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Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues 555 DEE 557 (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2 ΔDEE and CNM-linked DNM2 A618T mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2 WT ). However, DNM2 A618T forms cytoplasmic inclusions at concentrations lower than those of either DNM2 WT or DNM2 ΔDEE , suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.

Transthyretin (TTR) dissociation is the rate limiting step for both aggregation and subunit exchange. Kinetic stabilisers, small molecules that bind to the native tetrameric structure of TTR, slow TTR dissociation and inhibit aggregation. One such stabiliser is the non-steroidal anti-inflammatory drug (NSAID), diflunisal, which has been repurposed to treat TTR polyneuropathy. Previously, we compared the efficacy of diflunisal, tafamidis, tolcapone, and AG10 as kinetic stabilisers for transthyretin. However, we could not meaningfully compare diflunisal because we were unsure of its plasma concentration after long-term oral dosing. Herein, we report the diflunisal plasma concentrations measured by extraction, reversed phase HPLC separation, and fluorescence detection after long-term 250 mg BID oral dosing in two groups: a placebo-controlled diflunisal clinical trial group and an open-label Japanese polyneuropathy treatment cohort. The measured mean diflunisal plasma concentration from both groups was 282.2 μ M ±   143.7 μ M (mean ± standard deviation). Thus, quantification of TTR kinetic stabilisation using subunit exchange was carried out at 100, 200, 300, and 400  μM diflunisal concentrations, all observed in patients after 250 mg BID oral dosing. A 250 μ M diflunisal plasma concentration reduced the wild-type TTR dissociation rate in plasma by 95%, which is sufficient to stop transthyretin aggregation, consistent with the clinical efficacy of diflunisal for ameliorating transthyretin polyneuropathy.

Transthyretin (TTR) tetramer dissociation is rate limiting for aggregation and subunit exchange. Slowing of TTR tetramer dissociation via kinetic stabiliser binding slows cardiomyopathy progression. Quadruplicate subunit exchange comparisons of the drug candidate AG10, and the drugs tolcapone, diflunisal, and tafamidis were carried out at 1, 5, 10, 20 and 30 µM concentrations in 4 distinct pooled wild type TTR (TTRwt) human plasma samples. These experiments reveal that the concentration dependence of the efficacy of each compound at inhibiting TTR dissociation was primarily determined by the ratio between the stabiliser's dissociation constants from TTR and albumin, which competes with TTR to bind kinetic stabilisers. The best stabilisers, tafamidis (80 mg QD), AG10 (800 mg BID), and tolcapone (3 x 100 mg over 12 h), exhibit very similar kinetic stabilisation at the plasma concentrations resulting from these doses. At a 10 µM plasma concentration, AG10 is slightly more potent as a kinetic stabiliser vs. tolcapone and tafamidis (which are similar), which are substantially more potent than diflunisal. Dissociation of TTR can be limited to 10% of its normal rate at concentrations of 5.7 µM AG10, 10.3 µM tolcapone, 12.0 µM tafamidis, and 188 µM diflunisal. The potency similarities revealed by our study suggest that differences in safety, adsorption and metabolism, pharmacokinetics, and tissue distribution become important for kinetic stabiliser clinical use decisions.

Current laboratory methods for comprehensive thrombophilia status require large blood volumes and long turn-around times. We demonstrate the feasibility of performing thrombophilia panel testing of enzymatic functional assays on a microfluidic cartridge using low sample volume.Functional assays for Antithrombin III, Protein C, Factor VIII, and plasminogen were adapted on the digital microfluidic platform by developing novel fluorogenic substrates and establishing on-cartridge fluorescence (360/460 nm) detection. Cartridge vs. microtiter plate results were compared using samples obtained from pediatric patients. Linear regression and Bland-Altman plots were used to establish correlations. Results were not significantly different when performed on-cartridge compared to microtiter plates. Importantly, the sample volume required is significantly lower for all on-cartridge compared to microtiter plate assays (25 μL vs. 2 ml).This study demonstrates the feasibility of thrombophilia panel testing with high-fidelity using small plasma volume. The efficacy of this near-patient technology in clinical settings needs further investigation., (© American Society for Clinical Pathology 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.

As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

Objective: Leptin signaling is important in the establishment of pregnancy. We sought to determine if single nucleotide polymorphisms (SNPs) in the leptin and leptin receptor genes are associated with idiopathic recurrent pregnancy loss (RPL)., Study Design: We conducted a case-control study with cases defined as women with idiopathic RPL and controls as parous women without pregnancy losses. A total of 99 cases and 108 controls were genotyped for the leptin (-2548 G/A) SNP and the leptin receptor A223G SNP. Genotype and allele frequencies were compared between cases and controls using χ(2) test., Result: In this population, there was no significant difference in the genotype or allele frequencies for the leptin (-2548 G/A) or leptin receptor A223G SNPs between women with idiopathic RPL and controls., Conclusion: Although leptin signaling is critical to many aspects of reproduction, the maternal leptin and leptin receptor SNPs evaluated in this study are unlikely to have a clinically meaningful role in RPL.

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

Objective: The objective of the study was to determine whether single-nucleotide polymorphisms (SNPs) that influence the maternal innate immune response are associated with puerperal group A streptococcal sepsis., Study Design: Subjects with confirmed puerperal group A streptococal infection were prospectively identified in 2 tertiary care hospitals over 18 years. Controls were racially matched subjects with term, uncomplicated deliveries. Thirty-eight polymorphisms associated with the innate immune response to bacterial infection were analyzed. Allele and genotype frequencies for subjects and controls were compared., Results: Forty-eight women with puerperal group A streptococcal infection were identified. DNA was obtained for 28 subjects and 54 controls. Allele frequencies were significantly different between subjects and controls for polymorphisms in Toll-like receptor (TLR) 9-1486 (P = .03) and heat shock protein (HSP) 70-2 1267 (P = .003). Genotype frequencies were significantly different between subjects and controls for TLR9-1486 (P = .025), HSP70-2 1267 (P = .02), and interleukin (IL)-1beta-511 (P = .016)., Conclusion: Puerperal group A streptococcal sepsis may be associated with innate immune response gene polymorphisms in TLR9, HSP70-2, and IL1beta., (Copyright 2010 Mosby, Inc. All rights reserved.)

Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity are poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA-binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA-binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins.

Hox genes are crucial for body axis specification during embryonic development. Hoxa11 plays a role in anteroposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximodistal patterning of the limbs. Hoxa11 expression is also observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock-in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in-frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis., (Copyright (c) 2008 Wiley-Liss, Inc.)

A series of structurally novel stearoyl-CoA desaturase1 (SCD1) inhibitors has been identified via molecular scaffold manipulation. Preliminary structure-activity relationship (SAR) studies led to the discovery of potent, and orally bioavailable piperidine-aryl urea-based SCD1 inhibitors. 4-(2-Chlorophenoxy)-N-[3-(methyl carbamoyl)phenyl]piperidine-1-carboxamide 4c exhibited robust in vivo activity with dose-dependent desaturation index lowering effects.

C-Jun NH2 terminal kinases (JNKs) are important cell signaling enzymes. JNK1 plays a central role in linking obesity and insulin resistance. JNK2 and JNK3 may be involved in inflammatory and neurological disorders, respectively. Small-molecule JNK inhibitors could be valuable tools to study the therapeutic benefits of inhibiting these enzymes and as leads for potential drugs targeting JNKs. In this report, we disclose a series of potent and highly selective JNK inhibitors with good pharmacokinetic profiles.

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.

Ghrelin, a gut-derived orexigenic hormone, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Centrally administered ghrelin has been shown to cause hunger and increase food intake in rodents. Inhibition of ghrelin actions with ghrelin antibody, peptidyl GHS-R antagonists, and antisense oligonucleosides resulted in weight loss and food intake decrease in rodents. Here we report the effects of GHS-R antagonists, some of which were potent, selective, and orally bioavailable. A structure-activity relationship study led to the discovery of 8a, which was effective in decreasing food intake and body weight in several acute rat studies.

The synthesis and structure-activity relationships of the 4- and 6-substituents of 2,4-diaminopyrimidine-based growth hormone secretagogue receptor (GHS-R) antagonists are described. Diaminopyrimidines with 6-norbornenyl (4n) and 6-tetrahydrofuranyl (4p) substitutents were found to exhibit potent GHS-R antagonism and good selectivity (approximately 1000-fold) against dihydrofolate reductase.

The structure-activity relationship studies on a series of tetralin carboxamide growth hormone secretagogue receptor (GHS-R) antagonists are discussed. It was found that certain 2-alkoxycarbonylamino substituted tetralin carboxamides are potent, selective, and orally bioavailable GHS-R antagonists.

Farnesyltransferase inhibitors (FTIs) have been developed as potential anti-cancer agents due to their efficacy in blocking malignant growth in a variety of murine models of human tumors. To that end, we have developed a series of pyridone farnesyltransferase inhibitors with potent in vitro and cellular activity. The synthesis, SAR and biological properties of these compounds will be discussed.

Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered a series of aryl tetrahydropyridines that incorporate substituted glycine, phenylalanine and histidine residues. The design, synthesis, SAR and biological properties of these compounds will be discussed.

Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered bioavailable aryl tetrahydropyridines that are potent in cell culture. The design, synthesis, SAR and biological properties of these compounds will be discussed.

Purpose: We designed a study to determine whether increases in p53 protein in primary carcinomas of the colon or rectum correlate with overall survival. Mutations of the tumor suppressor gene p53 are detectable by immunocytochemical methods in colorectal cancers because of accumulation of nuclear p53 protein., Methods: IgG1 monoclonal antibody to human p53 protein (PAb 1801) was used to detect p53 in formalin-fixed, paraffin-embedded archival tumors resected from 84 patients with tumor limited to the bowel wall. A multivariate analysis was performed using five prognostic pathobiologic variables compared with the level of staining of the p53 product., Results: Nuclear p53 protein was observed in 52 (62 percent) of 84 colorectal cancer patients with Stage T2 or T3, N0, M0 disease. Patients with strong expression (3+ and 4+) of p53 appeared to die from their disease sooner than those with weak expression (1+ and 2+), although this was not statistically significant (P > 0.59). Thirty-two patients did not express nuclear p53 by immunocytochemical methods. When these patients were analyzed in combination with the strong p53 expressors, the trend toward decreased survival increased (P > 0.15)., Conclusions: This data suggest that lack of p53 expression may also predict an adverse outcome in colorectal cancer. However, before the immunocytochemical method can be used clinically as a prognostic indicator, the colorectal cancer patients with zero expression should be studied further to clarify the functional status of p53 in their tumors.

Local recurrences in the surgical bed after tumor resection may be due to residual tumor cells "dropping" into the wound. Irrigation with water is often used to remove these cells. We designed experiments to determine whether irrigation would prevent tumor recurrence. Surgical wounds of uniform size in C57BL/6 mice were seeded with 5 x 10(2), 5 x 10(3), 5 x 10(4), 5 x 10(5), or 5 x 10(6) viable syngeneic B16-F10 melanoma cells to test the hypothesis that irrigation with water would decrease local tumor recurrence. The tumor-contaminated wounds were irrigated with distilled water or with saline (0.9% NaCl) immediately or 5, 30, 60, 120, or 240 min after seeding. Control wounds were seeded but not irrigated. The technique of irrigation was altered in a second group of experiments such that the amount of time the tumor cells were exposed to the water or saline was 5, 10, or 15 min. To determine the rapidity and durability of tumor cell attachment to host tissue, 1 x 10(4) viable B16-F10 tumor cells were seeded in vitro onto freshly cut disks of syngeneic mouse dermis. The tissue was irrigated with saline or distilled water 0, 2, 5, 10, 15, 30, 60, 120, or 240 min later. Tumor growth was observed in all the mice and neither the mechanical action of irrigation nor the hypotonic effect of distilled water changed the rate of growth. Scanning electron microscopy (SEM) demonstrated stable and firm attachment to mouse tissue within seconds of seeding with no noticeable dislodgement or cytotoxicity by either saline or water irrigation. The data suggest that the commonly used technique of irrigating the bed of the resected tumor may not be of value in preventing local recurrences.

A malignant tumor that arose spontaneously in the subcutaneous tissue of the back of a C57BL/6 female mouse was found to metastasize spontaneously to the liver. The primary and metastatic tumors, SML (spontaneous metastasis to the liver) 1 and SML 2, were established in vitro in long-term cell suspension culture and were passaged 10 times in vivo for 18 months. When 100,000 cells were injected subcutaneously in the orthotopic position, tumor growth appeared in 60% of the SML 1 mice and 100% of the SML 2 mice. SML 1 did not grow when injected in the footpad, while SML 2 did. The median survival was 47 days for SML 1 and 48.5 days for SML 2 (P = 0.013). The pattern of metastasis was similar for both tumor cell lines, irrespective of intravenous or subcutaneous injection routes. Spontaneous metastasis of the SML 2 line occurred from both the orthotopic and heterotopic sites, while the SML 1 metastasized spontaneously from the orthotopic site only. Liver metastasis appeared in > 90% of the mice for both SML 1 and SML 2. Metastasis to the spleen occurred in about half the mice. Other sites of metastasis were the ovaries (36% and 52%, respectively, for SML 1 and SML 2), the kidneys (approximately 15%) and the small bowel (very rarely). Metastasis to the lungs did not occur except very rarely in the later passages of the SML 2 line. Histologic, immunohistochemical and electron microscopic studies showed a histiocytic tumor with macrophage characteristics. The cells exhibited chemotaxis toward liver extracellular matrix and reduced motility toward collagen IV, laminin and fibronectin compared to the B16-F10 melanoma line. This spontaneously occurring tumor should prove useful for the study of organ-specific metastasis to the liver.