Gene therapy for noninfectious uveitis.

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.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice.

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.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function.

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.

Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis.

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.

Assessment of ocular transduction using single-stranded and self-complementary recombinant adeno-associated virus serotype 2/8.

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.

Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12.

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.

EIAV vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental CNV.

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.

DFNA25, a novel locus for dominant nonsyndromic hereditary hearing impairment, maps to 12q21-24.

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).

A pilot randomized controlled trial of two regimens of fetal surveillance for small-for-gestational-age fetuses with normal results of umbilical artery doppler velocimetry.

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.

Outcome of newborn hearing screening by ABR compared with four different DPOAE pass criteria.

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.

Prevalence of binge eating disorder in obese adults seeking weight loss treatment.

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.

Compensation seeking status and psychometric assessment of combat veterans seeking treatment for PTSD.

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.

Clarifying body-image disturbance: analysis of a multidimensional model using structural modeling.

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.

Body image, body dysphoria, and dietary restraint: factor structure in nonclinical subjects.

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.

Congenital heart disease in infants with Down's syndrome.

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.

Additive effects of mood and eating forbidden foods upon the perceptions of overeating and binging in bulimia nervosa.

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.

Confirmatory factor analysis of a multidimensional model of 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.