Search

Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signaling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in coexisting lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes and by changing nanostructure size and topology. We demonstrate the functionality of our strategy with a bioinspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral redistribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.

The ectoparasite, Lepeophtheirus salmonis (Kroyer 1837), is effective at avoiding elimination from its host, Atlantic salmon, Salmo salar L., by inhibiting the recruitment of immune cells to the site of attachment. In other ectoparasitic arthropods, numerous factors have been identified that bind or neutralize chemokines preventing their interaction with receptors on the surfaces of immune cells. To determine if L. salmonis is utilizing a similar mechanism of immune modulation, the chemotactic activity of peripheral blood leukocytes (PBL) to leukotriene B4 (LTB4) and the secreted/excreted products (SEPs) of the sea louse were investigated in vitro. The results showed that incubation of LTB4 with SEPs reduced leukocyte migration compared to LTB4 immune stimulation alone. Data suggests that one of the mechanisms L. salmonis may be using to regulate immune cell recruitment in Atlantic salmon is by inhibiting or neutralizing the activity of chemokines., Competing Interests: Declaration of competing interest The authors declare they have no conflict of interest in this research., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The role of parasitic sea lice (Siphonostomatoida; Caligidae), especially Lepeophtheirus salmonis, in the epidemiology of Infectious Salmon Anemia Virus (ISAv) has long been suspected. The epidemiological studies conducted during the 1998 major Infectious Salmon Anaemia (ISA) outbreak in Scotland demonstrated a strong correlation between sea lice presence and ISAv positive sites or subsequent clinical outbreaks of ISA. The question posed from this observation was "do sea lice infestations on Atlantic salmon make them more susceptible to viral infections?" This study investigated the role that sea lice infestations have on the severity of ISAv infections and disease mortality in experimental populations of farmed Atlantic salmon (Salmo salar). A series of experiments was carried out that investigated the potential of sea lice to modify the outcome of an ISAv infection. Experimental populations of Atlantic salmon were established that had: no lice and no ISAv, a single infection with either ISAv or lice and a co-infection with lice then ISAV. The results were quite clear, the process of infestation by the parasite prior to ISAv exposure significantly increased the mortality and death rates of Atlantic salmon, when compared to uninfected controls and ISAv infected groups only. This was consistent over two source strains of Atlantic salmon (Pennobscot and Saint John River), but the severity and timing was altered. Immunological responses were also consistent in that pro-inflammatory genes were induced in lice only and co-infected fish, whereas the anti-viral response, Mx, MH class I β, Galectin 9 and TRIM 16, 25 genes were down-regulated by lice infection prior to and shortly after co-infection with ISAv. It is concluded that the sea lice settlement on Atlantic salmon and the parasite's subsequent manipulation of the host's immune system, which increases parasite settlement success, also increased susceptibility to ISAv., Competing Interests: The authors have declared that no competing interests exist.

Fungal infections are a major cause of animal and plant morbidity and mortality worldwide. Effective biological therapeutics could complement current antifungal drugs, but understanding of their in vivo mechanisms has been hampered by technical barriers to intravital imaging of host-pathogen interactions. Here we characterize the fungal infection of zebrafish as a model to understand the mechanism-of-action for biological antifungal therapeutics through intravital imaging of these transparent animals. We find that non-specific human IgG enhances phagocytosis by zebrafish phagocytes in vivo. Polyclonal anti-Candida antibodies enhance containment of fungi in vivo and promote survival. Analysis suggests that early phagocytic containment is a strong prognostic indicator for overall survival. Although polyclonal anti-Candida antibodies protect against disease, this is not necessarily the case for individual monoclonal anti-Candida antibodies. Thus, the zebrafish appears to provide a useful model host for testing if a biological therapeutic promotes phagocytosis in vivo and enhances protection against candidemia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The impact of many inflammatory diseases is influenced by age-related changes in the activation of resident and circulating myeloid cells. In the eye, a major sight-threatening consequence of age-related macular degeneration is the development of severe choroidal neovascularization (CNV). To identify the molecular pathways and myeloid cell populations involved in this increased neovascular response, we characterized the immune status of murine choroid and retina during aging and in the context of experimental CNV. In the choroid, but not in the retina, advancing age is associated with proinflammatory upregulation of CCL2-CCR2 signaling. Genetic excision of CCL2 diminishes age-related inflammatory changes in the choroid, with reduced recruitment of proinflammatory myeloid cells and attenuation of CNV. These findings indicate that CCL2-driven recruitment of myeloid cells contributes to increased severity of CNV with age. Similar mechanisms may be involved in other age-related inflammatory diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited., Methods: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings., Results: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG., Conclusions: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

Noninfectious intraocular inflammatory disease remains a significant cause of visual loss, even with current systemic immunosuppression. Alternative novel treatments are thus required, particularly for severe forms of posterior uveitis. Encouraging results from several phase I/II clinical trials of gene therapy for monogenic retinal disorders have paved the way for the development of this approach for other ocular conditions. Gene therapy for uveitis offers the possibility of potent, self-regulating, long-term disease control following a single treatment and without systemic side effects. To date, gene therapy approaches using interleukin-10, interleukin-1 receptor antagonist, interferon-alpha, soluble TNF-alpha receptors, and alpha-MSH gene transfer have been used successfully to attenuate experimental animal models of uveitis. This review evaluates these preclinical studies, considers the route to clinical application, and explores future targets and approaches.

Insertional mutagenesis following gene therapy with gammaretroviral vectors can cause the development of lymphoproliferation in children with X-linked severe combined immunodeficiency. In experimental studies, recombinant adeno-associated virus (rAAV) vectors have also been reported to increase susceptibility to carcinogenesis. The possibility of vector-induced transformation in quiescent ocular cells is probably significantly lower than in mitotically active cells, but given the increasing number of clinical applications of rAAV and lentiviral vectors for ocular disease, a specific assessment of their oncogenic potential in the eye is important. In this study, we investigated the effect of rAAV2/2 and integrating HIV-1 vectors upon the incidence of ocular neoplasia in p53 tumour-suppressor gene-knockout (p53(-/-)) mice, which are highly susceptible to intraocular malignant transformation. Subretinal injections of high titre rAAV2/2 or integrating HIV-1 vectors induced no tumours in p53(-/-) or p53(+/-) animals, nor significantly affected their natural longevity. We conclude that any insertional events arising from subretinal delivery of these vectors appear insufficient to cause intraocular malignancy, even in highly susceptible animals. These findings support the continued development of these vectors for ocular applications.

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse "knock-in" model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct.

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65-/- dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85-180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65-/- dog. This finding has important implications for the treatment of human LCA type II patients.

Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp(+ve) photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective., (Copyright © 2010 AlphaMed Press.)

Purpose: Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD., Methods: The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry., Results: The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice., Conclusions: These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

Background: Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product., Methods: We evaluated cellular and humoral responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression., Results: Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low-dose AAV.hRPE65.hRPE65 to both eyes of RPE65(-/-) mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye., Conclusions: These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent. Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration., ((c) 2009 John Wiley & Sons, Ltd.)

Uveitis is a sight threatening inflammatory disorder that remains a significant cause of visual loss. We investigated lentiviral gene delivery of interleukin 1 receptor antagonist (IL-1ra) or interleukin (IL)-10 to ameliorate murine endotoxin-induced uveitis (EIU). An human immunodeficiency virus-1-based vector containing the mIL-1ra or mIL-10 cDNA demonstrated high expression of biologically active cytokine. Following administration of Lenti.GFP into the anterior chamber, transgene expression was observed in corneal endothelial cells, trabecular meshwork and iris cells. To treat EIU, mice were injected with Lenti.IL-1ra, Lenti.IL-10 or a combination of these. EIU was induced 14 days after vector administration and mice were culled 12 h following disease induction. Lenti.IL-1ra or Lenti.IL-10-treated eyes showed significantly lower mean inflammatory cell counts in the anterior and posterior chambers compared with controls. The aqueous total protein content was also significantly lower in treated eyes, demonstrating better preservation of the blood-ocular barrier. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral-mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis.

To date adeno-associated viral (AAV) vectors are the only gene therapy vectors that have been shown to efficiently transduce photoreceptor cells and have thus become the most commonly used vector for ocular transduction. Various AAV serotypes have been evaluated in the eye, the first of which was AAV2, which is able to transduce photoreceptors, retinal pigment epithelium (RPE) and retinal ganglion cells. AAV serotypes 1 and 4, as well as AAV2 pseudotyped with these capsids, only transduce the RPE. AAV serotype 5 and AAV2/5 transduce the photoreceptors as well as RPE, but not retinal ganglion cells. Here, we assessed the capacity of the novel serotype AAV2/8 to transduce various ocular tissues of the adult murine retina by administering AAV2/8 green fluorescent protein intravitreally, subretinally and intracamerally. We also determined the kinetics and efficiency of self-complementary AAV (scAAV) vectors of serotypes 2/2, 2/5 and 2/8 and compared them with single-stranded AAV (ssAAV). We found that ssAAV2/8 transduces photoreceptors and RPE more efficiently than ssAAV2/2 and ssAAV2/5, and that scAAV2/8 had faster onset and higher transgene expression than ssAAV2/8. This improved transduction efficiency might facilitate the development of improved gene therapy protocols for inherited retinal degenerations, particularly those caused by defects in photoreceptor-specific genes.

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif.

Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma.

We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIAV.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compared with EIAV.null-injected eyes, EIAV.endostatin resulted in a 59.5% (P<0.001) reduction in CNV area and a reduction in hyperpermeability of 25.6% (P<0.05). Equine infectious anaemia virus.angiostatin resulted in a 50.0% (P<0.05) reduction in CNV area and a 23.9% (P<0.05) reduction in hyperpermeability. Equine infectious anaemia virus.endostatin, but not EIAV.angiostatin significantly augmented the frequency of apoptosis within the induced CNV as compared with injected controls. TdT-dUTP terminal nick end labeling analysis 5 weeks post-injection, and histological and retinal flatmount analysis 12 months post-injection revealed no evidence of vector- or transgene expression-related deleterious effects on neurosensory retinal cells, or mature retinal vasculature in non-lasered eyes. Highly expressing EIAV-based vectors encoding endostatin or angiostatin effectively control angiogenesis and hyperpermeability in experimental CNV without long-term deleterious effects, supporting the use of such a strategy in the management of patients with exudative age-related macular degeneration.

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

Objectives: To clinically characterize a family with nonsyndromic sensorineural hearing loss linked to the DFNA25 gene and to assess whether mitochondrial mutations influence the penetrance of the phenotype., Design: Longitudinal clinical and basic science molecular genetic study., Setting: Academic medical center and molecular genetic research laboratory., Participants: Members of a family with dominant high-frequency sensorineural hearing loss., Interventions: Questionnaires, serial audiograms, and interviews correlated with molecular genetic data., Main Outcome Measures: Symptoms, age at onset, serial audiometric data, and the presence or absence of 4 deafness-associated mitochondrial mutations., Results: Affected individuals typically manifest a high-frequency, slowly progressive sensorineural hearing loss in the postlingual period. The mode of inheritance is autosomal dominant with age-dependent penetrance. Male affected members tended to report an earlier onset of hearing loss than female members. In those inheriting the DFNA25-associated haplotype from an affected mother, hearing loss invariably developed by the second decade of life, whereas those inheriting the DFNA25 haplotype from an affected father often maintained hearing levels comparable to those of age-matched control subjects, even into the seventh decade of life. None of 4 deafness-associated mitochondrial mutations screened (1555A>G, 7445A>G, Cins7472, and 7511T>C) were found to segregate in the family., Conclusions: It is difficult to differentiate delayed-onset high-frequency sensorineural hearing loss inherited as a simple mendelian trait like DFNA25-associated hearing loss from that due to noise exposure or presbycusis, disorders that may also have a genetic component. An awareness of the clinical presentation of such hearing loss may help clinicians identify hearing loss attributable to genetic causes and improve care for these patients.

Research into population declines of North American bird species has mainly focused on the fragmentation of habitat on the breeding or wintering grounds [Robinson, S. K., Thompson, F. R., Donovan, T. M., Whitehead, D. R. & Faaborg, J. (1995) Science 267, 1987-1990]. In contrast, research into declines of European species has mainly focused on intensification of agriculture [Donald, P. F., Green, R. E. & Heath, M. F. (2001) Proc. R. Soc. London Ser. B 268, 25-29] and the role played by the atmospheric deposition of pollutants, in particular, acid rain [Graveland, J. (1998) Environ. Rev. 6, 41-54]. However, despite widespread unexplained declines of bird populations in regions of heavy wet acid ion deposition [Sauer, J. R., Hines, J. E. & Fallon, J. (2001) The North American Breeding Bird Survey Results and Analysis 1966-2000 (Patuxent Wildlife Research Center, Laurel, MD)], no North American studies have presented evidence linking such widespread terrestrial bird declines to acid rain. To address the question of the role played by acid rain in population declines of eastern North American songbird species, we combine data from several sources. We use a multiple logistic regression model to test for adverse effects of acid rain on the Wood Thrush, while controlling for regional abundance, landscape-level habitat fragmentation, elevation, soil pH, and vegetation. We show a strong, highly significant, negative effect of acid rain on the predicted probability of breeding by this species, and interactions with elevation, low pH soils, and habitat fragmentation that worsen these negative effects. Our results suggest an important role for acid rain in recent declines of some birds breeding in the eastern United States, particularly in high elevation zones with low pH soils, and show the need to consider other large-scale influences, in addition to habitat fragmentation, when addressing bird population declines.

Using linkage analysis, we identified a novel dominant locus, DFNA25, for delayed-onset, progressive, high-frequency, nonsyndromic sensorineural hearing loss in a large, multigenerational United States family of Czech descent. On the basis of recombinations in affected individuals, we determined that DFNA25 is located in a 20-cM region of chromosome 12q21-24 between D12S327 (centromeric) and D12S84 (telomeric), with a maximum two-point LOD score of 6.82, at recombination fraction.041, for D12S1030. Candidate genes in this region include ATP2A2, ATP2B1, UBE3B, and VR-OAC. DFNA25 may be the human ortholog of bronx waltzer (bv).

The purpose of this study is to compare the effectiveness and utility of distortion product otoacoustic emission (DPOAE) and auditory brain stem response (ABR) testing as screening methodologies suitable for universal application at a large birthing hospital. Five hundred sixty-nine neonates (1184 ears) without risk indicators for hearing loss underwent DPOAE and ABR screening before hospital discharge at birth. All ears (100%) passed the ABR screening. DPOAE results were categorized on the basis of the number of frequencies at which emissions were obtained as well as presence versus absence of a replicated response at each test frequency. Pass and refer rates varied widely, on the basis of whether the presence of DPOAE response at 2000 Hz or replication were required. With the most stringent criteria, only 64.44% of ears passed, whereas with the least stringent criteria 88.94% passed. Given that 100% of ears passed according to the gold standard of the ABR screening, these results indicate false-positive rates ranging from 11% to 35% by DPOAE screening. This discrepancy in pass and refer rates when various criteria are applied indicates the need for standardization and further comparison of appropriate pass criteria for newborn hearing screening programs.

Objective: This study was undertaken to determine whether the frequency of fetal surveillance could be safely reduced from twice weekly to fortnightly in the case of small-for-gestational-age fetuses with normal results of umbilical artery Doppler velocimetry studies., Study Design: Pregnant women between 24 and 36 weeks' gestation (n = 167) with small-for-gestational-age fetuses and normal results of umbilical artery Doppler velocimetry studies were randomly allocated to undergo twice-weekly or fortnightly fetal surveillance. Statistical analysis was carried out according to intention to treat., Results: Eighty-five women were randomly assigned to undergo twice-weekly fetal surveillance and 82 were randomly assigned to undergo fortnightly fetal surveillance. Those randomly assigned to twice-weekly surveillance were delivered 4 days earlier (264 vs 268 days; P =.04) and were more likely to have labor induced (n = 70, 82%, vs n = 54, 66%; P =.02) than those randomly assigned to fortnightly surveillance. Fifty-four babies (23%) were admitted to the neonatal nursery, but there were no differences in neonatal morbidity between the groups., Conclusions: Maternal intervention (induction) was more common in the twice-weekly group. No differences in neonatal outcomes were detected. A much larger trial is required to determine the safety and potential benefits of less frequent surveillance of small-for gestational-age fetuses with normal results of umbilical artery Doppler velocimetry studies.

Binge eating has been identified as a common problem in samples of obese persons. Earlier studies found that approximately 30% of participants presenting for weight loss treatment could be diagnosed with Binge Eating Disorder (BED). This study investigated the prevalence of BED using the Questionnaire on Eating and Weight Patterns (QEWP) and the Interview for the Diagnosis of Eating Disorders (IDED) in a sample of 468 obese adults seeking weight loss treatment at two research facilities. The study found that only a small percentage of the participants met Diagnostic and Statistical Manual for Mental Disorders, 4th Revision (DSM-IV) diagnostic criteria for BED using either the IDED (1.3%) or QEWP (7.3%). A larger percentage of the sample (10.7% based on the IDED and 20.5% based on the QEWP) reported binge eating, but did not endorse all criteria necessary to warrant a diagnosis of BED. The primary finding of the study was that the prevalence of BED in treatment seeking obese adults was much lower than was reported in previous studies. Also, there was significant discrepancy in prevalence rates of BED as defined by self-report and interview assessment methods, with the interview method yielding lower estimates of prevalence. These findings suggest that the prevalence of BED may be lower than estimates of earlier reports. We recommend that future studies of BED use reliable and valid interview methods and that this research focus on more diverse populations, including men and a variety of racial and ethnic groups.

Examined differences between compensation seeking (CS) veterans and noncompensation seeking (NCS) veterans on the Minnesota Multiphasic Personality Inventory-2 (MMPI-2) and other psychological measures in 142 combat veterans evaluated for posttraumatic stress disorder (PTSD) at an outpatient Veterans Affairs (VA) hospital PTSD clinic. Patients were grouped on the basis of their compensation seeking status, with 69% classified as CS for PTSD. The CS veterans achieved significantly more pathological scores across a wide range of psychological inventories and MMPI-2 validity indices, although they did not differ in frequency of PTSD diagnoses from NCS veterans. Implications of these findings are discussed, and clinicians are advised to be aware of the compensation seeking status of combat-veterans being evaluated for PTSD.

A multidimensional model of body-image disturbance was tested. The model incorporated the concepts of body-size distortion, preference for thinness, body dissatisfaction, and fear of fatness as predictors of restrictive eating. The LISREL 7 program was used to perform a structural modeling analysis of the theoretical model. A total of 175 women participated in the study (54 eating-disordered patients and 121 undergraduate students). The results supported the hypothesized four-dimensional model relative to alternative one-, two-, and three-dimensional models. Body dissatisfaction appeared to be directly affected by both body-size distortion and preference for thinness. Fear of fatness was found to be the best predictor of restrictive eating. The results appeared consistent across the clinical and nonclinical samples. These data may help resolve many of the current controversies in the body-image literature. The results also suggested the need to develop more sound assessment instruments for fear of fatness.

The principal aim of this study was to examine the factor structure of several assessment methods used to measure dietary restraint, body dissatisfaction, and body image. Factor analysis was employed to identify and confirm the primary constructs measured by these assessment methods. A total of 206 undergraduate women were recruited as subjects. This sample was divided into two subsets of 100 and 106 subjects. On the first subset, principle components analysis identified three factors: body dysphoria, dietary restraint, and body image. With the second subset of subjects, confirmatory factor analysis cross-validated this factor structure. A two factor solution, body dysphoria and dietary restraint, was identified and confirmed when the body image measure was converted to a self-minus-ideal discrepancy score. These findings are discussed in relation to the definition of control groups to be used in studies of anorexia and bulimia nervosa. Guidelines for the selection of measures for each of the three factors also are presented.

Medical records of 118 newborn infants with Down's syndrome were reviewed to document the types of congenital heart disease (CHD) in those having echocardiography. Of 102 infants having echocardiography, 49 (48%) had heart defects; 47 of these had trisomy 21 and 2 had unbalanced translocation karyotypes. Of the 53 (52%) who did not have heart defects, all had trisomy except 1 with a mosaic karyotype and 1 with a translocation karyotype. The most common heart malformation was an atrioventricular canal, followed in frequency by ventricular septal defect, atrial septal defect, patent ductus arteriosus, and tetralogy of Fallot. Benefits of echocardiography in such infants are early detection of CHD, with aggressive management to prevent future complications, and reassurance to parents if the infant does not have CHD.

We examined the relationship between actual caloric intake and subjective perceptions of amount eaten using self-monitoring data. Forty subjects participated in the study: 20 bulimia nervosa patients and 20 normal controls. All subjects monitored their eating for a 2-week period and rated each eating episode on a Likert-type scale ranging from an undereat to a binge. Estimates of actual caloric intake were compared with these subjective ratings. Bulimics were found to overrate the amount consumed, relative to controls. The effect increased as caloric intake increased. Bulimics' ratings of amount eaten and binging were found to be predicted by the estimate of the actual amount eaten, the type of foods eaten, and the subjects' mood prior to eating, while nonbulimics' ratings were predicted only by the estimated actual amount. Subjective ratings of amount were found to be the best predictor of purgative activity. The results are discussed in terms of a perceptual bias theory, treatment implications, and possible revisions to the current DSM criteria for bulimia nervosa.

In a recent investigation of the psychopathology of bulimia nervosa by Tobin, Johnson, Steinberg, Staats, and Dennis (1991), a multidimensional model for bulimia nervosa was presented, based on the results of an exploratory factor analysis. In the present investigation, these results and the multidimensional model were tested by means of confirmatory factor analysis with 100 women diagnosed as having bulimia nervosa. The results not only support the multidimensional model with the higher order dimensions Affective and Personality Disorder, Bulimic Behaviors, and Restricting Behaviors, but also demonstrate the importance of body dissatisfaction as a significant, and possibly independent, component of bulimia nervosa.