M1-like, but not M0- or M2-like, macrophages, reduce RSV infection of primary bronchial epithelial cells in a media-dependent fashion.

Respiratory syncytial virus (RSV) is a common childhood infection that in young infants can progress into severe bronchiolitis and pneumonia. Disease pathogenesis results from both viral mediated and host immune processes of which alveolar macrophages play an important part. Here, we investigated the role of different types of alveolar macrophages on RSV infection using an in vitro co-culture model involving primary tissue-derived human bronchial epithelial cells (HBECs) and human blood monocyte-derived M0-like, M1-like, or M2-like macrophages. It was hypothesized that the in vitro model would recapitulate previous in vivo findings of a protective effect of macrophages against RSV infection. It was found that macrophages maintained their phenotype for the 72-hour co-culture time period and the bronchial epithelial cells were unaffected by the macrophage media. HBEC infection with RSV was decreased by M1-like macrophages but enhanced by M0- or M2-like macrophages. The medium used during the co-culture also impacted the outcome of the infection. This work demonstrates that alveolar macrophage phenotypes may have differential roles during epithelial RSV infection, and demonstrates that an in vitro co-culture model could be used to further investigate the roles of macrophages during bronchial viral infection.

ETS proto-oncogene 1 modulates PTP1B expression to participate in high glucose-mediated endothelial inflammation.

Hyperglycemia-induced endothelial inflammation participates in the pathogenesis of cardiovascular complications in diabetics. Previous studies showed that protein tyrosine phosphatase 1B (PTP1B) and ETS proto-oncogene 1 (ets1) are involved in hyperglycemia-induced endothelial inflammation. In this study, we hypothesized that ets1 modulates PTP1B expression, thus playing a crucial role in hyperglycemia-induced vascular endothelial inflammation. Our results indicated that high glucose increases monocyte/endothelial adhesion, vascular cell adhesion molecule-1 (VCAM-1) expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose-mediated endothelial inflammation is reversed by PTP1B silencing. In addition, high glucose increases ets1 expression in HUVECs. silencing reverses high glucose-mediated endothelial inflammation. Furthermore, the effect of ets1 overexpression is similar to that of high glucose treatment, which is counteracted by si-PTP1B. The results from ChIP assays indicated that ets1 occupies the PTP1B promoter region. Ets1 overexpression enhances PTP1B promoter activity, which is disappeared after specific binding site mutation. experiments demonstrated that the expressions of VCAM-1, PTP1B, and ets1, as well as the phosphorylation of p65 are augmented in the aorta of diabetic rats. In conclusion, ets1 contributes to hyperglycemia-mediated endothelial inflammation via upregulation of PTP1B expression.

Alveolar-like Macrophages Attenuate Respiratory Syncytial Virus Infection.

Respiratory Syncytial Virus (RSV) is the leading cause of acute lower respiratory infections in young children and infection has been linked to the development of persistent lung disease in the form of wheezing and asthma. Despite substantial research efforts, there are no RSV vaccines currently available and an effective monoclonal antibody targeting the RSV fusion protein (palivizumab) is of limited general use given the associated expense. Therefore, the development of novel approaches to prevent RSV infection is highly desirable to improve pediatric health globally. We have developed a method to generate alveolar-like macrophages (ALMs) from pluripotent stem cells. These ALMs have shown potential to promote airway innate immunity and tissue repair and so we hypothesized that ALMs could be used as a strategy to prevent RSV infection. Here, we demonstrate that ALMs are not productively infected by RSV and prevent the infection of epithelial cells. Prevention of epithelial infection was mediated by two different mechanisms: phagocytosis of RSV particles and release of an antiviral soluble factor different from type I interferon. Furthermore, intratracheal administration of ALMs protected mice from subsequent virus-induced weight loss and decreased lung viral titres and inflammation, indicating that ALMs can impair the pathogenesis of RSV infection. Our results support a prophylactic role for ALMs in the setting of RSV infection and warrant further studies on stem cell-derived ALMs as a novel cell-based therapy for pulmonary viral infections.

Inflammatory epithelial cytokines after in vitro respiratory syncytial viral infection are associated with reduced lung function.

Respiratory syncytial virus (RSV) infections in early life predispose children with cystic fibrosis (CF) to more severe lung function decline in later life. The mechanisms explaining the associations between RSV and progression of CF lung disease are not clear. In this study, a human bronchial epithelial cell line and primary human nasal epithelial cells (PNECs) from individuals with CF and healthy control donors were infected with RSV. Real-time PCR, plaque assay, cytokine detection, immunofluorescence and Western blot analyses were performed. RSV is replicated to a higher degree in CF epithelial cells as compared to control cells; however, no defects in innate immune pathways were identified in CF cells. Rather, primary p.Phe508del cystic fibrosis transmembrane conductance regulator PNECs produced more cytokines after RSV infection than control cells. Moreover, interleukin-8 and tumour necrosis factor-α production post RSV negatively correlated with lung function (% predicted forced expiratory volume in 1 s) in the individuals who donated the cells. These data suggest that CF epithelium has a dysfunctional response to RSV allowing for enhanced viral replication and an exaggerated inflammatory response that ultimately may predispose to greater airway inflammation and reduced lung function.

Ketamine inhibits aerobic glycolysis in colorectal cancer cells by blocking the NMDA receptor-CaMK II-c-Myc pathway.

Aerobic glycolysis plays a crucial role in cancer progression. Ketamine is often used for cancer pain relief in clinical settings. Moreover, ketamine inhibits proliferation and induces apoptosis in many cancer cell types. However, the anti-tumour mechanism of ketamine is still poorly understood. In the present study, we survey whether and how ketamine inhibits aerobic glycolysis in colon cancer cells. Glycolysis of colon cancer cells was determined by detecting the extracellular acidification rate in HT29 and SW480 cells. Quantitative real-time PCR was employed to determine mRNA expression. Calcium levels were detected with a Fluo-3 AM fluorescence kit. Micro-positron emission tomography/computed tomography (microPET/CT) imaging was employed to assess glycolysis in the tumours of the xenograft model. Ketamine treatment inhibited colon cancer cell viability and migration in HT29 and SW480 cells. Moreover, ketamine decreased aerobic glycolysis and decreased the expression of glycolysis-related proteins in HT29 and SW480 cells. MicroPET/CT demonstrated that ketamine decreased 18F-FDG uptake in the xenograft model. In addition, ketamine inhibited c-Myc expression and CaMK II phosphorylation and decreased calcium levels. Further, dizocilpine (an NMDAR inhibitor), and KN93 (a CaMK II inhibitor), decreased CaMK II phosphorylation, c-Myc expression, and cancer cell glycolysis; these results were similar to those with ketamine treatment. Furthermore, the anti-tumour effect of ketamine was counteracted by rapastinel (an NMDAR activator). Ketamine inhibited aerobic glycolysis in colon cancer cells probably by blocking the NMDA receptor-CaMK II-c-Myc pathway, thus attenuating colon cancer cell viability and migration.

Ketamine inhibits colorectal cancer cells malignant potential via blockage of NMDA receptor.

Ketamine, a common N-methyl-d-aspartate receptor (NMDAR) antagonist, is an option for cancer pain treatment in clinical practice. Ketamine has been shown to have the capacity to attenuate cancer cells malignancy. However, the underlying mechanism remains elusive. In the present study, we reported that ketamine inhibited the malignant potential of colorectal cancer cells and investigated the possible mechanisms involved. Ketamine suppressed the expression of VEGF, HIF-1α, p-AKT, p-ERK, and p-CaMK II, and reduced intracellular Ca2+ level in a concentration dependent manner (1, 5, 10 µg/ml). Furthermore, AP5 and MK801 (NMDAR inhibitors), and KN93 (CaMK II inhibitor), decreased the expression of VEGF, HIF-1a, p-AKT, p-ERK, and p-CaMK II, which were similar to the effect of ketamine. Further, the anti-tumor effect of ketamine was reversed by d-serine (NMDAR activator). Ketamine did not affect NMDA receptor expression, however knockdown of NMDA receptor using siRNA attenuated the effect of ketamine on cell migration. Collectively, these findings demonstrated that ketamine attenuated the expression of VEGF and cell migration ability in colorectal cancer cells, probably via blockage of NMDA receptor.

Predictors of Long-Term Pulmonary Morbidity in Children with Congenital Diaphragmatic Hernia.

PURPOSE: The aim is to identify prognostic markers of long-term pulmonary morbidity among congenital diaphragmatic hernia (CDH) survivors. METHODS: A single-institution, retrospective review was performed on all CDH patients from 2000 and 2012 (REB#1000053383). Liver position, patch use, and pulmonary function tests (PFTs) (forced expiratory volume at 1 second [FEV1] and forced vital capacity [FVC] expressed as mean % predicted + SD) were recorded. Data were analyzed using analysis of variance. RESULTS: Patients with acceptable and reproducible PFT (n = 72 for 202 total PFT) with patch repair and liver up (n = 28) had significantly lower FEV1 (72.4 + 17.6) than those with no patch and liver down (n = 98, FEV1= 86.3 + 15.9, p = 0.002). Patients with patch repair and liver down (n = 40) also had significantly lower FEV1 (76.6 + 14.4) than those with liver down and no patch (p = 0.0463). Patients with liver up and patch repair had PFT results consistent with moderate reduction of lung function, while the remainder had mild to no decrease in lung function. All CDH patients older than 14 years had a reduction in FEV1/FVC consistent with obstructive phenotype, with a mean FEV1/FVC = 62.3 for patch repair group and FEV1/FVC = 76.1 in the no patch group. CONCLUSION: Decreased pulmonary function of CDH survivors correlated with the use of patch repair and liver position. CDH lung disease should be monitored in adulthood.

Targeting Intracellular Ion Homeostasis for the Control of Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a leading cause of mortality in infants and young children. Despite the RSV disease burden, no vaccine is available, and treatment remains nonspecific. New drug candidates are needed to combat RSV. Toward this goal, we screened over 2,000 compounds to identify approved drugs with novel anti-RSV activity. Cardiac glycosides, inhibitors of the membrane-bound Na+/K+-ATPase, were identified to have anti-RSV activity. Cardiac glycosides diminished RSV infection in human epithelial type 2 cells and in primary human airway epithelial cells grown at an air-liquid interface. Digoxin, a U.S. Food and Drug Administration-approved cardiac glycoside, was also able to inhibit infection of primary nasal epithelial cells with community isolates of RSV. Our results suggest that the antiviral effects of cardiac glycosides may be dependent on changes in the intracellular Na+ and K+ composition. Consistent with this mechanism, we demonstrated that the ionophoric antibiotics salinomycin, valinomycin, and monensin inhibited RSV in human epithelial type 2 cells and primary nasal epithelial cells. Our data indicate that the K+/Na+-sensitive steps in the RSV life cycle occur within the initial 4 hours of viral infection but do not include virus binding/entry. Rather, our findings demonstrated a negative effect on the RSV transcription and/or replication process. Overall, this work suggests that targeting intracellular ion concentrations offers a novel antiviral strategy.

Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges and Opportunities.

Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.

Pulmonary function and nutritional morbidity in children and adolescents with congenital diaphragmatic hernia.

BACKGROUND: Malnutrition is common among congenital diaphragmatic hernia (CDH) survivors and may result from elevated respiratory effort. We evaluated body mass index (BMI), measured resting energy expenditure (mREE) and pulmonary function test (PFT) results in children and adolescents with CDH to determine if there is a correlation. METHODS: With ethics approval (REB# 1000035323), anthropometrics, indirect calorimetry (IC) results and PFTs were collected from patients 5-17years of age during CDH clinic visits between 2000 and 2016. Malnutrition was defined as BMI z-scores <-2.0; mREE (as percent predicted REE) was measured using IC; z-scores for forced expiratory volume in 1s (FEV1) and forced vital capacity (FVC) were normal if <-1.64. STATISTICS: GraphPad Prism 6, San Diego, CA. RESULTS & DISCUSSION: Of 118 patients who attended clinic, 33 had reproducible PFTs, anthropometrics and IC results. Mean BMI z-score was -0.89±1.47 and 24% of patients were malnourished; mean FVC z-score (-1.32±1.39) was within normal range, whereas mean z-scores for FEV1 (-2.21±1.68) and FEV1/FVC ratio (-1.78±0.73) were below normal. A correlation was noted between BMI and PFTs (FEV1 r=0.70, P<0.0001; FVC r=0.74 P<0.0001). Mean mREE was 112%±12% of expected and 67% of patients were hypermetabolic (mREE<110% predicted). IC results did not correlate with z-scores for either FEV1 (r=0.10, P=0.57); or FVC (r=0.28, P=0.12). CONCLUSIONS: These preliminary results suggest that a correlation is present between BMI and lung function in CDH children and adolescents, whereas lung function does not seem to correlate with mREE. LEVEL OF EVIDENCE: II.

A prognostic risk model for patients with triple negative breast cancer based on stromal natural killer cells, tumor-associated macrophages and growth-arrest specific protein 6.

The aim of this study was to establish a prognostic risk model for patients with triple negative breast cancer (TNBC). A total of 278 specimens of human TNBC tissues were investigated by immunohistochemistry for growth-arrest specific protein 6 expression, infiltrations of stromal natural killer cells and tumor-associated macrophages. According to their prognostic risk scores based on the model, patients were divided into three groups (score 0, 1-2, 3). Correlations of prognostic risk scores, clinicopathologic features and overall survival (OS) were analyzed. To study the clinical value of this stratification model in early disease recurrence or metastasis, 177 patients were screened out for further analysis. Based on disease free survival (DFS), 90 patients fell within the DFS ≤3 years group and 87 patients within the DFS ≥5 years group. We analyzed the differences in prognostic risk scores between the two groups. The prognostic risk scores were negatively related to tumor size, lymph node metastasis and P53 status (P < 0.001 for all). Patients with low prognostic risk scores had longer OS (P = 0.001). Using multivariate analysis, it was determined that TNM stage (HR = 0.432, 95% confidence interval [CI] = 0.281-0.665, P = 0.003), FOXP3 positive lymphocytes (HR = 1.712, 95% CI = 1.085-2.702, P = 0.021) and prognostic risk scores (HR = 1.340, 95% CI = 1.192-1.644, P = 0.005) were independent prognostic factors for OS. Compared with the DFS ≥5 years group, the DFS ≤3 years group patients had significantly higher prognostic risk scores (P < 0.001). In conclusion, the prognostic risk score of the model was a significant indicator of prognosis for patients with TNBC.

Testing gene therapy vectors in human primary nasal epithelial cultures.

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity.

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Identification of nucleolin as a cellular receptor for human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) causes a large burden of disease worldwide. There is no effective vaccine or therapy, and the use of passive immunoprophylaxis with RSV-specific antibodies is limited to high-risk patients. The cellular receptor (or receptors) required for viral entry and replication has yet to be described; its identification will improve understanding of the pathogenesis of infection and provide a target for the development of novel antiviral interventions. Here we show that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro. We observed decreased RSV infection in vitro in neutralization experiments using nucleolin-specific antibodies before viral inoculation, in competition experiments in which virus was incubated with soluble nucleolin before inoculation of cells, and upon RNA interference (RNAi) to silence cellular nucleolin expression. Transfection of nonpermissive Spodoptera frugiperda Sf9 insect cells with human nucleolin conferred susceptibility to RSV infection. RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice (P = 0.0004), confirming that nucleolin is a functional RSV receptor in vivo.

Long-term terbutaline exposure stimulates alpha1-Na+-K+-ATPase expression at posttranscriptional level in rat fetal distal lung epithelial cells.

Transepithelial Na(+) transport through epithelial Na(+) channels (ENaC) on the apical membrane and Na(+)-K(+)-ATPase activity on the basolateral membrane of distal lung epithelial cells are critical for alveolar fluid clearance. Acute exposure to beta-adrenergic agonists stimulates lung fluid clearance by increasing Na(+) transport. We investigated the effects of chronic exposure to the beta(2)-adrenergic agonist terbutaline on the transepithelial Na(+) transport in rat fetal distal lung epithelia (FDLE). FDLE monolayers exposed to 10(-4) M terbutaline for 48 h had significantly increased propanolol-blockable transepithelial total and amiloride-sensitive short-circuit current (I(sc)); however, when these chronically exposed monolayers were acutely exposed to additional beta-agonists and intracellular cAMP upregulators, there was no further increase in I(sc). Monolayers exposed to terbutaline for >48 h had I(sc) similar to control cells. Ouabain-sensitive Na(+)-K(+)-ATPase activity was increased in 48-h terbutaline-exposed FDLE whose apical membranes were permeabilized with nystatin. In contrast, terbutaline did not increase amiloride-sensitive apical membrane I(sc) in FDLE whose basolateral membranes were permeabilized with nystatin. Terbutaline treatment did not affect alpha-, beta-, or gamma-ENaC mRNA or alpha-ENaC protein steady-state levels, but increased total cellular levels and rate of synthesis of alpha(1)-Na(+)-K(+)-ATPase protein in FDLE in the absence of any change in alpha(1)-Na(+)-K(+)-ATPase mRNA. Total cellular beta(1)-Na(+)-K(+)-ATPase mRNA and protein levels were not affected by terbutaline. These data suggest that FDLE have different responses from adult type II epithelial cells when chronically exposed to terbutaline, and their increased transepithelial Na(+) transport occurs via a posttranscriptional increase in alpha(1)-Na(+)-K(+)-ATPase expression.

Glucocorticoid-mediated repression of REDD1 mRNA expression in rat fetal distal lung epithelial cells.

REDD1 (Regulated in Development and DNA Damage-1) is a stress-response gene that represses mammalian target of rapamycin (mTOR) thus decreasing protein synthesis. In contrast to studies using cell lines and adult alveolar type II (ATII) cells, we find that REDD1 mRNA levels did not increase in rat fetal distal lung epithelia (FDLE) or fetal lung fibroblasts grown in primary cultures and then exposed to 3% O2. REDD1 mRNA expression was repressed by dexamethasone (DEX) in FDLE and ATII, but induced by DEX in fibroblasts. Lung epithelial cell lines, A549 and MLE-15, showed increases in REDD1 mRNA in response to hypoxia and DEX. The effect of DEX on REDD1 mRNA and protein in FDLE and fibroblasts was dose- and time-dependent. Inhibitor studies support repression of REDD1 mRNA by DEX in FDLE was mediated via glucocorticoid receptor and not by nongenomic effects of glucocorticoids via MAPK pathways. The half-life of REDD1 mRNA was shorter in DEX-exposed FDLE compared with hormone-free media suggesting that DEX reduced REDD1 mRNA stability in FDLE. These studies indicate that REDD1 expression in response to hypoxia and DEX is cell-type specific and that physiologically appropriate levels of PO2 should be used when investigating fetal lung development.

Pulmonary neuroendocrine cell-secreted factors may alter fetal lung liquid clearance.

The neuroendocrine system is most active at birth and may play a role in the transition from fetal to postnatal life, in particular in the lungs' transition from fluid secretion to fluid absorption. Pulmonary neuroendocrine cells do release dopamine (DA), serotonin, and gastrin-releasing peptide but their effects on lung ion and fluid transport are poorly understood. Therefore, we studied their effects on fetal distal lung explants and primary cultures of fetal distal lung epithelium (FDLE). We show that DA, but neither serotonin nor gastrin-releasing peptide, alters ion and fluid transport, in a dose-dependent manner. DAs effects were abrogated by D1/D2 receptor blockers in FDLE but not in explants. Propranolol abrogated DAs effects in both models. DA increased intracellular cAMP levels in FDLE. Terbutaline, forskolin, and isobutylmethylxanthine did not increase short circuit current (Isc) in DA-treated cells, despite a further increase in cAMP. We conclude that at least one, but not all mediators released by pulmonary neuroendocrine cells alter distal lung epithelial ion transport.

Steroid and oxygen effects on eIF4F complex, mTOR, and ENaC translation in fetal lung epithelia.

Fetal distal lung epithelium (FDLE) must increase amiloride-sensitive epithelial Na(+) channel (ENaC) activity during the perinatal period to increase Na(+) transport and fluid clearance. Glucocorticosteroid (GC) levels increase, there is a 7-fold increase in Po(2) at birth, and we have previously shown that dexamethasone (DEX)-induced alpha-ENaC mRNA is efficiently translated only under postnatal (21%) O(2) (Otulakowski et al., AJRCMB 2006;34:204-212). Translation of mRNAs with long GC-rich 5'UTRs, such as alpha-ENaC mRNA, are sensitive to the amount of eIF4F, the mRNA 5'-cap binding complex composed of eIF4E and eIF4G. We now show, by Western blotting and m(7)GTP-Sepharose pull-down experiments, that in FDLE cultured under 3% O(2), DEX decreases formation of eIF4F and increases association of eIF4E with its inhibitor 4E-BP by changing 4E-BP phosphorylation. Conversely, FDLE cultured at 21% O(2) expressed lower levels of 4E-BP and maintained eIF4E-eIF4G association independent of DEX. Phosphorylation of 4E-BP is regulated by the kinase mTOR. Under 3% O(2), DEX decreased abundance of phosphorylated forms of the mTOR effectors, S6 kinase and ribosomal protein S6. Neither effect was associated with changes in REDD1, an upstream regulator of mTOR. When mTOR was inhibited (3 nM rapamycin) there was reduced 4E-BP phosphorylation, fewer ribosomes on alpha-ENaC mRNA, and decreased amiloride-sensitive short-circuit current, but no change in ribosomal loading onto any of beta- or gamma-ENaC or cytokeratin 18 mRNAs. We speculate that at birth increased Po(2) acts with GC through an mTOR-related pathway to increase alpha-ENaC protein synthesis, thereby promoting lung fluid absorption.

A novel knock-in prostate cancer model demonstrates biology similar to that of human prostate cancer and suitable for preclinical studies.

Preclinical studies of prostate cancer (CaP) have employed a genetically engineered mouse model, since there is no naturally occurring CaP in rodents. We have previously reported a new knock-in mouse adenocarcinoma prostate (KIMAP) model. In this study, we demonstrate that the new model possesses a tumor architecture of heterogeneity and multifocality similar to that of human CaP, by utilizing a new compound scoring system to compare with the PSP94 (approved gene symbol Msmb) gene-directed transgenic mouse CaP model (TGMAP). KIMAP mice showed a balanced distribution of tumor extent, which penetrated the prostate gland. Comparative studies on cDNA microarrays demonstrated that KIMAP tumors were upregulated with higher contents of immunoresponse genes, whereas PSP-TGMAP tumors had neuroendocrine (NE) differentiation. The majority of KIMAP mice did not progress to NE CaP, which was observed only at a very late stage and a low frequency. Several tumor marker genes characteristic of human CaP were uniquely identified in KIMAP tumors, including hepsin, maspin, Nkx3.1, CD10 and PSP94 (similar to PSA), etc. The differences between these two CaP models are attributed to the introduction of a single endogenous knock-in mutation. Due to the similarities between human CaP tumors and the PSP-KIMAP tumors, this preclinical model may supplement the current transgenic models to study CaP more accurately.

Knockin of SV40 Tag oncogene in a mouse adenocarcinoma of the prostate model demonstrates advantageous features over the transgenic model.

Prostate cancer (CaP) is the most common cancer in adult men in North America. Since there is no naturally occurring prostate cancer in the mouse, preclinical studies stipulate for the establishment of a genetically manipulated mouse CaP model with features close to the human situation. In view of the limitations of transgenic technique-derived CaP models, herein we report the first application of knockin technology to establish a new mouse adenocarcinoma prostate model (PSP-KIMAP) by targeting of SV40 Tag to a prostate tissue-specific gene, PSP94 (prostate secretory protein of 94 amino acids). In order to demonstrate its novelty, we compared KIMAP to a PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP) model. The CaP development of the PSP-KIMAP mice started almost immediately after puberty at 10 weeks of age from mouse prostatic intraepithelial neoplasia (mPIN) with microinvasion to well-differentiated CaP, and demonstrated a close-to-human kinetics of prolonged tumor growth and a predominance of well and moderately differentiated tumors. The invasive nature of KIMAP model was demonstrated by multitissue metastases (lymph node, lung and liver etc) and also by immunohistochemical study of multiple invasive prostate tumor markers. PSP-KIMAP model is responsive to androgen deprivation (castration). The knockin technology in our KIMAP model demonstrates highly predictive CaP development procedures and many advantageous features, which the traditional transgenic technique-derived CaP models could not reach for both basic and clinical studies. These features include the high stability of both phenotype and genotype, highly synchronous prostate cancer development, high and precise prostate tissue targeting and with no founder line variation. The differences between the two CaP models were attributed to the introduction of a single endogenous knockin mutation, resulting in a CaP model self-regulated and controlled by a prostate gene promoter/enhancer of PSP94.