Coupling between spontaneous pupillary fluctuations and brain activity relates to inattentiveness.

Autonomic activity in neurological and psychiatric disorders is often dysregulated, particularly in the context of attentional behaviors. This suggests that interplay between the autonomic nervous system and aspects of the central nervous system subserving attention may be disrupted in these conditions. Better understanding these interactions and their relationship with individual variation in attentional behaviors could facilitate development of mechanistic biomarkers. We identified brain regions defined by trait-sensitive central-autonomic coupling as a first step in this process. As spontaneous neural activity measured during the resting state is sensitive to phenotypic variability, unconfounded by task performance, we examined whether spontaneous fluctuations in brain activity and an autonomic measure, pupil diameter, were coupled during the resting state, and whether that coupling predicted individual differences in attentional behavior. By employing concurrent pupillometry and fMRI during the resting state, we observed positive coupling in regions comprising cingulo-opercular, default mode, and fronto-parietal networks, as well as negative coupling with visual and sensorimotor regions. Individuals less prone to distractibility in everyday behavior demonstrated stronger positive coupling in cingulo-opercular regions often associated with sympathetic activity. Overall, our results suggest that individuals less prone to distractibility have tighter intrinsic coordination between specific brain areas and autonomic systems, which may enable adaptive autonomic shifts in response to salient environmental cues. These results suggest that incorporating autonomic indices in resting-state studies should be useful in the search for biomarkers for neurological and psychiatric disorders.

A CCL24-dependent pathway augments eosinophilic airway inflammation in house dust mite-challenged Cd163(-/-) mice.

CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell-surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163(-/-) mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163(-/-) mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides pteronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163(-/-) mice had the same phenotype as HDM-challenged Cd163(-/-) mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163(-/-) mice, but not BMMΦs from wild-type (WT) mice. Finally, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ's from Cd163(-/-) mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163(-/-) mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.

Detrusor myotomy: a 5-year review in unstable and non-compliant bladders.

OBJECTIVE: To identify urodynamic factors that might determine the clinical outcome of detrusor myotomy in incontinent children. PATIENTS AND METHODS: Six girls and three boys (aged 5-14 years) underwent detrusor myotomy for severe urinary incontinence. Seven children had spina bifida, one had traumatic paraplegia and one had low bladder compliance. The patients were followed for a minimum of 5 years. RESULTS: Urodynamic studies before surgery showed that three patients had normal compliance with grossly unstable detrusor contractions, and six had low bladder compliance with few phasic detrusor contractions. Detrusor leak-point pressures were > 40 cmH2O in five patients and < 40 cmH2O in four. Only two patients, both with grossly unstable detrusor contractions and leak-point pressures of > 40 cmH2O, had a successful 5-year outcome. The other seven patients remained incontinent; six underwent further surgery and one died from unrelated causes. CONCLUSION: Detrusor myotomy appears to have the best outcome in those patients with marked phasic unstable detrusor contractions with a competent urethral sphincter. In this group it may have distinct advantages over more commonly used procedures.

Preclinical evaluation of the penciclovir analog 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET.

UNLABELLED: The gene for herpes simplex virus thymidine kinase (HSV-tk) is widely used as a suicide gene in experimental gene therapy of cancer. 9-(4-Fluoro-3-hydroxymethylbutyl)guanine (FHBG) is an antiviral nucleoside analog that is rapidly phosphorylated by viral thymidine kinase but is a poor substrate for mammalian thymidine kinase. Recently, FHBG labeled in the 4-fluoro position with (18)F has shown promise relative to other similar compounds for imaging in vivo expression of HSV-tk using PET. In this study, we evaluated the uptake of [(18)F]FHBG in vitro and in vivo using transduced and wild-type human colon cancer cells (HT-29). We also imaged [(18)F]FHBG and measured the radioactivity concentrations of circulating [(18)F]FHBG and its metabolites in monkeys. METHODS: Sterile, pyrogen-free [(18)F]FHBG was produced routinely in good yields. Cells were transduced with the retroviral vector G1Tk1SvNa containing HSV-tk gene. In vitro uptake studies were performed by incubating cells with [(18)F]FHBG at 37 degrees C for 1 and 5 h. Biodistribution studies were performed at 2 and 5 h after injection in nude mice bearing tumors grown from wild-type or transduced cells. Sequential, whole-body PET scans of cynomolgus monkeys were obtained over a period of >2 h after intravenous injection of [(18)F]FHBG. Arterial plasma samples obtained from monkeys 15-120 min after intravenous injection were subjected to acid extraction, and the acid-soluble fractions were analyzed by high-performance liquid chromatography. RESULTS: In vitro studies showed 31 and 71 (P < 0.001) times higher uptake of the probe at 1 and 5 h, respectively, in transduced cells compared with nontransduced cells. In vivo studies in mice showed that tumor uptake of the radiotracer was 4-fold (P < 0.05) and 13-fold (P < 0.001) higher at 2 and 5 h, respectively, in tumors grown from transduced cells compared with control cells. Transduced tumor-to-normal tissue ratios ranged from 2 to 25 at 2 h and from 2 to 22 at 5 h. Recirculating labeled metabolites had only a minor effect on the biodistribution of radiolabel from [(18)F]FHBG in monkeys. CONCLUSION: These results indicate that [(18)F]FHBG may yield high-contrast PET images of HSV-tk expression in tumors and, therefore, it is a very promising radiotracer for monitoring of gene therapy of cancer with PET.

Retroviral vectors bearing IgG-binding motifs for antibody-mediated targeting of vascular endothelial growth factor receptors.

Targeting retroviral vectors to tumor vasculature is an important goal of cancer gene therapy. In this study, we report a novel targeting approach wherein IgG-binding peptides were inserted into the Moloney murine leukemia virus (MuLV) envelope (env) protein. The modifications on the viral env included replacement of the entire receptor binding region of the viral env with protein A (or ZZ) domains. The truncated env incorporating IgG-binding motifs (known as proteins) provided the targeting function, while the co-expressed wild-type (WT) env protein enabled viral fusion and cell entry. An anti-human VEGF receptor (Flk-1/KDR) antibody served as a molecular bridge, directing the retroviral vector to the endothelial cell. Hence, the IgG-targeted vectors bound to the Flk-1/KDR antibody which in turn bound to VEGF receptors on Kaposi sarcoma, KSY1, endothelial cells. The net effect was increased viral fusion and infectivity of IgG-bound retroviral vectors when compared to non-targeted vectors bearing WT env alone. These data provide the proof of concept that IgG-binding vector/VEGF receptor antibody complexes may be used to enhance retroviral gene delivery to activated endothelial cells.

Lesion-targeted injectable vectors for vascular restenosis.

Pathologic lesions caused by catheter-based revascularization procedures for occlusive artery disease include disruption of the endothelium, exposure of extracellular matrix (ECM) proteins, and proliferation of vascular smooth muscle cells, which lead to neointima formation and restenosis. We have developed matrix-collagen-targeted retroviral vectors that are able to accumulate at sites of vascular injury (Hall et al., Hum. Gene Ther. 1997;8:2183-2192; Hall et al., Hum. Gene Ther. 2000;11:983-993). The primary tissue-targeting motif, adapted from the physiological surveillance sequence found in von Willebrand factor, served to localize and concentrate the vector within vascular lesions. In the present study, we evaluated the efficiency of this vector-targeting system in rats with nonligated balloon-injured carotid arteries. Both intraarterial (by retrograde femoral artery catheterization) and intravenous (via femoral vein) injection of a matrix-targeted vector enhanced transduction of neointimal cells ( approximately 20%) at severely denuded areas when compared with the nontargeted vector (<1%). Further, intraarterial instillation of a matrix-targeted, but not a nontargeted, vector bearing an antisense cyclin G1 construct inhibited neointima lesion formation in the injured carotid arteries. Taken together, these data indicate that strategic targeting of retroviral vectors to vascular lesions would have therapeutic potential in the management of vascular restenosis and many other disorders of uncontrolled proliferation where endothelial disruption, ECM remodeling, and collagen deposition form the nexus for preferential vector localization and concentration in vivo.

Long term inhibition of neointima formation in balloon-injured rat arteries by intraluminal instillation of a matrix-targeted retroviral vector bearing a cytocidal mutant cyclin G1 construct.

Restenosis from neointimal proliferation is a frequent complication of intracoronary stenting and catheter-based revascularization procedures. Currently, there is no known therapeutic strategy that has been sufficiently effective to warrant its widespread use. In the present study, the anti-proliferative properties of a matrix (collagen)-targeted retroviral vector bearing a mutant cyclin G1 (DNT 41-249) construct was evaluated in vitro and in vivo. In controlled one-month efficacy studies, the intraluminal instillation of the mutant cyclin G1 vector significantly inhibited neointima lesion formation in balloon-injured rat arteries without neointimal growth, associated necrosis or intense inflammatory reaction. Taken together, these data extend the potential utility of the matrix-targeted mutant cyclin G1 retroviral vector for management of vascular restenosis.

Phenotypic differentiation of TGF-beta1-responsive pluripotent premesenchymal prehematopoietic progenitor (P4 stem) cells from murine bone marrow.

On the horizon of modern molecular medicine is the requisite technology to capture multipotent human stem cells that are capable of self-renewal and to direct these stem cells along defined lineages for therapeutic purposes. In this article, we describe the hematopoietic and mesenchymal differentiation potential of a unique population of transforming growth factor-beta1 (TGF-beta1)-responsive stem cells derived from murine bone marrow. Stringent selection of the stem cells was accomplished under low serum conditions by virtue of an inherent survival response to a TGF-beta1-vWF fusion protein that was bound to collagen matrices. The TGF-beta1-responsive stem cells initially exhibited a non-adherent and uniformly blastoid morphology, underwent expansion into colonies upon serum reconstitution, and were capable of overt cytodifferentiation along fibrogenic, osteogenic, chondrogenic, or adipogenic lineages upon growth factor stimulation. Remarkably, these stem cells also underwent rapid expansion in the presence of either hematopoietic stem cell factor (SCF) or interleukin3 (IL-3), and differentiated into myeloid and lymphoid phenotypes upon exposure to the latter. Taken together, these results support the hypothesis that pluripotent premesenchymal prehematopoietic progenitor cells, designated P4 stem cells, are present postnatally in murine bone marrow and, thus, may be summarily isolated for various cell-based experimental therapies.

Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice.

Targeting cytocidal vectors to tumors and associated vasculature in vivo is a long-standing goal of human gene therapy. In the present study, we demonstrated that intravenous infusion of a matrix (i.e., collagen)-targeted retroviral vector provided efficacious gene delivery of a cytocidal mutant cyclin G1 construct (designated Mx-dnG1) in human cancer xenografts in nude mice. A nontargeted CAE-dnG1 vector (p = 0.014), a control matrix-targeted vector bearing a marker gene (Mx-nBg; p = 0.004), and PBS served as controls (p = 0.001). Enhanced vector penetration and transduction of tumor nodules (35.7 +/- 1.4%, mean +/- SD) correlated with therapeutic efficacy without associated toxicity. Kaplan-Meier survival studies were conducted in mice treated with PBS placebo, the nontargeted CAE-dnG1 vector, and the matrix-targeted Mx-dnG1 vector. Using the Tarone log-rank test, the overall p value for comparing all three groups simultaneously was 0.003, with a trend that was significant to a level of 0.004, indicating that the probability of long-term control of tumor growth was significantly greater with the matrix-targeted Mx-dnG1 vector than with the nontargeted CAE-dnG1 vector or PBS placebo. The present study demonstrates that a matrix-targeted retroviral vector deployed by peripheral vein injection (1) accumulated in angiogenic tumor vasculature within 1 hr, (2) transduced tumor cells with high-level efficiency, and (3) enhanced therapeutic gene delivery and long-term efficacy without eliciting appreciable toxicity.

Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis.

In the present study, the mature epidermal growth factor (EGF) protein was engineered to incorporate a high affinity collagen-binding domain (CBD) derived from co-agulation von Willebrand factor, to specifically target EGF to colonic lesions. The fusion protein was expressed in an E. coli bacterial expression system, purified by metal chelate chromatography, and renatured by oxidative refolding into a soluble biologically active growth factor. The EGF-CBD fusion protein bound tightly to collagen matrices under conditions in which native non-targeted EGF was washed away. In biologic assays, the EGF-CBD fusion protein stimulated NIH3T3 cell proliferation with near wild-type biological activity. In vivo binding studies showed that the collagen-targeted EGF, but not the non-targeted EGF, accumulated at areas of exposed collagen on the luminal surface of the inflamed colon. Finally, a single colonic instillation of the collagen-targeted EGF-induced a more rapid regeneration of intestinal crypts 24 h after treatment (no. of crypts = 89.2+/-8.1) compared to the non-targeted EGF (no. of crypts = 52.2+/-29.8; p=0.027), and the PBS control (no. of crypts = 24. 0+/-22.9; p=0.001). Taken together, these findings indicate that intracolonic delivery of collagen-targeted EGF represents a potentially effective therapeutic strategy for acute or chronic inflammatory bowel disease.

Characterization of differential gene expression in monkey arterial neointima following balloon catheter injury.

Vaso-occlusive sequelae following percutaneous transluminal coronary angioplasty (PTCA), including smooth muscle cell migration, proliferation, and attendant extracellular matrix production, often results in restenosis of the treated artery. To further understand the molecular mechanisms governing progressive intimal hyperplasia, we performed a molecular screen using differential display PCR on total RNA prepared from injured and normal carotid arterial segments to identify a subset of differentially expressed genes at t=7 days post-balloon catheter injury in a non-human primate. DNA sequence analysis of selected differentially expressed RNA by this procedure using 240 combinations of random primer pairs yielded 41 distinct cDNA sequences: 22 of which have significant sequence homology to previously identified meta-zoan genes, 15 GEMS (genes expressed in monkey neointima), and 4 GSMS (genes suppressed in monkey neointima) that have little homology to reported sequences. Among the up-regulated homologues include i) secreted growth regulatory factors, ii) membrane receptors, iii) transcription factors, iv) cell adhesion molecules, and v) extracellular matrix proteins; some of which have not been previously linked to vascular restenosis. In particular, Cyr61, a known angiogenesis inducer, was found to be highly expressed in the neointima lesion of the balloon-injured monkey artery. This finding provides the first links of Cyr61 to the pathogenesis of vascular restenosis, and identifies a novel locus for potential therapeutic intervention. These studies identified a number of known and unknown genes, whose up- or down-regulated expression during the proliferative phase of vascular restenosis makes them potential targets for therapeutic intervention.

Site-directed ligand discovery.

We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

Inhibition of metastatic tumor growth in nude mice by portal vein infusions of matrix-targeted retroviral vectors bearing a cytocidal cyclin G1 construct.

Tumor invasion and associated angiogenesis evoke a remodeling of extracellular matrix components. Retroviral vectors bearing auxiliary matrix-targeting motifs (ie., collagen-binding polypeptides) accumulate at sites of newly exposed collagen, thus promoting tumor site-specific gene delivery. In this study, we assessed the antitumor effects of serial portal vein infusions of matrix-targeted vectors bearing a mutant cyclin G1 (dnG1) construct in a nude mouse model of liver metastasis. The size of tumor foci was dramatically reduced in dnG1 vector-treated mice compared with that in control vector- or PBS-treated animals (P = 0.0002). These findings represent a definitive advance in the development of targeted injectable vectors for metastatic cancer.

Incorporation of tumor vasculature targeting motifs into moloney murine leukemia virus env escort proteins enhances retrovirus binding and transduction of human endothelial cells.

Adhesion receptors expressed on the surfaces of tumor-activated endothelial cells provide an advantageous locus for targeting gene therapy vectors to angiogenic tissues and/or tumor vasculature. In this study, we engineered a series of Asn-Gly-Arg (NGR)-containing congeners of the presumptive cell binding motif contained within the ninth type III repeat of fibronectin and displayed these tumor vasculature targeting motifs (TVTMs) within the context of Moloney murine leukemia envelope "escort" proteins. Comparative studies of envelope incorporation into viral particles and evaluation of the cell binding properties of the targeted vectors revealed critical structural features, thus identifying a subset of optimal TVTMs. Utilizing a modified ELISA to evaluate viral binding to target cells, we observed a significant down-regulation of TVTM-virion binding to human endothelial cells following sustained (48-h) exposure to VEGF. Normalized for equivalent titers (10(6) CFU/ml), as assayed on NIH 3T3 cells, vectors displaying TVTM escort proteins significantly enhanced the transduction efficiency from 12.2 to 37.4% in human KSY-1 endothelial cell cultures (P < 0.001) and from 0.4 to 4.1% in human umbilical vein endothelial cell (HUVEC) cultures (P < 0.001). In summary, these studies utilized an engineering approach to identify a subset of TVTMs that are stably incorporated as envelope "escort" proteins into retroviral vectors and that, by functioning to improve the binding efficiency and transduction of both HUVEC and KSY1 endothelial cells, may have therapeutic potential for targeting gene delivery to the tumor-associated vasculature.

Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor.

A major obstacle that limits the potential of human gene therapy is the inefficiency of gene delivery to appropriate sites in vivo. Previous studies demonstrated that the physiological surveillance function performed by von Willebrand factor (vWF) could be incorporated into retroviral vectors by molecular engineering of the MuLV ecotropic envelope (Env) protein. To advance the application of vWF targeting technology beyond laboratory animals, we prepared an extensive series of Env proteins bearing modified vWF-derived matrix-binding sequences and assembled these chimeric proteins into targeted vectors that are capable of transducing human cells. Initially, a dual envelope configuration was utilized, which required coexpression of a wild-type amphotropic Env. Subsequently, streamlined "escort" Env proteins were constructed wherein the inoperative receptor-binding domain of the targeting partner was replaced by the vWF-derived collagen-binding motif. Ultimately, an optimal construct was developed that exhibited properties of both extracellular matrix (ECM)-targeting and near wild-type amphotropic infectivity, and could be arrayed as a single envelope on a retroviral particle. On intraarterial instillation, enhanced focal transduction of neointimal cells (approximately 20%) was demonstrated in a rat model of balloon angioplasty. Moreover, transduction of tumor foci (approximately 1-3%) was detected after portal vein infusion of a matrix-targeted vector in a nude mouse model of liver metastasis. We conclude that the unique properties of these targeted injectable retroviral vectors would be suitable for improving therapeutic gene delivery in numerous clinical applications, including vascular restenosis, laser and other surgical procedures, orthopedic injuries, wound healing, ischemia, arthritis, inflammatory disease, and metastatic cancer.

Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs.

The aim of this study is to evaluate the potential of gene transfer of cell cycle control genes as treatment of corneal haze or secondary cataract formation. The guiding hypothesis is that strategic modulation of the cyclin G1 or MAT1 gene by retrovirus-mediated gene transfer will inhibit proliferation of rabbit keratocytes (RabK) and fetal human lens epithelial (FHLEpi) cells in vitro. RabK and FHLEpi cell cultures were transduced in triplicate with retroviral vectors bearing either a nuclear-targeted beta-galactosidase, an antisense cyclin G1 (aG1), an antisense MAT1 (aMAT1) construct, or the neo(r) gene. The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while cyclin G1 and MAT1 protein expression levels were evaluated by Western analysis. Proliferation of RabKs and FHLEpi cells was analyzed by counting the number of cells in the aG1 and aMAT1 vector-transduced cultures over 5 days. The mean transduction efficiency was 34.4% (SD 1.41) for RabKs and 19.7% (SD 1.83) for FHLEpi cells. Downregulation of cyclin G1 and MAT1 protein expression was noted 24 hr after transduction of RabK cultures with the respective vectors. Cytostatic effects of the aG1 and aMAT1 vectors in both RabKs and FHLEpi cells were most pronounced on the fifth day (RabKs, p < 0.0007; FHEpi cells, p < 0.001). An increased incidence of apoptosis was identified in both aG1 and MAT1-transduced FHLEpi cells. Taken together, these data suggest the potential utility of developing aG1 and aMAT1 retroviral vectors in gene therapy protocols for corneal haze and secondary cataract formation.

Evaluation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]-FHPG) in vitro and in vivo as a probe for PET imaging of gene incorporation and expression in tumors.

Preparation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) for clinical use, and its evaluation as a positron emission tomography (PET) imaging agent for gene incorporation and expression in tumors are reported. In vitro studies in human colon cancer cells, HT-29, transduced with the retroviral vector G1Tk1SvNa and nontransduced (wild type) showed 4, 8, 12, and 15 times higher uptake of the probe in 1, 3, 5, and 7 h, respectively, in transduced cells compared with the controls. In vivo studies in tumor-bearing nude mice demonstrated that the tumor uptake of the radiotracer is three and six-fold higher in 2 and 5 h, respectively, in transduced cells compared with the control cells. These results suggest that [18F]-FHPG is a potential in vivo PET imaging agent for monitoring gene incorporation and expression in gene therapy of cancer.