Widespread correction of central nervous system disease after intracranial gene therapy in a feline model of Sandhoff disease.

Sandhoff disease (SD) is caused by deficiency of N-acetyl-ß-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

Low-loss passive alignment of single-mode fibers in low-temperature cofired ceramics using CO2 laser fabricated U-grooves.

We report a step toward optoelectronic integration on low-temperature cofired ceramics substrates in the form of fiber alignment U-grooves. The precision of the CO2 laser machining of green state ceramic for this purpose is investigated. The groove-writing process with a speed of 1 mm/s was carried out in air at room temperature and ambient pressure. The process is to a large extent self-cleaning without any gas jet assist. By analysis of groove profiles after firing of a near-zero shrinkage green tape formulation, it is shown that the cutting accuracy is approximately 2 microm, which is at the fundamental limit set by the particle size (1-5 microm). We demonstrate low-loss butt coupling of <1 dB for single-mode fibers using the laser written U-grooves. The technique exhibits a potential for fabrication of low-cost fiber ribbon and fiber array connectors.

Localized CO2 laser damage repair of fused silica optics.

A technique has been developed for the localized treatment of laser damage sites in fused silica optics by CO2 laser melt-flow smoothing, by using a 50 to 125 microm diameter beam in a regime that avoids mass removal by ablation. A detailed calibration of the laser irradiance for the threshold ablation of craters was carried out for a range of beam diameters and pulses in the 20 micros to 200 ms range. The results agree with a thermal model that also provides estimates of the melt depth for the different irradiation conditions. Smoothing trials for glass melting at irradiance values just below the ablation threshold irradiance were conducted to determine the optimum conditions and limits for the smoothing process. The technique has been found to remove damage pits up to a depth of 0.5 microm, while the small melt depth associated with localized treatment limits the smoothing to a

Correction of beam errors in high power laser diode bars and stacks.

The beam errors of an 11 bar laser diode stack fitted with fast-axis collimator lenses have been corrected by a single refractive plate, produced by laser cutting and polishing. The so-called smile effect is virtually eliminated and collimator aberration greatly reduced, improving the fast-axis beam quality of each bar by a factor of up to 5. The single corrector plate for the whole stack ensures that the radiation from all the laser emitters is parallel to a common axis. Beam-pointing errors of the bars have been reduced to below 0.7 mrad.

High-average-power Nd:YAG planar waveguide laser that is face pumped by 10 laser diode bars.

A planar waveguide Nd:YAG laser is pumped with 430 W of power from 10 laser diode bars to produce a multimode output power of 150 W at an optical efficiency of 35%. Use of a hybrid resonator of the positive-branch confocal unstable type for the lateral axis and of one of the near-case I waveguide type for the transverse axis increased the laser brightness by a factor of ~26 with only 12% less power than in the multimode case.

Use of dual energy x-ray absorptiometry for noninvasive body composition measurements in clinically normal dogs.

OBJECTIVE: To determine quantitative values for components of body composition in clinically normal dogs of various breeds by use of dual energy x-ray absorptiometry (DEXA) and validate the precision and accuracy of DEXA technology in dogs. ANIMALS: 103 clinically normal sexually intact adult dogs. PROCEDURE: In a cross-sectional study, Beagles, Pembroke Welsh Corgis, Golden Retrievers, Great Danes, Pointers, Rottweilers, and nonpurebred dogs received total body DEXA scans. For the validation portion of the study, the results of DEXA scans of 6 dogs were compared with values obtained by chemical analyses of tissues from euthanatized dogs to determine the accuracy of this modality in dogs. RESULTS: Results (coefficient of variation) of the precision tests ranged from 0.10% for lean tissue to 5.19% for fat tissue, whereas accuracy tests revealed a difference between percentage bone mineral content and ash values. Body composition differed by sex, such as higher lean tissue and bone mineral content in males within some breeds, and among breeds. Regardless of body size or weight, the percentage of body weight that was bone mineral ranged from 3 to 4.0% [corrected]. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this cross-sectional study provide valuable body composition data for clinically normal adult dogs, which may have research and clinical applications.

Body composition of growing and adult cats as measured by use of dual energy X-ray absorptiometry.

BACKGROUND AND PURPOSE: Total body scans were performed on 89 domestic cats of various ages, using dual energy x-ray absorptiometry (DEXA) to determine body composition, including fat, lean, and bone mineral content. Bone mineral density results from scans also are presented. METHODS: This cross sectional study included data for cats from a closed colony and from privately owned cats. Data were grouped by age and were analyzed by sex and reproductive status to provide information as to the rate of growth of the individual components of body composition. RESULTS: The results indicate that the rate of accretion of bone mineral, fat, and lean tissue differs throughout maturation and by sex. Regressions are provided to highlight age- and sex-related differences. CONCLUSIONS: The results of this study emphasize the benefits of examining the growth of each component of body composition when studying the effects of nutrition, disease processes, or therapeutic interventions.

Intracavity Beam Behavior in Hybrid Resonator Planar-Waveguide CO(2) Lasers.

We describe a combined computer simulation and experimental investigation of the intracavity spatial beam profile characteristics of a planar-waveguide rf-excited CO(2) laser that incorporates a hybrid waveguide confocal unstable negative-branch resonator. The study includes results for the intracavity lateral beam intensity profile and output power of the laser as a function of resonator mirror misalignment. In addition, the behavior of the unstable resonator, observed experimentally and predicted by the simulation, in generating localized high intensity hot-spots when it is subjected to relatively large misalignment angles is reported.

Alterations in the growth hormone/insulin-like growth factor I pathways in feline GM1 gangliosidosis.

Cats affected with feline GM1 gangliosidosis, an autosomal, recessively inherited, lysosomal enzymopathy, have progressive neurological dysfunction, premature thymic involution, stunted growth, and premature death. Although increased membrane GM1 gangliosides can result in increased apoptosis of thymocytes, there is not a direct correlation between thymocyte surface GM1 and thymic apoptosis in vivo, suggesting that other factors may be important to the pathogenesis of thymic involution in affected cats. Because GH and insulin-like growth factor I (IGF-I) are important hormonal peptides supporting thymic function and affecting growth throughout the body, particularly in the prepubescent period, several components of the GH/IGF-I pathway were compared in GM1 mutant and normal age-matched cats. GM1 mutant cat serum IGF-I concentrations were reduced significantly compared with those in normal cats by 150 days of age, and GM1 mutant cats had no peripubertal increase in serum IGF-I. Additionally, IGF-binding protein-3 was reduced, and IGF-binding protein-2 was elevated significantly in GM1 mutant cats more than 200 days of age. Liver IGF-I messenger RNA and pituitary GH messenger RNA both were reduced significantly in GM1 mutant cats. After stimulation by exogenous recombinant canine GH, serum IGF-I levels increased significantly in GM1 mutant cats, indicating that GH/IGF-I signaling pathways within the liver remain intact and suggesting that alterations are external to the liver.

The transport of polymeric microspheres across the ciliated epithelia of the bullfrog.

The influence of some hydrophilic polymers on the clearance of particles across the ciliated epithelium of the bullfrog palate has been examined. The polymers studied were Carbopol 907 cross-linked with maltose to provide microspheres of varying cross-link density, Carbopol 934P, hydroxypropylmethylcellulose, chitosan and poly(vinyl alcohol). Transport rates were determined relative to glass spheres. The polymers in dilute solution (0.1 and 0.5% w/v) resulted in a reduction in the transport rate of the glass spheres. For non-cross-linked microspheres, Carbopol 934P exhibited a lower transport rate than the more slowly hydrating chitosan. The cross-linked poly(acrylic acid) microspheres showed clearance rates which were dependent on the cross-link density. Incorporation of some preservatives (EDTA, methylhydroxybenzoate, chlorbutol and chlorocresol), known to reversibly retard clearance, into the cross-linked poly(acrylic acid) microspheres produced effects dependent on cross-link density: lightly cross-linked microspheres were cleared more slowly than the preservative-free microspheres whilst for more heavily cross-linked particles the converse was observed.

Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human carcinoembryonic antigen.

A nucleic acid vaccine encoding human carcinoembryonic antigen (CEA) was administered to 10 juvenile dogs, 10-15 weeks of age, by four parenteral routes. The routes tested were intramuscular injection using a needle and syringe, intramuscular injection using the Biojector needleless injection device, intradermal injection or intravenous injection. All groups received 150 micrograms of plasmid DNA on weeks 0, 4, 7 and 13. All dogs were bled weekly for 17 weeks and tested for antibody against human CEA. Dogs given plasmid intramuscularly either by needle and syringe or Biojector showed significant antibody responses by week 9 which peaked by week 15. Dogs receiving plasmid intravenously showed slight, unsustained increases in antibody titers while dogs receiving plasmid intradermally had significant titers, but at levels approximately one log less than those induced by intramuscular injection. The five dogs immunized by intramuscular delivery of plasmid DNA were examined for cellular immune responses to human CEA by lymphoblast transformation (LBT) assay. All five showed significant CEA-specific lymphoproliferation when compared with unvaccinated dogs. Physical examination, clinical chemistry, hematology and histopathology examinations revealed no abnormalities associated with nucleic acid immunization.

Evaluation of the effects of omega-3 fatty acid-containing diets on the inflammatory stage of wound healing in dogs.

OBJECTIVES: To ascertain the effects of dietary omega-3 (n-3) fatty acids on biochemical and histopathologic components of the inflammatory stage of wound healing. ANIMALS: 30 purpose-bred Beagles. PROCEDURE: Dogs were allotted to 5 groups of 6. Each group was fed a unique dietary fatty acid ratio of omega-6 to n-3--diet A, 5.3:1; diet B, 10.4:1; diet C, 24.1:1; diet D, 51.6:1; and diet E, 95.8:1. Dogs were fed once daily for 12 weeks, then biopsy specimens were taken from 4-day-old wounds of each dog and analyzed by gas chromatography-mass spectrometry for: prostaglandin E2 (PGE2) metabolites, and ratios of omega-6 to n-3 fatty acids, arachidonic acid (AA) to eicosapentaenoic acid (EPA), adrenic acid to docosahexaenoic acid, and PGE2 to prostaglandin E3 (PGE3) metabolites. RESULTS: Qualitative analysis was carried out on AA, EPA, adrenic acid, docosahexaenoic acid, and the major metabolite from the PGE2 and PGE3 pathway. These molecules were further quantified with respect to diet to determine significant differences. By analysis of the AA-to-EPA ratio, diet A was different from diets D and E and diets B and C were different from diet E (P < 0.05). By analysis of the PGE2-to-PGE3 metabolite ratio, diet A was different from diet E (P < 0.05). Though biochemical analysis indicated dietary dependence, histopathologic data indicated no significant difference with respect to diet groups. CONCLUSION: The biochemical component of the inflammatory stage of wound healing can be manipulated by diet. CLINICAL RELEVANCE: Omega-3 fatty acid-enriched diets can be used to control inflammation associated with dermatologic conditions.

Thymic alterations in feline GM1 gangliosidosis.

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.

Gangliosides enhance apoptosis of thymocytes.

Monosialogangliosides, normal components of cell membranes, regulate cell development and differentiation in several organs. Our previous observation of dramatic premature thymic involution in cats with feline GM1 gangliosidosis, whose thymocytes have abnormally high cell surface gangliosides, suggested that excess GM1 ganglioside (GM1) could modulate thymocyte apoptosis in this disease (Cox et al., "Thymic Alterations in Feline GM1 Gangliosidosis," submitted). In these studies, we added exogenous GM1 to murine primary thymocyte cultures and demonstrated enhanced apoptosis in treated cells by DNA fragmentation, apoptotic body, and electrophoretic analyses. GM1-enhanced apoptosis was blocked by common apoptotic pathway inhibitors including aurintricarboxylic acid (inhibitor of endonuclease activity), actinomycin D (inhibitor of RNA transcription), and cycloheximide (inhibitor of protein synthesis). GM1 treatment primarily affected the immature CD4+ CD8+ subset, as shown by flow cytometric evaluation of fetal thymic organ culture and primary thymocyte cultures. Apoptosis also could be induced by GM2, GM3, and GT1b, whereas asialo-GM1 failed to do so, suggesting that the sialic acid moiety may play an important role in the induction of thymocyte apoptosis.

Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus.

Nucleic acid vaccines (NAVs) use expression vectors encoding one or more antigen genes to transfect host cells inducing both humoral and cellular immunity against the expressed antigen. NAV offers major advantages over conventional vaccines for the protection of humans and animals. This study shows that a plasmid DNA (pGT36VP1) encoding the full length VP1 region of canine parvovirus (CPV) induces immunity that protects dogs against challenge with virulent virus. Five dogs without anti-CPV antibodies were injected at 9 months of age with increasing doses of pGT36VP1 or saline. NAV vaccinated dogs showed an increase of serum IgG titer starting 1 week post-injection which peaked at week 2 and remained detectable for at least 14 weeks. A second dose of NAV resulted in an anamnestic response within 1 week. IgG titers peaked at week 3 and 4 after the second injection. All pGT36VP1 vaccinated dogs were protected against infection after virulent CPV challenge regardless of dose and the unvaccinated control dog was fully susceptible. This study demonstrated for the first time that a NAV can protect dogs against an infectious disease.

Profile of electrodiagnostic abnormalities in cats with GM1 gangliosidosis.

OBJECTIVE: To determine which electrodiagnostic tests yield abnormal findings in cats with GM1 gangliosidosis, and to determine the approximate age of onset of electrodiagnostic abnormalities. ANIMALS: Cats (28 to 335 days old) affected with GM1 gangliosidosis (n = 11) and unaffected controls (n = 14). PROCEDURE: Cats were grouped by age: group 1, < or = 90 days, group 2, 91 to 200 days; and group 3, > 200 days. Electrodiagnostic tests were conducted, including needle electromyography, motor and sensory nerve conduction velocity, spinal evoked potentials, and brainstem auditory evoked potentials. Results for control and affected cats were compared, using the general linear model for ANOVA and Scheffé's test for multiple comparisons. RESULTS: Needle electromyography did not reveal abnormal spontaneous activity in skeletal muscles of any cat; furthermore, statistical analysis did not indicate significant difference between affected and control groups for nerve conduction velocity, confirming that degeneration of peripheral nerve fibers is not a feature of this disease. However, spinal evoked potentials were abnormal in group-3 cats; conduction velocity within sensory pathways in the cranial part of the spinal cord was significantly slower in GM1-affected cats (P = 0.0002). Brainstem auditory evoked responses also were abnormal: wave V (generated in the region of the pons) had prolonged latency in cats of groups 2 and 3 (P = 0.0003 and 0.0001, respectively, at 90 decibels sound pressure level). In the oldest cats, latencies for earlier waves within the auditory pathway also were prolonged; wave I (generated by the cochlear nerve) was prolonged in group-3 cats (P = 0.0423). CONCLUSIONS: Motor and sensory nerve conduction velocities remained within normal limits in GM1-affected cats. However, spinal evoked potentials indicated slowing in conduction velocity along the cranial part of the spinal cord in group 3 cats. Brainstem auditory evoked responses indicated prolonged latencies in cats of groups 2 and 3.

Improvement of hemispatial neglect with cold-water calorics: an electrophysiological test of the arousal hypothesis of neglect.

Introducing ice-water into the left ear of right-brain-damaged patients attenuates unilateral neglect symptoms. By examining EEG changes over each hemisphere during this procedure, we were able to test a hypothesis concerning the mechanism of cold-water calorics and the attention-arousal hypothesis of hemispatial neglect. We present a case study of an 83-year-old woman with a massive right-hemisphere CVA exhibiting severe hemispatial neglect. Caloric stimulation produced a leftward eye deviation to central position, and a temporary partial remission of neglect symptoms. Significant changes in EEG activation indicated a central mechanism associated with the regularization of eye gaze. Caloric stimulation also produced a significant interaction between EEG frequency band and hemisphere, indicating that while both hemispheres increased in cortical activation, the right hemisphere increase was significantly greater. This supports the activation-arousal hypothesis of neglect over the mutual inhibition model.

Face pumping of thin, solid-state slab lasers with laser diodes.

A new technique for face pumping of slab lasers uses transfer of light from 10 quasi-cw laser diode bars through a slotted mirror into a rectangular, highly ref lective pump chamber, giving efficient multipass pumping of a thin Nd:glass slab laser. A slope efficiency of 28% and a maximum pulse energy of 65 mJ have been obtained, and gain and loss measurements with thickness t = 0.45-1.04 mm have confirmed the 1/t scaling of gain in thin slabs and the high efficiency of pump light transfer.