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Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A 2A receptor (A 2A R). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A 2A R with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A 2A R are superior to shRNA mediated knockdown or pharmacological blockade of A 2A R. Mechanistically, human A 2A R-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A 2A R deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A 2A R for the improvement of CAR T cell function in the clinic.

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8 + CD62L + TCF7 + IRF4 - population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7 + phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

Background: Adverse drug reactions (ADRs) are one of the leading causes of morbidity and mortality in health care. Understanding which drug targets are linked to ADRs can lead to the development of safer medicines., Methods: Here, we analyse in vitro secondary pharmacology of common (off) targets for 2134 marketed drugs. To associate these drugs with human ADRs, we utilized FDA Adverse Event Reports and developed random forest models that predict ADR occurrences from in vitro pharmacological profiles., Findings: By evaluating Gini importance scores of model features, we identify 221 target-ADR associations, which co-occur in PubMed abstracts to a greater extent than expected by chance. Amongst these are established relations, such as the association of in vitro hERG binding with cardiac arrhythmias, which further validate our machine learning approach. Evidence on bile acid metabolism supports our identification of associations between the Bile Salt Export Pump and renal, thyroid, lipid metabolism, respiratory tract and central nervous system disorders. Unexpectedly, our model suggests PDE3 is associated with 40 ADRs., Interpretation: These associations provide a comprehensive resource to support drug development and human biology studies., Funding: This study was not supported by any formal funding bodies., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The Ras signaling pathway is commonly dysregulated in human malignant peripheral nerve sheath tumors (MPNSTs). It is well known that galectin-1 (Gal-1) is essential to stabilize membrane Ras and thereby induce the activation of Ras. However, the role of Gal-1 in MPNST progression remains unknown. The aim of this study was to examine whether Gal-1 knockdown could have an effect on the Ras signaling pathway., Methods: Cell viability, apoptosis assay, and colony formation were performed to examine the effects of inhibition of Gal-1 in MPNST cells. We used a human MPNST xenograft model to assess growth and metastasis inhibitory effects of Gal-1 inhibitor LLS2., Results: Gal-1 was upregulated in MPNST patients and was highly expressed in MPNST cells. Knockdown of Gal-1 by small interfering (si)RNA in Gal-1 expressing MPNST cells significantly reduces cell proliferation through the suppression of C-X-C chemokine receptor type 4 (CXCR4) and the rat sarcoma viral oncogene homolog (RAS)/extracellular signal-regulated kinase (ERK) pathway, which are important oncogenic signaling in MPNST development. Moreover, Gal-1 knockdown induces apoptosis and inhibits colony formation. LLS2, a novel Gal-1 allosteric small molecule inhibitor, is cytotoxic against MPNST cells and was able to induce apoptosis and suppress colony formation in MPNST cells. LLS2 treatment and Gal-1 knockdown exhibited similar effects on the suppression of CXCR4 and RAS/ERK pathways. More importantly, inhibition of Gal-1 expression or function by treatment with either siRNA or LLS2 resulted in significant tumor responses in an MPNST xenograft model., Conclusion: Our results identified an oncogenic role of Gal-1 in MPNST and that its inhibitor, LLS2, is a potential therapeutic agent, applied topically or systemically, against MPNST., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The interaction of neurotransmitters and genes that control the release of dopamine is the Brain Reward Cascade (BRC). Variations within the BRC, whether genetic or epigenetic, may predispose individuals to addictive behaviors and altered pain tolerance. This discussion authored by a group of concerned scientists and clinicians examines the Genetic Addiction Risk Score (GARS), the first test to accurately predict vulnerability to pain, addiction, and other compulsive behaviors, defined as Reward Deficiency Syndrome (RDS). Innovative strategies to combat epidemic opioid, iatrogenic prescription drug abuse and death, based on the role of dopaminergic tone in pain pathways, are proposed. Sensitivity to pain may reside in the mesolimbic projection system, where genetic polymorphisms associate with a predisposition to pain vulnerability or tolerance. They provide unique therapeutic targets that could assist in the treatment of pain, and identify risk for subsequent addiction. Pharmacogenomic testing of candidate genes like CB1, mu receptors, and PENK might result in pharmacogenomic, personalized solutions, and improved clinical outcomes. Genetically identifying risk for all RDS behaviors, especially in compromised populations, may be a frontline tool to assist municipalities to provide better resource allocation.

While there is a considerable body of literature correlating the role of dopaminergic genes and obesity, body mass index, body type, overeating, carbohydrate binging, energy expenditure and low dopamine D2 receptor (D2R) receptor density, there is a paucity of research concerning the dopamine D2 receptor gene (DRD2) variants and percent body fat. We report here the potential association of DRD2 genotypes and the percent fat phenotype. In this study we genotyped 122 obese/overweight (O/OW) Caucasian subjects and 30 non-obese Caucasian controls, screened to exclude substance abuse. The subjects were assessed for weight, body mass index (BMI; kg m(-2)) and percent body fat using dual energy X-ray absorptiometry (DEXA). The sample was separated into two independent groups; those with the Taq1 A1 allele (A1/A1 or A1/A2) and those without the A1 allele (A2/A2). The controls had a normal range of body fat (25-31% for females and 18-25% for males). The O/OW subjects had a percent body fat value of over 32% for females and over 25% for males. For the O/OW subjects, the mean BMI was 29.3 ± 6.25 kg m(-2), mean body fat was 42.1 ± 7.5% and mean weight was 82.7 ± 21.7 kg. The DRD2 Taq1 A1 allele was present in 67% of the O/OW subjects compared to 3.3% of super controls (A group), 33.3% of screened (for drug abuse and obesity) controls (B group) and unscreened literature controls 29.4% (P≤ 0.001). Comparing all cases with more than 34% body fat, utilizing logistic regression analysis, the DRD2 A1 allele accounts for 45.9% of the variance, which is statistically significant (χ(2) = 43.47, degrees of freedom (df) = 1, P < 0.0001). These results are consistent with a role for the DRD2 gene in obesity, as measured by percent body fat as well as by weight and BMI.

Introduction: Approximately 15% (more than 2 million individuals, based on these estimates) of all people with diabetes will develop a lower-extremity ulcer during the course of the disease. Ultimately, between 14% and 20% of patients with lower-extremity diabetic ulcers will require amputation of the affected limb. Analysis of the 1995 Medicare claims revealed that lower-extremity ulcer care accounted for $1.45 billion in Medicare costs. Therapies that promote rapid and complete healing and reduce the need for expensive surgical procedures would impact these costs substantially. One such example is the electrotherapeutic modality utilizing the H-Wave(R) device therapy and program.It has been recently shown in acute animal experiments that the H-Wave(R) device stimulation induces a nitric oxide-dependent increase in microcirculation of the rat Cremaster skeletal muscle. Moreover, chronic H-wave(R) device stimulation of rat hind limbs not only increases blood flow but induces measured angiogenesis. Coupling these findings strongly suggests that H-Wave(R) device stimulation promotes rapid and complete healing without need of expensive surgical procedures., Case Presentation: We decided to do a preliminary evaluation of the H-Wave(R) device therapy and program in three seriously afflicted diabetic patients. Patient 1 had chronic venous stasis for 6 years. Patient 2 had chronic recurrent leg ulcerations. Patient 3 had a chronic venous stasis ulcer for 2 years. All were dispensed a home H-Wave(R) unit. Patient 1 had no other treatment, patient 2 had H-Wave(R) therapy along with traditional compressive therapy, and patient 3 had no other therapy.For patient 1, following treatment the ulcer completely healed with the H-Wave(R) device and program after 3 months. For patient 2, by one month complete ulcer closure occurred. Patient 3 had a completely healed ulcer after 9 months., Conclusions: While most diabetic ulcers can be treated successfully on an outpatient basis, a significant proportion will persist and become infected. Based on this preliminary case series investigation we found that three patients prescribed H-Wave(R) home treatment demonstrate accelerated healing with excellent results. While these results are encouraging, additional large scale investigation is warranted before any interpretation is given to these interesting outcomes.

Unlabelled: The hypothesis that the H-Wave(R) device (Electronic Waveform Lab, Inc., Huntington Beach, CA), a small-diameter fiber stimulator, is a paradigm shift of electrotherapeutic treatment of pain associated with human neuropathies and sports injuries is based on a number of its properties. The primary effect of H-Wave(R) device stimulation (HWDS) is the stimulation of "red-slow-twitch" skeletal muscle fibers. The authors propose, based on the unique waveform, that the H-Wave(R) device specifically and directly stimulates the small smooth muscle fibers within the lymphatic vessels ultimately leading to fluid shifts and reduced edema. In unpublished rat studies, it has been observed that HWDS induces protein clearance. The H-Wave(R) device was designed to stimulate an ultra low frequency (1-2 Hz), low tension, nontetanizing, and nonfatiguing contraction, which closely mimics voluntary or natural muscle contractions. The H-Wave(R) device can stimulate small fibers due in part to its exponentially decaying waveform and constant current generator activity. The main advantage of these technologies over currently applied electrical stimulators (eg, transcutaneous electrical nerve stimulator [TENS], interferential [IF], neuromuscular electrical stimulation [NMES], high-volt galvanic, etc.) is that H-Wave\'s(R) small fiber contraction does not trigger an activation of the motor nerves of the large white muscle fibers or the sensory delta and C pain nerve fibers, thus eliminating the negative and painful effects of tetanizing fatigue, which reduces transcapillary fluid shifts. Another function of the H-Wave(R) device is an anesthetic effect on pain conditions, unlike a TENS unit which in the short term activates a hypersensory overload effect (gate theory) to stop pain signals from reaching the thalamic region of the brain. When the H-Wave(R) device is used at high frequency (60 Hz), it acts intrinsically on the nerve to deactivate the sodium pump within the nerve fiber, leading to a long-lasting anesthetic/analgesic effect due to an accumulative postsynaptic depression. Moreover, HWDS produces a nitric oxide (NO)-dependent enhancement of microcirculation and angiogenesis in rats. Thus, the authors hypothesize that because of these innate properties of the H-Wave(R) device, it may provide a paradigm shift for the treatment of both short- and long-term inflammatory conditions associated with pain due to sports injuries. A recent meta-analysis found a moderate-to-strong effect of the H-Wave(R) device in providing pain relief, reducing the requirement for pain medication, and increasing functionality. The most robust effect was observed for improved functionality, suggesting that the H-Wave(R) device may facilitate a quicker return to the field., Keywords: H-Wave(R) device; sportsmedicine, nitric oxide-dependent blood flow; analgesia; angiogenesis.