Gene therapy-emulating small molecule treatments in cystic fibrosis airway epithelial cells and patients.

BACKGROUND: Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy. METHODS: IB3-1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[wildtype]CFTR-treated IB3-1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients. RESULTS: CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures. CONCLUSIONS: Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.

Gene-expression data integration to squamous cell lung cancer subtypes reveals drug sensitivity.

BACKGROUND: Squamous cell lung cancer (SqCC) is the second most common type of lung cancer in the United States. Previous studies have used gene-expression data to classify SqCC samples into four subtypes, including the primitive, classical, secretory and basal subtypes. These subtypes have different survival outcomes, although it is unknown whether these molecular subtypes predict response to therapy. METHODS: Here, we analysed RNAseq data of 178 SqCC tumour samples and characterised the features of the different SqCC subtypes to define signature genes and pathway alterations specific to each subtype. Further, we compared the gene-expression features of each molecular subtype to specific time points in models of airway development. We also classified SqCC-derived cell lines and their reported therapeutic vulnerabilities. RESULTS: We found that the primitive subtype may come from a later stage of differentiation, whereas the basal subtype may be from an early time. Most SqCC cell lines responded to one of five anticancer drugs (Panobinostat, 17-AAG, Irinotecan, Topotecan and Paclitaxel), whereas the basal-type cell line EBC-1 was sensitive to three other drugs (PF2341066, AZD6244 and PD-0325901). CONCLUSION: Compared with the other three subtypes of cell lines, the secretory-type cell lines were significantly less sensitive to the five most effective drugs, possibly because of their low proliferation activity. We provide a bioinformatics framework to explore drug repurposing for cancer subtypes based on the available genomic profiles of tumour samples, normal cell types, cancer cell lines and data of drug sensitivity in cell lines.

Different cellular p16(INK4a) localisation may signal different survival outcomes in head and neck cancer.

BACKGROUND: Recently, the management of head and neck squamous cell carcinoma (HNSCC) has focused considerable attention on biomarkers, which may influence outcomes. Tests for human papilloma infection, including direct assessment of the virus as well as an associated tumour suppressor gene p16, are considered reproducible. Tumours from familial melanoma syndromes have suggested that nuclear localisation of p16 might have a further role in risk stratification. We hypothesised p16 staining that considered nuclear localisation might be informative for predicting outcomes in a broader set of HNSCC tumours not limited to the oropharynx, human papilloma virus (HPV) status or by smoking status. METHODS: Patients treated for HNSCC from 2002 to 2006 at UNC (University of North Carolina at Chapel Hill) hospitals that had banked tissue available were eligible for this study. Tissue microarrays (TMA) were generated in triplicate. Immunohistochemical (IHC) staining for p16 was performed and scored separately for nuclear and cytoplasmic staining. Human papilloma virus staining was also carried out using monoclonal antibody E6H4. p16 expression, HPV status and other clinical features were correlated with progression-free (PFS) and overall survival (OS). RESULTS: A total of 135 patients had sufficient sample for this analysis. Median age at diagnosis was 57 years (range 20-82), with 68.9% males, 8.9% never smokers and 32.6% never drinkers. Three-year OS rate and PFS rate was 63.0% and 54.1%, respectively. Based on the p16 staining score, patients were divided into three groups: high nuclear, high cytoplasmic staining group (HN), low nuclear, low cytoplasmic staining group (LS) and high cytoplasmic, low nuclear staining group (HC). The HN and the LS groups had significantly better OS than the HC group with hazard ratios of 0.10 and 0.37, respectively, after controlling for other factors, including HPV status. These two groups also had significantly better PFS than the HC staining group. This finding was consistent for sites outside the oropharynx and did not require adjustment for smoking status. CONCLUSION: Different p16 protein localisation suggested different survival outcomes in a manner that does not require limiting the biomarker to the oropharynx and does not require assessment of smoking status.

Cocaine-associated chest pain: how common is myocardial infarction?

OBJECTIVE: Prior studies addressing the incidence of acute myocardial infarction (AMI) in patients with cocaine-associated chest pain have found divergent results. Previous prospective studies, which found approximately a 6% incidence of AMI, have been criticized for selection bias. This study sought to determine the rate of AMI in patients with cocaine-associated chest pain. METHODS: All patients seen in an urban university-affiliated hospital between July 1996 and February 1998 were identified by ICD-9 medical records search for cocaine use and chest pain/ acute coronary syndromes. In this system, all faculty admit all patients with cocaine-associated chest pain for at least 23-hour observation periods. Data collected included demographics, medical and cocaine use history, presenting characteristics, hospital course, cardiovascular complications, and diagnostic tests using a 119-item closed-question data instrument with high interrater reliability. The main outcome measure was AMI according to World Health Organization (WHO) criteria. RESULTS: There were 250 patients identified with a mean age of 33.5 +/- 8.5 years; 77% were male; 84% were African American. Of 196 patients tested, 185 had cocaine or cocaine metabolites in the urine (94%). The incidence of cardiac risk factors were: hypercholesterolemia, 8%; diabetes, 6%; family history, 34%; hypertension, 26%; tobacco use, 77%; prior MI, 6%; and prior chest pain, 40%. Seventy-seven percent admitted to cocaine use in the preceding 24 hours: crack, 85%; IV, 2%; nasal, 6%. Twenty-five patients (10%) had electrocardiographic evidence of ischemia. A total of 15 patients experienced an AMI (6%; 95% CI = 4.1% to 8.9%) using WHO criteria. Complications were infrequent: bradydysrrhythmias, 0.4%; congestive heart failure, 0.4%; supraventricular tachycardia, 1.2%; sustained ventricular tachycardia, 0.8%. CONCLUSION: The incidence of AMI was 6% in patients with cocaine-associated chest pain. This result is identical to that found in prior prospective studies.

A combination of midazolam and ketamine for procedural sedation and analgesia in adult emergency department patients.

OBJECTIVE: To describe the clinical characteristics of a combination of midazolam and ketamine for procedural sedation and analgesia in adult emergency department (ED) patients. METHODS: This was a prospective, observational trial, conducted in the ED of an urban level II trauma center. Patients > or = 18 years of age requiring procedural sedation and analgesia were eligible, and enrolled patients received 0.07 mg/kg of intravenous midazolam followed by 2 mg/kg of intravenous ketamine. Vital signs were recorded at regular intervals. The adequacy of sedation, adverse effects, patient satisfaction, and time to reach discharge alertness were determined. Descriptive statistics were calculated using statistical analysis software. RESULTS: Seventy-seven patients were enrolled. Three were excluded due to protocol violations, three due to lack of documentation, and one due to subcutaneous infiltration of ketamine, leaving 70 patients for analysis. The average age was 31 years, and 41 (59%) were female. Indications for procedural sedation and analgesia included abscess incision and drainage (66%), fracture/joint reduction (26%), and other (8%). The mean dose of midazolam was 5.6 +/- 1.4 mg and the mean dose of ketamine was 159 +/- 42 mg. The mean time to achieve discharge criteria was 64 +/- 24 minutes. Five patients experienced mild emergence reactions, but there were no episodes of hallucinations, delirium, or other serious emergence reactions. Eighteen (25%) patients recalled dreaming while sedated; twelve (17%) were described as pleasant, two (3%) unpleasant, three (4%) both pleasant and unpleasant, and one (1%) neither pleasant nor unpleasant. There were four (6%) cases of respiratory compromise, two (3%) episodes of emesis, and one (1%) case of myoclonia. All of these were transient and did not result in a change in the patient's disposition. Only one (1%) patient indicated that she was not satisfied with the sedation regimen. CONCLUSIONS: The combination of midazolam and ketamine provides effective procedural sedation and analgesia in adult ED patients, and appears to be safe.

A randomized comparison of helium-oxygen mixture (Heliox) and racemic epinephrine for the treatment of moderate to severe croup.

OBJECTIVE: To compare the additive effect of a helium-oxygen mixture (Heliox) or racemic epinephrine (RE) on croup scores (CSs) in children with moderate to severe croup treated with humidified oxygen and steroids. Design. A prospective, randomized, double-blind trial. SETTING: Emergency department and pediatric intensive care unit of an urban level I trauma center. PARTICIPANTS: Randomly assigned, consecutive children ages 6 months to 3 years presenting with moderate to severe croup (CS: >/=5). Interventions. After cool humidified oxygen and 0.6 mg/kg of intramuscular dexamethasone, patients were randomized to receive either Heliox or RE. Vital signs, oxygen saturation, and CSs were recorded at regular intervals. OUTCOME/ANALYSIS: Reductions in CSs were compared using repeated-measures analysis of variance. RESULTS: Thirty-three patients were enrolled. Three were excluded because of protocol violations, and 1 was excluded because of lack of documentation, leaving 29 patients for final analysis. The average age was 24.2 months, 20 were male (68.8%). Both Heliox and RE were associated with improvement in CSs over time. There were no significant differences in mean CS, oxygen saturation, respiratory rate, or heart rate between groups at baseline or at the end of the treatment period. CONCLUSION: In patients with moderate to severe croup, the administration of Heliox resulted in similar improvements in CS compared with patients given RE.