Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity.

Cutaneous TRPV1+ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1+ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1+ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1+ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1+ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

The American Heart Association 2030 Impact Goal: A Presidential Advisory From the American Heart Association.

Each decade, the American Heart Association (AHA) develops an Impact Goal to guide its overall strategic direction and investments in its research, quality improvement, advocacy, and public health programs. Guided by the AHA's new Mission Statement, to be a relentless force for a world of longer, healthier lives, the 2030 Impact Goal is anchored in an understanding that to achieve cardiovascular health for all, the AHA must include a broader vision of health and well-being and emphasize health equity. In the next decade, by 2030, the AHA will strive to equitably increase healthy life expectancy beyond current projections, with global and local collaborators, from 66 years of age to at least 68 years of age across the United States and from 64 years of age to at least 67 years of age worldwide. The AHA commits to developing additional targets for equity and well-being to accompany this overarching Impact Goal. To attain the 2030 Impact Goal, we recommend a thoughtful evaluation of interventions available to the public, patients, providers, healthcare delivery systems, communities, policy makers, and legislators. This presidential advisory summarizes the task force's main considerations in determining the 2030 Impact Goal and the metrics to monitor progress. It describes the aspiration that these goals will be achieved by working with a diverse community of volunteers, patients, scientists, healthcare professionals, and partner organizations needed to ensure success.

Phase 1 multicenter dose-escalation study of ezatiostat hydrochloride (TLK199 tablets), a novel glutathione analog prodrug, in patients with myelodysplastic syndrome.

Phase 1 testing of ezatiostat, a glutathione S-transferase P1-1 inhibitor, for the treatment of myelodysplastic syndrome was conducted in a multidose-escalation study. Patients received 10 dose levels (200, 400, 1000, 1400, 2000, 2400, 3000, 4000, 5000, and 6000 mg) of ezatiostat tablets in divided doses on days 1 to 7 of a 21-day cycle for a maximum of 8 cycles. The safety and pharmacokinetics of ezatiostat were evaluated. Forty-five patients with low to intermediate-2 International Prognostic Scoring System risk myelodysplastic syndrome were enrolled. No dose-limiting toxicities were observed. The most common grade 1 or 2, respectively, treatment-related adverse events were nonhematologic: nausea (56%, 9%), diarrhea (36%, 7%), vomiting (24%, 7%), abdominal pain (9%, 0%), constipation (4%, 9%), anorexia (3%, 7%), and dyspepsia (3%, 7%). Concentration of the primary active metabolite, TLK236, increased proportionate to ezatiostat dosage. Seventeen hematologic improvement (HI) responses by International Working Group criteria were observed at dose levels of 200 to 6000 mg/day with 11 HI responses at doses of 4000 to 6000 mg/day. HI responses occurred in all lineages including 3 bilineage and 1 complete cytogenetic response. Decreased number of red blood cell and platelet transfusions and in some cases transfusion independence were attained. Extended dose schedules of ezatiostat tablets are under investigation.

Randomized phase III study of canfosfamide in combination with pegylated liposomal doxorubicin compared with pegylated liposomal doxorubicin alone in platinum-resistant ovarian cancer.

OBJECTIVE: To evaluate the safety and efficacy of canfosfamide in combination with pegylated liposomal doxorubicin (PLD) in platinum-resistant ovarian cancer (OC). METHODS: Patients with platinum-refractory or -resistant (primary or secondary) OC were randomized to receive canfosfamide at 1000 mg/m² and PLD at 50 mg/m² intravenously or PLD alone at 50 mg/m2 intravenously on day 1 every 28 days until tumor progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). Other end points were objective response rate and safety. The study was originally planned for 244 patients. The trial was temporarily placed on hold after 125 patients were randomized while the results of another trial were being reviewed and the sponsor decided not to resume enrollment. The interim analysis became the final analysis. RESULTS: The median PFS was 5.6 months for canfosfamide + PLD (n = 65) versus 3.7 months for PLD (n = 60) (hazards ratio, 0.92; P = 0.7243). A preplanned subgroup analysis showed that 75 patients with platinum-refractory or primary platinum-resistant OC had a median PFS of 5.6 months for canfosfamide + PLD versus 2.9 months for PLD (hazards ratio, 0.55; P = 0.0425). Hematologic adverse events were 66% on the canfosfamide + PLD arm versus 44% on the PLD arm, manageable with dose reductions. Nonhematologic adverse events were similar for both arms. The incidence of palmar-plantar erythrodysesthesia and stomatitiswas lower on canfosfamide + PLD(23%, 31%, respectively) versus (39%, 49%, respectively) on PLD. CONCLUSIONS: Overall median PFS showed a positive trend but was not statistically significant. The median PFS in the platinum-refractory and primary platinum-resistant OC patients was significantly longer for canfosfamide + PLD versus PLD. Canfosfamide may ameliorate the palmar-plantar erythrodysesthesia and stomatitis known to be associated with PLD. Further study of this active well-tolerated regimen in platinum-refractory and primary platinum-resistant OC is planned. This study was registered at www.clinicaltrials.gov: NCT00350948.

Facile coupling of synthetic peptides and peptide-polymer conjugates to cartilage via transglutaminase enzyme.

Covalent attachment of synthetic and biological molecules to tissue surfaces can be used to enhance local drug delivery, reduce adhesions after surgery, and attach reconstructive biomaterials and tissue-engineered matrices to tissues. We present here a mild approach to coupling polymers to tissue surfaces through an enzyme catalyzed reaction between peptide modified polymer and native protein components of the tissue extracellular matrix (ECM). Tissue transglutaminase (tTG), a Ca2+-dependent enzyme that catalyzes the reaction between lysine and glutamine residues to form a epsilon(gamma-glutaminyl) lysine isopeptide bond, was incubated with cartilage in the presence of lysine (FKG-NH2) and glutamine (GQQQLG-NH2) peptides as well as peptide functionalized poly(ethylene glycol) (PEG). Immunohistochemistry was used to detect the presence of covalently bound PEG polymer at the tissue surface as well as to a depth of as much as 10 microm below the surface. Collagen II, fibronectin, osteopontin and osteonectin were found to react with the peptides and peptide modified PEG in the presence of tTG in solution, suggesting these cartilage ECM components as being substrates in the tissue reaction. The results illustrate the use of tTG as a simple, effective and biologically compatible method of coupling synthetic and biological molecules to cartilage and other tissues containing ECM proteins that are substrates of tTG.

Radiofrequency septal reduction in symptomatic hypertrophic obstructive cardiomyopathy.

BACKGROUND: Alcohol septal ablation remains the only approved nonsurgical therapeutic alternative for patients with drug-resistant hypertrophic obstructive cardiomyopathy (HOCM). Radiofrequency (RF) ablation offers another option for treating HOCM. OBJECTIVE: The purpose of this study was to determine whether irrigated RF ablation can reduce symptomatic outflow tract obstruction in adults with HOCM. METHODS: Patients with symptomatic HOCM and an outflow gradient of >50 mm Hg despite medication were offered RF ablation. In 11 patients, the hypertrophied interventricular septum was localized on a 3-dimensional mapping system and ablated via a transmitral or retrograde aortic approach. RESULTS: Ten of 11(91%) patients had a significant and persistent reduction in resting and provocable left ventricular outflow tract gradients. Mean resting gradients at 12 months postprocedure were reduced by 85% (66.7 mm Hg at baseline to 10.0 mm Hg at 12 months); mean provocable gradients were reduced by 85% from baseline (136.2 mm Hg at baseline to 20.0 mm Hg at 12 months). Functional New York Heart Association heart classification improved from class 3.0 ± 0.0 in all patients to class mean of 1.8 ± 0.8. CONCLUSION: RF septal ablation for the treatment of HOCM is a safe therapeutic option that allows for significant reduction in left ventricular outflow tract gradients, improvement in symptoms, and increase in efficacy rates comparable to reported rates for alcohol septal ablation.

Intrathecal cannabinoid-1 receptor agonist prevents referred hyperalgesia in acute acrolein-induced cystitis in rats.

We investigated the capacity of intrathecal arachidonyl-2'-chloroethylamide (ACEA), a cannabinoid-1 receptor (CB1R) agonist, to inhibit referred hyperalgesia and increased bladder contractility resulting from acute acrolein-induced cystitis in rats. 24 female rats were divided into 4 groups: 1) intrathecal vehicle/intravesical saline; 2) intrathecal vehicle/intravesical acrolein; 3) intrathecal ACEA/intravesical saline; and 4) intrathecal ACEA/intravesical acrolein. Bladder catheters were placed 4-6 days prior to the experiment. On the day of the experiment, rats were briefly anesthetized with isoflurane to recover the external end of the cystostomy catheter. After recovery from anesthesia, pre-treatment cystometry was performed, and mechanical sensitivity of the hindpaws was determined. Rats were again briefly anesthetized with isoflurane to inject ACEA or vehicle into the intrathecal space between L5-L6. Beginning 10 minutes after intrathecal injection, saline or acrolein was infused into the bladder for 30 minutes. Post-treatment cystometry and mechanical sensitivity testing were performed. Rats were euthanized, and bladders were collected, weighed, and fixed for histology. The intrathecal vehicle/intravesical acrolein group developed mechanical hyperalgesia with post-treatment mechanical sensitivity of 6 ± 0.3 g compared to pretreatment of 14 ± 0.4 g (p < 0.01). Pre- and post-treatment hind paw mechanical sensitivity was statistically similar in rats that received intrathecal ACEA prior to intravesical infusion of acrolein (15 ± 0.2 g and 14 ± 0.4 g, respectively). Acrolein treatment increased basal bladder pressure and maximal voiding pressure and decreased intercontraction interval and voided volume. However, intrathecal ACEA was ineffective in improving acrolein-related urodynamic changes. In addition, bladder histology demonstrated submucosal and muscularis edema that was similar for all acrolein-treated groups, irrespective of ACEA treatment. Intravesical saline had no effect on results of cystometry or mechanical sensitivity of the hind paws, regardless of intrathecal treatment. Intrathecal ACEA prevented referred hyperalgesia associated with acute acrolein-induced cystitis. However, in this experimental model, ACEA did not ameliorate the associated urodynamic changes. These findings suggest that pain arising from cystitis may be inhibited by activation of spinal CB1R but the acute local response of the bladder appeared to be unaffected by stimulation of spinal CB1R.

Phase 2 study of canfosfamide in combination with pegylated liposomal doxorubicin in platinum and paclitaxel refractory or resistant epithelial ovarian cancer.

BACKGROUND: Canfosfamide is a novel glutathione analog activated by glutathione S-transferase P1-1. This study evaluated the safety and efficacy of canfosfamide in combination with pegylated liposomal doxorubicin (PLD) in patients with platinum resistant ovarian cancer. Patients with platinum resistant ovarian carcinoma and measurable disease received canfosfamide at 960 mg/m2 in combination with PLD at 50 mg/m2, intravenously day 1 in every 28 day cycles until tumor progression or unacceptable toxicities. The primary endpoints were objective response rate (ORR) and progression-free survival (PFS). RESULTS: Canfosfamide plus PLD combination therapy was administered at 960/50 mg/m2, respectively. Thirty-nine patients received a median number of 4 cycles (range 1.0-18.0). The ORR was 27.8% (95% CI, 14.2-45.2) with a disease stabilization rate of 80.6% (95% CI, 64.0-91.8) in the evaluable population. The CA-125 marker responses correlated with the radiological findings of complete response or partial response. The median PFS was 6.0 months (95% CI, 4.2-7.9) and median survival was 17.8 months. The combination was well tolerated. Myelosuppression was managed with dose reductions and growth factor support. Grade 3 febrile neutropenia was observed in 2 patients (5.1%). Non-hematologic adverse events occurred at the expected frequency and grade for each drug alone, with no unexpected or cumulative toxicities. CONCLUSIONS: Canfosfamide in combination with PLD is well tolerated and active in platinum and paclitaxel refractory or resistant ovarian cancer. A randomized phase 3 study was conducted based on this supportive phase 2 study.

Phase 1-2a multicenter dose-escalation study of ezatiostat hydrochloride liposomes for injection (Telintra, TLK199), a novel glutathione analog prodrug in patients with myelodysplastic syndrome.

BACKGROUND: Ezatiostat hydrochloride liposomes for injection, a glutathione S-transferase P1-1 inhibitor, was evaluated in myelodysplastic syndrome (MDS). The objectives were to determine the safety, pharmacokinetics, and hematologic improvement (HI) rate. Phase 1-2a testing of ezatiostat for the treatment of MDS was conducted in a multidose-escalation, multicenter study. Phase 1 patients received ezatiostat at 5 dose levels (50, 100, 200, 400 and 600 mg/m2) intravenously (IV) on days 1 to 5 of a 14-day cycle until MDS progression or unacceptable toxicity. In phase 2, ezatiostat was administered on 2 dose schedules: 600 mg/m2 IV on days 1 to 5 or days 1 to 3 of a 21-day treatment cycle. RESULTS: 54 patients with histologically confirmed MDS were enrolled. The most common adverse events were grade 1 or 2, respectively, chills (11%, 9%), back pain (15%, 2%), flushing (19%, 0%), nausea (15%, 0%), bone pain (6%, 6%), fatigue (0%, 13%), extremity pain (7%, 4%), dyspnea (9%, 4%), and diarrhea (7%, 4%) related to acute infusional hypersensitivity reactions. The concentration of the primary active metabolites increased proportionate to ezatiostat dosage. Trilineage responses were observed in 4 of 16 patients (25%) with trilineage cytopenia. Hematologic Improvement-Erythroid (HI-E) was observed in 9 of 38 patients (24%), HI-Neutrophil in 11 of 26 patients (42%) and HI-Platelet in 12 of 24 patients (50%). These responses were accompanied by improvement in clinical symptoms and reductions in transfusion requirements. Improvement in bone marrow maturation and cellularity was also observed. CONCLUSION: Phase 2 studies of ezatiostat hydrochloride liposomes for injection in MDS are supported by the tolerability and HI responses observed. An oral formulation of ezatiostat hydrochloride tablets is also in phase 2 clinical development. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00035867.

Phase 1-2a multicenter dose-ranging study of canfosfamide in combination with carboplatin and paclitaxel as first-line therapy for patients with advanced non-small cell lung cancer.

INTRODUCTION: We aimed to evaluate the safety and efficacy of canfosfamide in combination with carboplatin and paclitaxel as first-line therapy in patients with locally advanced or metastatic non-small cell lung cancer. METHODS: This was a phase 1-2a, multicenter, dose-ranging trial that enrolled patients with stage IIIB or IV non-small cell lung cancer with measurable disease. Patients received canfosfamide in doses ranging from 400 to 1000 mg/m2 intravenously (IV) with carboplatin at area under the curve 6 IV and paclitaxel at 200 mg/m2 IV day 1 every 3 weeks. The primary end point was objective response rate, and the secondary endpoints were safety and progression-free survival. RESULTS: One hundred twenty-nine patients were treated with canfosfamide at dose levels of 400 (n = 3), 500 (n = 51), 750 (n = 54), and 1000 mg/m2 (n = 21). Objective tumor responses by RECIST were observed in 40 patients [34% (95% confidence interval [CI], 26-44)], the median progression-free survival was 4.3 months (95% CI, 3.7-5.2) and the median survival 9.9 months (95% CI, 7.7-11.9). The percent of patients alive at 1 year was 43.1%. The overall safety profile of the combination was acceptable and consistent with the profiles of the individual agents. In an exploratory analysis, patients receiving the optional maintenance canfosfamide therapy had a prolonged median survival of 16.8 months compared with those eligible for but not receiving maintenance therapy at 8.8 months (hazard ratio = 0.38, p < 0.001). CONCLUSIONS: The combination of canfosfamide with carboplatin and paclitaxel chemotherapy is well tolerated and active. Maintenance canfosfamide may further improve outcomes. This regimen is worthy of additional study.

Method for screening and MALDI-TOF MS sequencing of encoded combinatorial libraries.

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.

Thermal gelation and tissue adhesion of biomimetic hydrogels.

Marine and freshwater mussels are notorious foulers of natural and manmade surfaces, secreting specialized protein adhesives for rapid and durable attachment to wet substrates. Given the strong and water-resistant nature of mussel adhesive proteins, significant potential exists for mimicking their adhesive characteristics in bioinspired synthetic polymer materials. An important component of these proteins is L-3,4-dihydroxylphenylalanine (DOPA), an amino acid believed to contribute to mussel glue solidification through oxidation and crosslinking reactions. Synthetic polymers containing DOPA residues have previously been shown to crosslink into hydrogels upon the introduction of oxidizing reagents. Here we introduce a strategy for stimuli responsive gel formation of mussel adhesive protein mimetic polymers. Lipid vesicles with a bilayer melting transition of 37 degrees C were designed from a mixture of dipalmitoyl and dimyristoyl phosphatidylcholines and exploited for the release of a sequestered oxidizing reagent upon heating from ambient to physiologic temperature. Colorimetric studies indicated that sodium-periodate-loaded liposomes released their cargo at the phase transition temperature, and when used in conjunction with a DOPA-functionalized poly(ethylene glycol) polymer gave rise to rapid solidification of a crosslinked polymer hydrogel. The tissue adhesive properties of this biomimetic system were determined by in situ thermal gelation of liposome/polymer hydrogel between two porcine dermal tissue surfaces. Bond strength measurements showed that the bond formed by the adhesive hydrogel (mean = 35.1 kPa, SD = 12.5 kPa, n = 11) was several times stronger than a fibrin glue control tested under the same conditions. The results suggest a possible use of this biomimetic strategy for repair of soft tissues.