Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

The effectiveness of workplace smoking cessation programmes: a meta-analysis of recent studies.

OBJECTIVE: Using meta-analytic procedures, we compare the effectiveness of recent controlled trials of worksite smoking cessation during the 1990s with a previous meta-analysis of programmes conducted in the 1980s. DATA SOURCES: ABI/Inform, BRS, CHID, Dissertation Abstracts International, ERIC, Medline, Occupational Health and Safety Database, PsycInfo, Smoking and Health Database, SSCI, and Sociological Abstracts. STUDY SELECTION: Controlled smoking cessation interventions at the workplace with at least six months follow up published from 1989 to 2001 and reporting quit rates (QRs). DATA EXTRACTION: Two reviewers independently scanned titles/abstracts of relevant reports, and we reached consensus regarding inclusion/exclusion of the full text reports by negotiation. A third reviewer resolved disagreements. Two reviewers extracted data according to a coding manual. Consensus was again reached through negotiation and the use of a third reviewer. DATA SYNTHESIS: 19 journal articles were found reporting studies conforming to the study's inclusion criteria. Interventions included self help manuals, physician advice, health education, cessation groups, incentives, and competitions. A total of 4960 control subjects were compared with 4618 intervention subjects. The adjusted random effects odds ratio was 2.03 (95% confidence interval 1.42 to 2.90) at six months follow up, 1.56 (95% CI 1.17 to 2.07) at 12 months, and 1.33 (95% CI 0.95 to 1.87) at more than 12 months follow up. Funnel plots were consistent with strong publication bias at the first two follow ups but not the third. In Fisher et al's 1990 study, the corresponding ORs were 1.18, 1.66, and 1.18. CONCLUSIONS: Smoking cessation interventions at the worksite showed initial effectiveness, but the effect seemed to decrease over time and was not present beyond 12 months. Compared to the Fisher (1990) analysis, the effectiveness was higher for the six month follow up. Disappointingly, we found methodological inadequacies and insufficient reporting of key variables that were similar to those found in the earlier meta-analysis. This prevented us from determining much about the most effective components of interventions. It is advisable for researchers conducting studies in the future to report data on attrition and retention rates of participants who quit, because these variables can affect QRs.

AAV-encoded expression of TRAIL in experimental human colorectal cancer leads to tumor regression.

Gene transfer vectors based on the adeno-associated virus (AAV) are used for various experimental and clinical therapeutic approaches. In the present study, we demonstrate the utility of rAAV as a tumoricidal agent in human colorectal cancer. We constructed an rAAV vector that expresses tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) and used it to transduce human colorectal cancer cells. TRAIL belongs to the TNF superfamily of cytokines that are involved in various immune responses and apoptotic processes. It has been shown to induce cell death specifically in cancer cells. Transduction with AAV.TRAIL gave rise to rapid expression of TRAIL, followed by induction of apoptosis, which could be inhibited by the caspase inhibitor z-VAD.fmk, in several human colon cancer cell lines. The apoptotic mechanism included activation of caspase-3, as well as cytochrome c release from mitochondria. The outgrowth of human colorectal tumors grown in mice was completely blocked by transduction with AAV.TRAIL in vitro, while in vivo transduction significantly inhibited the growth of established tumors. AAV vectors could provide a safe method of gene delivery and offer a novel method of using TRAIL as a therapeutic protein.

Long-term protection of retinal structure but not function using RAAV.CNTF in animal models of retinitis pigmentosa.

The present study aimed to determine whether intravitreal administration of an adeno-associated virus (AAV) carrying ciliary neurotrophic factor (CNTF) can achieve long-term morphological and physiological rescue of photoreceptors in animal models of retinitis pigmentosa, and whether injection of this virus after degeneration begins is effective in protecting the remaining photoreceptors. We injected rAAV.CNTF.GFP intravitreally in early postnatal Prph2(Rd2/Rd2) (formerly rds/rds) mice and in adult P23H and S334ter rhodopsin transgenic rats. Contralateral eyes received an intravitreal injection of rAAV.GFP or a sham injection. We evaluated the eyes at 6 months (rats) and 8.5 to 9 months (mice) postinfection and looked for histological and electoretinographic (ERG) evidence of photoreceptor rescue and CNTF-GFP expression. Intravitreal administration of rAAV resulted in efficient transduction of retinal ganglion cells in the Prph2(Rd2/Rd2) retina, and ganglion, Muller, and horizontal/amacrine cells in the mutant rat retinas. Transgene expression localized to the retinal region closest to the injection site. We observed prominent morphological protection of photoreceptors in the eyes of all animals receiving rAAV.CNTF.GFP. We found the greatest protection in regions most distant from the CNTF-GFP-expressing cells. The Prph2(Rd2/Rd2) ERGs did not exhibit interocular differences. Eyes of the rat models administered rAAV.CNTF.GFP had lower ERG amplitudes than those receiving rAAV.GFP. The discordance of functional and structural results, especially in the rat models, points to the need for a greater understanding of the mechanism of action of CNTF before human application can be considered.

An evaluation of the effects of Tai Chi exercise on physical function among older persons: a randomized contolled trial.

This study was designed to determine whether a 6-month Tai Chi exercise program can improve self-reported physical functioning limitations among healthy, physically inactive older individuals. Ninety-four community residents ages 65 to 96 (Mage = 72.8 years, SD = 5.1) volunteered to participate in the study. Participants were randomly assigned to either a 6-month experimental (Tai Chi) group (n = 49), which exercised twice per week for 60 min, or a wait-list control group (n = 45). A 6-item self-report physical functioning scale, assessing the extent of behavioral dysfunction caused by health problems, was used to evaluate change in physical functioning limitations as a result of Tai Chi intervention. Results indicated that compared to the control group, participants in the Tai Chi group experienced significant improvements in all aspects of physical functioning over the course of the 6-month intervention. Overall, the experimental group had 65% improvement across all 6 functional status measures ranging from daily activities such as walking and lifting to moderate-vigorous activities such as running. It was concluded that the 6-month Tai Chi exercise program was effective for improving functional status in healthy, physically inactive older adults. A self-paced and self-controlled activity such as Tai Chi has thepotential to be an effective, low-cost means of improving functional status in older persons.

AAV-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse.

Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. The present study aimed to test whether adeno-associated virus (AAV)-mediated delivery of the gene encoding CNTF delays photoreceptor death in the rhodopsin knockout (opsin(-/-)) mouse, an animal model of RP. The vector was made to express a secretable form of CNTF in tandem with a marker GFP. Cultured 293 cells transduced with this virus expressed both CNTF and GFP. The conditioned media from such cells supported the survival of chick dorsal root ganglion neurons in the same manner as recombinant CNTF. Subretinal administration of this virus led to efficient transduction of photoreceptors as indicated by GFP fluorescence and CNTF immunostaining. Histologic examination showed significant photoreceptor preservation in the injected quadrant of the retina. This protection lasted through termination of the experiment (3 months). AAV-mediated delivery of CNTF may have implications for the treatment of human retinal degeneration.

Sub-ppt detection limits for copper ions with Gly-Gly-His modified electrodes.

An electrochemical metal ion sensor has been developed with a detection limit of less than 0.2 ppt by the covalent attachment of the tripeptide Gly-Gly-His as a recognition element to a 3-mercaptopropionic acid modified gold electrode.

Tai Chi, self-efficacy, and physical function in the elderly.

Using Tai Chi as an exercise mode, this study examined the association between self-efficacy and physical function. Ninety-four healthy, physically inactive older adults (M age = 72.8 years, SD = 5.1) were randomly assigned to either a 6-month, twice a week, Tai Chi condition or a wait-list control condition. Outcome variables included self-reports of movement efficacy and physical function assessed at baseline, middle, and termination of the study. Multisample latent curve analyses revealed a significant rate of change attributable to the Tai Chi intervention in both self-efficacy and physical function, with participants experiencing significant improvements over the course of the intervention. Analyses also showed a positive association between self-efficacy and physical function, indicating that improvements in older adults' self-efficacy of movement as a function of Tai Chi were related to increased levels of perceived physical capability. This study uncovered the need for further exploration of the relationship between exercise self-efficacy and physical function for enhancing health-related quality of life in older adults.

Induction of circular episomes during rescue and replication of adeno-associated virus in experimental models of virus latency.

The synthesis of linear duplex replicative structures (monomers, head-to-head, and tail-to-tail dimers) is an important hallmark of the productive phase of the adeno-associated virus (AAV) life cycle. These structures are generated by a strand-displacement replication mechanism and believed to be a reservoir for single-stranded DNA genomes. During the course of studies with recombinant versions of AAV (rAAV), we discovered the assembly of circular duplex provirus derivatives in latently infected cell lines under conditions permissive for replication (i.e., helper virus dependent). These novel structures were cloned by bacterial trapping revealing a markedly homogeneous structure that included a single copy of the rAAV genome joined head-to-tail about the inverted terminal repeats (ITR). Restriction and sequence analysis of the point of circularization revealed a so-called "TRT" domain, consisting of a single ITR hairpin palindrome flanked by 5' and 3' D sequence elements. The circular conformation was additionally characterized by Southern blotting and confirmed by purification on an ethidium bromide-CsCl gradient where the buoyant density was consistent with circular supercoiled DNA. These findings suggest that AAV replication is accompanied by the assembly of circular duplex structures.

The effect of wrapping scarred nerves with autogenous vein graft to treat recurrent chronic nerve compression.

The purpose of this study was to determine the efficacy of vein wrapping of scarred nerves in a chronic nerve compression model in rats. The ultimate goal was to provide experimental evidence for application of the technique of vein wrapping of nerves for the treatment of recurrent compressive neuropathy. The chronic nerve compression model was created in 100 rats. After 8 months the nerves were decompressed. In 50 rats the nerves were wrapped with an opened femoral vein graft; the remaining 50 animals served as controls. The sciatic nerves of both groups were evaluated at 4, 8, 12, 24, and 48 weeks after surgery. Functionally, the sciatic nerves in the vein-wrapped group showed greater improvement than those in the non-vein-wrapped group. For electrophysiologic testing the latency was significantly shorter in the vein-wrapped group. Histologic evaluation showed marked nerve degeneration and scar tissue formation around the nerves in the non-vein-wrapped group but not in the vein-wrapped group. The results indicate that the vein graft could improve the recovery of nerve function by protecting the nerve from surrounding scar and is an effective and feasible technique for the surgical treatment of recurrent compressive neuropathy. (J Hand Surg 2000; 25A:93-103.

Healthy behaviors, lifestyle, and reasons for quitting smoking among out-of-school youth.

STUDY OBJECTIVE: To investigate the relationships among healthy behavior, healthy values, social influences, and quitting smoking in adolescents not attending school. DESIGN: Following screening procedures, young smokers independently completed a self-report questionnaire administered by trained staff. SETTING: Vocational (TAFE) colleges and Commonwealth Employment Offices (CES) from varying socioeconomic localities were selected as sites to intercept smoking adolescents on their attitudes about quitting smoking. SUBJECTS: Youth attending vocational colleges or CES. RESULTS AND CONCLUSIONS: There were no differences between the two groups of smokers (vocational students and unemployed youth). The decision to quit smoking among these youth is based on a number of factors including social and personal reasons. Health-oriented values were found to be more highly associated with quitting behavior than social influences. Programs for smoking cessation need to be focused on an overall health and improvement approach rather than only a quit-smoking approach.

Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV.

Recombinant adeno-associated virus (rAAV) is a promising vector for retinal application as it transduces photoreceptors and retinal pigment epithelium cells efficiently and in a stable fashion. Because rAAV also transduces retinal ganglion cells, we reasoned that ocular application of rAAV might result in delivery of transgenic protein to the CNS. Here we describe high levels of green fluorescent protein (GFP) persisting at least 6 months in optic nerves and brains of mice and dogs after intravitreal delivery of rAAV-GFP. There was no clinical or histological evidence of inflammatory response although a mild humoral Th-2 response to viral capsid proteins was detected. These findings have important implications with respect to therapeutic applications of rAAV.

Beliefs about smoking cessation among out-of-school youth.

Although the majority of adolescents in the 13-18 age range are at school, there is a need to target specific groups of young smokers such as unemployed youth. For those young people who are not at school, few directed programs are available in either prevention or cessation and information is needed about the design and delivery of appropriate programs for this population. This report presents the results from a survey of unemployed youth and students at vocational colleges about various aspects of smoking cessation. The majority of out-of-school youth smokers had not tried to quit, but 52% were contemplating action to quit. Only a quarter of the smokers had quit for more than a week. Few young smokers would use a recognised program though more females would change to a lower nicotine brand, quit with the help of a friend or participate in a group quit program. The method of quitting most would recommend to peers is 'use of will power'. Incentives to quit were attractive to only a third of the smokers, and many enhancing and inhibiting factors for participation in programs were identified. In particular, efforts to quit increased their confidence in quitting, supporting the need to assist those who are contemplating action to quit. Programs need to incorporate input from youth and be tailored for them but not necessarily for different groups such as non-secondary school students and unemployed youth.

Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene expression.

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5' viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rfm) and dimer (Rfd) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.

Quantification of left ventricular diastolic wall motion by Doppler tissue imaging in healthy cats and cats with cardiomyopathy.

OBJECTIVE: To assess Doppler tissue imaging (DTI) for evaluating left ventricular diastolic wall motion in healthy cats and cats with cardiomyopathy. ANIMALS: 20 healthy cats, 9 cats with hypertrophic cardiomyopathy (HCM), and 9 cats with unclassified cardiomyopathy (UCM). PROCEDURE: A pulsed wave DTI sample gate was positioned at a subendocardial region of the left ventricular free wall in the short axis view and at the lateral mitral annulus in the apical 4-chamber view. Indices of diastolic wall motion were measured, including peak diastolic velocity (PDV), mean rate of acceleration and deceleration of the maximal diastolic waveform (MDWaccel and MDWdecel, respectively), and isovolumetric relaxation time (IVRT). RESULTS: The PDV of cats with HCM and 6 of 9 cats with UCM was significantly decreased, compared with that of healthy cats. In the 3 cats with UCM that had a PDV that was not different from healthy cats, MDWaccel and MDWdecel were greater, and IVRT was shorter than those of healthy cats. The IVRT in cats with HCM was longer than that of other cats. CONCLUSIONS AND CLINICAL RELEVANCE: Indices of diastolic function in cats with HCM, and in many cats with UCM, differed from those of healthy cats and were similar to those reported in humans with HCM and restrictive cardiomyopathy, respectively. However, the hemodynamic abnormality was not the same for all cats with UCM; some cats with an enlarged left atrium and a normal left ventricle (ie, UCM) had abnormal left ventricular wall motion consistent with restrictive cardiomyopathy while others did not.

Sustained production of beta-glucuronidase from localized sites after AAV vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice.

The lysosomal storage disorders are a large group of inherited diseases that involve central nervous system degeneration. The disease in the brain has generally been refractory to treatment, which will require long-term correction of lesions dispersed throughout the central nervous system to be effective. A promising approach is somatic gene therapy but the methods have so far been inadequate because they have only achieved short-term or localized improvements. A potential approach to overcome these limitations is to obtain sustained high level expression and secretion of the missing normal enzyme from a small group of cells for export to neighboring diseased cells, which might allow the therapeutic protein to reach distal sites. We tested this in a mouse model of mucopolysaccharidosis VII (Sly disease) using an adeno-associated virus vector. After a single treatment the vector continuously produced the normal enzyme from infected cells at the injection sites. The secreted enzyme was disseminated along most of the neuraxis, resulting in widespread reversal of the hallmark pathology. An extensive sphere of correction surrounding the transduction sites was created, suggesting that a limited number of appropriately spaced sites of gene transfer may provide overlapping spheres of enzyme diffusion to cover a large volume of brain tissue.

Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue.

Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver.

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.

Immune responses to vector-corrected cells have limited the application of gene therapy for treatment of chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus (AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to neoantigenic transgene products such as beta-galactosidase, which differs from the experience with recombinant adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells activated following intramuscular administration of adenovirus expressing lacZ (AdlacZ) can destroy AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV gene therapy. Adoptive transfer of dendritic cells infected with AdlacZ leads to immune mediated elimination of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate beta-galactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.

Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus.

We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (10(12)-10(13) vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 1 mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200-350 ng/ml) of F. IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.