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Activin receptor-like kinase 2 (ALK2) has been implicated as a key target in multiple rare diseases. Herein, we describe the design of a novel bicyclic lactam series of potent and selective ALK2 inhibitors. This manuscript details an improvement in potency of two orders of magnitude from the initial bicyclic structure as well as a two-fold improvement in cellular potency from the original monocyclic inhibitor. Furthermore, we provide a detailed strategy for progressing this project in the future.

Pharmacological modulation of the Janus kinase (JAK) family has achieved clinically meaningful therapeutic outcomes for the treatment of inflammatory and hematopoietic diseases. Several JAK1 selective compounds are being investigated clinically to determine their anti-inflammatory potential. We used recombinant enzymes and primary human lymphocytes to assess the JAK1 specificity of itacitinib (INCB039110) and study inhibition of signal transducers and activators of transcription (STAT) signaling. Rodent models of arthritis and inflammatory bowel disease were subsequently explored to elucidate the efficacy of orally administered itacitinib on inflammatory pathogenesis. Itacitinib is a potent and selective JAK1 inhibitor when profiled against the other JAK family members. Upon oral administration in rodents, itacitinib achieved dose-dependent pharmacokinetic exposures that highly correlated with STAT3 pharmacodynamic pathway inhibition. Itacitinib ameliorated symptoms and pathology of established experimentally-induced arthritis in a dose-dependent manner. Furthermore, itacitinib effectively delayed disease onset, reduced symptom severity, and accelerated recovery in three distinct mouse models of inflammatory bowel disease. Low dose itacitinib administered via cannula directly into the colon was highly efficacious in TNBS-induced colitis but with minimal systemic drug exposure, suggesting localized JAK1 inhibition is sufficient for disease amelioration. Itacitinib treatment in an acute graft-versus-host disease (GvHD) model rapidly reduced inflammatory markers within lymphocytes and target tissue, resulting in a marked improvement in disease symptoms. This is the first manuscript describing itacitinib as a potent and selective JAK1 inhibitor with anti-inflammatory activity across multiple preclinical disease models. These data support the scientific rationale for ongoing clinical trials studying itacitinib in select GvHD patient populations.

Antigen recognition and T-cell receptor (TCR) activation are fundamental processes that drive anti-tumor T cell responses. HPK1 has been identified as a negative regulator of TCR activation, as well as BCR activation, and is a potential anticancer target for immuno-oncology. We show that genetic ablation of HPK1 in human T cells resulted in increased cytokine production upon cell stimulation. Jurkat cells with genetic HPK1 knockout were unable to phosphorylate SLP76, a direct downstream target of HPK1, upon cell stimulation which was associated with increased IL-2 production. Genetic deletion of HPK1 in human RAMOS B cells reduced phosphorylation of BLNK upon IgM stimulation and led to increased TNF alpha and TNF beta production. Treatment of HPK1 knockout primary human T cells with pembrolizumab enhanced IFN gamma secretion compared to the knockout cells alone. Based on these genetic data, HPK1 may be an attractive target for immuno-oncology. We describe herein the in vitro and in vivo profile of several small molecule HPK1 inhibitors and report a surprising disparity between in vitro and in vivo findings. In vitro, the HPK1 inhibitors tested phenocopied the genetic data by potently inhibiting pSLP76 and enhancing IL-2 production in Jurkat cells following stimulation. Similarly, HPK1 inhibitors enhanced IL-2 production in human PBMCs, and increased IFN gamma production in combination with atezolizumab in a primary T cell co-culture assay. In vivo, the HPK1 small molecule inhibitors inhibited tumor growth in the MC38 model, which was further enhanced in combination with anti-PD-L1 in 3 different syngeneic models (MC38, CT26 and MBT-2). Despite these results, tumor growth inhibition was not observed in the GL261 glioma syngeneic model whose growth was recently shown to be inhibited when implanted into HPK1 kinase-dead mice. Further, although several in vitro assays demonstrated increased functional cytokine production with HPK1 inhibitor treatment, the compounds did not increase in vivo cytokine production in tumors. Moreover, combining HPK1 inhibitors with a PD-L1 antibody in vivo abolished the anti-PD-L1-induced production of IFN gamma in the CT26 model. In summary, while our genetic data support the role of HPK1 as a negative regulator of T and B cells, the in vitro activity of HPK1 kinase activity inhibitors was not correlated with functional effects in in vivo syngeneic tumor models. These results underline the complexity of interpreting HPK1 biology and also highlight challenges for the development of clinically active compounds targeting this pathway. Citation Format: Yaoyu Chen, Jonathan Rios-Doria, Michelle Pusey, Kerri Lasky, Min Ye, Pramod Thekkat, Karen Gallagher, Kristine Stump, Patricia Conlen, Christine Gardiner, Hui Wang, Alexander Sokolsky, Mark Rupar, Luping Lin, Elham Behshad, Maryanne Covington, Holly Koblish, Oleg Vechorkin, Wenqing Yao, Sunkyu Kim, Yingnan Chen. The role of HPK1 in the regulation of T cell function and anti-tumor immune activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4513.

Obesity promotes insulin resistance associated with liver inflammation, elevated glucose production, and type 2 diabetes. Although insulin resistance is attenuated in genetic mouse models that suppress systemic inflammation, it is not clear whether local resident macrophages in liver, denoted Kupffer cells (KCs), directly contribute to this syndrome. We addressed this question by selectively silencing the expression of the master regulator of inflammation, NF-κB, in KCs in obese mice. We used glucan-encapsulated small interfering RNA particles (GeRPs) that selectively silence gene expression in macrophages in vivo. Following intravenous injections, GeRPs containing siRNA against p65 of the NF-κB complex caused loss of NF-κB p65 expression in KCs without disrupting NF-κB in hepatocytes or macrophages in other tissues. Silencing of NF-κB expression in KCs in obese mice decreased cytokine secretion and improved insulin sensitivity and glucose tolerance without affecting hepatic lipid accumulation. Importantly, GeRPs had no detectable toxic effect. Thus, KCs are key contributors to hepatic insulin resistance in obesity and a potential therapeutic target for metabolic disease.—Tencerova, M., Aouadi, M., Vangala, P., Nicoloro, S. M., Yawe, J. C., Cohen, J. L., Shen, Y., Garcia-Menendez, L., Pedersen, D. J., Gallagher-Dorval, K., Perugini, R. A., Gupta, O. T., Czech, M. P. Activated Kupffer cells inhibit insulin sensitivity in obese mice.

Monoclonal antibodies against PD-L1 or PD-1 have been approved for the treatment of multiple tumor histologies by virtue of their ability to restore T cell effector function, increase T cell proliferation and enhance infiltration of tumor-reactive T cells. A small molecule approach to PD-(L)1 axis blockade may offer distinct benefits over the use of monoclonal antibodies including improved tissue penetration, titratability, absence of immunogenicity, ease of administration, and potential for fixed dose oral-oral therapeutic combinations. We have identified a novel class of small molecule PD-L1 antagonists that are capable of functional PD-(L)1 axis blockade by virtue of their ability to induce PD-L1 internalization. In vitro, select small molecules demonstrate high affinity to human PD-L1, potently disrupt the PD-L1:PD-1 interaction ( Citation Format: Liang-Chuan S. Wang, Holly Koblish, Yue Zhang, Ashwini Kulkarni, Maryanne Covington, Karen Gallagher, Gengjie Yang, Jonathan Rios-Doria, Christina Stevens, Michael Hansbury, Sybil O'Connor, Yan-ou Yang, Sharon Diamond, Krista Burke, Kaijiong Xiao, Jingwei Li, Wenqing Yao, Liangxing Wu, Peggy Scherle, Gregory Hollis, Reid Huber. Preclinical characterization of potent and selective oral PD-L1 small-molecule antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4480.

Purpose The purpose of this study was to determine whether detailed analysis of story recall performance reveals significant differences between veterans with and without history of mild traumatic brain injury (mTBI). Method Twenty-one military veterans participated, with 7 reporting history of mTBI. All participants were administered the Logical Memory I and II subtests from the Wechsler Memory Scale–Fourth Edition (Wechsler, 2009). Responses were scored for total correct ideas (TCI) and total words produced (TWP). Results Although the groups did not differ in scaled scores, other measures did reveal significant differences. After a delay, the mTBI group showed a greater drop in TCI relative to the control group. Additionally, the control group showed an increase in TWP when the recall was delayed versus immediate; a pattern not observed for the mTBI group. Conclusions The mTBI and control groups did not significantly differ in scaled scores. However, group differences were observed in TCI and TWP. The findings suggest that, relative to the control group, the mTBI group were less successful in retrieving episodic information and eliciting self-cueing. Small sample size limited data interpretation, and larger sample sizes are needed to confirm the findings. The results indicate that veterans with mTBI may present with symptoms persisting beyond the acute state of the injury.

Interdisciplinary projects offer a strategy for German programs to become more visible and valued in the university context. Freedom Without Walls at Chapman University offers a case study of a project that situates the German program within an institution's larger goals. When building a project, the German program should look first to existing expertise, interests, and resources on campus. Connection with the institution's mission and vision is also essential. With funding from the German government to U.S. universities, Freedom Without Walls encompassed events in art, poetry, film, communications, and other disciplines as well as a gala and a team in the 5K run/walk.

There is a growing emphasis on delivering services for persons with early-stage dementia (ie, ‘‘persons with memory loss,’’ or PWMLs) and their family members (care partners). The goal of this evaluation was to determine whether participation in the Memory Club, a 10- to 13-session joint support group, would result in decreased distress, enhanced preparation for care, and improved feelings of confidence managing the challenges of early-stage dementia. The single group, pre-/post-test evaluation included 63 PWMLs and 61 care partners who participated in three Memory Club sites in Minnesota. Paired T-test results found that care partners reported significant (P < .05) increases in preparation activities, feelings of preparation, and confidence in managing memory loss. The results suggest that the Memory Club can fill an important gap in early-stage dementia care by offering care partners the opportunity to plan, prepare, and increase coping skills in the face of early dementia progression.

Background Obesity is a strong risk factor for resistance to insulin-mediated glucose disposal, a precursor of type 2 diabetes and other disorders. However, not all obese individuals are insulin resistant. We sought to identify the molecular pathways that might cause obesity-associated insulin resistance in humans by studying the morbidly obese who were insulin sensitive versus insulin resistant, thereby eliminating obesity as a variable. Methods Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from similarly obese patients undergoing gastric bypass surgery. Results Gene sets related to chemokine activity and chemokine receptor binding were identified as most highly expressed in the omental tissue from insulin-resistant compared with insulin-sensitive subjects, independent of the body mass index. These upregulated genes included chemokines (C-C motif) ligand 2, 3, 4, and 18 and interleukin-8/(CC-X motif) ligand 8 and were not differentially expressed in the subcutaneous adipose tissues between the 2 groups of subjects. Insulin resistance, but not the body mass index, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size, in these morbidly obese subjects. Conclusion Our findings have demonstrated that inflammation of the omental adipose tissue is strongly associated with insulin resistance in human obesity even in subjects with similar body mass index values.

Background Obesity is associated with a pathologic predominance of sympathetic over parasympathetic tone. With respect to the heart, this autonomic dysfunction presents as a decreased heart rate variability (HRV), which has been associated with increased cardiovascular morbidity. Gastric bypass (GB) reduces cardiovascular mortality, and, thus, could beneficially affect the HRV. We sought to identify the factors predictive of HRV in a severely obese population of undergoing GB at a university hospital in the United States. Methods The data of all patients presenting for GB were included in a prospective database. The homeostatic model of assessment (HOMA) was used to calculate the insulin resistance and glucose disposition index. A 24-hour Holter monitor was used to assess the HRV. Measurements were repeated at 2 weeks and 6 months postoperatively. The correlations between variables were determined using linear mixed models. Results We studied 30 patients undergoing GB. All exhibited some degree of reduced HRV that improved postoperatively. The HOMA-insulin resistance inversely correlated with the HRV, and the HOMA-glucose disposition index directly correlated with the parameters of HRV in our longitudinal models. Weight, body mass index, excess body weight, gender, and age did not correlate with HRV. Improvements in HRV correlated with reductions in the average heart rate, underscoring a postoperative increase in relative vagal tone. Conclusion HRV in the severely obese is better predicted by the degree of insulin resistance, than by the degree of obesity, age, or gender. GB led to an improvement in HRV, the magnitude of which correlated with the change in insulin resistance and glucose disposition index, but not with weight loss.

The chemokine receptor 2 (CCR2) directs migration of monocytes and has been proposed to be a drug target for chronic inflammatory diseases. INCB3344 was first published as a small molecule nanomolar inhibitor of rodent CCR2. Here, we show that INCB3344 can also bind human CCR2 (hCCR2) with high affinity, having a dissociation constant (Kd) of approximately 5 nM. The binding of the compound to the receptor is rapid and reversible. INCB3344 potently inhibits hCCR2 binding of monocyte chemoattractant protein-1 (MCP-1) and MCP-1-induced signaling and function in hCCR2-expressing cells, including ERK phosphorylation and chemotaxis, and is competitive against MCP-1 in vitro. INCB3344 also blocks MCP-1 binding to monocytes in human whole blood, with potency consistent with in vitro studies. The whole blood binding assay described here can be used for monitoring pharmacodynamic activity of CCR2 antagonists in both preclinical models and in the clinic.

Background As the number of laparoscopic adjustable gastric bands (LAGBs) placed has increased, the number of patients requiring removal of the device has also increased. Methods The data from our institution, a U.S. university medical center, were reviewed to determine the feasibility, patient characteristics, and early results of converting patients from LAGB to laparoscopic Roux-en-Y gastric bypass. Results A total of 350 patients underwent LAGB placement at our institution from 2001 to 2008. Of these, 26 required conversion to laparoscopic Roux-en-Y gastric bypass for the following reasons: slippage, poor weight loss, LAGB intolerance, esophageal dilation, infection, and gastric ischemia. All conversions were completed laparoscopically. The average operating time and length of stay was 160 minutes and 3 days, respectively. Three complications developed. The average interval to conversion was 29 months. The average follow-up after conversion was 18 months. The average percentage of excess body weight loss at conversion was 23%. At 12 months after conversion, the patients had achieved an average percentage of excess body weight loss of 56% from their pre-LAGB weight. Conclusion The increasing popularity of the LAGB has led to a considerable number of revisions of the device. Our early experience has shown that converting patients from LAGB to laparoscopic Roux-en-Y gastric bypass is feasible and safe and can offer patients substantial additional weight loss.

Background/Objective: Identifying which patients with varicose veins are at risk of progressing to more severe forms of chronic venous disease could help in assigning clinical priorities and targeting appropriate treatments. The aim of this study was to determine, in subjects with varicose veins, the characteristics of venous disease and other factors associated with an increased risk of ulceration.Methods: One hundred twenty subjects with varicose veins and an open or healed venous leg ulcer were compared with 120 controls with varicose veins and no history of venous ulcer on this case control study. Subjects were recruited from hospital settings and primary care. Each subject completed a questionnaire on life-style and medical history and underwent an examination comprising of clinical classification of venous disease (CEAP), duplex scanning, quantitative digital photoplethysmography, and measurement of dorsiflexion. Multiple logistic regression analyses and calculation of receiver operating characteristic (ROC) curves were performed to identify the combination of factors which most accurately predicted which patients with varicose veins will develop leg ulcers.Results: An increased risk of ulceration was associated with the severity of clinical venous disease, especially with the presence of skin changes (P < .0001). Other significant risk factors included history of deep vein thrombosis (DVT) (P = .001), higher body mass index (BMI) (P = .006), smoking (P = .009), and reflux in the deep veins (P = .0001). Ulceration was associated with reduced volume of blood displaced as reflected by photoplethysmography and a limited range of ankle movement (not wholly due to the effects of an active ulcer) (both P < .05). Multivariate analyses showed that skin changes including lipodermatosclerosis (odds ratio [OR] 8.90, 95% confidence interval [CI] 1.44-54.8),corona phlebectatica (OR 4.52, 95% CI 1.81-11.3) and eczema (OR 2.87, 95% CI 1.12-7.07), higher BMI (OR 1.08, 95% CI 1.01-1.15), and popliteal vein reflux(OR 2.82, 95% CI 1.03-7.75) remained independently associated with increased risk of ulceration while good dorsiflexion of the ankle (OR 0.88, 95% CI 0.81-0.97) and an effective calf muscle pump (OR 0.96, 95% CI 0.92-0.99) remained protective factors. ROC curve analyses indicated that a model based on clinical observation of skin changes, duplex scanning for popliteal reflux, and calf muscle pump tests would be the most accurate in determining which patients with varicose veins develop leg ulcers.Conclusions: The results of this study confirm that, in patients with varicose veins, those with skin changes of chronic venous insufficiency and deep vein incompetence are at greatly increased risk of ulceration. However, the risks may also be increased in those who smoke, are obese, and have restricted ankle movement and reduced calf muscle pump power. (J Vasc Surg 2009;49:1490-8.)

A team‐taught interdisciplinary undergraduate unit in Sustainable Development has been developed and run over the past two years at the University of Bristol. This has been a unique initiative for this university to take. As in most other research‐intensive higher education institutions, teaching generally follows rather traditional disciplinary conventions, operating within departmental bounds. The initiative was unusual – and indeed ambitious – enough to gain the Higher Education Environmental Performance Improvement (HEEPI) Green Gown Award in teaching for 2007 (HEEPI is a project supported by the Higher Education Founding Council for England; http://www.heepi.org.uk/green_gown_awards.htm). There are both challenges and pleasures in designing and delivering a team‐taught unit in a traditional university setting. This experience is outlined and evaluated here, giving consideration to both the practical and the more fundamentally philosophical issues encountered in the process.

The development and widespread use of small Unmanned Aerial Vehicles (UAVs), such as quadcopters with GoPro cameras, is leading a revolution in a wide range of hobby, commercial and potential research applications of the technology. Within urban areas the variety and pace of changes occurring with both human and natural systems have significant impacts on efforts to address and manage sustainability. A wide range of opportunities and challenges exist in considering how UAVs may effectively be used as a spatial tool in examining urban areas, including buildings, infrastructure, ecosystem features and processes, natural areas, and environmental health. This chapter examines the technical, practical, policy and legal implications of the use of UAVs to assess and monitor urban sustainability and includes highlighting examples of recent research applications. Uses of scale, resolution, accuracy and reliability are noted along with how improving technologies and the growth in the widespread familiarity and use of UAVs represent an expanding field for urban research applications. Challenges including privacy, federal regulations governing the commercial use of UAVs are discussed. A series of recommendation research applications are presented in terms of the greatest potential where UAVs can be employed to further research, understanding, and decision-making in the area of urban sustainability.

The class 1A phosphatidylinositol 3-kinase enzymes consist of a number of heterodimeric complexes of regulatory and catalytic subunits and have been implicated in a number of cellular responses. While platelet-derived growth factor (PDGF)-induced chemotaxis of human vascular smooth muscle cells (HVSMC) is inhibited by both wortmannin and LY294002, DNA synthesis is only inhibited by LY294002. Serum-induced DNA synthesis however is inhibited by LY294002, wortmannin and rapamycin. Similarly PDGF-induced protein kinase B (PKB) activation is inhibited by LY294002 but not by wortmannin or rapamycin. In conclusion PDGF-induced DNA synthesis appears to occur through a phosphatidylinositol 3-kinase (PI3-K)-dependent, but wortmannin-insensitive, PKB/Akt pathway.

Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking alpha(v)beta(3) antibody, a function-blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking alpha(3)beta(1) antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis.

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents.

SummaryThe sulphated polysaccharides fucoidan and heparin both inhibit vascular smooth muscle cell (VSMC) proliferation. In this study we compared their actions on mitogenesis and ERK1/ERK2 activation in human VSMC. Although they displaced cell surface [3H]-heparin binding with similar affinity, they exerted clearly distinguishable actions. Fucoidan potently inhibited DNA synthesis stimulated by foetal calf serum, PDGF-BB and thrombospondin-1. Heparin inhibited the mitogenic action of serum and thrombospondin-1 (though less potently than fucoidan), but failed to inhibit PDGF-BB-induced DNA synthesis. In parallel studies, fucoidan, but not heparin, inhibited ERK1/ERK2 activation by PDGF-BB. Moreover, fucoidan inhibited serum-induced mitogenesis in “heparin resistant” VSMC, which are refractory to heparin’s antimitogenic action. In summary, the structurally different polysaccharides, heparin, fucoidan (and fucans) have distinguishable effects on mitogenesis and ERK1/ERK2 activation, suggesting that different mechanism(s) mediate these actions. The potent antimitogenic action of fucoidan and its efficacy in heparin resistant VSMC emphasise the need to further investigate its mechanism of action in human VSMC and suggest this agent could have therapeutic potential.

A novel analytical method has been developed for direct quantification of intracellular nucleoside triphosphates (NTPs). Lysates of human peripheral blood mononuclear cells (PBMCs) were extracted by protein precipitation, and the filtered extracts were analyzed by weak anion exchange liquid chromatography (WAX-LC) coupled to detection by mass spectrometry (MS). Compared with ion pairing (IP)-LC/MS/MS, the only MS-compatible direct detection method for NTPs currently available, the new method completely avoids the usage of ion-pairing reagents and has a shorter analytical time of only 2 min. The method was validated and is being used to determine the amount of the triphosphate metabolite of D-D4FC (DPC817), an investigational HIV nucleoside reverse transcriptase inhibitor (NRTI), in human PBMC samples from clinical studies. By using a PE Sciex API 4000 triple quadrupole instrument operating in positive ion MRM mode, the method was able to achieve a lower limit of quantitation (LLOQ) of 5 fmol/10(6) cells in samples containing 3 x 10(6) lysed cells (6 fmol on-column). With minor adaptation, the method described here may be suitable for analyzing other NTPs. This paper also provides a discussion of the unique retention characteristics of WAX-LC, the principles of which may prove to be valuable for designing other forms of directly coupled ion-exchange (IX)-LC/MS methods suited for high sensitivity quantitative analysis.

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. FGFR4 signaling contributes to the development and progression of subsets of cancer: in approximately 10 percent of hepatocellular carcinoma (HCC), genetic amplification of FGF19, encoding an endocrine FGF ligand that activates FGFR4-KLB receptors, has been reported. In models with this alteration, FGF19-FGFR4 signaling is oncogenic and antagonism of the FGF19-FGFR4 axis has been shown to be efficacious suggesting that selective targeting of FGFR4 may be an effective strategy for malignancies with FGFR4 activation. We describe the preclinical characterization of INCB062079 a potent and selective inhibitor of the FGFR4 kinase. In biochemical assays INCB062079 inhibited FGFR4 with low nM potency and exhibited at least 250-fold selectivity against other FGFR kinases and greater than 800-fold selectivity against a large kinase panel. This selectivity derives from the ability of INCB062079 to bind irreversibly to Cys552, a residue within the active site of FGFR4 that is non-conserved among other FGFR receptors. Covalent binding of INCB062079 to Cys552 was demonstrated using a LC/MS/MS-based proteomic analysis that confirmed specificity for the target Cys. In assays using HCC cells with autocrine production of FGF19, INCB062079 inhibited the autophosphorylation of FGFR4 and blocked signal transduction by FGFR4 to downstream markers of pathway activation. Cancer cell lines that have amplification and expression of FGF19 are uniquely sensitive to growth inhibition by INCB062079 (EC50 less than 200 nM) compared with HCC cell lines or normal cells without FGF19-FGFR4 dependence (EC50 > 5000 nM) confirming selectivity for FGFR4. In vivo, oral administration of INCB062079 inhibited the growth and induced significant regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity at doses that were well-tolerated (10-30 mg/kg BID) and did not result in a significant increase in serum phosphate levels which is observed with FGFR1/2/3 inhibition. Suppression of tumor growth correlated with pharmacodynamic inhibition of FGFR4 signaling. Collectively, these preclinical studies demonstrate that INCB062079 potently and selectively inhibits models of FGF19-FGFR4-dependent cancers in vitro and in vivo, supporting clinical evaluation in patients harboring oncogenic FGFR4 activation. Citation Format: Phillip C.C. Liu, Liang Lu, Kevin Bowman, Matthew C. Stubbs, Liangxing Wu, Darlise DiMatteo, Sindy Condon, Ronald Klabe, Ding-Quan Qian, Xiaoming Wen, Paul Collier, Karen Gallagher, Michael Hansbury, Xin He, Bruce Ruggeri, Yan-ou Yang, Maryanne Covington, Timothy C. Burn, Sharon Diamond-Fosbenner, Richard Wynn, Reid Huber, Wenqing Yao, Swamy Yeleswaram, Peggy Scherle, Gregory Hollis. Selective inhibition of FGFR4 by INCB062079 is efficacious in models of FGF19- and FGFR4-dependent cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2100. doi:10.1158/1538-7445.AM2017-2100

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML. Citation Format: Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4712.

Restenosis remains the largest single obstacle to the long-term success of invasive vascular interventions. Lovastatin, an HMG-CoA reductase inhibitor, has been shown to reduce myointimal hyperplasia in animal models of restenosis and in one clinical coronary restenosis trial. We have assessed the effect of lovastatin on the growth of cultured human vascular smooth muscle cells derived from saphenous vein and vascular graft stenoses. Lovastatin (2 microM) inhibited proliferation over 14 days in saphenous vein (and graft stenoses) derived vascular smooth muscle cells by 42% and 32% respectively: this was not significantly different. Lovastatin (10 microM) reduced [methyl 3H]-thymidine uptake by 51% in saphenous vein-derived cells. These concentrations were significantly higher than those achieved in plasma during therapeutic dosage. Lovastatin-induced inhibition of vascular smooth muscle cell proliferation and [methyl 3H]-thymidine uptake was completely reversed by adding mevalonate (100 microM) but cholesterol (10-40 micrograms ml-1) had no effect. Isopentenyl adenine (25-50 microM) did not affect the inhibition of [methyl 3H]-thymidine uptake by lovastatin (10 microM), but farnesol (20 microM), another isoprenoid precursor of cholesterol synthesis, reversed the antiproliferative effect.

Vascular restenosis after invasive interventions is an important clinical problem for which no preventive pharmacologic therapy exists. Calcium channel blockers have been shown to inhibit myointimal hyperplasia in animal models of restenosis and in some small and flawed clinical coronary restenosis trials. We examined the inhibitory effect of amlodipine, verapamil, and diltiazem on the growth of cultured human vascular smooth muscle cells (VSMC) derived from saphenous vein (n = 20) and graft stenoses (n = 7), in 14-day proliferation assays and [methyl 3H]thymidine uptake studies. Amlodipine and verapamil produced significant inhibition (30%) of VSMC proliferation and DNA synthesis at 10 microM but not at 500 nM-1 microM. To our knowledge, this is the first study to examine the antiproliferative effect of calcium channel blockers in VSMC derived from human graft stenoses. Growth inhibition of VSMC from graft stenoses was not significantly different from that of control saphenous vein-derived cells. We conclude, therefore, that calcium channel blockers inhibit human VSMC proliferation in vitro, regardless of whether the cells were grown from graft stenoses or saphenous vein. However, the concentrations at which these calcium channel blockers elicit antiproliferative effects may not be attainable during therapeutic dosing in humans.

Objective: The aim was to assess the growth inhibitory effect of fibrates on human vascular smooth muscle cells. Restenosis is the most important factor limiting the long term success of invasive vascular interventions and there is as yet no effective preventive treatment. Platelet derived growth factor (PDGF) is considered to be an important growth promoting agent for vascular smooth muscle cells (VSMC) and fenofibric acid (a hypolipidaemic drug) has been reported to be a PDGF antagonist. Methods: The effect of the fibrate drugs fenofibrate, clofibrate, bezafibrate, and gemfibrozil were examined on the proliferation of cultured human vascular smooth muscle cells derived from saphenous vein (n = 20) and graft stenoses (n = 7). Results: Fenofibrate (100 μM) produced potent inhibition (48%) of VSMC proliferation at a concentration equivalent to that of its circulating metabolite fenofibric acid, but none of the other drugs produced any significant effect on growth. VSMC derived from graft stenoses were equally sensitive to inhibition as saphenous vein derived controls, in contrast to our previous work which reported that graft stenosis derived VSMC were resistant to growth inhibition by the physiological inhibitor heparin. The antiproliferative effect of fenofibrate was independent of inhibition of cellular cholesterol synthesis or toxicity. Fenofibrate inhibited VSMC growth induced by 15% fetal calf serum, PDGF, and basic fibroblast growth factor to a similar degree, indicating that it is not a specific PDGF antagonist. Conclusions: Fenofibrate is not a specific PDGF antagonist. Fenofibric acid, one of the principal metabolites of fenofibrate, did not produce any inhibition of growth, suggesting that oral administration of fenofibrate would not be efficacious. Fenofibrate is the first potent inhibitor to be described for VSMC derived from human myo-intimal hyperplastic lesions. Cardiovascular Research 1994; 28 :615-620

Alterations of video monitor and laparoscopic camera position may create perceptual distortion of the operative field, possibly leading to decreased laparoscopic efficiency. We aimed to determine the influence of monitor/camera position on the laparoscopic performance of surgeons of varying skill levels.Twelve experienced and 12 novice participants performed a one-handed task with their dominant hand in a modified laparoscopic trainer. Initially, the camera was fixed directly in front of the participant (0 degrees) and the monitor location was varied between three positions, to the left of midline (120 degrees), directly across from the participant (180 degrees), and to the right of the midline (240 degrees). In the second experiment monitor position was constant straight across from the participant (180 degrees) while the camera position was adjusted between the center position (0 degrees), to the left of midline (60 degrees), and to the right of midline (300 degrees). Participants completed five trials in each monitor/camera setting. The significance of the effects of skill level and combinations of camera and monitor angle were evaluated by analysis of variance (ANOVA) for repeated measures using restricted maximum likelihood estimation.Experienced surgeons completed the task significantly faster at all monitor/camera positions. The best performance in both groups was observed when the monitor and camera were located at 180 degrees and 0 degrees, respectively. Monitor positioning to the right of midline (240 degrees) resulted in significantly worse performance compared to 180 degrees for both experienced and novice surgeons. Compared to 0 degrees (center), camera position to the left or the right resulted in significantly prolonged task times for both groups. Novice subjects also demonstrated a significantly lower ability to adjust to suboptimal camera/monitor positions.Experienced subjects demonstrated superior performance under all study conditions. Optimally, the camera should be directly in front and the monitor should be directly across from a surgeon. Alternatively, the monitor/camera could be placed opposite to the surgeon's non-dominant hand. The suboptimal camera/monitor conditions are especially difficult to overcome for inexperienced subjects. Monitor and camera positioning must be emphasized to ensure optimal laparoscopic performance.

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. Genomic analyses of squamous cell lung, gastric and urothelial tumors have revealed recurrent genetic alterations in FGFR1, FGFR2 and FGFR3 genes, respectively. FGFR proteins contribute to the development of malignancies by promoting tumor cell proliferation, survival, and migration and supporting angiogenesis. Therefore targeting FGFR kinases may provide therapeutic benefit to patients with cancers that have genetic alterations in genes encoding components of the FGF-FGFR axis. INCB054828 is a potent inhibitor of FGFR1, FGFR2, and FGFR3 that has selective pharmacological activity against cancer cells with FGFR alterations. In vitro, INCB054828 potently inhibited the kinase activity of recombinant FGFR1, FGFR2 and FGFR3 enzymes and was highly selective against a panel of kinases including VEGFR2. In cellular assays, INCB054828 inhibited the autophosphorylation of FGFR proteins with low nanomolar IC50 values and blocked signal transduction by FGFR to downstream markers of pathway activation. Cancer cell lines that have genetic alterations in FGFR1, FGFR2 and FGFR3 were uniquely sensitive to growth inhibition by INCB054828, with IC50 values generally in the range of 3-50 nM, compared with cancer cell lines or normal cells without FGFR dependence (IC50 > 1500 nM). In vivo, once-daily oral administration of INCB054828 inhibited the growth of tumors that are dependent upon FGFR1, FGFR2 and FGFR3 activity at tolerated doses. Suppression of tumor growth was dose-dependent and correlated with pharmacodynamic inhibition of FGFR. Collectively, these preclinical studies demonstrate that INCB054828 potently and selectively inhibits models of FGFR-dependent cancers in vitro and in vivo, supporting the compound's clinical evaluation in patients harboring oncogenic FGFR activation. Citation Format: Phillip CC Liu, Liangxing Wu, Holly Koblish, Kevin Bowman, Yue Zhang, Ronald Klabe, Lynn Leffet, Darlise DiMatteo, Mark Rupar, Karen Gallagher, Michael Hansbury, Colin Zhang, Chunhong He, Paul Collier, Maryanne Covington, Richard Wynn, Swamy Yeleswaram, Kris Vaddi, Timothy Burn, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Preclinical characterization of the selective FGFR inhibitor INCB054828. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2015-771

The PIM (Proviral Integration sites for Moloney virus) family of constitutively active serine/threonine kinases consists of three members (PIM1, PIM2 and PIM3) that phosphorylate substrates involved in both apoptosis and cellular metabolism. As downstream targets of the JAK/STAT pathway and by having overlapping substrates with the PI3K/AKT pathway, PIM kinases integrate signals from multiple pathways important for the survival and proliferation of malignant cells. Overexpression of PIM kinases has been reported in human hematological cancers with each isoform showing a distinct expression pattern among the various malignancy subtypes, including MM, AML, ALL and NHL. Although the three family members possess overlapping functions that are mutually compensatory, mice deficient for all three PIM kinases exhibit minor phenotypic abnormalities, with smaller overall size at maturity and decreased cellular responses to cytokines. These data suggest that pan-inhibition of the PIM kinase family will have a favorable toxicity profile and may be required for therapeutic benefit in hematological malignancies. INCB053914 is a novel, ATP-competitive, small molecule, pan-inhibitor of PIM kinases. In biochemical assays, INCB053914 is potent against PIM1, PIM2 and PIM3 and highly selective. In cell proliferation assays, INCB053914 is active as a single agent in the majority of cell lines derived from different hematological malignancies, including MM, AML, DLBCL, MCL and T-ALL, with IC50 values ranging from 3 - 300 nM. INCB053914 synergizes with a variety of cytotoxic and targeted agents, reducing the viability of a panel of hematological tumor cell lines. In pharmacodynamic assays, INCB053914 inhibits phosphorylation of S6RP, P70S6K, 4E-BP-1 and BAD, known PIM kinase targets. Following treatment with INCB053914, induction of PIM kinase protein levels was noted in human cancer cell lines. Importantly, this compensatory response does not reduce the activity of the molecule, as effects on downstream signaling and efficacy remain intact when PIM kinase levels are elevated. Induction of PIM kinases after INCB53914 treatment was also evident in whole blood from healthy volunteers and in primary blast cells isolated from patients diagnosed with AML, further supporting the need for pan-PIM kinase inhibition. Taken together, these results indicate that INCB053914 has potent and selective activity against PIM kinases and may be useful as monotherapy or in combination with other agents for treating hematologic malignancies that are dependent on PIM kinase activity for growth and survival. Citation Format: Niu Shin, Maryanne Covington, Richard Wynn, Yanlong Li, Alexander Margulis, Qian Wang, Kathy Wang, Cindy Marando, Patricia Feldman, Lynn Leffet, Karen Gallagher, Xin He, Hong Chang, He Zhang, Hao Feng, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Timothy Burn, Kris Vaddi, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Gregory Hollis, Robert Newton, Reid Huber, Peggy Scherle, Holly Koblish. Characterization of INCB053914, a novel pan-PIM kinase inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5397. doi:10.1158/1538-7445.AM2015-5397

Hypothesis The use of smaller instruments during laparoscopic cholecystectomy (LC) has been proposed to reduce postoperative pain and improve cosmesis. However, despite several recent trials, the effects of the use of miniaturized instruments for LC are not well established. We hypothesized that LC using miniports (M-LC) is safe and produces less incisional pain and better cosmetic results than LC performed conventionally (C-LC). Design A patient- and observer-blinded, randomized, prospective clinical trial. Setting A tertiary care, university-based hospital. Patients Seventy-nine patients scheduled for an elective LC who agreed to participate in this trial were randomized to undergo surgery using 1 of the 2 instrument sets. The criteria for exclusion were American Society of Anesthesiologists class III or IV, age older than 70 years, liver or coagulation disorders, previous major abdominal surgical procedures, and acute cholecystitis or acute choledocholithiasis. Intervention Laparoscopic cholecystectomy performed with either conventional or miniaturized instruments. Main Outcome Measures Patients’ age, sex, operative time, operative blood loss, intraoperative complications, early and late postoperative incisional pain, and cosmetic results. Results Thirty-three C-LCs and 34 M-LCs were performed and analyzed. There were 8 conversions (24%) to the standard technique in the M-LC group. No intraoperative or major postoperative complications occurred in either group. The average incisional pain score on the first postoperative day was significantly less in the M-LC group (3.9 vs 4.9; P = .04). No significant differences occurred in the mean scores for pain on postoperative days 3, 7, and 28. However, 90% of patients in the M-LC group and only 74% of patients in the C-LC group had no pain (visual analog scale score of 0) at 28 days postoperatively ( P = .05). Cosmetic results were superior in the M-LC group according to both the study nurse’s and the patients’ assessments (38.9 vs 28.9; P P = .001, respectively). Conclusions Laparoscopic cholecystectomy can be safely performed using 10-mm umbilical, 5-mm epigastric, 2-mm subcostal, and 2-mm lateral ports. The use of mini-laparoscopic techniques resulted in decreased early postoperative incisional pain, avoided late incisional discomfort, and produced superior cosmetic results. Although improved instrument durability and better optics are needed for widespread use of miniport techniques, this approach can be routinely offered to many properly selected patients undergoing elective LC.

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC50 = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.

Nucleoside reverse transcriptase inhibitors (NRTIs) represent the cornerstone of highly active antiretroviral therapy when combined with non-nucleoside reverse transcriptase inhibitors (NNRTIs) or HIV-1 protease inhibitors (PIs). Unlike the NNRTIs and PIs, NRTIs must be successively phosphorylated by cellular kinases to a triphosphate form, which represents the active metabolite possessing antiviral activity. Emergence of viral resistance to NRTIs has severely hampered treatment options for persons infected with HIV-1. As such, there is an urgent need to develop NRTIs capable of suppressing NRTI-resistant strains of HIV-1. We have recently reported that the cytidine analogue D-d4FC (DPC817, Reverset™) effectively inhibits clinically prevalent resistant strains of HIV-1. In this report, we have extended these findings and now describe a detailed resistance profile for this novel NRTI. By examining a panel of 50 viruses carrying RTs derived from HIV-1 clinical isolates displaying a wide range of NRTI resistance mutations, we report that the median fold increase in effective antiviral concentration for such a panel of viruses is 3.2, which is comparable to tenofovir (2.8-fold) and didanosine (2.4-fold). D-d4FC is highly effective at inhibiting subsets of lamivudine-and zidovudine-resistant variants but, like other NRTIs, seems less potent against multi-NRTI-resistant viruses, particularly those carrying the Q151M complex of mutations. Finally, in vitro selections for HIV-1 mutants capable of replicating in the presence of D-d4FC yielded a mutant carrying the RT K65R mutation. This mutation confers 5.3- to 8.7-fold resistance to D-d4FC in vitro. These findings suggest that D-d4FC may represent an alternative NRTI for the treatment of individuals infected with lamivudine- and zidovudine-resistant strains of HIV-1.

The backbone of effective highly active antiretroviral therapy regimens for the treatment of HIV infections currently contains at least two nucleosides. Among the features that influence the potency of each component of a regimen and the overall efficacy of the combination are the cellular uptake and bioconversion of nucleoside analogues to their active triphosphate form, and the extent of possible interactions in these steps that might occur when more than one nucleoside is used in a regimen. D-d4FC (Reverset™), a new cytidine analogue with the ability to inhibit many nucleoside-resistant viral variants, was examined for these parameters. In phytohemaglutinin-stimulated human peripheral blood mononuclear cells, D-d4FC was taken up in a rapid (8 h to 50% maximal value), saturable (plateau above 10 μM parent nucleoside concentration) process, resulting in levels of D-d4FC triphosphate that should provide potent antiviral activity against a variety of virus genotypes. Based on measurement of antiviral effects in cell culture, additive and in some cases, synergistic interactions were observed with protease inhibitors, non-nucleoside reverse transcriptase inhibitors or other nucleosides, including cytidine analogues.

Efavirenz (also known as DMP 266 or SUSTIVA) is a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and of HIV-1 replication in vitro and in vivo. Most patients on efavirenz-containing regimens have sustained antiviral responses; however, rebounds in plasma viral load have been observed in some patients in association with the emergence of mutant strains of HIV-1. Virus isolates from the peripheral blood mononuclear cells (PBMCs) of patients with such treatment failures, as well as recombinant viruses incorporating viral sequences derived from patient plasma, show reduced in vitro susceptibility to efavirenz in association with mutations in the RT gene encoding K103N, Y188L, or G190S/E substitutions. Patterns of RT gene mutations and in vitro susceptibility were similar in plasma virus and in viruses isolated from PBMCs. Variant strains of HIV-1 constructed by site-directed mutagenesis confirmed the role of K103N, G190S, and Y188L substitutions in reduced susceptibility to efavirenz. Further, certain secondary mutations (V106I, V108I, Y181C, Y188H, P225H, and F227L) conferred little resistance to efavirenz as single mutations but enhanced the level of resistance of viruses carrying these mutations in combination with K103N or Y188L. Viruses with K103N or Y188L mutations, regardless of the initial selecting nonnucleoside RT inhibitor (NNRTI), exhibited cross-resistance to all of the presently available NNRTIs (efavirenz, nevirapine, and delavirdine). Some virus isolates from nevirapine or delavirdine treatment failures that lacked K103N or Y188L mutations remained susceptible to efavirenz in vitro, although the clinical significance of this finding is presently unclear.

Abstract —Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1–stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1–induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure.

Purpose: The mechanisms whereby restenoses occur at discrete sites within the vasculature remain uncertain. We have recently reported that vascular smooth muscle cells (VSMC) derived from patients with graft stenoses are resistant to growth inhibition by heparin. In this study, we have examined whether VSMC proliferation rates and responses to inhibition by heparin vary according to the individual or the anatomic site of origin. Methods: Long saphenous veins from seven patients were divided into proximal, middle, and distal portions, and VSMC were cultured separately from each. VSMC proliferation in response to 15% fetal calf serum ± 100 μg/ml heparin was measured by counting triplicate samples at 0, 3, 7, 10, and 14 days. This experiment was repeated from the second to the sixth passage ( n = 6) and for artery and vein pairs derived from four additional patients. Results: Differences between vein segment cultures of individual veins were found not to differ significantly from experimental error for either proliferation or heparin inhibition and were not altered by repeated passage (ANOVA). There were, however, significant differences in sensitivity to heparin inhibition between patients ( p = 0.02) (ANOVA). There were no significant differences between paired samples of artery and vein for either proliferation or heparin inhibition (Mann-Whitney test). Conclusions: VSMC growth characteristics reflect the individual patient and are maintained in cell culture. (J VASC SURG 1994;20:482-7.)