during pregnancy and postpartum.

Pancreatic ß-cell mass expands during pregnancy and regresses in the postpartum period in conjunction with dynamic metabolic demands on maternal glucose homeostasis. To understand transcriptional changes driving these adaptations in ß-cells and other islet cell types, we performed single-cell RNA sequencing on islets from virgin, late gestation, and early postpartum mice. We identified transcriptional signatures unique to gestation and the postpartum in ß-cells, including induction of the AP-1 transcription factor subunits and other genes involved in the immediate-early response (IEGs). In addition, we found pregnancy and postpartum-induced changes differed within each endocrine cell type, and in endothelial cells and antigen-presenting cells within islets. Together, our data reveal insights into cell type-specific transcriptional changes responsible for adaptations by islet cells to pregnancy and their resolution postpartum.

PGAT: a multistrain analysis resource for microbial genomes.

MOTIVATION: The Prokaryotic-genome Analysis Tool (PGAT) is a web-based database application for comparing gene content and sequence across multiple microbial genomes facilitating the discovery of genetic differences that may explain observed phenotypes. PGAT supports database queries to identify genes that are present or absent in user-selected genomes, comparison of sequence polymorphisms in sets of orthologous genes, multigenome display of regions surrounding a query gene, comparison of the distribution of genes in metabolic pathways and manual community annotation. AVAILABILITY AND IMPLEMENTATION: The PGAT website may be accessed at http://nwrce.org/pgat. CONTACT: mbrittna@uw.edu.

Changes in fecal microbiota with CFTR modulator therapy: A pilot study.

Studies have demonstrated that people with CF with pancreatic insufficiency (PI) have fecal dysbioses. Evidence suggests the causes of these dysbioses are multifactorial, and that important drivers include antibiotic exposure, dietary intake, and CF gastrointestinal tract dysfunction, including nutrient malabsorption. In this pilot study, we tested whether initiation of the CFTR modulator treatments ivacaftor (in a cohort of pancreatic sufficient (PS) people with CF and an R117H CFTR variant) or lumacaftor/ivacaftor (in a cohort of PI people with CF and an F508del variant) changed fecal measures of malabsorption or fecal microbiomes. While we identified no statistically significant fecal changes with either treatment, we detected trends in the PI cohort when initiating lumacaftor/ivacaftor towards decreased fecal fat content and towards fecal microbiomes that more closely resembled the fecal microbiota of people without PI. While these findings support a model in which nutrient malabsorption resulting from CF-induced PI drives fecal dysbiosis, they must be validated in future, larger studies of fecal microbiome and malabsorption outcomes with highly effective CFTR modulator therapies.

Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bi-transgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in ß-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. The utility of this model is enhanced by the ability to form tumor-derived allografts. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

Dual inhibition of EGFR and mTOR pathways in small cell lung cancer.

BACKGROUND: In this report we investigated the combination of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) pathway inhibition as a possible new therapeutic strategy for small cell lung cancer (SCLC). METHODS: EGFR, p-AKT, p-ERK, p-mTOR and p-p70s6K protein expressions were studied by immunohistochemistry in 107 small cell lung carcinomas and correlated with clinicopathological parameters. Cells of SCLC were treated with erlotinib+/-RAD001 and analysed for cell viability, proliferation, autophagy, and pathway regulation. RESULTS: Epidermal growth factor receptor, p-AKT, p-ERK, p-mTOR, and p-p70s6K were expressed in 37, 24, 13, 55 and 91% of the tumour specimens of all SCLC patients, respectively, and were not associated with disease-free or overall survival. The expression of EGFR was lower in neoadjuvant-treated patients (P=0.038); mTOR pathway activation was higher in the early stages of disease (P=0.048). Coexpression of EGFR/p-mTOR/p-p70s6K was observed in 28% of all patients . EGFR immunoreactivity was associated with p-ERK and p-mTOR expression (P=0.02 and P=0.0001); p-mTOR immunoreactivity was associated with p-p70s6K expression (P=0.001). Tumour cells comprised a functional EGFR, no activating mutations in exons 18-21, and resistance to RAD001 monotherapy. We found synergistic effects of erlotinib and RAD001 combination therapy on the molecular level, cell viability, proliferation and autophagy. CONCLUSIONS: The combined inhibition of EGFR/mTOR pathways could be a promising approach to treat SCLC.

Complete Genome Sequence of Citrobacter freundii Myophage Maleficent.

Citrobacter freundii, a member of the Enterobacteriaceae family, has been linked to opportunistic infections in neonates and immunocompromised adults. Here, we report the complete genome sequence of a T4-like myophage, Maleficent, which infects C. freundii.

Prolactin Receptor Signaling Regulates a Pregnancy-Specific Transcriptional Program in Mouse Islets.

Pancreatic ß-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram ß-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where ß-cell-specific PRLR knockout (ßPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within ß-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either ßPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent ß-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for ß-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.

How much atopy is attributable to common childhood environmental exposures? A population-based birth cohort study followed to adulthood.

Background: The rising prevalence of atopic diseases implies a strong influence of environmental determinants. Epidemiological studies have identified several early life exposures that appear to influence the risk of developing atopic sensitization, but the combined influence of these exposures is unknown. We sought to estimate the proportion of atopy that could be attributed to common childhood exposures associated with atopic sensitization in adolescence and young adulthood. Methods: Atopic sensitization was measured by skin-prick tests for common aeroallergens in a population-based New Zealand birth cohort at ages 13 and 32 years. The independent effects of previously identified risk and protective factors for atopic sensitization were assessed using multiple logistic regression. Population attributable fractions were calculated for atopic sensitization in childhood and adulthood. Results: Tobacco smoke exposure, dog and cat ownership, nail-biting and thumb-sucking, attending pre-school day care, and household crowding were associated with a lower risk of atopic sensitization whereas breastfeeding was associated with a higher risk. Population attributable fractions for combined effects of these environmental factors suggest that they may account for 58% of atopic sensitization at age 13 and 49% at age 32 years. Conclusions: A substantial proportion of atopic sensitization appears to be attributable to common childhood environmental and lifestyle factors, and the influence of these exposures persists into adulthood. The absolute risks attributable to these exposures will be different in other cohorts and we cannot assume that these associations are necessarily causal. Nevertheless, the findings suggest that identifiable childhood environmental factors contribute substantially to atopic sensitization.

Interferon-free regimens improve portal hypertension and histological necroinflammation in HIV/HCV patients with advanced liver disease.

BACKGROUND: HIV/HCV co-infected patients show accelerated fibrosis progression and higher risk for complications of portal hypertension (PHT). AIM: To assess the effects of interferon-free therapy on portal pressure, liver histology and plasma biomarkers in HIV/HCV-coinfected patients with PHT. METHODS: Twenty-two patients with paired hepatic venous pressure gradient (HVPG) measurements prior and after successful treatment (SVR) with interferon-free regimens were included. Liver stiffness was assessed by transient elastography and biopsies were scored according to METAVIR. Plasma biomarkers were determined by ELISA. RESULTS: Overall, HVPG decreased from 10.7 ± 4.1 mmHg at baseline to 7.4 ± 4.2 mmHg after HCV treatment (Δ:-3.3 ± 2.7 mmHg; p < 0.001). In patients with clinically significant PHT (HVPG≥10 mmHg, n = 11), HVPG decreased from 14.1 ± 2.9 to 10.4 ± 3.9 mmHg (Δ:-3.7 ± 3.3 mmHg; p = 0.004) and a haemodynamic response (HVPG decrease ≥10%) was observed in 73%. In 64% of patients with subclinical PHT (HVPG 6-9 mmHg, n = 11), portal pressure normalised at SVR. Mean liver stiffness decreased from 20.8 kPa to 11.5 kPa (Δ:-8.8 ± 7.4 kPa; p < 0.001). Fifty percent (7/14) of patients with cirrhosis were re-classified as METAVIR ≤F3 and all patients with decompensated cirrhosis improved their Child-Pugh stage. After successful HCV treatment, 39% still had persistent histological necroinflammatory activity (METAVIR A1), which correlated with less HVPG response and more steatosis. While most biomarkers improved with SVR, METAVIR A1 patients had significantly higher plasma levels of fibrogenic (PDGF, TGF-ß) and angiogenic (VEGF, Angiopoietin1) biomarkers. CONCLUSIONS: Interferon-free therapy reduces PHT and halts histological necroinflammatory activity in the majority of HIV/HCV-coinfected patients after SVR, which may lead to re-compensation of liver function in cirrhosis. Biomarkers could identify patients with persisting hepatic necroinflammation.

Major Gene Resistance to Blackleg in Brassica napus Overcome Within Three Years of Commercial Production in Southeastern Australia.

The infection by Leptosphaeria maculans of Brassica napus cultivars with major gene resistance derived from Brassica rapa subsp. sylvestris was studied in southeastern Australia. Following the commercial release of these cultivars in Australia in 2000, plants with stem cankers were first reported in 2002 at two geographically isolated regions in South Australia and New South Wales. In 2003, this study showed that the major gene resistance had been overcome in an area of approximately 50,000 ha in South Australia and in two fields in New South Wales (0.5 and 30 ha). There was no relationship between disease severity and incidence in 2003 and the proximity to the sites where resistance breakdown occurred in 2002. At some locations, the frequency of isolates able to overcome the B. rapa subsp. sylvestris-derived resistance had increased between 2002 and 2003. Isolates cultured from canola cultivars with either B. rapa subsp. sylvestris-derived resistance or polygenic resistance showed host specificity when inoculated onto cultivars with B. rapa subsp. sylvestris-derived or polygenic resistance, respectively. The most likely cause of the resistance breakdown was the rapid increase in frequency of L. maculans isolates virulent on this particular resistance source. The selection pressure leading to this increased frequency was probably mediated by the planting of cultivars harboring the major resistance gene in the same locations for a 3-year period, and the ability of the pathogen to produce large numbers of asexual and sexual spores.

The Genetic Structure of Australian Populations of Mycosphaerella musicola Suggests Restricted Gene Flow at the Continental Scale.

ABSTRACT Mycosphaerella musicola causes Sigatoka disease of banana and is endemic to Australia. The population genetic structure of M. musicola in Australia was examined by applying single-copy restriction fragment length polymorphism probes to hierarchically sampled populations collected along the Australian east coast. The 363 isolates studied were from 16 plantations at 12 sites in four different regions, and comprised 11 populations. These populations displayed moderate levels of gene diversity (H = 0.142 to 0.369) and similar levels of genotypic richness and evenness. Populations were dominated by unique genotypes, but isolates sharing the same genotype (putative clones) were detected. Genotype distribution was highly localized within each population, and the majority of putative clones were detected for isolates sampled from different sporodochia in the same lesion or different lesions on a plant. Multilocus gametic disequilibrium tests provided further evidence of a degree of clonality within the populations at the plant scale. A complex pattern of population differentiation was detected for M. musicola in Australia. Populations sampled from plantations outside the two major production areas were genetically very different to all other populations. Differentiation was much lower between populations of the two major production areas, despite their geographic separation of over 1,000 km. These results suggest low gene flow at the continental scale due to limited spore dispersal and the movement of infected plant material.