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Background and objective Pulmonary arterial hypertension (PAH) continues to be a fatal disease and is associated with downregulation of bone morphogenetic protein receptor type-2 (BMPR2). Our approach is to upregulate BMPR2 in the pulmonary vasculature allowing us to examine the changes in endothelial cell signalling and better understand what pathways are altered when disease is attenuated using this treatment approach. Methods We used gene delivery of BMPR2 to human pulmonary endothelial cells to investigate downstream signalling, then assessed the impact of this approach on downstream signalling in vivo in rats with PAH using the monocrotaline (MCT) model. Results Gene delivery of BMPR2 leads to an increase in BMPR2 protein expression, and this is associated with increased Smad1/5/8 and reduced Smad2/3 signalling. Additionally, we have found that BMPR2 modulation has effects on non-Smad signalling with increases found in phosphoinositide-3 kinase (PI3K) and a decrease in phosphorylated-p38-mitogen activated protein kinase (p38-MAPK) in vivo. These findings are associated with amelioration of PAH (reduced right ventricular, mean pulmonary artery pressures and Fulton Index). Conclusion These results indicate that the therapeutic effect of BMPR2 gene delivery on PAH is associated with a switch between TGF-β-Smad2/3 signalling to BMPR2-Smad1/5/8 signalling. This supports the further development of this treatment approach.

Pulmonary arterial hypertension (PAH) continues to be a fatal disease and is associated with downregulation of bone morphogenetic protein receptor type-2 (BMPR2). Our approach is to upregulate BMPR2 in the pulmonary vasculature allowing us to examine the changes in endothelial cell signalling and better understand what pathways are altered when disease is attenuated using this treatment approach.We used gene delivery of BMPR2 to human pulmonary endothelial cells to investigate downstream signalling, then assessed the impact of this approach on downstream signalling in vivo in rats with PAH using the monocrotaline (MCT) model.Gene delivery of BMPR2 leads to an increase in BMPR2 protein expression, and this is associated with increased Smad1/5/8 and reduced Smad2/3 signalling. Additionally, we have found that BMPR2 modulation has effects on non-Smad signalling with increases found in phosphoinositide-3 kinase (PI3K) and a decrease in phosphorylated-p38-mitogen activated protein kinase (p38-MAPK) in vivo. These findings are associated with amelioration of PAH (reduced right ventricular, mean pulmonary artery pressures and Fulton Index).These results indicate that the therapeutic effect of BMPR2 gene delivery on PAH is associated with a switch between TGF-β-Smad2/3 signalling to BMPR2-Smad1/5/8 signalling. This supports the further development of this treatment approach.

Matrix metalloproteinases (MMPs) are essential for extracellular matrix (ECM) breakdown and repair, and have been implicated in the development of metastases. TIMP-3 was initially identified as a potent inhibitor of MMPs, however it also has several properties that are unique and not related to its ability to abrogate MMPs. We studied the effects of overexpression of tissue inhibitor of metalloproteinases-3 (TIMP-3) on lung cancer cells and explored the mechanisms involved in apoptosis-induction in susceptible cells and subsequently, the therapeutic effect in vivo. Overexpression of TIMP-3 resulted in apoptosis of A549 lung cancer cells and AdCMVTIMP3 up-regulated the expression of p53, Fas ligand, TNFR1 and TNFR2 on these cells. Adenoviral delivery of TIMP-3 gene inhibited the growth of pre-established A549 tumours in Balb/c nude mice, and was associated with a greater therapeutic effect than either TIMP-1 or -2 gene delivery. There was no evidence of increased hepatic toxicity following the delivery of TIMP-3 either from intra-tumoural or intravenous injection. Thus, at least in cells showing in vitro susceptibility, TIMP-3 gene therapy offers a therapeutic advantage over TIMPs 1 and 2. These findings establish the potential of adenoviral gene delivery of TIMP3 as a therapeutic agent for selected lung cancers.

Objective: Tachykinins are mediators of airway hyper-reactivity and inflammation. There is in vitro evidence that ovine responses to tachykinins correlate closely to human responses. This study was designed to characterize the effect of intravenously administered tachykinins on sheep lung resistance in vivo to determine the effect of dose timing on reproducibility of responses and the induction of tachyphylaxis. We then used this information to help further characterize the response with several pharmacological agents. Methodology: Substance P (SP) was administered by infusion to conscious merino ewes and lung resistance (RL) was measured. Infusions were given at 30, 60, 120 min and 24 h intervals. The effect of various agents on the response to SP was then assessed. Results: Substance P led to a transient increase in RL, mean (± SEM) 754.8 (± 139)% of baseline, with marked tachyphylaxis at 30, 60 and 120 min. Phosphoramidon increased the peak response to 1151.5 ± 196%. Atropine and CP 96 345 abolished the response to SP, while indomethacin, sodium cromoglycate and pyrilamine had no significant effect. Substance P had a greater effect on RL than did neurokinin A. Conclusions: Substance P increases RL in sheep via a cholinergic mechanism which is mediated by NK-1 receptors, and is subject to tachyphylaxis. These findings have implications for the design of studies using the ovine model in the evaluation of tachykinin antagonists as potential therapeutic agents.

R ecent epidemiologic studies' estimate that at least 2 per 1,000 children have autistic spectrum disorders; that is, autism or its milder variant, pervasive developmental disorder-not otherwise specified (PDD-NOS) or atypical autism. Thus, most primary care pediatricians will find some children with autistic spectrum disorders in their practice panels. Given the reported efficacy of early interventions for children with autism,2 particularly relative to children with other developmental disabilities,3 early recognition of these children by primary care pediatricians is essential to enhance outcomes for these children and their families. Despite its high prevalence, it is probably safe to say that many pediatricians feel somewhat ill at

OBJECTIVE In vivo airway hyperresponsiveness has been demonstrated following inhalation of specific cytokines in normal individuals. Whether this airway hyperresponsiveness results from a direct effect of cytokines on airway smooth muscle contractility, or via changes in airway wall structure is not known. The aim of the present study was to determine the effect of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) on airway smooth muscle contractility in vitro. METHODOLOGY Ovine tracheal smooth muscle strips were incubated for 18 h at room temperature in Dulbecco's modified Eagle's medium, supplemented with antibiotics, with IL-1beta (10 ng/mL) and TNF-alpha (100 ng/mL), in an atmosphere of 5%CO2:95%O2. Following incubation cumulative concentration-response curves to acetylcholine (ACh) and neurokinin A (NKA) were obtained. Antagonist affinity studies were performed to determine whether the cytokine-induced enhanced contractility to ACh and NKA resulted from a functional alteration to specific M3 and NK2 receptors. Cumulative concentration-response curves to NKA were performed in the presence of phosphoramidon to determine if the enhanced contractility to NKA following cytokine exposure was due to a reduction in endogenous neutral endopeptidase activity. To assess the calcium dependence of the hyperresponsiveness, cumulative concentration-responses to ACh were conducted in calcium-free Krebs'-Henseleit solution. RESULTS Pre-incubation with TNF-alpha and IL-1beta caused a significant leftward shift, and an increase in the magnitude, of the concentration-response curves to both ACh and NKA. No difference in M3 and NK2 receptor antagonist affinity (pA2) values between the control and cytokine-treated tissue was observed. Neurokinin A contractility in the presence of phosphoramidon indicated that the enhanced contractility following cytokine exposure was not due to a reduction in endogenous neutral endopeptidase activity. Removal of extracellular calcium ions attenuated the contractile response to low concentrations of ACh in the control and cytokine-pretreated tissue. However, enhanced contractility following TNF-alpha and IL-1beta pretreatment was still present. CONCLUSION Pro-inflammatory cytokines induce in vitro hyperresponsiveness in normal airway smooth muscle via a mechanism involving intracellular calcium mobilization.

We review the early identification and long-term outcome of children with specific reading disabilities. The current widely held theory is that reading disabilities are language-based. Recent literature suggests that specific aspects of early language development, such as phonological processing, are important in learning to read, and that deficiencies in them hinder the process. Deficits in language, attention, and behavior have been found to be nonspecific risk factors for later development of reading disabilities. There is consensus that reading disabilities persist into adulthood. They not only effect academic skills and post-secondary educational and employment opportunities, but also social, emotional, and behavioral function. © 1996 Wiley-Liss, Inc.

Pulmonary arterial hypertension (PAH) remains a fatal disease despite modern pharmacotherapy. Mutations in the gene for bone morphogenetic protein receptor type II (BMPR2) lead to reduced BMPR2 expression, which is causally linked to PAH. BMPR2 is predominantly expressed on pulmonary endothelium and has complex interactions with transforming growth factor (TGF)-β signalling mechanisms. Our objectives were to assess the effect on PAH of upregulating BMPR2 by targeted adenoviral BMPR2 gene delivery to the pulmonary vascular endothelium. We used two established rat models of PAH: chronic hypoxia and monocrotaline (MCT). In both hypertensive models, those receiving BMPR2 had less right ventricular hypertrophy, less pulmonary vascular resistance, improved cardiac function and reduced vascular remodelling. In the MCT model, there was an increase in TGF-β, which was prevented by BMPR2 treatment. In vitro, TGF-β1-induced endothelial-mesenchymal transition (EndMT) in human pulmonary microvascular endothelial cells, which was associated with reduced BMPR2 expression. EndMT was partially ameliorated by stimulating BMPR2 signalling with appropriate ligands even in the ongoing presence of TGF-β1. Collectively, these results indicate therapeutic potential for upregulation of the BMPR2 axis in PAH, which may be, in part, mediated by countering the remodelling effects of TGF-β.

Idiopathic pulmonary arterial hypertension (PAH) is characterized by proliferation of pulmonary vascular endothelial and smooth muscle cells causing increased vascular resistance and right heart failure. Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) are believed to cause the familial form of the disease. Reduced expression of BMPR2 is also noted in secondary PAH. Recent advances in the therapy of PAH have improved quality of life and survival, but many patients continue to do poorly. The possibility of treating PAH via improving BMPR2 signaling is thus a rational consideration. Such an approach could be synergistic with or additive to current treatments. We developed adenoviral vectors containing the BMPR2 gene. Transfection of cells in vitro resulted in upregulation of SMAD signaling and reduced cell proliferation. Targeted delivery of vector to the pulmonary vascular endothelium of rats substantially reduced the pulmonary hypertensive response to chronic hypoxia, as reflected by reductions in pulmonary artery and right ventricular pressures, right ventricular hypertrophy, and muscularization of distal pulmonary arterioles. These data provide further evidence for a role for BMPR2 in PAH and provide a rationale for the development of therapies aimed at improving BMPR2 signaling.

In vitro studies were conducted to characterize the contractile effects of tachykinins in normal ovine trachea with a view in the future to compare tachykinin contractile responses in allergic tissue. Tracheal smooth muscle strips were prepared for in vitro studies of isometric contraction in response to cumulative addition of carbachol, acetylcholine, histamine, neuropeptide gamma, substance P, neurokinin A, neurokinin B, [Sar9, Met(O2)11]substance P, [Nle10]neurokinin A-(4-10), and [Succinyl-Asp6, Me-Phe8]substance P-(6-11) (senktide). The rank order of potency was neuropeptide gammacarbacholneurokinin Aor = [Nle10]neurokinin A-(4-10)acetylcholineor = histamine. Phosphoramidon enhanced the contractile response to neurokinin A and substance P, but not to neuropeptide gamma, [Sar9, Met(O2)11]substance P or senktide. Repeated cumulative concentration responses for acetylcholine, substance P, neurokinin A, [Sar9, Met(O2)11]substance P and histamine were also conducted to test for tachyphylaxis. No tachyphylaxis to acetylcholine, substance P, or neurokinin A was observed, however, [Sar9, Met(O2)11]substance P and histamine did exhibit tachyphylaxis. Atropine had no effect on tracheal contractions to neurokinin A and substance P, while [Sar9, Met(O2)11]substance P contractions were atropine sensitive. Pyrilamine did not affect substance P-induced tracheal smooth muscle contractions, indicating that the response to substance P was not mediated by histamine release. These results show that, in vitro, natural tachykinins induce tracheal smooth muscle contraction predominantly by a direct effect mediated by tachykinin NK2 receptors, and a small tachykinin NK1 receptor mediated cholinergic mechanism.

Idiopathic pulmonary arterial hypertension is a fatal disease characterized by vascular remodeling and right ventricular failure. Modern vasodilator therapies improve prognosis but ultimately most patients succumb or require heart-lung transplantation. We previously developed targeted adenoviral (Ad) strategies for pulmonary vascular gene delivery based on the use of a bispecific conjugate (Fab-9B9) that targets Ad infection to angiotensin converting enzyme (ACE) expressed on pulmonary vascular endothelium. In the current study we determined whether targeting achieved therapeutic gains when using Ad gene delivery of endothelial nitric oxide synthase (eNOS), then evaluated a more novel approach using targeted delivery of the gene for bone morphogenetic protein receptor type 2, based on the knowledge that mutations in this receptor and defects in BMPR2 signaling account for many of the inherited forms of IPAH. Rats (n=6) were injected with 5|[times]|109 pfu of either irrelevant virus, AdCMVeNOS, AdCMVeNOS+Fab9B9 or saline (PBS), then exposed to 10% oxygen atmosphere for three weeks, then right ventricular systolic pressure (RVSP), mean pulmonary artery pressure (PAP) and right ventricular hypertrophy (Fulton Index, FI) were determined. A concurrent group of rats maintained in normoxic conditions was used as a control. Hypoxia lead to increased RVSP (42.2|[plusmn]|4.6mmHg), PAP (34.5|[plusmn]|2.8 mmHg) and FI (0.48|[plusmn]|0.02) vs normoxic controls (22.3|[plusmn]|0.8, 19.4|[plusmn]|1.2 and 0.26|[plusmn]|0.01 respectively). AdCMVeNOS attenuated RVSP(33.9|[plusmn]|1.9), PAP(27|[plusmn]|2.3) and FI(0.41|[plusmn]|.03), with a further therapeutic gain seen with Fab-9B9 targeting (RVSP 28.0|[plusmn]|1.8). A separate experiment using irrelevant virus+Fab-9B9, AdCMVBMPR2+Fab-9B9 or PBS showed that BMPR2 gene delivery attenuated the hypoxia-induced increase in RVSP, PAP and FI by 27%, 34% and 50% respectively. Irrelevant Ad alone or with Fab-9B9 had no effect. Thus, the novel findings are that Ad-mediated eNOS delivery attenuates chronic hypoxia-induced PHT, and this effect is improved with targeting. This is also the first demonstration that upregulation of BMPR2 signaling attenuates pulmonary hypertension, suggesting a therapeutic strategy for patients with BMPR2 mutations.

Lung cancer causes more cancer deaths than cancers related to colon, breast and prostate combined. More primary lung cancers are now diagnosed in former smokers than current smokers and this trend is set to continue. Despite advances in chemotherapeutic agents current treatment strategies are not effective enough in reducing the burden of disease, producing survival benefit or improving quality of life. There is no doubt that new treatment strategies are necessary to improve the outlook from this disease. Gene therapy exploits our expanding knowledge of the molecular basis of cancer using a number of cancer modifying techniques. The extracellular matrix (ECM) provides the scaffolding for all cells and is the environment in which all tissues and cells exist. In order for tissues to change their form the ECM must be degraded and the tissue remodelled. Matrix Metalloproteinases (MMPs) are the proteins that regulate ECM functions. Through a variety of complex mechanisms they control cell differentiation, proliferation and migration. The Tissue inhibitors of Metalloproteinases (TIMPs) are a family of multifunctional proteins named for their ability to inhibit MMPs. TIMP 3 also has the unique ability to directly induce apoptosis in certain tumour types. Hypothesis: In vitro delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells, that AdCMVTIMP-3 has a significant bystander effect and in vivo delivery of AdCMVTIMP-3 to implanted subcutaneous A549 tumours reduces tumour growth. Aims: To demonstrate the in vitro and in vivo apoptotic and cell killing effects of AdCMVTIMP-3. Methods: Lung cancer cells A549 were plated at known concentrations and then infected with adenoviral vectors carrying the genes for TIMP-1, -2 and 3. Changes consistent with apoptosis were observed at 48-66 hours post infection. A variety of flow cytometric analyses including Annexin V, Propidium Iodide, 7AAD and Single Stranded DNA confirm that AdCMVTIMP-3 induces apoptosis and cell death. Elevated Caspace, p53, BAX/Bcl2 and Fas levels show activation of the caspase cascade that regulates programmed cell death. MTT assays confirm reductions in cell numbers and demonstrate the significant bystander effects of AdCMVTIMP-3. In vivo analysis of A549 tumours implanted subcutaneously in nude mice shows delivery of AdCMVTIMP-3 inhibits tumour growth. TUNEL analysis of paraffin embedded tissue sections of these tumours shows evidence of increased numbers of apoptotic cells when compared to uninjected controls and those injected with control virus. Conclusion: Delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells and this can be demonstrated in vitro and in vivo.

Early Pediatric Neurodevelopmental Profile and Laboratory Workup of Children with Autism and Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS)

Correction for prematurity in early developmental assessment of high risk infants continues to be a dilemma. Correction for prematurity in the first year of life is a generally accepted practice, however, correction in the second year is more controversial. To determine whether “catch up” still occurs in the second year of life, 75 premature infants born prior to 31 weeks gestation (mean gestational age = 28 weeks with a range of 24 to 31 weeks) were followed serially at 4 month intervals during the first 2 years of life. The Clinical Adaptive Test (CAT)/Clinical Linguistic and Auditory Milestone Scale (CLAMS) and the Gross Motor scale of the Revised Gesell Developmental Screening Inventory were used to determine each infant's developmental function in visual problem solving, language and gross motor domains at each visit. Interval gain in the first year was compared with interval gain in the second year using a paired t test for each of the developmental domains. The rate of developmental progress in the second year was greater than in the first year in all domains. Differences between rates of progress in year one versus year two were significant for language(mean interval gain in year one = 84%, mean interval gain in year two = 111%, N = 64, t = -4.865, p

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are essential components of the structural lipids in neuronal cell membranes. Fortification of infant formula with these fatty acids has been reported to have a positive impact on neurodevelopmental outcome and visual function in preterm infants. Stockard, et al (Pediatric Research 1996; 39: 320A) reported that ABR latencies were longer in rat pups whose dams were fed fish oil (high in DHA) vs corn oil or chow throughout pregnancy and lactation. However, no data are available in human infants. Since preterm infants routinely have ABR's performed prior to discharge, a retrospective study was conducted in preterm infants who, as part of another study, were fed formula with either 1% or 3.2% of fat as ALA (the precursor of DHA) and had plasma phospholipid levels of DHA and AA determined prior to discharge. Stepwise multiple regression analysis was used to determine the best set of predictors of ABR I-V interpeak intervals at hospital discharge. The best such equation was