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AB598 is a CD39 inhibitory antibody being pursued for the treatment of solid tumors in combination with chemotherapy and immunotherapy. CD39 metabolizes extracellular adenosine triphosphate (eATP), an alarmin capable of promoting antitumor immune responses, into adenosine, an immuno-inhibitory metabolite. By inhibiting CD39, the consumption of eATP is reduced, resulting in a proinflammatory milieu in which eATP can activate myeloid cells to promote antitumor immunity. The preclinical characterization of AB598 provides a mechanistic rationale for combining AB598 with chemotherapy in the clinic. Chemotherapy can induce ATP release from tumor cells and, when preserved by AB598, both chemotherapy-induced eATP and exogenously added ATP promote the function of monocyte-derived dendritic cells via P2Y11 signaling. Inhibition of CD39 in the presence of ATP can promote inflammasome activation in in vitro-derived macrophages, an effect mediated by P2X7. In a MOLP8 murine xenograft model, AB598 results in full inhibition of intratumoral CD39 enzymatic activity, an increase in intratumoral ATP, a decrease of extracellular CD39 on tumor cells, and ultimately, control of tumor growth. In cynomolgus monkeys, systemic dosing of AB598 results in effective enzymatic inhibition in tissues, full peripheral and tissue target engagement, and a reduction in cell surface CD39 both in tissues and in the periphery. Taken together, these data support a promising therapeutic strategy of harnessing the eATP generated by standard-of-care chemotherapies to prime the tumor microenvironment for a productive antitumor immune response., (©2024 The Authors; Published by the American Association for Cancer Research.)

We provide the complete genome sequence for a novel Pseudomonas fluorescens bacteriophage named UNO-G1W1. This phage was isolated from a single ice cover sampling. The genome was sequenced on the Nanopore MinION, generated with the direct terminal repeat-phage-pipeline and polished with Illumina short reads. Sequence identity classifies the phage as an otagovirus ., Competing Interests: The authors declare no conflict of interest.

In 2012, the U.S. Food & Drug Administration (FDA) published an established list of 93 harmful and potentially harmful constituents (HPHCs) targeting four tobacco product types (cigarettes, cigarette tobacco, roll-your-own tobacco, smokeless tobacco). In 2016, the FDA finalized the deeming rule to regulate electronic nicotine delivery systems (ENDS). However, knowledge gaps exist regarding whether certain HPHCs are present in ENDS e-liquids and aerosols. We identified and addressed these gaps by conducting literature searches and then experimentally quantifying HPHCs in the e-liquid and aerosol of 37 ENDS brands based on gaps in the literature. The literature searches identified 66 e-liquid HPHCs and 68 aerosol HPHCs that have limited to no information regarding the quantifiability of these constituents. A contracted ISO 17025 accredited laboratory performed the HPHC quantifications. The availability of validated analytical methods in the contracted laboratory determined the HPHCs included in the study scope (63/66 for e-liquids, 64/68 for aerosols). Combining the results from the quantifications and literature searches, 36 (39%) and 34 (37%) HPHCs were found quantifiable (≥limit of quantification [LOQ]) in ENDS e-liquids and aerosols, respectively, with 25 HPHCs being quantifiable in both matrices. Quantifiability results imply potential HPHC transfers between matrices, leaching from components, or formations from aerosol generation. The study results can inform the scientific basis for manufacturers and regulators regarding regulatory requirements for HPHC reporting. The HPHC quantities can also inform evaluations of the public health impact of ENDS and public communications regarding ENDS health risks.

T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) is an inhibitory receptor on immune cells that outcompetes an activating receptor, CD226, for shared ligands. Tumor-infiltrating lymphocytes express TIGIT and CD226 on regulatory T cells (Treg) and on CD8+ T cells with tumor-reactive or exhausted phenotypes, supporting the potential of therapeutically targeting TIGIT to enhance antitumor immunity. To optimize the efficacy of therapeutic antibodies against TIGIT, it is necessary to understand IgG Fc (Fcγ) receptor binding for therapeutic benefit. In this study, we showed that combining Fc-enabled (Fce) or Fc-silent (Fcs) anti-TIGIT with antiprogrammed cell death protein 1 in mice resulted in enhanced control of tumors by differential mechanisms: Fce anti-TIGIT promoted the depletion of intratumoral Treg, whereas Fcs anti-TIGIT did not. Despite leaving Treg numbers intact, Fcs anti-TIGIT potentiated the activation of tumor-specific exhausted CD8+ populations in a lymph node-dependent manner. Fce anti-TIGIT induced antibody-dependent cell-mediated cytotoxicity against human Treg in vitro, and significant decreases in Treg were measured in the peripheral blood of patients with phase I solid tumor cancer treated with Fce anti-TIGIT. In contrast, Fcs anti-TIGIT did not deplete human Treg in vitro and was associated with anecdotal objective clinical responses in two patients with phase I solid tumor cancer whose peripheral Treg frequencies remained stable on treatment. Collectively, these data provide evidence for pharmacologic activity and antitumor efficacy of anti-TIGIT antibodies lacking the ability to engage Fcγ receptor., Significance: Fcs-silent anti-TIGIT antibodies enhance the activation of tumor-specific pre-exhausted T cells and promote antitumor efficacy without depleting T regulatory cells., (©2024 American Association for Cancer Research.)

Purpose: Menthol is known to elicit opposing thermoregulatory and perceptual alterations during intense exercise. The current purpose was to determine the thermoregulatory and perceptual effects of topical menthol application prior to walking in the heat., Methods: Twelve participants walked (1.6 m s -1 , 5% grade) for 30 min in the heat (38 °C, 60% relative humidity) with either a 4% menthol or control gel on the upper (shoulder to wrist) and lower (mid-thigh to ankle) limbs. Skin blood flow (SkBF), sweat (rate, composition), skin conductivity, heart rate, temperature (skin, core), and thermal perception were measured prior to and during exercise., Results: Skin conductivity expressed as time to 10, 20, 30, and 40 µS was delayed due to menthol (559 ± 251, 770 ± 292, 1109 ± 301, 1299 ± 335 s, respectively) compared to the control (515 ± 260, 735 ± 256, 935 ± 300, 1148 ± 298 s, respectively, p = 0.048). Sweat rate relative to body surface area was lower due to menthol (0.55 ± 0.16 L h -1 m (2)-1 ) than the control (0.64 ± 0.16 L h -1 m (2)-1 , p = 0.049). Core temperature did not differ at baseline between the menthol (37.4 ± 0.3 °C) and control (37.3 ± 0.4 °C, p = 0.298) but was higher at 10, 20, and 30 min due to menthol (37.5 ± 0.3, 37.7 ± 0.2, 38.1 ± 0.3 °C, respectively) compared to the control (37.3 ± 0.4, 37.4 ± 0.3, 37.7 ± 0.3 °C, respectively, p < 0.05). The largest rise in core temperature from baseline was at 30 min during menthol (0.7 ± 0.3 °C) compared to the control (0.4 ± 0.2 °C, p = 0.004). Overall, the menthol treatment was perceived cooler, reaching "slightly warm" whereas the control treatment reached "warm" (p < 0.001)., Conclusion: Menthol application to the limbs impairs whole-body thermoregulation while walking in the heat despite perceiving the environment as cooler., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We discovered dibenzannulated medium-ring keto lactams (11,12-dihydro-5 H -dibenzo[ b , g ]azonine-6,13-diones) as a new antimalarial chemotype. Most of these had chromatographic LogD 7.4 values ranging from <0 to 3 and good kinetic solubilities (12.5 to >100 μg/mL at pH 6.5). The more polar compounds in the series (LogD 7.4 values of <2) had the best metabolic stability (CL int values of <50 μL/min/mg protein in human liver microsomes). Most of the compounds had relatively low cytotoxicity, with IC 50 values >30 μM, and there was no correlation between antiplasmodial activity and cytotoxicity. The four most potent compounds had Plasmodium falciparum IC 50 values of 4.2 to 9.4 nM and in vitro selectivity indices of 670 to >12,000. They were more than 4 orders-of-magnitude less potent against three other protozoal pathogens ( Trypanosoma brucei rhodesiense , Trypanosoma cruzi , and Leishmania donovani ) but did have relatively high potency against Toxoplasma gondii , with IC 50 values ranging from 80 to 200 nM. These keto lactams are converted into their poorly soluble 4(1 H )-quinolone transannular condensation products in vitro in culture medium and in vivo in mouse blood. The similar antiplasmodial potencies of three keto lactam-quinolone pairs suggest that the quinolones likely contribute to the antimalarial activity of the lactams.

Purpose: Determine if topical capsaicin, a transient receptor potential vanilloid heat thermoreceptor activator, alters thermoregulation and perception when applied topically prior to thermal exercise., Methods: Twelve subjects completed 2 treatments. Subjects walked (1.6 m s -1 , 5% grade) for 30 min in the heat (38 °C, 60% relative humidity) with either a capsaicin (0.025% capsaicin) or control cream applied to the upper (shoulder to wrist) and lower (mid-thigh to ankle) limbs covering ∼50% body surface area. Skin blood flow (SkBF), sweat (rate, composition), heart rate, temperature (skin, core), and perceived thermal sensation were measured prior to and during exercise., Results: The relative change in SkBF was not different between treatments at any time point (p = 0.284). There were no differences in sweat rate between the capsaicin (1.23 ± 0.37 L h -1 ) and control (1.43 ± 0.43 L h -1 , p = 0.122). There were no differences in heart rate between the capsaicin (122 ± 38 beats·min -1 ) and control (125 ± 39 beats·min -1 , p = 0.431). There were also no differences in weighted surface (p = 0.976) or body temperatures (p = 0.855) between the capsaicin (36.0 ± 1.7 °C, 37.0 ± 0.8 °C, respectively) and control (36.0 ± 1.6 °C, 36.9 ± 0.8 °C, respectively). The capsaicin treatment was not perceived as hotter than the control treatment until minute 30 of exercise (2.8 ± 0.4, 2.5 ± 0.5, respectively, p = 0.038) CONCLUSIONS: Topical capsaicin application does not alter whole-body thermoregulation during acute exercise in the heat despite perceiving the treatment as hotter late in exercise., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The U.S. Food and Drug Administration established a list of 93 harmful and potentially harmful constituents (HPHCs) in tobacco products. While HPHCs are required to be submitted for tobacco products, knowledge gaps exist regarding which tobacco-containing tobacco product (TCTP, i.e., tobacco products that contain tobacco(s) as a component) types (cigarettes, cigars, roll-your-own tobaccos [RYOs], pipe tobaccos [pipes], smokeless tobacco products [STPs], waterpipe tobaccos [waterpipes]) and matrices (filler, smoke) contain which HPHCs. This study identified and addressed such gaps by conducting literature searches and measuring the amount of HPHCs in TCTP types and matrices. First, literature searches, performed for cigarettes, RYOs, and STPs for publications up to 2014 and for cigars, pipes, and waterpipes for publications up to 2016, identified knowledge gaps for the 93 HPHCs (or 119 HPHCs if cresols [ o -, m -, p -cresol] are counted as 3 and chlorinated dioxins/furans as 25) across TCTP types and matrices. Then, three ISO 17025 accredited laboratories including two subcontracted laboratories performed the HPHC quantifications. Inclusion of the HPHCs, TCTP types, and matrices in the study scope was also determined by the availability of validated analytical methods in each laboratory. Eleven (9%) HPHCs are quantifiable in all brands for all TCTP types and matrices, 33 (28%) HPHCs are not quantifiable in any brands of any TCTP type and matrix, and 74 (63%) HPHCs are quantifiable only in some brands across TCTP types and matrices examined. Understanding the quantifiability of HPHCs in each TCTP type and matrix can inform the scientific basis for manufacturers regarding the regulatory requirements for reporting HPHCs. The quantity of HPHCs observed can also inform the evaluation of the public health impact of HPHCs and public communications regarding the health risks of tobacco products., Competing Interests: The authors declare no competing financial interest., (Not subject to U.S. Copyright. Published 2022 by American Chemical Society.)

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes., (©2022 The Authors; Published by the American Association for Cancer Research.)

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

The COVID-19 pandemic poses unique challenges within the austere clinical setting, and the time between patient presentation and deterioration is a critical opportunity for intervention. In some cases, this may be a life-saving transfer to a higher level of care. US Central Command (CENTCOM) has provided valuable guidance for COVID-19 management in the operational environment,1 and has proposed the National Early Warning System 2 (NEWS2) scoring tool as a useful adjunct to gauging illness severity. NEWS2, however, does not consider co-morbidities, such as diabetes or chronic cardiac disease, which could worsen the clinical course of SARS-CoV-2 patients. Thus, NEWS2 fails to address such factors during the risk stratification of patients to a higher level of care. To address this concern, June 2020, 3rd Medical Brigade, Operation Spartan Shield (OSS) developed the COVID-19 Army Rapid Assessment Tool (CARAT) with inputs from clinicians and researchers (The Team). The CARAT is a clinical scoring system, modified from the NEWS2, which combines the effects of co-morbid disease with the current physiological condition of a COVID-19 patient. The Team obtained clinical data for 105 patients from the CENTCOM area of responsibility (AOR), who presented to a military treatment facility (MTF) symptomatic for, and testing positive for SARS-CoV-2, during the time period of June to mid-August 2020. Each patient was retrospectively assigned a CARAT score based on his or her initial presentation. Preliminary review of data suggested a CARAT value of 4 or greater was an indicator for risk of further deterioration. Patients were then grouped into two categories: patients who received transfer to a higher level of care, versus "stay-in-place" supportive care. Results showed that 100% of patients with a score ≥4 had been transferred to a higher echelon of care, compared to 2% of patients with scores less than 4. A Fisher's exact test demonstrated a statistically significant difference between these two groups (p is less than 0.001). Interestingly, when compared with the NEWS2 score, the CARAT identified 9 individuals for transfer to a higher level of care, of whom only one patient was identified by the NEWS2, clearly underscoring the significance of CARAT despite small sample size. We therefore recommend that CARAT be further validated in predicting disease severity and need for emergent evacuation in larger patient settings.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent ( K i = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1 H -pyrazol-4-yl)pyrazolo[1,5- a ]pyrimidin-5-yl]-2-[(1 S )-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1 H -isoindol-1-one ( 4 , IC 50 = 0.064 μM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

The successful application of immunotherapy in the treatment of cancer relies on effective engagement of immune cells in the tumor microenvironment. Phosphoinositide 3-kinase γ (PI3Kγ) is highly expressed in tumor-associated macrophages, and its expression levels are associated with tumor immunosuppression and growth. Selective inhibition of PI3Kγ offers a promising strategy in immuno-oncology, which has led to the development of numerous potent PI3Kγ inhibitors with variable selectivity profiles. To facilitate further investigation of the therapeutic potential of PI3Kγ inhibition, we required a potent and PI3Kγ-selective tool compound with sufficient metabolic stability for use in future in vivo studies. Herein, we describe some of our efforts to realize this goal through the systematic study of SARs within a series of 7-azaindole-based PI3Kγ inhibitors. The large volume of data generated from this study helped guide our subsequent lead optimization efforts and will inform further development of PI3Kγ-selective inhibitors for use in immunomodulation., Competing Interests: The authors declare the following competing financial interest(s): All authors are current or former employees of Arcus Biosciences.

Study Objective: Moderate hypothermia (e.g., 34.5 °C) causes surgical site infections, but it remains unknown whether mild hypothermia (34.6 °C-35.9 °C) causes infection. Therefore, the objective of this study was to evaluate the relationship between intraoperative time-weighted average core temperature and a composite of serious wound and systemic infections in adults having colorectal surgery over a range of near-normal temperatures., Design: Retrospective, single center study., Setting: The operating rooms of the Cleveland Clinic Foundation from January 2005 to December 2014., Patients: Adult patients having colorectal surgery at least 1 h in length who received both general anesthesia and esophageal core temperature monitoring., Intervention(s): Time weighted average intraoperative core temperature., Measurements: Our primary outcome was a composite of serious infections obtained from a surgical registry and billing codes. Average intraoperative esophageal temperatures and the composite of serious 30-day complications were assessed with logistic regression, adjusted for potential confounding factors., Main Results: A total of 7908 patients were included in the analysis. A 0.5 °C decrease in time-weighted average intraoperative core temperature ≤ 35.4 °C was associated with an increased odds of serious infection (OR = 1.38, P = .045); that is, hypothermia below 35.4 °C progressively worsened infection risk. Additionally, at higher core temperatures, the odds of serious infection increased slightly with each 0.5 °C increase in average temperature (OR = 1.10, P = .047)., Conclusions: Below 35.5 °C, hypothermia was associated with increased risk of serious infectious complications. Why composite complications increased at higher temperatures remains unclear, but the highest temperatures may reflect febrile patients who had pre-existing infections. Avoiding time-weighted average core temperatures <35.5 °C appears prudent from an infection perspective, but higher temperatures may be needed to prevent other hypothermia-related complications., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Adenosine suppresses antitumor immune responses via A 2a and A 2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5'-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A 2a R/A 2b R antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 μM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.

Objective: To investigate the associations between lifestyle and vascular risk factors and changes in Alzheimer's disease (AD) biomarkers (beta-amyloid load via 11 C-PiB PET, glucose metabolism via 18 F-FDG PET and neurodegeneration via structural MRI) and global cognition in middle-aged asymptomatic participants at risk for AD., Design: Prospective, longitudinal., Setting: The study was conducted at New York University Langone/Weill Cornell Medical Centres in New York City., Participants: Seventy cognitively normal participants from multiple community sources, aged 30-60 years with lifestyle measures (diet, intellectual activity and physical activity), vascular risk measures and two imaging biomarkers visits over at least 2 years, were included in the study., Outcome Measures: We examined MRI-based cortical thickness, fluoro-deoxy-glucose (FDG) glucose metabolism and PiB beta-amyloid in AD-vulnerable regions. A global cognitive z-score served as our summary cognition measure. We used regression change models to investigate the associations of clinical, lifestyle and vascular risk measures with changes in AD biomarkers and global cognition., Results: Diet influenced changes in glucose metabolism, but not amyloid or cortical thickness changes. With and without accounting for demographic measures, vascular risk and baseline FDG measures, lower adherence to a Mediterranean-style diet was associated with faster rates of FDG decline in the posterior cingulate cortex (p≤0.05) and marginally in the frontal cortex (p=0.07). None of the other lifestyle variables or vascular measures showed associations with AD biomarker changes . Higher baseline plasma homocysteine was associated with faster rates of decline in global cognition, with and without accounting for lifestyle and biomarker measures (p=0.048). None of the lifestyle variables were associated with cognition., Conclusions: Diet influenced brain glucose metabolism in middle-aged participants, while plasma homocysteine explained variability in cognitive performance. These findings suggest that these modifiable risk factors affect AD risk through different pathways and support further investigation of risk reduction strategies in midlife., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Correlations are made between mainstream cigarette smoke deliveries of individual PAHs over multiple years. Average overall PAH deliveries in mainstream cigarette smoke by study year, mentholation, ring size, and manufacturer are compared., Methods: Mainstream smoke deliveries were determined by GC/MS for 14 polycyclic aromatic hydrocarbons (PAHs) from selected cigarettes on the US market in 2002, 2004, 2007, and 2011. The mainstream smoke PAH emissions were measured under international standardization organization (ISO) smoking conditions. Pearson product-moment correlation was used to examine the linear relationship among the PAHs over multiple years., Results: A number of the PAH analytes were statistically highly correlated with each other. The overall average for mainstream smoke deliveries of PAHs did not change significantly between study years. Similar levels in average PAH deliveries were seen for mentholated and non-mentholated cigarettes., Conclusions: The strong correlations between PAH compounds over multiple years show that a limited set of PAHs can predict deliveries of others with confidence over multiple years. A more limited panel of analytes may be considered when designing studies involving PAH measurements in mainstream smoke., Competing Interests: Conflict of Interest Statement None.

Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11c + CD206 + immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c + CD206 + myeloid cells correlated with systemic insulin resistance. CD11c + CD206 + cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11b + CD11c + F4/80 + monocytes, which are functional homologs of human CD11c + CD206 + cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11c + CD206 + myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.

A new tobacco filler Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST) in September 2016 with certified and reference mass fraction values for nicotine, N-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and volatiles. The constituents have been determined by multiple analytical methods with measurements at NIST and at the Centers for Disease Control and Prevention, and with confirmatory measurements by commercial laboratories. This effort highlights the development of the first SRM for reduced nicotine and reduced tobacco-specific nitrosamines with certified values for composition.

Objective: We investigated the differences in TNCO (tar, nicotine, and carbon monoxide) smoke yields generated under the International Organization of Standardization (ISO) and Federal Trade Commission (FTC) Cambridge Filter Test (CFT) smoking regimens., Methods: Twenty-nine commercial cigarette products from the US marketplace were acquired in 2015 and tested by measuring the TNCO smoke yields generated under these 2 nonintense smoking regimens., Results: Data obtained demonstrated a linear relationship between the TNCO yields produced under the 2 smoking regimens (R 2 > 0.99). TNCO yields produced by each product were higher under the CFT smoking regimen than the ISO smoking regimen., Conclusion: We found that tar, nicotine, and carbon monoxide yields were consistently 10% to 13% higher under the CFT smoking regimen than under the ISO smoking regimen. This strong correlation indicates that the 2 smoking regimens can be used to apply a correlation correction to CFT TNCO data and allow its comparison to ISO TNCO data in tobacco product marketing applications., Competing Interests: Conflict of Interest Statement The authors report no conflict of interest to disclose.

There are an estimated 8 million users of smokeless tobacco products (STPs) in the United States, and yet limited data on microbial populations within these products exist. To better understand the potential microbiological risks associated with STP use, a study was conducted to provide a baseline microbiological profile of STPs. A total of 90 samples, representing 15 common STPs, were purchased in metropolitan areas in Little Rock, AR, and Washington, DC, in November 2012, March 2013, and July 2013. Bacterial populations were evaluated using culture, pyrosequencing, and denaturing gradient gel electrophoresis (DGGE). Moist-snuff products exhibited higher levels of bacteria (average of 1.05 × 10 6 CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 × 10 1 CFU/g STP) and some chewing tobacco products (average of 2.54 × 10 5 CFU/g STP). The most common species identified by culturing were Bacillus pumilus, B. licheniformis, B. safensis, and B. subtilis, followed by members of the genera Oceanobacillus, Staphylococcus, and Tetragenococcus. Pyrosequencing analyses of the 16S rRNA genes identified the genera Tetragenococcus, Carnobacterium, Lactobacillus, Geobacillus, Bacillus, and Staphylococcus as the predominant taxa. Several species identified are of possible concern due to their potential to cause opportunistic infections and reported abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. This report provides a microbiological baseline to help fill knowledge gaps associated with microbiological risks of STPs and to inform potential regulations regarding manufacture and testing of STPs., Importance: It is estimated that there 8 million users of smokeless tobacco products (STPs) in the United States; however, there are limited data on microbial populations that exist within these products. The current study was undertaken to better understand the potential microbiological risks associated with STP use and provide a baseline microbiological profile of STPs. Several bacterial species were identified that are of possible concern due to their potential to cause opportunistic infections. In addition, some species have abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. Overall, this report provides a microbiological baseline to help fill knowledge gaps related to the microbiological risks of STPs and to inform potential regulations regarding the manufacture and testing of STPs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Introduction: A significant portion of the increased risk of cancer and respiratory disease from exposure to cigarette smoke is attributed to volatile organic compounds (VOCs). In this study, 21 VOCs were quantified in mainstream cigarette smoke from 50U.S. domestic brand varieties that included high market share brands and 2 Kentucky research cigarettes (3R4F and 1R5F)., Methods: Mainstream smoke was generated under ISO 3308 and Canadian Intense (CI) smoking protocols with linear smoking machines with a gas sampling bag collection followed by solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) analysis., Results: For both protocols, mainstream smoke VOC amounts among the different brand varieties were strongly correlated between the majority of the analytes. Overall, Pearson correlation (r) ranged from 0.68 to 0.99 for ISO and 0.36 to 0.95 for CI. However, monoaromatic compounds were found to increase disproportionately compared to unsaturated, nitro, and carbonyl compounds under the CI smoking protocol where filter ventilation is blocked., Conclusions: Overall, machine generated "vapor phase" amounts (µg/cigarette) are primarily attributed to smoking protocol (e.g., blocking of vent holes, puff volume, and puff duration) and filter ventilation. A possible cause for the disproportionate increase in monoaromatic compounds could be increased pyrolysis under low oxygen conditions associated with the CI protocol., Implications: This is the most comprehensive assessment of volatile organic compounds (VOCs) in cigarette smoke to date, encompassing 21 toxic VOCs, 50 different cigarette brand varieties, and 2 different machine smoking protocols (ISO and CI). For most analytes relative proportions remain consistent among U.S. cigarette brand varieties regardless of smoking protocol, however the CI smoking protocol did cause up to a factor of 6 increase in the proportion of monoaromatic compounds. This study serves as a basis to assess VOC exposure as cigarette smoke is a principle source of overall population-level VOC exposure in the United States., (Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The aim of this study is to explore how differences in cigarette physical design parameters influence tar, nicotine, and carbon monoxide (TNCO) yields in mainstream smoke (MSS) using the International Organization of Standardization (ISO) smoking regimen. Standardized smoking methods were used to evaluate 50 U.S. domestic brand cigarettes and a reference cigarette representing a range of TNCO yields in MSS collected from linear smoking machines using a nonintense smoking regimen. Multivariate statistical methods were used to form clusters of cigarettes based on their ISO TNCO yields and then to explore the relationship between the ISO generated TNCO yields and the nine cigarette physical design parameters between and within each cluster simultaneously. The ISO generated TNCO yields in MSS are 1.1-17.0 mg tar/cigarette, 0.1-2.2 mg nicotine/cigarette, and 1.6-17.3 mg CO/cigarette. Cluster analysis divided the 51 cigarettes into five discrete clusters based on their ISO TNCO yields. No one physical parameter dominated across all clusters. Predicting ISO machine generated TNCO yields based on these nine physical design parameters is complex due to the correlation among and between the nine physical design parameters and TNCO yields. From these analyses, it is estimated that approximately 20% of the variability in the ISO generated TNCO yields comes from other parameters (e.g., filter material, filter type, inclusion of expanded or reconstituted tobacco, and tobacco blend composition, along with differences in tobacco leaf origin and stalk positions and added ingredients). A future article will examine the influence of these physical design parameters on TNCO yields under a Canadian Intense (CI) smoking regimen. Together, these papers will provide a more robust picture of the design features that contribute to TNCO exposure across the range of real world smoking patterns.

This investigation provides an updated survey measuring the levels of N'-nitrosonornicotine (NNN) and water content of a select number of smokeless tobacco products sold in the United States in 2015. A total of 34 smokeless tobacco products were collected and analyzed for NNN and water content using LC-MS/MS and GC-TCD, respectively. Smokeless tobacco products were chosen to obtain a representative sample of the different types of products on the U.S. market. These smokeless products represent 12 of the 25 top-selling smokeless tobacco products according to 2013 Nielsen net sales data while five of the smokeless tobacco products are of lower selling smokeless tobacco products. The NNN levels and the water content of the smokeless tobacco products were determined and compared to previous studies. Although the range of NNN levels found was broad for the examined smokeless tobacco products (0.64-12.0 μg/g dry weight), dry snuff had the highest levels of NNN observed (>5 μg/g dry weight). We observed a general decrease in NNN levels for the same six moist snuff products that were analyzed in 2004 compared to our current 2015 study. The water content of the smokeless tobacco products surveyed ranged from 3.92 to 54.8%.

This work developed a UHPLC-MS/MS method for the detection and quantitation of aflatoxins in smokeless tobacco products, which was then used to determine aflatoxin B1 concentrations in 32 smokeless tobacco products commercially available in the United States. Smokeless tobacco products were dried, milled, and amended with (13)C17-labeled internal standards, extracted in water/methanol solution in the presence of a surfactant, isolated through use of immunoaffinity column chromatography, and reconstituted in mobile phase prior to UHPLC-MS/MS analysis. The method was capable of baseline separation of aflatoxins B1, B2, G1, and G2 in a 2.5 min run by use of a fused core C18 column and a water/methanol gradient. MS/MS transition (m/z) 313.3 → 241.2 was used for aflatoxin B1 quantitation, with 313.3 → 285.1 used for confirmation. The limit of detection (LOD) for aflatoxin B1 was 0.007 parts per billion (ppb). Method imprecision for aflatoxin B1 (expressed as coefficient of variation) ranged from 5.5 to 9.4%. Spike recoveries were 105-111%. Aflatoxin B1 concentrations in the smokeless tobacco products analyzed ranged from

While it has long been established that the chemokine receptor CCR9 and its ligand CCL25 are essential for the movement of leukocytes into the small intestine and the development of small-intestinal inflammation, the role of this chemokine-receptor pair in colonic inflammation is not clear. Toward this end, we compared colonic CCL25 protein levels in healthy individuals to those in patients with ulcerative colitis. In addition, we determined the effect of CCR9 pharmacological inhibition in the mdr1a(-/-) mouse model of ulcerative colitis. Colon samples from patients with ulcerative colitis had significantly higher levels of CCL25 protein compared to healthy controls, a finding mirrored in the mdr1a(-/-) mice. In the mdr1a(-/-) mice, CCR9 antagonists significantly decreased the extent of wasting and colonic remodeling and reduced the levels of inflammatory cytokines in the colon. These findings indicate that the CCR9:CCL25 pair plays a causative role in ulcerative colitis and suggest that CCR9 antagonists will provide a therapeutic benefit in patients with colonic inflammation.

Background: In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed., Methods: We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro., Results: CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs., Conclusions: These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

The concentration of CXCL12/SDF-1 in the bloodstream is tightly regulated, given its central role in leucocyte and stem/progenitor cell egress from bone marrow and recruitment to sites of inflammation or injury. The mechanism responsible for this regulation is unknown. Here we show that both genetic deletion and pharmacological inhibition of CXCR7, a high-affinity CXCL12 receptor, caused pronounced increases in plasma CXCL12 levels. The rise in plasma CXCL12 levels was associated with an impairment in the ability of leucocytes to migrate to a local source of CXCL12. Using a set of complementary and highly sensitive techniques, we found that CXCR7 protein is expressed at low levels in multiple organs in both humans and mice. In humans, CXCR7 was detected primarily on venule endothelium and arteriole smooth muscle cells. CXCR7 expression on venule endothelium was also documented in immunodeficient mice and CXCR7(+/lacZ) mice. The vascular expression of CXCR7 therefore gives it immediate access to circulating CXCL12. These studies suggest that endothelial CXCR7 regulates circulating CXCL12 levels and that CXCR7 inhibitors might be used to block CXCL12-mediated cell migration for therapeutic purposes., (© 2013 John Wiley & Sons Ltd.)

Objective: CCR2 inhibition has produced promising experimental and clinical anti-hyperglycemic effects. These results support the thesis that insulin resistance and Type 2 diabetes (T2D) are associated with chronic unresolved inflammation. The aim of this study was to provide a broad analysis of the various physiological changes occurring in mouse models of T2D in connection with pharmacological CCR2 inhibition., Materials/methods: A mouse-active chemical analogue of the clinical candidate CCX140-B was tested in diet-induced obese (DIO) mice and db/db mice. Measurements included: adipose tissue inflammatory macrophage counts; peripheral blood glucose levels at steady-state and after glucose and insulin challenges; peripheral blood insulin and adiponectin levels; 24-h urine output and urinary glucose levels; pancreatic islet number and size; hepatic triglyceride and glycogen content; and hepatic glucose-6-phosphatase levels., Results: In DIO mice, the CCR2 antagonist completely blocked the recruitment of inflammatory macrophages to visceral adipose tissue. The mice exhibited reduced hyperglycemia and insulinemia, improved insulin sensitivity, increased circulating adiponectin levels, decreased pancreatic islet size and increased islet number. It also reduced urine output, glucose excretion, hepatic glycogen and triglyceride content and glucose 6-phosphatase levels. Similar effects were observed in the db/db diabetic mice., Conclusions: These data indicate that pharmacological inhibition of CCR2 in models of T2D can reduce inflammation in adipose tissue, alter hepatic metabolism and ameliorate multiple diabetic parameters. These mechanisms may contribute to the promising anti-diabetic effects seen in humans with at least one CCR2 antagonist., (© 2013.)

Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans., (2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd)

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

Recent literature indicates that mice deficient in the chemokine receptor CCR9 (CCR9(-/-) mice) are unable to generate oral tolerance. The present report describes how such inability can be overcome by increasing the dose of oral antigen. Pharmacological inhibition of CCR9 did not affect the generation of oral tolerance, regardless of antigen dose. These results highlight the inadequacy of genetic deletion of CCR9 when predicting the effects of pharmacological CCR9 inhibition on intestinal biology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: Chemokines may directly participate in the pathogenesis of neonatal chronic hypoxia-induced pulmonary hypertension (PH). Although stromal-derived factor-1 (SDF-1) has been shown to be involved in PH, the role of its most recently discovered receptor, chemokine receptor type 7 (CXCR7), remains unclear. We sought to determine whether antagonism of the CXCR7 receptor would decrease pulmonary vascular remodeling in newborn mice exposed to chronic hypoxia by decreasing pulmonary vascular cell proliferation., Methods: Neonatal mice were exposed to hypoxia (fractional inspired oxygen concentration = 0.12) or room air (RA) for 2 wk. After 1 wk of exposure, mice received daily injections of placebo or a CXCR7 antagonist (CCX771) from postnatal day 7 (P7) to P14. Right ventricular systolic pressure (RVSP), the ratio of the weight of the right ventricle to left ventricle + septum (RV/LV + S), and pulmonary vascular cell proliferation and remodeling were determined at P14., Results: As compared with mice exposed to RA, hypoxia placebo mice had a significant increase in the lung protein expression of CXCR7. Although hypoxic placebo-treated mice had a significant increase in RVSP, RV/LV+S, and pulmonary vascular cell proliferation and remodeling, the administration of CCX771 markedly decreased these changes., Discussion: These results indicate that antagonism of CXCR7 may be a potent strategy to decrease PH and vascular remodeling.

A goal for developers of immunomodulatory drugs has long been a systemically administered small molecule that can selectively inhibit inflammation in specific tissues. The chemokine receptor CCR9 is an attractive target for this approach, as entry of T cells into the small intestine from blood requires interaction between CCR9 and its ligand CCL25. We have tested the ability of a small molecule CCR9 antagonist, CCX8037, to inhibit antigen-mediated T cell accumulation in the intestine. This compound prevented accumulation of gut-imprinted antigen-specific CD8 T cells within epithelium of the small intestine. Interestingly, the antagonist did not affect the robust generation of gut-imprinted CD8 T cells within mesenteric lymph nodes. To distinguish "gut-selective" from "general" T cell inhibition, we tested the drug's ability to influence accumulation of T cells within skin, a tissue in which CCR9 plays no known role, and we found no appreciable effect. This study demonstrates the feasibility of creating systemically-administered pharmaceuticals capable of tissue-selective immune modulation. This proof of concept is of utmost importance for designing effective treatments against various autoimmune disorders localized to a specific tissue.

The initiation of atherosclerosis involves retention of colloidal atherogenic lipoproteins, primarily low density lipoprotein (LDL), in the arterial intima. This retention occurs when LDL binds to smooth muscle cell extracellular matrix (SMC ECM), and is enhanced by lipoprotein lipase (LpL) and sphingomyelinase (Smase). Here we use a fluorescence assay and dynamic light scattering to study the individual and combined effects of these two enzymes on LDL aggregation. Our results show: (1) LpL is self-sufficient to induce LDL aggregation with aggregate sizes up to ~400 nm; (2) Smase induces LDL aggregation due to generation of ceramide and subsequent hydrophobic interactions; (3) Smase hydrolysis of LpL-induced LDL aggregates does not cause further aggregation and results in a ~3-fold diminished production of ceramide, while LpL treatment of Smase-induced aggregates does enhance aggregation; (4) The simultaneous addition of LpL and Smase causes increased variability in aggregation with final sizes ranging from 50 to 110 nm. Our data suggest a new proatherogenic function for LpL, namely, bridging between LDL particles causing their aggregation and consequently enhanced retention by SMC ECM. The mechanism of LpL-and-Smase-mediated LDL aggregation and binding to SMC ECM provides specific points of intervention to design novel effective antiatherogenic therapeutics., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. Naproxen exhibits gastrointestinal toxicity, but its cardiovascular toxicity may be reduced compared with other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis, and x-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition, and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH(2) for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and x-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 Å resolution, respectively. The combination of mutagenesis, structure analysis, and x-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2.

This paper illustrates how principles of colloid science are useful in studying atherosclerosis. Accumulation of foam cells in the arterial intima is a key step in atherogenesis. The extent of foam cell formation is enhanced by low density lipoprotein (LDL) aggregates, and we have previously shown that the size of sphingomyelinase (Smase)-hydrolysis-induced aggregates depends directly on the concentration of ceramide generated in the LDL phospholipid monolayer, mediated by the hydrophobic effect. Here, we focus on the effect of LDL aggregate particle sizes on their subsequent uptake by macrophages. Our data show the first direct measurement of uptake as a function of aggregate size and the first direct comparison of uptake after Smase-catalyzed and vortex-mixing-mediated aggregation. Vortex-mixed aggregates with radii 20-77 nm showed maximal uptake approximately 118 microg sterol/mg protein at a 53 nm intermediate size, consistent with a mathematical model describing competition between aggregate surface area and volume. Smase-treated aggregates with radii 25-211 nm also showed maximal uptake at an intermediate size, approximately 58 microg sterol/mg protein for 132 nm particles, and fit a modified model that incorporated ceramide concentration expressed as aggregate size. This study shows that particle size is significant and composition may also be a factor in LDL uptake., (Copyright 2010 Elsevier Inc. All rights reserved.)

The chemokine system represents a diverse group of G protein-coupled receptors responsible for orchestrating cell recruitment under both homeostatic and inflammatory conditions. Chemokine receptor 9 (CCR9) is a chemokine receptor known to be central for migration of immune cells into the intestine. Its only ligand, CCL25, is expressed at the mucosal surface of the intestine and is known to be elevated in intestinal inflammation. To date, there are no reports of small-molecule antagonists targeting CCR9. We report, for the first time, the discovery of a small molecule, CCX282-B, which is an orally bioavailable, selective, and potent antagonist of human CCR9. CCX282-B inhibited CCR9-mediated Ca(2+) mobilization and chemotaxis on Molt-4 cells with IC(50) values of 5.4 and 3.4 nM, respectively. In the presence of 100% human serum, CCX282-B inhibited CCR9-mediated chemotaxis with an IC(50) of 33 nM, and the addition of α1-acid glycoprotein did not affect its potency. CCX282-B inhibited chemotaxis of primary CCR9-expressing cells to CCL25 with an IC(50) of 6.8 nM. CCX282-B was an equipotent inhibitor of CCL25-directed chemotaxis of both splice forms of CCR9 (CCR9A and CCR9B) with IC(50) values of 2.8 and 2.6 nM, respectively. CCX282-B also inhibited mouse and rat CCR9-mediated chemotaxis. Inhibition of CCR9 with CCX282-B results in normalization of Crohn's disease such as histopathology associated with the TNF(ΔARE) mice. Analysis of the plasma level of drug associated with this improvement provides an understanding of the pharmacokinetic/pharmacodynamic relationship for CCR9 antagonists in the treatment of intestinal inflammation.

Sulindac sulfide is a benzylidene-indene that is a potent, time-dependent inhibitor of cyclooxygenases-1 and -2. Removal of the 2'-methyl group from the indene ring dramatically reduces time-dependent inhibition of both enzymes but also changes the geometry of the benzylidene double bond from Z to E. Herein, we explore the importance of double bond geometry on cyclooxygenase inhibition. The Z-isomer of 2'-des-methyl sulindac sulfide was synthesized by reduction of a bromoindene precursor or by photoisomerization of the E-isomer. The Z-isomer inhibited both cyclooxygenases, but with diminished potency compared to sulindac sulfide. Thus, although the 2'-methyl group is a major determinant of time-dependent cyclooxygenase inhibition, the geometry of the benzylidene double bond plays a role as well.

This study addresses the response-to-retention hypothesis, which states that the subendothelial retention of atherogenic lipoproteins is the necessary and sufficient condition for the initiation of atherosclerosis. Here we focus on the relationship between the generation of ceramide in the low-density lipoprotein (LDL) phospholipid monolayer and the resulting aggregation of LDL particles. This study provides the first measurement of neutral, Mg (2+)-dependent Sphingomyelinase (Smase)-mediated ceramide formation from LDL-sphingomyelin and does so for a range of enzyme concentrations (0-0.22 units Smase/mL). The kinetics of ceramide generation was measured using a fluorescence assay for the above enzyme concentrations with a fixed substrate concentration (0.33 mg LDL/mL). The kinetics of LDL aggregate formation was measured by dynamic light scattering (DLS, method of cumulants) for identical enzyme concentrations. Ceramide concentration profiles were fit with a modification of the Michaelis-Menten model ( k a = 1.11 x 10 (-1) microM (-1) min (-1), k -a = 6.54 x 10 (2) microM (-1) min (-1), k 1 = 3.33 x 10 (1) microM (-1) min (-1), k -1 = 1.41 x 10 (-2) min (-1), k cat = 8.05 x 10 (1) min (-1), K M = 2.418 microM, k deact = 4.66 x 10 (-2) microM (-1) min (-1)) that accounts for the effects of enzyme attachment to the LDL monolayer and for deactivation of Smase due to product inhibition. LDL aggregation is described by a mass action model as explained in previous studies. A key result of this work is the finding that LDL aggregate size depends directly on ceramide concentration and is independent of enzyme concentration. This study demonstrates how principles of colloid science are relevant to important biomedical problems.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.

A procedure for the preparation of optically pure alpha-keto-gamma-hydroxy carboxylic acids through stereospecific aldol addition catalyzed by pyruvate aldolases from the Entner-Doudoroff and the DeLey-Doudoroff glycolytic pathways is described. This highly versatile fragment serves as a precursor for a variety of commonly encountered functionalities, including beta-hydroxy aldehydes and carboxylic acids, alpha-amino-gamma-hydroxy carboxylic acids and alpha,gamma-dihydroxy carboxylic acids. The protocol described here uses recombinant His6-tagged KDPG aldolase for the synthesis of (S)-4-hydroxy-2-keto-4-(2'-pyridyl)butyrate. A protocol for evaluating enantiomeric excess through formation of the gamma-lactone of the dithioacetal followed by chiral-phase gas-liquid chromatography is also described. Enzyme expression and enzymatic synthesis can be accomplished in approximately 1 week. The enzymatic aldol addition proceeds in nearly quantitative yields with enantiomeric excesses greater than 99.7%.

Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smoking-induced pathophysiology is often resistant to the anti-inflammatory effects of glucocorticoids. The nature of cigarette smoke-induced inflammation is still not defined, although neutrophil recruitment and activation seem to be consistent features. In the current study, we have used a range of approaches to demonstrate that cigarette smoke activates human monocytes and macrophages to release the CXC chemokine CXCL8 [(interleukin-8 (IL-8)]. Furthermore, we show for the first time that cigarette smoke synergizes with proinflammatory cytokines IL-1beta and tumor necrosis factor-alpha, and it is this interaction that confers steroid resistance to smoke-induced CXCL8 release. We go on to show that smoke-induced activation of human cells is an oxidant-mediated phenomenon acting through activator protein-1, but not nuclear factor kappaB, pathway. These observations add significantly to our understanding of smoke as an inflammatory stimulus that has implications for potential the development of treatments of smoking or related disease.

Endothelin-1 is a potent vasoconstrictor with mitogenic properties. This 21-amino-acid protein, released in the vasculature by endothelial and smooth muscle cells, has been implicated in pulmonary hypertension. More recently, evidence has accumulated for a role of the heme oxygenase system in pulmonary hypertension. Heme oxygenase catalyses the breakdown of heme to produce carbon monoxide, biliverdin and free iron. Here we show that a carbon monoxide-releasing molecule, but not biliverdin, inhibits endothelin-1 release from serum-stimulated human pulmonary artery smooth muscle cells. Under certain conditions, carbon monoxide appears to act as an endogenous break on endothelin-1 release.