Trehalose Uptake through P2X7 Purinergic Channels Provides Dehydration Protection

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors (Steinberg et al., 1987; Sung et al., 1985), and with sustained application of ATP it has been shown that dyes as large as 831 daltons can permeate the cell membrane (Steinberg et al, 1987). The current study explores the kinetics of loading α,α-trehalose (342 daltons) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor (Steinberg et al., 1987). Cells that were incubated at 37 ̊C in a 50 mM phosphate buffer (pH 7.0) contailing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of ~50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 ̊C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 oC (\u3e60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 oC to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49% ± 12 % by Trypan Blue exclusion, 56 ± 20% by Alamar BlueTM assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells

Sox2 Cooperates with Lkb1 Loss in a Mouse Model of Squamous Cell Lung Cancer

Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer. With limited treatment options, the 5-year survival rate of SCC is only 15%. Although genomic alterations in SCC have been characterized, identifying the alterations that drive SCC is critical for improving treatment strategies. Mouse models of SCC are currently limited. Using lentiviral delivery of Sox2 specifically to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Expression of Sox2, frequently amplified in human SCC, specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Mouse tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. This model may be utilized to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery

Accelerated Resolution Therapy for Treatment of Pain Secondary to Symptoms of Combat-Related Posttraumatic Stress Disorder

Background: As many as 70% of veterans with chronic pain treated within the US Veterans Administration (VA) system may have posttraumatic stress disorder (PTSD), and conversely, up to 80% of those with PTSD may have pain. We describe pain experienced by US service members and veterans with symptoms of PTSD, and report on the effect of Accelerated Resolution Therapy (ART), a new, brief exposure-based therapy, on acute pain reduction secondary to treatment of symptoms of PTSD. Methods: A randomized controlled trial of ART versus an attention control (AC) regimen was conducted among 45 US service members/veterans with symptoms of combat-related PTSD. Participants received a mean of 3.7 sessions of ART. Results: Mean age was 41.0 + 12.4 years and 20% were female. Most veterans (93%) reported pain. The majority (78%) used descriptive terms indicative of neuropathic pain, with 29% reporting symptoms of a concussion or feeling dazed. Mean pre-/post-change on the Pain Outcomes Questionnaire (POQ) was −16.9±16.6 in the ART group versus −0.7±14.2 in the AC group (p=0.0006). Among POQ subscales, treatment effects with ART were reported for pain intensity (effect size = 1.81, p=0.006), pain-related impairment in mobility (effect size = 0.69, p=0.01), and negative affect (effect size = 1.01, p=0.001). Conclusions: Veterans with symptoms of combat-related PTSD have a high prevalence of significant pain, including neuropathic pain. Brief treatment of symptoms of combat-related PTSD among veterans by use of ART appears to acutely reduce concomitant pain

Accelerated Resolution Therapy for Treatment of Pain Secondary to Symptoms of Combat-Related Posttraumatic Stress Disorder

Background: As many as 70% of veterans with chronic pain treated within the US Veterans Administration (VA) system may have posttraumatic stress disorder (PTSD), and conversely, up to 80% of those with PTSD may have pain. We describe pain experienced by US service members and veterans with symptoms of PTSD, and report on the effect of Accelerated Resolution Therapy (ART), a new, brief exposure-based therapy, on acute pain reduction secondary to treatment of symptoms of PTSD. Methods: A randomized controlled trial of ART versus an attention control (AC) regimen was conducted among 45 US service members/veterans with symptoms of combat-related PTSD. Participants received a mean of 3.7 sessions of ART. Results: Mean age was 41.0 + 12.4 years and 20% were female. Most veterans (93%) reported pain. The majority (78%) used descriptive terms indicative of neuropathic pain, with 29% reporting symptoms of a concussion or feeling dazed. Mean pre-/post-change on the Pain Outcomes Questionnaire (POQ) was −16.9±16.6 in the ART group versus −0.7±14.2 in the AC group (p=0.0006). Among POQ subscales, treatment effects with ART were reported for pain intensity (effect size = 1.81, p=0.006), pain-related impairment in mobility (effect size = 0.69, p=0.01), and negative affect (effect size = 1.01, p=0.001). Conclusions: Veterans with symptoms of combat-related PTSD have a high prevalence of significant pain, including neuropathic pain. Brief treatment of symptoms of combat-related PTSD among veterans by use of ART appears to acutely reduce concomitant pain

The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor