Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Rapid and inexpensive bedside diagnosis of RAN binding protein 2-associated acute necrotizing encephalopathy.

Acute necrotizing encephalopathy 1 (ANE1) is a very rare disorder associated with a dominant heterozygous mutation in the RANBP2 (RAN binding protein 2) gene. ANE1 is frequently triggered by a febrile infection and characterized by serious and irreversible neurological damage. Although only a few hundred cases have been reported, mutations in RANBP2 are only partially penetrant and can occur de novo, suggesting that their frequency may be higher in some populations. Genetic diagnosis is a lengthy process, potentially delaying definitive diagnosis. We therefore developed a rapid bedside qPCR-based tool for early diagnosis and screening of ANE1 mutations. Primers were designed to specifically assess RANBP2 and not RGPD (RANBP2 and GCC2 protein domains) and discriminate between wild-type or mutant RANBP2. Nasal epithelial cells were obtained from two individuals with known RANBP2 mutations and two healthy control individuals. RANBP2-specific reverse transcription followed by allele-specific primer qPCR amplification confirmed the specific detection of heterozygously expressed mutant RANBP2 in the ANE1 samples. This study demonstrates that allele-specific qPCR can be used as a rapid and inexpensive diagnostic tool for ANE1 using preexisting equipment at local hospitals. It can also be used to screen non-hospitalized family members and at risk-population to better establish the frequency of non-ANE-associated RANBP2 mutations, as well as possible tissue-dependent expression patterns. Systematic review registration: The protocol was registered in the international prospective register of systematic reviews (PROSPERO- CRD42023443257).

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.

Statin-mediated disruption of Rho GTPase prenylation and activity inhibits respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory tract infections in children. To uncover new antiviral therapies, we developed a live cell-based high content screening approach for rapid identification of RSV inhibitors and characterized five drug classes which inhibit the virus. Among the molecular targets for each hit, there was a strong functional enrichment in lipid metabolic pathways. Modulation of lipid metabolites by statins, a key hit from our screen, decreases the production of infectious virus through a combination of cholesterol and isoprenoid-mediated effects. Notably, RSV infection globally upregulates host protein prenylation, including the prenylation of Rho GTPases. Treatment by statins or perillyl alcohol, a geranylgeranyltransferase inhibitor, reduces infection in vitro. Of the Rho GTPases assayed in our study, a loss in Rac1 activity strongly inhibits the virus through a decrease in F protein surface expression. Our findings provide new insight into the importance of host lipid metabolism to RSV infection and highlight geranylgeranyltransferases as an antiviral target for therapeutic development.

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.

Publisher Correction: IGF1R is an entry receptor for respiratory syncytial virus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

IGF1R is an entry receptor for respiratory syncytial virus.

Pneumonia resulting from infection is one of the leading causes of death worldwide. Pulmonary infection by the respiratory syncytial virus (RSV) is a large burden on human health, for which there are few therapeutic options1. RSV targets ciliated epithelial cells in the airways, but how viruses such as RSV interact with receptors on these cells is not understood. Nucleolin is an entry coreceptor for RSV2 and also mediates the cellular entry of influenza, the parainfluenza virus, some enteroviruses and the bacterium that causes tularaemia3,4. Here we show a mechanism of RSV entry into cells in which outside-in signalling, involving binding of the prefusion RSV-F glycoprotein with the insulin-like growth factor-1 receptor, triggers the activation of protein kinase C zeta (PKCζ). This cellular signalling cascade recruits nucleolin from the nuclei of cells to the plasma membrane, where it also binds to RSV-F on virions. We find that inhibiting PKCζ activation prevents the trafficking of nucleolin to RSV particles on airway organoid cultures, and reduces viral replication and pathology in RSV-infected mice. These findings reveal a mechanism of virus entry in which receptor engagement and signal transduction bring the coreceptor to viral particles at the cell surface, and could form the basis of new therapeutics to treat RSV infection.

Ketamine attenuates high-glucose-mediated endothelial inflammation in human umbilical vein endothelial cells.

Hyperglycemia mediates oxidative stress, thus inducing transcription factor nuclear factor kappa B (NF-κB) activation, increasing endothelial adhesion molecule expression and monocyte/endothelial interaction, and resulting in endothelial injury. Ketamine was reported to attenuate oxidative stress in many cases. In this research, we determined whether and how ketamine protects against high-glucose-mediated augmentation of monocyte/endothelial interaction and endothelial adhesion molecule expression in human umbilical vein endothelial cells. High glucose augmented monocyte/endothelial adhesion and endothelial adhesion molecule expression. High glucose induced reactive oxygen species (ROS) production and augmented phospho-protein kinase C (p-PKC) ßII expression and PKC activity. Moreover, high glucose inhibited the inhibitory subunit of nuclear factor-κBα (IκBα) expression in the cytoplasm and induced NF-κB nuclear translocation. Importantly, the effects induced by high glucose were counteracted by ketamine treatment. Further, CGP53353, a PKC ßII inhibitor, inhibited high-glucose-mediated NF-κB nuclear translocation, attenuated adhesion molecule expression, and reduced monocyte/endothelial interaction. Further, these effects of ketamine against high-glucose-induced endothelial injury were inhibited by phorbol 12-myristate 13-acetate, a PKC ßII activator. In conclusion, ketamine, via reducing ROS accumulation, inhibited PKC ßII Ser660 phosphorylation and PKC and NF-κB activation and reduced high-glucose-induced expression of endothelial adhesion molecules and monocyte/endothelial interaction.

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.

Dexmedetomidine attenuates LPS-mediated BV2 microglia cells inflammation via inhibition of glycolysis.

Microglia inflammation induces pro-inflammatory cytokines and pro-inflammatory enzymes expression, thus leading to inflammation-mediated neuronal cell death. Increased intracellular glycolysis participates in LPS-mediated microglia inflammation. Dexmedetomidine exhibits neuroprotective effects in some situations. In this study, we mainly focused on whether and how dexmedetomidine inhibits LPS-mediated cellular glycolysis and inflammation in BV2 cells. LPS induced pro-inflammatory cytokines and pro-inflammatory enzymes expression, and increased glycolysis capacity in BV2 cells. Moreover, inhibition of glycolysis by 2DG attenuated LPS-induced pro-inflammatory cytokines and pro-inflammatory enzymes expression. Moreover, LPS upregulated hypoxia-inducible factor 1α (HIF1α) expression and decreased sirt1 expression. Dexmedetomidine counteracted these effects induced by LPS. Further, 2-methoxyestradiol, a HIF1α inhibitor, could inhibit LPS-mediated glycolysis and inflammation in BV2 cells, which was similar to the effects of dexmedetomidine. In addition, these effects of dexmedetomidine could be reversed by EX527, a sirt1 inhibitor. The present study indicated that dexmedetomidine, via upregulation of sirt1 expression, inhibited HIF-1α expression and glycolysis, thus reducing LPS-mediated inflammation in BV2 cells.

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.

Propofol inhibits high glucose-induced PP2A expression in human umbilical vein endothelial cells.

Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis, dysfunction and inflammation, resulting in endothelial injury. Propofol is a widely used anesthetic drug in clinical settings. Our previous studies indicated that propofol, via inhibiting high glucose-induced phosphatase A2 (PP2A) expression, attenuated high glucose-induced reactive oxygen species (ROS) accumulation, thus improving endothelial apoptosis, dysfunction and inflammation. However, the mechanisms by which propofol attenuated high glucose-induced PP2A expression is still obscure. In the present study, we examined how propofol attenuates high glucose-induced endothelial PP2A expression. Compared with 5mM glucose treatment, 15mM glucose up-regulated expression and activity of PP2A, increased cAMP response element binding protein (CREB), Ca2+-calmodulin dependent kinase II (CaMK II) phosphorylation and Ca2+ accumulation. More importantly, propofol decreased PP2A expression and activity, attenuated CREB, CaMK II phosphorylation and Ca2+ accumulation in a concentration-dependent manner. Moreover, we demonstrated that the effect of propofol was similar to that of MK801, an inhibitor of NMDA receptor. In contrast, rapastinel, an activator of NMDA receptor, antagonized the effect of propofol. Also, the effect of KN93, an inhibitor of CaMK II, was similar to that of propofol, except KN93 had no effect on 15mM glucose-mediated Ca2+ accumulation. Our data indicated that propofol, via inhibiting NMDA receptor, attenuated 15mM glucose-induced Ca2+ accumulation, CaMK II and CREB phosphorylation, thus inhibiting PP2A expression and improving 15mM glucose-induced endothelial dysfunction and inflammation.

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.