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.

Amino-terminal dimerization of an erythropoietin mimetic peptide results in increased erythropoietic activity.

BACKGROUND: Erythropoietin (EPO), the hormone involved in red blood cell production, activates its receptor by binding to the receptor's extracellular domain and presumably dimerizing two receptor monomers to initiate signal transduction. EPO-mimetic peptides, such as EMP1, also bind and activate the receptor by dimerization. These mimetic peptides are not as potent as EPO, however. The crystal structure of the EPO receptor (EBP) bound to EMP1 reveals the formation of a complex consisting of two peptides bound to two receptors, so we sought to improve the biological activity of EPO-mimetic peptides by constructing covalent dimers of EMP1 and other peptide mimetics linked by polyethylene glycol (PEG). RESULTS: The potency of the PEG-dimerized EPO peptide mimetics both in vitro and in vivo was improved up to 1,000-fold compared to the corresponding peptide monomers. The dimers were constructed using peptide monomers which have only one reactive amine per molecule, allowing us to conclude that the increase in potency can be attributed to a structure in which two peptides are linked through their respective amino termini to the difunctional PEG molecule. In addition, an inactive peptide was converted into a weak agonist by PEG-induced dimerization. CONCLUSIONS: The potency of previously isolated peptides that are modest agonists of the EPO receptor was dramatically increased by PEG-induced dimerization. The EPO receptor is thought to be dimerized during activation, so our results are consistent with the proposed 2:2 receptor : peptide stoichiometry. The conversion of an inactive peptide into an agonist further supports the idea that dimerization can mediate receptor activation.

Posttraumatic stress disorder in adolescents after Hurricane Andrew.

OBJECTIVE: To examine rates and correlates of posttraumatic stress disorder (PTSD) in adolescents after Hurricane Andrew. METHOD: A random-digit dialing sample of 158 Hispanic, 116 black, and 104 white adolescent-parent pairs were surveyed in high- and low-impact areas within Dade County, Florida, 6 months after Hurricane Andrew. Subjects completed a structured telephone interview focused on within-disaster experiences and emotional reaction, disaster-related losses, lifetime exposure to violent or traumatic events, recent stressful experiences, and psychiatric symptomatology. RESULTS: Approximately 3% of males (95% confidence interval 0.4 to 5.3) and 9% of females (95% confidence interval 4.6 to 13.7) met the criteria for PTSD. Rates were highest among blacks (8.3%, 95% confidence interval 2.3 to 14.2) and Hispanics (6.1%, 95% confidence interval 2.2 to 9.9) and increased with age (odds ratio of 1.34, 95% confidence interval 1.04 to 1.72) and the number of undesirable events reported (odds ratio of 1.38, 95% confidence interval 1.21 to 1.57). CONCLUSIONS: While only a relatively small percentage of adolescents reported symptoms consistent with a diagnosis of PTSD, most reported some posttraumatic symptoms. Postdisaster planning should recognize that common stressful events occurring after disasters may be more strongly associated with PTSD than magnitude of contact with the actual disaster.

Los Angeles County after the 1992 civil disturbances: degree of exposure and impact on mental health.

The impact of the 1992 Los Angeles (L.A.) civil disturbances on psychosocial functioning was assessed as part of a larger project investigating the views and attitudes of residents in L.A. County. Random digit dialing methodology identified a household probability sample of 1,200 adults (age 18 or older) from L.A. County. Respondents completed a telephone interview 6 to 8 months after the disturbances. Respondents' degree of exposure to the disturbances, mental health impact of the disturbances, and mental health effects of chronic versus acute exposure to violence were assessed. Consistent with hypotheses, the impact of the disturbances was the worst in the South Central communities. Higher rates of posttraumatic stress disorder (PTSD; both diagnostic level and subclinical symptomatology) were found among respondents who reported disturbance-related experiences. Exposure to an acute event (i.e., the disturbances) was predictive of current PTSD symptomatology after controlling for demographics, lifetime trauma, and other types of stressful events.

Psychosocial resource loss as a mediator of the effects of flood exposure on psychological distress and physical symptoms.

This study used the Conservation of Resources stress model to examine the role of psychosocial resource loss in the aftermath of Midwest flooding. Questionnaires were distributed through churches and completed by 131 adults in flood-affected communities 6 weeks and 6 months after the flood's crest. Frequent psychosocial losses included losses of routine, sense of control, sense of optimism, accomplishing goals, and time with loved ones. Path analysis revealed that psychosocial resource loss mediated the effects of flood exposure on both psychological distress and physical symptoms at 6 months post-flood. The findings suggest that interventions designed to prevent psychosocial resource loss may reduce the long-term effects of disasters.

Identification of disulfide bridges in a cardiotoxic peptide by electrospray ionization.

A new method was developed for subjecting a peptide to a specified number of Edman degradation cycles on an automated polypeptide sequencer and desorbing the residual peptide for further investigations. The procedure was applied in combination with electrospray ionization mass spectrometry to identify the four disulfide bridges present in a small tightly bound peptide. The task was accomplished using only a few nanomoles of the intact peptide.

Understanding acute psychological distress following natural disaster.

A household probability sample of 229 adults was interviewed four to seven months after the Sierra Madre earthquake (June 28, 1991; Los Angeles County). The study predicted psychological distress from these variables: demographics, traumatic event history, low magnitude event history, earthquake related threat perceptions, and earthquake related resource loss. Based on the Conservation of Resources (COR) stress model, it was predicted that resource loss would be central in predicting psychological distress. Three major hypotheses were supported: (1) resource loss was positively associated with psychological distress; (2) resource loss predicted psychological distress when other predictors were statistically controlled; and (3) resource loss was associated with mild to moderate elevations in of psychological distress. The findings support COR stress theory. Theoretical and practical implications are discussed.