Reaching rural veterans: a new mechanism to connect rural, low-income US Veterans with resources and improve food security.

BACKGROUND: Rural, low-income US veterans face additional barriers to accessing food and resources compared to urban veterans. Based on both social-ecological and cultural competence approaches, the Reaching Rural Veterans (RRV) pilot intervention built on the existing infrastructure of food pantries to improve food security and connect rural, low-income veterans with resources. This article describes the process of implementing and evaluating RRV. METHODS: Five rural food pantries within each of two states, Indiana and Kentucky, received training in cultural competence and held monthly outreach events where food and services were offered to veterans. Veteran adult participants completed an assessment at baseline and 3-month follow-up that measured food security using the US Household Food Security Survey Module and self-reported resource enrollment. Repeated measures logistic regression models evaluated the odds of improving food security and resource enrollment from baseline to follow-up (significance P < 0.05). RESULTS: RRV recruited 234 participants; 53% completed the follow-up assessment. At follow-up, the odds of household (P = 0.009) and adult (P = 0.01) food security increased, as did enrollment in one or more of the following resources: Temporary Assistance for Needy Families, Supplemental Security Income, General Assistance or Assistance from the Township Trustee (P = 0.005). CONCLUSIONS: RRV yielded promising preliminary results of improved food security and resource use.

Transcriptional activation of genes involved in oxidative stress in Salmo salar challenged with Piscirickettsia salmonis.

Piscirickettsiosis caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. The objective of this study was to evaluate the modulation of genes involved in the oxidative stress in the liver and muscle of Salmo salar challenge with low dosage of P. salmonis. The treatment (in duplicate) were as follows: Control injection (culture medium) and P. salmonis injection (1 × 102 PFU/mL) with sampling (liver and muscle) at several time-points during the 42-days experimental period (dpi). In liver, the gene expression of superoxide dismutase (SOD) and acetylcholinesterase (AChE) had differences with the control group only at 7 dpi, compared with glutathione-S-transferase (GST) and heat shock protein 70 (HSP70) that presented increases at 7 and 21 dpi. The glutathione peroxidase (GPx) and catalase (CAT) mRNAs were elevated at 13 and 21 dpi, respectively. While glutathione reductase (GR) and cytochrome P450 (P450) did not show variations in their expression during the experimental course. In muscle, the expression of CAT and AChE was higher than in the control condition at 2 and 42 dpi, respectively. While the number of transcripts SOD, GPx, GR, GST, P450 and HSP70 showed increases at 7- and 42-days post injection. The results suggest a transcriptional activation of genes involved in oxidative stress in both liver and muscle, with expression profiles that were tissue-specific and dependent on the time. This is the first study that reveals the transcriptional participation of all these genes associated with oxidative stress in response to the injection of P. salmonis.

Ectoparasite Caligus rogercresseyi modifies the lactate response in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch).

Although Caligus rogercresseyi negatively impacts Chilean salmon farming, the metabolic effects of infection by this sea louse have never been completely characterized. Therefore, this study analyzed lactate responses in the plasma, as well as the liver/muscle lactate dehydrogenase (LDH) activity and gene expression, in Salmo salar and Oncorhynchus kisutch infested by C. rogercresseyi. The lactate responses of Atlantic and Coho salmon were modified by the ectoparasite. Both salmon species showed increasing in plasma levels, whereas enzymatic activity increased in the muscle but decreased in the liver. Gene expression was overexpressed in both Coho salmon tissues but only in the liver for Atlantic salmon. These results suggest that salmonids need more energy to adapt to infection, resulting in increased gene expression, plasma levels, and enzyme activity in the muscles. The responses differed between both salmon species and over the course of infection, suggesting potential species-specific responses to sea-lice infection.

Intra-articular therapy with recombinant human GDF5 arrests disease progression and stimulates cartilage repair in the rat medial meniscus transection (MMT) model of osteoarthritis.

OBJECTIVE: Investigation of osteoarthritis (OA) risk alleles suggests that reduced levels of growth and differentiation factor-5 (GDF5) may be a precipitating factor in OA. We hypothesized that intra-articular recombinant human GDF5 (rhGDF5) supplementation to the OA joint may alter disease progression. METHODS: A rat medial meniscus transection (MMT) joint instability OA model was used. Animals received either one intra-articular injection, or two or three bi-weekly intra-articular injections of either 30 µg or 100 µg of rhGDF5 beginning on day 21 post surgery after structural pathology had been established. Nine weeks after MMT surgery, joints were processed for histological analysis following staining with toluidine blue. Control groups received intra-articular vehicle injections, comprising a glycine-buffered trehalose solution. OA changes in the joint were evaluated using histopathological end points that were collected by a pathologist who was blinded to treatment. RESULTS: Intra-articular rhGDF5 supplementation reduced cartilage lesions on the medial tibial plateau in a dose-dependent manner when administered therapeutically to intercept OA disease progression. A single 100 µg rhGDF5 injection on day 21 slowed disease progression at day 63. A similar effect was achieved with two bi-weekly injections of 30 µg. Two bi-weekly injections of 100 µg or three bi-weekly injections of 30 µg stopped progression of cartilage lesions. Importantly, three biweekly injections of 100 µg rhGDF5 stimulated significant cartilage repair. CONCLUSIONS: Intra-articular rhGDF5 supplementation can prevent and even reverse OA disease progression in the rat MMT OA model. Collectively, these results support rhGDF5 supplementation as an intra-articular disease modifying OA therapy.

Disruption of host-seeking behaviour by the salmon louse, Lepeophtheirus salmonis, using botanically derived repellents.

The potential for developing botanically derived natural products as novel feed-through repellents for disrupting settlement of the salmon louse, Lepeophtheirus salmonis (Caligidae) upon farmed Atlantic salmon, Salmo salar, was investigated using an established laboratory vertical Y-tube behavioural bioassay for assessing copepodid behaviour. Responses to artificial sea water conditioned with the odour of salmon, or to the known salmon-derived kairomone component, α-isophorone, in admixture with selected botanical materials previously known to interfere with invertebrate arthropod host location were recorded. Materials included oils extracted from garlic, Allium sativum (Amaryllidaceae), rosemary, Rosmarinus officinalis (Lamiaceae), lavender, Lavandula angustifolia (Lamiaceae), and bog myrtle, Myrica gale (Myricaceae), and individual components (diallyl sulphide and diallyl disulphide from garlic; allyl, propyl, butyl, 4-pentenyl and 2-phenylethyl isothiocyanate from plants in the Brassica genus). Removal of attraction to salmon-conditioned water (SCW) or α-isophorone was observed when listed materials were presented at extremely low parts per trillion (ppt), that is picograms per litre or 10-12 level. Significant masking of attraction to SCW was observed at a level of 10 ppt for diallyl disulphide and diallyl sulphide, and allyl isothiocyanate and butyl isothiocyanate. The potential of very low concentrations of masking compounds to disrupt Le. salmonis copepodid settlement on a host fish has been demonstrated in vitro.

Contrasting expression of immune genes in scaled and scaleless skin of Atlantic salmon infected with young stages of Lepeophtheirus salmonis.

Atlantic salmon skin tissues with and without scales were taken from two preferred sites of salmon louse (Lepeophtheirus salmonis) attachment, behind the dorsal fin (scaled) and from the top of the head (scaleless), respectively. Tissues were profiled by qPCR of 32 genes to study responses to copepodids, 4 days post infection (dpi), and during the moult of copepodids to the chalimus stage, at 8 dpi. Basal/constitutive differences were found for many immune-related genes between the two skin sites; e.g., mannose binding protein C was over 100 fold higher expressed in the scaled skin from the back in comparison to the skin without scales from the head. With lice-infection, at 4 dpi most genes in both tissues showed lower values than in the non-infected control. By 8 dpi, the majority of responses increased towards the control levels, including cytokines of Th1, Th17 and Th2 pathways. Immunohistochemistry of three immune factors revealed an even distribution of MHC class II positive cells throughout epidermis, including the top layer of keratinocytes, marked compartmentalization of Mx+ and CD8α+ cells close to stratum basale, and an increase in numbers of CD8α+ cells in response to infection. In conclusion, suppression of immune genes during the copepodid stage likely sets off a beneficial situation for the parasite. At the moult to chalimus stage 8 dpi, only few genes surpassed the non-infected control levels, including CD8α. The gene expression pattern was reflected in the increased number of CD8α expressing cells, thus revealing a relatively minor activation of skin T-cell defenses in Atlantic salmon in response to L. salmonis infection.

Differential genetic etiology of reading difficulties as a function of IQ: an update.

In order to test the hypothesis that the genetic etiology of reading disability differs as a function of IQ, composite reading performance data from 308 pairs of identical (monozygotic, MZ) twins and 440 pairs of fraternal (dizygotic, DZ) twins (254 same-sex and 186 opposite-sex) in which at least one member of each pair was classified as reading-disabled were subjected to multiple regression analysis (DeFries and Fulker, Behav Genet 15:467-473, 1985; Acta Genet Med Gemellol 37:205-216, 1988). In the total sample, heritability of the group deficit in reading performance (h(g)(2)) was .61 (±.06). However, results of fitting an extended regression model to reading performance and IQ data suggested that the genetic etiology of reading disability differs as a linear function of IQ (p ≤ .04). When the basic regression model was fitted separately to data from twin pairs with Wechsler (Examiner's manual: Wechsler intelligence scale for children-revised, 1974; Examiner's manual: Wechsler adult intelligence scale-revised, 1981) Full Scale IQ scores in the upper and lower 25% of the sample, resulting estimates of h(g)(2) were .75 (±.12) and .50 (±.10), respectively (p ≤ .045). These results suggest that reading difficulties in children with a higher IQ are due substantially to genetic influences and may require intensive remediation efforts.

The heritability of general cognitive ability increases linearly from childhood to young adulthood.

Although common sense suggests that environmental influences increasingly account for individual differences in behavior as experiences accumulate during the course of life, this hypothesis has not previously been tested, in part because of the large sample sizes needed for an adequately powered analysis. Here we show for general cognitive ability that, to the contrary, genetic influence increases with age. The heritability of general cognitive ability increases significantly and linearly from 41% in childhood (9 years) to 55% in adolescence (12 years) and to 66% in young adulthood (17 years) in a sample of 11 000 pairs of twins from four countries, a larger sample than all previous studies combined. In addition to its far-reaching implications for neuroscience and molecular genetics, this finding suggests new ways of thinking about the interface between nature and nurture during the school years. Why, despite life's 'slings and arrows of outrageous fortune', do genetically driven differences increasingly account for differences in general cognitive ability? We suggest that the answer lies with genotype-environment correlation: as children grow up, they increasingly select, modify and even create their own experiences in part based on their genetic propensities.

Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization.

Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.

Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida.

Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1beta, IL-8, TNF-alpha and IFN-gamma was increased in the proximal intestine. TGF-beta was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1beta, IL-8, TNF-alpha and TGF-beta. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.

Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes.

Glucagon-like peptide 1 (GLP-1) is a promising candidate for the treatment of type II diabetes. However, the short in vivo half-life of GLP-1 has made peptide-based treatments challenging. Gene therapy aimed at achieving continuous GLP-1 expression presents one way to circumvent the rapid turnover of GLP-1. We have created a GLP-1 minigene that can direct the secretion of active GLP-1 (amino acids 7-37). Plasmid and adenoviral expression vectors encoding the 31-amino-acid peptide linked to leader sequences required for secretion of GLP-1 yielded sustained levels of active GLP-1 that were significantly greater than endogenous levels. Systemic administration of expression vectors to animals using two diabetic rodent models, db/db mice and Zucker Diabetic Fatty (ZDF) rats, yielded elevated GLP-1 levels that lowered both the fasting and random-fed hyperglycemia present in these animals. Because the insulinotropic actions of GLP-1 are glucose dependent, no evidence of hypoglycemia was observed. Improved glucose homeostasis was demonstrated by improvements in %HbA1c (glycated hemoglobin) and in glucose tolerance tests. GLP-1-treated animals had higher circulating insulin levels and increased insulin immunostaining of pancreatic sections. GLP-1-treated ZDF rats showed diminished food intake and, in the first few weeks following vector administration, a diminished weight gain. These results demonstrate the feasibility of gene therapy for type II diabetes using GLP-1 expression vectors.

Effects of a p38 MAP kinase inhibitor on bone ingrowth and tissue differentiation in rabbit chambers.

The effects of an oral p38 mitogen-activated protein kinase (MAPK) inhibitor and polyethylene particles separately and together on tissue differentiation in the bone harvest chamber (BHC) in rabbits over a 3-week treatment period were investigated. The harvested tissue was analyzed histomorphometrically for markers of bone formation (percentage of bone area), osteoblasts (alkaline phosphatase staining), and osteoclasts (CD51, the alpha chain of the vitronectin receptor). Polyethylene particles decreased the percentage of bone ingrowth and staining for alkaline phosphatase. The p38 MAPK inhibitor alone decreased alkaline phosphatase staining. When the oral p38 MAPK inhibitor was given and the chamber contained polyethylene particles, there was a suppression of bone ingrowth and alkaline phosphatase staining. In contrast to oral non-steroidal anti-inflammatory drugs (NSAIDs) and local Interleukin-1 receptor antagonist (IL-1ra) administration, the oral p38 MAPK inhibitor alone did not suppress bone formation when given during the initial phase of tissue differentiation. Particle-induced inflammation and the foreign body reaction were not curtailed when the p38 MAPK inhibitor was given simultaneously with particles. Additional experiments are needed to establish the efficacy of p38 MAPK inhibitor administration on mitigating an established inflammatory and foreign body reaction that parallels the clinical situation more closely.

Genetic and environmental influences on age at sexual initiation in the Colorado Adoption Project.

Whereas the majority of research on adolescent sexual initiation has focused solely on environmental factors, the present study used behavioral genetic analyses to investigate the relative contributions of genetic and environmental influences. Structural equation models were fitted to data from adoptive and non-adoptive sibling pairs (231 biologically related pairs and 169 unrelated pairs) from the Colorado Adoption Project. Information from censored individuals who had not yet experienced sexual initiation was maximized by adapting the twin survival analysis method of Pickles et al. (Behav Genet 24(5):457-468, 1994) to accommodate adoptive and non-adoptive siblings. Point estimates of variance components from an ACE model, including additive genetic (A), shared environmental (C), and non-shared environmental (E) influences were 28%, 24%, and 48%, respectively. Despite the lower point estimate for shared environmental effects than additive genetic effects, a CE model provided the best fit to the data. However, because adoptive siblings provide a direct estimate of shared environmental influences there is greater power to detect shared environmental effects in adoption designs. Evidence for genetic influences from our data were somewhat lower than those obtained in previous twin studies, possibly reflecting a return to more socially conservative sexual attitudes, changing sexual behaviors, or ambiguities in the wording of questions commonly used in research on adolescent sexuality.

A role for schwann cells in the neuroregenerative effects of a non-immunosuppressive fk506 derivative, jnj460.

UNLABELLED: FK506 and its non-immunosuppressive derivatives represent a class of pharmacological agents referred to as immunophilin ligands that have been reported to promote neuroregeneration and survival in several experimental models; however their cellular and molecular mechanisms of action have not been well established. Here we characterize a new immunophilin ligand that interacts with both FK506 binding protein 12 (FKBP12) and FKBP52, and demonstrate that JNJ460 induces neurite outgrowth from freshly explanted dorsal root ganglia (DRG) in a Schwann cell-dependent manner. Purified cultures of neurons fail to respond to these drugs, but cultures containing Schwann cells and neurons respond with neurite outgrowth, as do neurons grown in conditioned medium from JNJ460-treated Schwann cells. Using microarray analysis and a transcription reporter assay, we show that JNJ460 induces a series of transcriptional changes that occur in a temporal cascade. Among the Schwann cell-expressed genes upregulated following JNJ460 treatment is the POU transcription factor SCIP, which has been shown to regulate Schwann cell gene transcription and differentiation. JNJ460 potentiated transforming growth factor beta (TGF-beta)-induced transcriptional activation and SCIP induction in Schwann cells, by altering the interaction between FKBP12 and the TGF-beta type I receptor, TbetaR1. Finally, to test whether JNJ460 enhances neurite regeneration in vivo, we treated animals with JNJ460 for 30 days following mechanical transection of the sciatic nerve and demonstrated myelin and axonal hypertrophy at the ultrastructural level. Collectively, these data suggest that Schwann cells play an important role in the biological effects of immunophilin ligands by affecting neuron-glial signaling during regeneration. SUMMARY: The cellular and molecular mechanisms responsible for the regenerative effects of immunophilin ligands are not well understood. Here we show that the neuritogenic effects of JNJ460 in a DRG model depend on interactions between neurons and Schwann cells. Treatment of purified Schwann cells with JNJ460 alters Schwann cell gene expression, and promotes the generation of factors that act on neurons. These data indicate that Schwann cells play an important role in the actions of immunophilin ligands.

Parent-offspring resemblance for reading performance at 7, 12 and 16 years of age in the Colorado Adoption Project.

BACKGROUND: The study aimed to conduct the first analysis of CAP parent-offspring resemblance for reading performance in children aged 7, 12 and 16 years, and to assess the etiology of individual differences in reading performance of children at 16 years of age. METHOD: The Reading Recognition subtest of the Peabody Individual Achievement Test was administered to children in the Colorado Adoption Project (CAP) at 7, 12 and 16 years of age, and to their adoptive and nonadoptive parents when the children were 7 years of age. RESULTS: Resulting parent-offspring correlations in adoptive families were not significant at any age, but correlations between scores of nonadoptive control parents and their offspring were significant at all three ages. CONCLUSIONS: Results obtained from behavioral genetic model-fitting analyses of data from parents and their children tested at age 16 are consistent with results of studies of twins and siblings indicating that individual differences in reading performance are due substantially to genetic influences. In contrast, environmental transmission from parents to offspring was negligible, suggesting that environmental influences on individual differences in the reading performance of children are largely independent of parental reading performance.

Kinetics of small molecule inhibitor binding to p38 kinase.

p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.

Stability of genetic and environmental influences on reading performance at 7, 12, and 16 years of age in the Colorado Adoption Project.

The etiology of the longitudinal stability of reading performance was assessed by analyzing data from adoptive and nonadoptive sibling pairs (206 pairs at age 7, 195 pairs at age 12, and 110 pairs at age 16) tested in the Colorado Adoption Project (CAP). Results of longitudinal behavioral genetic analyses confirmed previous findings of moderate genetic influence on individual differences in reading performance at 7 and 12 years of age (a2 = .44 and .38, respectively), with somewhat higher heritability at age 16 (a2 = .57). Corresponding shared environmental influences were negligible (c2 = .07, .09, and .07). Moreover, common genetic influences were responsible for 66% of the observed stability (rp) between ages 7 and 12 (.62), 62% of that between ages 12 and 16 (rp = .74), and 88% of that between ages 7 and 16 (rp = .55). Of particular interest, no new heritable variation was detected at either 12 or 16 years of age, suggesting that genetic influences at 7 years of age are amplified at the later ages. In contrast, new nonshared environmental influences (including measurement error) were manifested at each age, suggesting the possible importance of nonshared environmental factors (e.g., instructional methods, teachers, peers) for the development of individual differences in reading performance between 7 and 16 years of age.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium.

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications.

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells.

Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.