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.

Skeletal contribution of cyclic adenosine monophosphate in response to parathyroid hormone and calcitonin in vivo in the rat.

Cyclic adenosine monophosphate (cAMP) is thought to be a second messenger for the actions of both parathyroid hormone (PTH) and calcitonin (CT). We examined the release of cAMP from rat bone in vivo after administration of synthetic rat PTH-(1-34) (rPTH), synthetic human PTH-(1-34) (hPTH), or synthetic human CT (hCT). Blood from the venous effluent of the femoral bone of rats (bone blood) was drawn at 5 and 10 minutes after the administration of hormones. The cAMP content of the bone blood was then compared to the cAMP content of arterial blood. In both kidney-clamped and non-kidney-clamped rats, hCT led to a significantly greater concentration of cAMP in the bone blood than in the arterial blood. We interpret this to be due to bone production and release of cAMP. Neither hPTH nor rPTH produced a significantly greater amount of cAMP in the bone blood than in arterial blood. These data do not preclude the possibility that there was a production of cAMP within the bone tissue itself after PTH but suggest that there was no release of cAMP from the bone into the bone blood.

Squamous cell carcinomas often produce more than a single bone resorption-stimulating factor: role of interleukin-1 alpha*.

Several cultured human squamous cell carcinoma cell lines (SCC-4, SCC-12B2, SCC-12F2, EC-GI-10, and BEN) and one normal keratinocyte line (Epy-1) were investigated for the production of bone resorption-stimulating activity (BRSA). Conditioned medium (CM) from each of the six cell lines stimulated bone resorption in neonatal mouse calvariae in culture. The BRSA of SCC-12F2 and EC-GI-10 was inhibited completely by antibody to interleukin-1 alpha (IL-1 alpha), whereas the BRSA in CM from the BEN, SCC-4, SCC-12B2, and Epy-1 cell lines was only partially inhibited by anti-IL-1 alpha. Addition of indomethacin to the calvarial cultures also partially inhibited the BRSA from EC-GI-10, SCC-4, SCC-12B2, and Epy-1 cells; the BRSA from BEN and SCC-12F2 cells was inhibited completely by indomethacin. cAMP production by calvariae was determined after a 60-min incubation with CM. CM from EC-GI-10, BEN, SCC-4, and Epy-1 stimulated cAMP production by bone. Preincubation of CM from BEN, EC-GI-10, SCC-4, and Epy-1 cells with two antisera against PTH-related protein [PTHrP; one specific for two PTHrP-(1-141), the other recognizing both PTHrP-(1-40) and PTHrP-(1-141)] completely inhibited the cAMP-stimulating activity. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in CM of the SCC-4, SCC-12B2, SCC-12F2, and Epy-1 cell lines. IL-1 beta was undetectable (less than 0.1 ng/ml) in CM from all cell lines. Our findings indicate that the BRSA secreted by SCC-12F2 cells can be accounted for largely or entirely by IL-1 alpha, while the activity produced by SCC-12B2 includes IL-1 alpha and another unknown factor(s). The BRSA produced by EC-GI-10, BEN, SCC-4, and Epy-1 cells includes both IL-1 alpha and PTHrP. We conclude that IL-1 alpha may be a more prevalent and biologically significant component of the BRSA produced by SCCs than previously recognized.

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.

Molecular markers of glial tumors: current targeting strategies.

Diagnosis and therapy for malignant gliomas represents one of the most challenging problems in clinical oncology. Current treatment of malignant glioma is multimodal, involving surgical resection, radiotherapy and chemotherapy. Even with these combined therapies, patients usually die within 1 to 2 years after onset of symptoms. Clearly, improved strategies for selective delivery of therapeutic agents to gliomas are needed to combat these devastating and usually fatal cancers. This review summarizes current knowledge concerning targetable molecular markers on the surface of glial tumor cells and tumor vasculature. Such markers are altered or up-regulated in gliomas compared to normal tissues, or they might be glioma-restricted. These markers include growth factor receptors, cell-surface adhesion molecules, and membrane-type matrix metalloproteinases. Current approaches that utilize growth factor peptides and peptide/antibodies identified via phage display technology as carrier ligands for targeting malignant gliomas are discussed.

Clofazimine analogues active against a clofazimine-resistant organism.

Clofazimine analogues active against a strain of Mycobacterium smegmatis 607 made resistant to the antileprosy agent have been synthesized. Activity (i.e., less than or equal to 2 micrograms/mL causing complete inhibition of growth) requires that there be a basic nitrogen in the "rimino" side chain and that the spacer distance between this nitrogen and the imino nitrogen be at least three carbon atoms. The nitrogen may be primary, secondary, or tertiary and may be part of an open chain or enclosed in a ring compound. Provided that the criteria of basicity and spacer distance are satisfied, all are active in vitro against both the sensitive and resistant strains. Substitution elsewhere in the molecule had little effect on the activity. The compounds have been shown to have growth inhibitory activity against human-derived Mycobacterium leprae in murine macrophages in culture.

Differences in binding affinities of human PTH(1-84) do not alter biological potency: a comparison between chemically synthesized hormone, natural and mutant forms.

The purpose of this study was to evaluate receptor binding affinities and biological properties in vitro and in vivo of various recombinant hPTH(1-84) forms representing the natural hormone and a mutagenized hPTH form, [Gln26]hPTH(1-84) (QPTH), after expression in E. coli and Saccharomyces cerevisiae. In LLC-PK1 cells stably transformed with the rat PTH/PTHrP receptor, chemically synthesized hPTH(1-84) and QPTH showed a reduced binding affinity (apparent Kd 18 and 23 nM, respectively) than the recombinant, hPTH(1-84) (apparent Kd 9.5 nM). All recombinant hPTH forms showed a similar potency to stimulate cellular cAMP production (EC50 1.5 nM) and significantly better than chemically synthesized hPTH (EC50 5.7 nM). All hormone forms showed an about equipotent activity in causing elevation in serum calcium, increased excretion of urine phosphate, and cAMP. Thus, the natural recombinant PTH forms showed higher binding affinities and adenylate cyclase activation potencies in LLC-PK1 cells, but the reduced receptor binding affinity exerted by QPTH did not transcend differences in cAMP generation and in vivo biological activities.

Plural medicine in Sri Lanka: do Ayurvedic and Western medical practices differ?

In Sri Lanka, as in India, two formally structured systems of medicine exist side by side. While Western-style biomedicine is believed to be useful, Ayurvedic medicine is well established and commonly used. Underlying one explanation for the persistence of such plural medical systems is a functional theory, suggesting that each system is used for different treatments, diseases, or for the ideological, linguistic or social characteristics of the physician. In part, Ayurvedic and Western medicine may persist because their practitioners provide distinctly different services. We tested part of this functional explanation by sending trained 'pseudo-patients' to 764 Ayurvedic and allopathic physicians across Sri Lanka. 'Patients' reported symptoms of common cold, diarrhea or back pain, and recorded after leaving the clinic many aspects of history-taking, diagnostic procedures and physician-patient interaction. Medicines prescribed were later analyzed by a laboratory. We found, basically, no significant differences between the medical practices of sampled Ayurvedic and Western-style physicians, with one exception. While both types spend 3-4 min asking four questions and doing two or three physical examination procedures, and while both prescribe, overwhelmingly, only Western medicines, the allopathic physicians give drugs, that, from the point of view of Western medicine, either 'help' or 'harm' and Ayurvedic physicians prescribe 'neutral' medicines. While we have not directly tested the entire functional explanation we suggest that a structural explanation of the persistence of two systems of medicine may be more valid. Ayurvedic and Western medicine continue in Sri Lanka because they, as institutions, are linked to the social, economic and political structure of the society. Thus, survival is based, not on what a physician does in his practice but upon the power of his medical profession to control medical territory.

Evaluation of GM1 ganglioside-mediated apoptosis in feline thymocytes.

Cats with inherited GM1 gangliosidosis (GM1 mutant cats) have premature thymic involution characterized by decreased total thymocytes primarily affecting the CD4+ CD8+ subpopulation. While GM1 mutant cats have increased cell surface GM1 gangliosides, as determined by cholera toxin B binding, on both thymocytes and peripheral lymph node cells only thymocytes show increased apoptosis. To determine if GM1 gangliosides can increase the occurrence of apoptosis in feline thymocytes directly, we added exogenous GM1 ganglioside (GM1) to feline thymocyte primary cultures and compared the results to apoptotic changes seen in untreated cells or in cells treated with dexamethasone (Dex), a known inducer of thymocyte apoptosis in other species. Incorporation of exogenous GM1 into thymocyte cytoplasmic membranes was confirmed by flow cytometric analyses of cholera toxin B labelling. Apoptosis in feline thymocytes was analyzed by electron microscopy, spectrophotometric evaluation of DNA fragmentation, flow cytometric enumeration of apoptotic nuclei, and gel electrophoretic analysis of degraded DNA. Alterations in percentages of thymocyte immunophenotype following GM1 incorporation were determined by flow cytometric analyses of labelled cell surface markers for feline CD4 and CD8. Because in vitro addition of GM1 gangliosides has been reported in other species to decrease surface expression of CD4 on both thymocytes and peripheral lymphocytes, we evaluated GM1-associated down-regulation of CD4 on the surface of feline thymocytes and peripheral lymph node cells by flow cytometry. Additionally, we compared the apoptotic response of the more mature peripheral lymph node cells to the less mature thymocytes. Our results indicate that incorporation of exogenous GM1 into feline thymocyte cell membranes produces a dose-dependent increase of apoptotic cell death. Although, CD4 expression on both feline thymocyte and lymph node cell membranes was abruptly decreased after introducing exogenous GM1, enhanced apoptotic death was observed only in thymocytes, not in lymph node cells at the same GM1 concentration. Enhancement of thymocyte apoptosis appears to be age-related since cells derived from cats <3 months of age were more vulnerable than those from cats >3 months of age.

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.

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.

Factors influencing oral health in long term care facilities.

In a stratified random sample of 41 long term care (LTC) facilities in Vancouver, 653 residents were chosen to investigate oral health needs and demands for treatment. All of the 603 dentists in the same area were questioned to assess their interest in attending the residents of the institutions. The information from each source was reviewed to identify factors influencing the oral health services to this predominantly elderly and medically compromised population. The majority (60%) of the residents were edentulous and they made infrequent demands on dentists. Two-thirds of those interviewed said that there was nothing wrong with their mouths, but most of those who were aware of a problem wanted it treated, preferably within the institution. They complained about loose or uncomfortable dentures most frequently, and many were dissatisfied with previous dental treatment. The oral mucosal lesions seen on examination were usually symptomless and associated with poor hygiene, while structurally defective dentures and deep carious lesions were not uncommon. The responding 334 dentists indicated that they enjoyed treating elderly patients, 19% had attended an LTC facility, usually to provide an emergency service, and 37% were willing to provide this service if asked. Interest, however, in the service was curtailed by pressures from private practice, concerns about inadequate training and the small demand and poor conditions in the facilities. Although the demand for treatment was not extensive from the residents, they did have problems that were not receiving care.

Opinions of dentists on the treatment of elderly patients in long-term care facilities.

The high prevalence of disorders in the mouths of elderly residents in long-term care (LTC) facilities suggests that the dental services available to the residents are inadequate. A questionnaire was sent to most of the dentists practicing in Vancouver (British Columbia) to solicit their opinions on treating older patients in LTC facilities, and a response was obtained from 334 (55%). It indicated that 19 percent of the respondents had treated old people within this context, although few of them felt they had been educated adequately for the service. Many reasons were offered to explain the small participation. Most respondents had never been asked to attend a facility. Many felt that it interfered with their practice and leisure, and they were concerned about the limited options available for treatment. Dentists who had attended facilities were motivated by a sense of professional or public responsibility, but they were uneasy about the limited options and about the inadequate space and equipment available. In general, the dentists were not interested in attending an institutionalized geriatric population, and they felt ill-prepared for the service.

The influence of social, economic, and professional considerations on services offered by dentists to long-term care residents.

Many studies have focused on the disabilities and behavior of the elderly population in an attempt to explain the frequent reports of poor oral health among residents of long-term care (LTC) facilities, but little attention has been given to the experiences and opinions of dentists relating to the problem. This study was conducted to discover how dentists feel about older patients and about working in LTC facilities. A response to a questionnaire was obtained from 334 (55%) of the 603 dentists in Vancouver who treated adults. The responses were subjected to bivariate and multivariate analyses. Three models were constructed from factors that might interest a dentist in attending a patient in a facility; the factors in each model were ranked in order of importance. Interest was associated significantly with lack of concern for time lost in practice, with training in managing medically compromised patients, and with a positive attitude toward elderly patients. Dentists with fewer years in practice were attracted by the economic potential of the service, while the older and busier dentists were less involved because of the disruption to their practice and leisure. Dentists who made home visits also were more likely to be interested. The model based on professional considerations was superior to either the social or economic model in explaining an interest in the service.

Effect of locally injected medications on healing of pad wounds in dogs.

OBJECTIVE: To ascertain the effects of locally injected immunostimulant and tripeptide-copper complex (TCC) on improving healing of pad wounds. DESIGN: Wounds in pads of large dogs were injected with either medication or physiologic saline solution (controls). Healing was evaluated. ANIMALS: 12 mature English Pointers. PROCEDURE: Full-thickness 6 x 8-mm wounds in metatarsal and third and fourth digital pads were injected with immunostimulant or TCC at 0, 3, and 6 days after wounding. Wounds on control dogs were injected with physiologic saline solution. Using planimetric measurements at 0, 3, 6, 14, and 21 days, rates of healing were evaluated. Biopsy of the digital pad wounds at 3, 6, and 14 days was used to evaluate collagen content by hydroxyproline analysis. Biopsy specimens were also evaluated for type-I and type-III collagen, using Sirius red differential staining. RESULTS: Effect on healing rate and hydroxyproline content was best during the first week for immunostimulant. Immunostimulant- and TCC-injected wounds had more type-I collagen than did controls at 6 days; TCC-injected wounds had the most type-I collagen. At 14 days, the amount of type-I collagen in TCC-injected wounds was significantly greater than that in other wounds. CONCLUSIONS: Tested medications had positive effects on healing of pad wounds. CLINICAL RELEVANCE: Intralesional injection of medications helps ensure their presence for enhancement of wound healing. The benefit could be lost with topical use in a bandage if the bandage is lost or becomes wet.

Peptide phage display: opportunities for development of personalized anti-cancer strategies.

Personalized medicine is critical for cancer patients, because (1) cancer is a highly heterogeneous disease with major molecular differences in the expression and distribution of tumor cell surface markers among patients with the same type and grade of cancer, (2) cellular mutations tend to accumulate as cancer progresses, further increasing tumor heterogeneity, and (3) currently used cancer therapies often are toxic to normal cells, causing severe side effects rarely seen in other diseases. To prevent side effects and to improve effectiveness, cytotoxic therapies should be targeted and each patient should be profiled for the presence of cancer targets before the therapy is administered. Phage display technology utilizes combinatorial libraries of proteins expressed on phage particles that can be selected for specific binding to cancer cells. Such cancer-specific molecules can be used in a variety of applications, including identification of cell-specific targeting molecules; identification of cell surface biomarkers; profiling of specimens obtained from individual cancer patients, and the design of peptide-based anti-cancer therapeutics for personalized treatments. This review is focused on peptide phage display strategies that target cell surfaces because many biomarkers important in cancer are differentially expressed molecules located on the outside of the cell membranes.

Restoration of T-cell responsiveness by thymosin: development of antituberculous resistance in BCG-infected animals.

T-cell-depleted (adult thymectomized, lethally irradiated, bone marrow-reconstituted [THXB]), sham-thymectomized (XB) and normal control mice were injected daily with 3 mg of calf thymosin for 16 days. On day 8 of the treatment, the mice, together with untreated controls, were infected intravenously with 4 X 10(6) viable Mycobacterium bovis (BCG Montreal). Growth of the BCG in the lungs and spleens was compared quantitatively for up to 100 days. Thymosin treatment reversed the progressive weight loss seen in BCG-infected THXB mice and prevented their death due to the ongoing mycobacteriosis that developed in the T-cell-depleted animal. There was a late-developing anti-mycobacterial response in the thymosin-treated THXB mice, which resulted in a progressive decline in viability for the lung and spleen populations over the 40- to 80-day period, when the corresponding counts for the untreated THXB mice remained relatively constant. The histopathology of the lung and the increased antibacterial activity seen in the thymosin-treated THXB mice correlated with decreased [3H]deoxyribonucleic acid levels seen in the lungs and spleen compared with that present in the T-cell-depleted controls.