The gait profile score characterises walking performance impairments in young stroke survivors.

BACKGROUND: The Gait Profile Score (GPS) provides a composite measure of the quality of joint movement during walking, but the relationship between this measure and metabolic cost, temporal (e.g. walking speed) and spatial (e.g. stride length) parameters in stroke survivors has not been reported. RESEARCH QUESTION: The aims of this study were to compare the GPS (paretic, non-paretic, and overall score) of young stroke survivors to the healthy able-bodied control and determine the relationship between the GPS and metabolic cost, temporal (walking speed, stance time asymmetry) and spatial (stride length, stride width, step length asymmetry) parameters in young stroke survivors to understand whether the quality of walking affects walking performance in stroke survivors. METHODS: Thirty-nine young stroke survivors aged between 18 and 65years and 15 healthy age-matched able-bodied controls were recruited from six hospital sites in Wales, UK. Joint range of motion at the pelvis, hip, knee and ankle, and temporal and spatial parameters were measured during walking on level ground at self-selected speed with calculation of the Gait Variable Score and then the GPS. RESULTS: GPS for the paretic leg (9.40° (8.60-10.21) p < 0.001), non-paretic leg (11.42° (10.20-12.63) p < 0.001) and overall score (11.18° (10.26-12.09) p < 0.001)) for stroke survivors were significantly higher than the control (4.25° (3.40-5.10), 5.92° (5.11 (6.73)). All parameters with the exception of step length symmetry ratio correlated moderate to highly with the GPS for the paretic, non-paretic, and/or overall score (ρ = <-0.732 (p < 0.001)). SIGNIFICANCE: The quality of joint movement during walking measured via the GPS is directly related to the speed and efficiency of walking, temporal (stance time symmetry) and spatial (stride length, stride width) parameters in young stroke survivors.

Sensory-Motor Mechanisms Increasing Falls Risk in Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is associated with peripheral sensory and motor nerve damage that affects up to half of diabetes patients and is an independent risk factor for falls. Clinical implications of DPN-related falls include injury, psychological distress and physical activity curtailment. This review describes how the sensory and motor deficits associated with DPN underpin biomechanical alterations to the pattern of walking (gait), which contribute to balance impairments underpinning falls. Changes to gait with diabetes occur even before the onset of measurable DPN, but changes become much more marked with DPN. Gait impairments with diabetes and DPN include alterations to walking speed, step length, step width and joint ranges of motion. These alterations also impact the rotational forces around joints known as joint moments, which are reduced as part of a natural strategy to lower the muscular demands of gait to compensate for lower strength capacities due to diabetes and DPN. Muscle weakness and atrophy are most striking in patients with DPN, but also present in non-neuropathic diabetes patients, affecting not only distal muscles of the foot and ankle, but also proximal thigh muscles. Insensate feet with DPN cause a delayed neuromuscular response immediately following foot-ground contact during gait and this is a major factor contributing to increased falls risk. Pronounced balance impairments measured in the gait laboratory are only seen in DPN patients and not non-neuropathic diabetes patients. Self-perception of unsteadiness matches gait laboratory measures and can distinguish between patients with and without DPN. Diabetic foot ulcers and their associated risk factors including insensate feet with DPN and offloading devices further increase falls risk. Falls prevention strategies based on sensory and motor mechanisms should target those most at risk of falls with DPN, with further research needed to optimise interventions.

Maternal thiopurine metabolism during pregnancy in inflammatory bowel disease and clearance of thiopurine metabolites and outcomes in exposed neonates.

BACKGROUND: Azathioprine and mercaptopurine are considered safe during pregnancy. However, the pharmacokinetic effects of pregnancy on thiopurine metabolism are undefined. AIMS: To characterise thiopurine metabolism in pregnancy and measure infant metabolite levels and outcomes. METHODS: Women with IBD who were taking a thiopurine and pregnant or trying to conceive were recruited. Maternal thiopurine metabolites were measured pre-conception, in each trimester, at delivery and post-partum. Infant metabolite levels, full blood examination and liver function testing were performed at birth, and repeated until levels undetectable and haematological and biochemical abnormalities resolved. RESULTS: Forty patients were included with measurements on at least two occasions, and two with only mother-baby levels at delivery. The median maternal 6-TGN level dropped in the second trimester compared with post-partum (179.0 vs 323.5 pmol/8 × 108 RBCs, P < 0.001) and the median 6-MMP level increased in the second trimester compared with post-partum (1103.0 vs 329.5 pmol/8 × 108 RBCs, P < 0.01). At delivery, the median 6-TGN level was lower in infants (n = 20) than mothers (78.5 vs 217 pmol/8 × 108 RBCs) (P < 0.001). Metabolites were not detected at 6 weeks in any infants. Anaemia was not seen, but thrombocytosis and abnormal liver biochemistry were detected in 80% of infants from 6 weeks, which gradually improved. CONCLUSIONS: 6-TGN levels decrease and 6-MMP levels increase in the second trimester of pregnancy. Infants are exposed to thiopurine metabolites at low levels with clearance by 6 weeks and no anaemia. The cause of infant thrombocytosis and abnormal liver biochemistry in the absence of metabolites is unclear.

Infliximab, adalimumab and vedolizumab concentrations across pregnancy and vedolizumab concentrations in infants following intrauterine exposure.

BACKGROUND: The impact of pregnancy on levels of biologic agents in patients with IBD is undefined and time to elimination in vedolizumab-exposed infants is unknown. AIMS: To determine the effect of pregnancy on infliximab, adalimumab and vedolizumab levels and to study infant vedolizumab clearance METHODS: In a prospective observational study, maternal drug levels were measured pre-conception, in each trimester, at delivery and postpartum. The association between drug levels and gestation in weeks was assessed using generalised estimating equation modelling. Infant vedolizumab levels were performed at birth (cord blood), 6 weeks and 3 months or until undetectable. RESULTS: We included 50 IBD patients (23 on infliximab, 15 on adalimumab and 12 on vedolizumab) with at least two intrapartum observations, plus 5 patients on vedolizumab with only mother and baby samples at delivery. Modelling showed no change in adalimumab levels, an increase in infliximab levels of 0.16 (95% CI 0.08-0.24) µg/L/week (P < 0.001) and a decrease of 0.18 (95% CI: -0.33 to -0.02) µg/L/week (P = 0.03) for vedolizumab. In 17 mother-baby pairs, median infant vedolizumab levels at birth were lower than maternal levels (P < 0.05) with an infant:maternal ratio of 0.7 (IQR 0.5-0.9). Vedolizumab was undetectable between 15 and 16 weeks of age in all 12 infants completing follow-up testing. CONCLUSIONS: During pregnancy, adalimumab levels remain stable, while infliximab levels increase and vedolizumab levels decrease. However, the increments were small suggesting that intrapartum therapeutic drug monitoring and dose adjustment are not indicated. Unlike infliximab and adalimumab, infant vedolizumab levels are lower in cord blood than in mothers and appear to clear rapidly.

Transgenerational impacts of herbivory and inbreeding on reproductive output in Solanum carolinense.

PREMISE: Plant maternal effects on offspring phenotypes are well documented. However, little is known about how herbivory on maternal plants affects offspring fitness. Furthermore, while inbreeding is known to reduce plant reproductive output, previous studies have not explored whether and how such effects may extend across generations. Here, we addressed the transgenerational consequences of herbivory and maternal plant inbreeding on the reproduction of Solanum carolinense offspring. METHODS: Manduca sexta caterpillars were used to inflict weekly damage on inbred and outbred S. carolinense maternal plants. Cross-pollinations were performed by hand to produce seed from herbivore-damaged outbred plants, herbivore-damaged inbred plants, undamaged outbred plants, and undamaged inbred plants. The resulting seeds were grown in the greenhouse to assess emergence rate and flower production in the absence of herbivores. We also grew offspring in the field to examine reproductive output under natural conditions. RESULTS: We found transgenerational effects of herbivory and maternal plant inbreeding on seedling emergence and reproductive output. Offspring of herbivore-damaged plants had greater emergence, flowered earlier, and produced more flowers and seeds than offspring of undamaged plants. Offspring of outbred maternal plants also had greater seedling emergence and reproductive output than offspring of inbred maternal plants, even though all offspring were outbred. Moreover, the effects of maternal plant inbreeding were more severe when plant offspring were grown in field conditions. CONCLUSIONS: This study demonstrates that both herbivory and inbreeding have fitness consequences that extend across generations even in outbred progeny.

Anti-TNF Therapy in Pregnant Women With Inflammatory Bowel Disease: Effects of Therapeutic Strategies on Disease Behavior and Birth Outcomes.

BACKGROUND: Active inflammatory bowel disease (IBD) adversely affects pregnancy outcomes. Little is known about the risk of relapse after stopping anti-tumor necrosis factor (anti-TNF) treatment during pregnancy. We assessed the risk of relapse before delivery in women who discontinued anti-TNF treatment before gestational week (GW) 30, predictors of reduced infant birth weight, a marker associated with long-term adverse outcomes, and rates and satisfaction with counseling. METHODS: Pregnant women with IBD receiving anti-TNF treatment were prospectively invited to participate in an electronic questionnaire carried out in 22 hospitals in Denmark, Australia, and New Zealand from 2011 to 2015. Risk estimates were calculated, and birth weight was investigated using t tests and linear regression. RESULTS: Of 175 women invited, 153 (87%) responded. In women in remission, the relapse rate did not differ significantly between those who discontinued anti-TNF before GW 30 (1/46, 2%) compared with those who continued treatment (8/74, 11%; relative risk, 0.20; 95% confidence interval [CI], 0.02 to 1.56; P = 0.08). Relapse (P = 0.001) and continuation of anti-TNF therapy after GW 30 (P = 0.007) were independently associated with reduced mean birth weight by 367 g (95% CI, 145 to 589 g; relapse) and 274 g (95% CI, 77 to 471 g; anti-TNF exposure after GW 30). Of 134 (88%) women who received counseling, 116 (87%) were satisfied with the information provided. CONCLUSIONS: To minimize fetal exposure in women in remission, discontinuation of anti-TNF before GW 30 seems safe. Relapse and continuation of anti-TNF therapy after GW 30 were each independently associated with lower birth weight, although without an increased risk for birth weight <2500 g. Most women received and were satisfied with counseling.

Magnetic resonance enterography for predicting the clinical course of Crohn's disease strictures.

BACKGROUND AND AIMS: Strictures are the most common Crohn's disease complication, but their natural history is unknown. This study aimed to characterize inflammation, predict prognosis, and understand the impact of drug therapy using magnetic resonance enterography (MRE). METHODS: Patients with a stricture diagnosed on MRE over a 5-year period were reviewed for MRE disease extent and inflammation, clinical course, C-reactive protein, response to anti-TNF therapy, endoscopic dilatation, hospitalization, and surgery. RESULTS: 136 patients had 235 strictures (77, one and 59, ≥ 2 strictures). TREATMENT: 46% of patients underwent surgery after a median 6 months; median follow-up for those not requiring surgery was 41 months. Predictors of surgery: Hospitalization because of obstruction predicted subsequent surgery (OR 2.50; 95% CI 1.06-5.90) while anti-TNF therapy commenced at stricture diagnosis was associated with a reduced risk (OR 0.23; 95% CI 0.05-0.99). MRE characteristics associated with surgery were proximal bowel dilatation ≥ 30-mm diameter (OR 2.98; 95% CI 1.36-6.55), stricture bowel wall thickness ≥ 10-mm (OR 2.42; 95% CI 1.11-5.27), and stricture length > 5-cm (OR 2.56; 95% CI 1.21-5.43). 81% of patients with these three adverse MRE features required surgery versus 17% if none were present (P < 0.001). Accuracy for these three MRE variables predicting surgery was high (AUC 0.76). CONCLUSION: Magnetic resonance enterography findings in Crohn's disease strictures are highly predictive of the disease course and the need for future surgery. MRE may also identify who would benefit from treatment intensification. Anti-TNF therapy is associated with reduced risk of surgery and appears to alter the natural history of this complication.

Return to Employment After Stroke in Young Adults: How Important Is the Speed and Energy Cost of Walking?

Background and Purpose- A quarter of individuals who experience a stroke are under the age of 65 years (defined as young adults), and up to 44% will be unable to return to work poststroke, predominantly because of walking difficulties. No research study has comprehensively analyzed walking performance in young adult's poststroke. The primary aim of this study is to investigate how a stroke in young adults affects walking performance (eg, walking speed and metabolic cost) compared with healthy age-matched controls. The secondary aim is to determine the predictive ability of walking performance parameters for return to employment poststroke. Methods- Forty-six individuals (18-40 years: n=6, 41-54 years: n=21, 55-65 years: n=19) who have had a stroke and 15 healthy age-matched able-bodied controls were recruited from 6 hospital sites in Wales, United Kingdom. Type, location, cause of stroke, and demographic factors (eg, employment status) were recorded. Temporal and spatial walking parameters were measured using 3-dimensional gait analysis. Metabolic energy expenditure and metabolic cost of walking were captured during 3 minutes of walking at self-selected speed from measurements of oxygen consumption. Results- Stroke participants walked slower (P<0.004) and less efficiently (P<0.002) than the controls. Only 23% of stroke participants returned to employment poststroke. Walking speed was the strongest predictor (sensitivity, 0.90; specificity, 0.82) for return to work (P=0.004) with a threshold of 0.93 m/s identified: individuals able to walk faster than 0.93 m/s were significantly more likely to return to work poststroke than those who walked slower than this threshold. Conclusions- This study is the first to capture walking performance parameters of young adults who have had a stroke and identifies slower and less efficient walking. Walking speed emerged as the strongest predictor for return to employment. It is recommended that walking speed be used as a simple but sensitive clinical indicator of functional performance to guide rehabilitation and inform readiness for return to work poststroke.

Combined exercise and visual gaze training improves stepping accuracy in people with diabetic peripheral neuropathy.

INTRODUCTION: Patients with diabetes and diabetic peripheral neuropathy (DPN) place their feet with less accuracy whilst walking, which may contribute to the increased falls-risk. This study examines the effects of a multi-faceted intervention on stepping accuracy, in patients with diabetes and DPN. METHODS: Forty participants began the study, of which 29 completed both the pre and post-intervention tests, 8 patients with DPN, 11 patients with diabetes but no neuropathy (D) and 10 healthy controls (C). Accuracy of stepping was measured pre- and post-intervention as participants walked along an irregularly arranged stepping walkway. Participants attended a one-hour session, once a week, for sixteen weeks, involving high-load resistance exercise and visual-motor training. RESULTS: Patients who took part in the intervention improved stepping accuracy (DPN: +45%; D: +36%) (p < 0.05). The diabetic non-intervention (D-NI) group did not display any significant differences in stepping accuracy pre- to post- the intervention period (-7%). DISCUSSION: The improved stepping accuracy observed in patients with diabetes and DPN as a result of this novel intervention, may contribute towards reducing falls-risk. This multi-faceted intervention presents promise for improving the general mobility and safety of patients during walking and could be considered for inclusion as part of clinical treatment programmes.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

Design and Synthesis of a Novel and Selective Kappa Opioid Receptor (KOR) Antagonist (BTRX-335140).

κ opioid receptor (KOR) antagonists are potential pharmacotherapies for the treatment of migraine and stress-related mood disorders including depression, anxiety, and drug abuse, thus the development of novel KOR antagonists with an improved potency/selectivity profile and medication-like duration of action has attracted the interest of the medicinal chemistry community. In this paper, we describe the discovery of 1-(6-ethyl-8-fluoro-4-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)quinolin-2-yl)- N-(tetrahydro-2 H-pyran-4-yl)piperidin-4 amine (CYM-53093, BTRX-335140) as a potent and selective KOR antagonist, endowed with favorable in vitro ADMET and in vivo pharmacokinetic profiles and medication-like duration of action in rat pharmacodynamic experiments. Orally administered CYM-53093 showed robust efficacy in antagonizing KOR agonist-induced prolactin secretion and in tail-flick analgesia in mice. CYM-53093 exhibited a broad selectivity over a panel of off-target proteins. This compound is in phase 1 clinical trials for the treatment of neuropsychiatric disorders wherein dynorphin is thought to contribute to the underlying pathophysiology.

Anti-TNF Therapeutic Drug Monitoring in Postoperative Crohn's Disease.

BACKGROUND: Anti-TNF prevents postoperative Crohn's disease recurrence in most patients but not all. This study aimed to define the relationship between adalimumab pharmacokinetics, maintenance of remission and recurrence. METHODS: As part of a study of postoperative Crohn's disease management, some patients undergoing resection received prophylactic postoperative adalimumab. In these patients, serum and fecal adalimumab concentration and serum anti-adalimumab antibodies [AAAs] were measured at 6, 12 and 18 months postoperatively. Levels of Crohn's disease activity index [CDAI], C-reactive protein [CRP] and fecal calprotectin [FC] were assessed at 6 and 18 months postoperatively. Body mass index and smoking status were recorded. A colonoscopy was performed at 6 and/or 18 months. RESULTS: Fifty-two patients [32 on monotherapy and 20 on combination therapy with thiopurine] were studied. Adalimumab concentration did not differ significantly between patients in endoscopic remission vs recurrence [Rutgeerts ≥ i2] [9.98µg/mL vs 8.43 µg/mL, p = 0.387]. Patients on adalimumab monotherapy had a significantly lower adalimumab concentration [7.89 µg/mL] than patients on combination therapy [11.725 µg/mL] [p = 0.001], and were significantly more likely to have measurable AAA [31% vs 17%, p = 0.001]. Adalimumab concentrations were lower in patients with detectable AAA compared with those without [3.59 µg/mL vs 12.0 µg/mL, p < 0.001]. Adalimumab was not detected in fecal samples. Adalimumab serum concentrations were lower in obese patients compared with in non-obese patients [p = 0.046]. CONCLUSION: Adalimumab concentration in patients treated with adalimumab to prevent symptomatic endoscopic recurrence postoperatively is, for most patients, well within the therapeutic window, and is not significantly lower in patients who develop recurrence compared with in those who remain in remission. Mechanisms of anti-TNF failure to prevent postoperative recurrence remain to be determined in these patients.

Enhancing Organic Semiconductor-Surface Plasmon Polariton Coupling with Molecular Orientation.

Due to strong electric field enhancements, surface plasmon polaritons (SPPs) are capable of drastically increasing light-molecule coupling in organic optoelectronic devices. The electric field enhancement, however, is anisotropic, offering maximal functional benefits if molecules are oriented perpendicular to the interface. To provide a clear demonstration of this orientation dependence, we study SPP dispersion and SPP-mediated photoluminescence at a model Au/small-molecule interface where identical molecules can be deposited with two very different molecular backbone orientations depending on processing conditions. First, we demonstrate that thin films of p-SIDT(FBTTh2)2 can be deposited with either all "in-plane" (parallel to substrate) or a 50/50 mix of in-plane/"out-of-plane" (perpendicular to substrate) optical transition dipoles by the absence or presence, respectively, of diiodooctane during spin-coating. In contrast to typical orientation control observed in organic thin films, for this particular molecule, this corresponds to films with conjugated backbones purely in-plane, or with a 50/50 mix of in-plane/out-of-plane backbones. Then, using momentum-resolved reflectometry and momentum-resolved photoluminescence, we study and quantify changes in SPP dispersion and photoluminescence intensity arising solely from changes in molecular orientation. We demonstrate increased SPP momentum and a 2-fold enhancement in photoluminescence for systems with out-of-plane oriented transition dipoles. These results agree well with theory and have direct implications for the design and analysis of organic optoelectronic devices.

Serologic antibodies in relation to outcome in postoperative Crohn's disease.

BACKGROUND AND AIM: Disease recurs frequently after Crohn's disease resection. The role of serological antimicrobial antibodies in predicting recurrence or as a marker of recurrence has not been well defined. METHODS: A total of 169 patients (523 samples) were prospectively studied, with testing peri-operatively, and 6, 12 and 18 months postoperatively. Colonoscopy was performed at 18 months postoperatively. Serologic antibody presence (perinuclear anti-neutrophil cytoplasmic antibody [pANCA], anti-Saccharomyces cerevisiae antibodies [ASCA] IgA/IgG, anti-OmpC, anti-CBir1, anti-A4-Fla2, anti-Fla-X) and titer were tested. Quartile sum score (range 6-24), logistic regression analysis, and correlation with phenotype, smoking status, and endoscopic outcome were assessed. RESULTS: Patients with ≥ 2 previous resections were more likely to be anti-OmpC positive (94% vs 55%, ≥ 2 vs < 2, P = 0.001). Recurrence at 18 months was associated with anti-Fla-X positivity at baseline (49% vs 29%; positive vs negative, P = 0.033) and 12 months (52% vs 31%, P = 0.04). Patients positive (n = 28) for all four antibacterial antibodies (anti-CBir1, anti-OmpC, anti-A4-Fla2, and anti-Fla-X) at baseline were more likely to experience recurrence at 18 months than patients negative (n = 32) for all four antibodies (82% vs 18%, P = 0.034; odds ratio 6.4, 95% confidence interval 1.16-34.9). The baseline quartile sum score for all six antimicrobial antibodies was higher in patients with severe recurrence (Rutgeert's i3-i4) at 18 months, adjusted for clinical risk factors (odds ratio 1.16, 95% confidence interval 1.01-1.34, P = 0.039). Smoking affected antibody status. CONCLUSIONS: Anti-Fla-X and presence of all anti-bacterial antibodies identifies patients at higher risk of early postoperative Crohn's disease recurrence. Serologic screening pre-operatively may help identify patients at increased risk of recurrence.

Model-blind characterization of thin-film optical constants with momentum-resolved reflectometry.

Determining optical constants of thin material films is important for characterizing their electronic excitations and for the design of optoelectronic devices. Spectroscopic ellipsometry techniques have emerged as the predominant approach for measuring thin-film optical constants. However, ellipsometry methods suffer from complications associated with highly model-dependent, multi-parameter spectral fitting procedures. Here, we present a model-blind, momentum-resolved reflectometry technique that yields accurate and precise optical constants, with quantifiable error estimates, even for film thicknesses less than 50 nm. These capabilities are demonstrated by interrogating an optical absorption resonance in films of the polymer P(NDI2OD-T2). We show that this approach produces exceptional agreement with UV-Vis-NIR absorption measurements, while simultaneously avoiding the need to construct complicated multi-oscillator spectral models. Finally, we use this procedure to resolve subtle differences in the out-of-plane optical properties of different film morphologies that were previously obscured in ellipsometry measurements.

Chemical Proteomic Profiling of Human Methyltransferases.

Methylation is a fundamental mechanism used in Nature to modify the structure and function of biomolecules, including proteins, DNA, RNA, and metabolites. Methyl groups are predominantly installed into biomolecules by a large and diverse class of S-adenosyl methionine (SAM)-dependent methyltransferases (MTs), of which there are ∼200 known or putative members in the human proteome. Deregulated MT activity contributes to numerous diseases, including cancer, and several MT inhibitors are in clinical development. Nonetheless, a large fraction of the human MT family remains poorly characterized, underscoring the need for new technologies to characterize MTs and their inhibitors in native biological systems. Here, we describe a suite of S-adenosyl homocysteine (SAH) photoreactive probes and their application in chemical proteomic experiments to profile and enrich a large number of MTs (>50) from human cancer cell lysates with remarkable specificity over other classes of proteins. We further demonstrate that the SAH probes can enrich MT-associated proteins and be used to screen for and assess the selectivity of MT inhibitors, leading to the discovery of a covalent inhibitor of nicotinamide N-methyltransferase (NNMT), an enzyme implicated in cancer and metabolic disorders. The chemical proteomics probes and methods for their utilization reported herein should prove of value for the functional characterization of MTs, MT complexes, and MT inhibitors in mammalian biology and disease.

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation: DRUGGABILITY OF THE SMALL GTPase Rab27a.

Neutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the up-regulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic inflammation.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.

Design, Synthesis, and In Vitro and In Vivo Evaluation of an (18)F-Labeled Sphingosine 1-Phosphate Receptor 1 (S1P1) PET Tracer.

Sphingosine 1-phosphate receptor 1 (S1P1) plays a pivotal signaling role in inflammatory response; because S1P1 modulation has been identified as a therapeutic target for various diseases, a PET tracer for S1P1 would be a useful tool. Fourteen fluorine-containing analogues of S1P ligands were synthesized and their in vitro binding potency measured; four had high potency and selectivity for S1P1 (S1P1 IC50 < 10 nM, >100-fold selectivity for S1P1 over S1P2 and S1P3). The most potent ligand, 28c (IC50 = 2.63 nM for S1P1) was (18)F-labeled and evaluated in a mouse model of LPS-induced acute liver injury to determine its S1P1-binding specificity. The results from biodistribution, autoradiography, and microPET imaging showed higher [(18)F]28c accumulation in the liver of LPS-treated mice than controls. Increased expression of S1P1 in the LPS model was confirmed by immunohistochemical analysis (IHC). These data suggest that [(18)F]28c is a S1P1 PET tracer with high potential for imaging S1P1 in vivo.