Necrotizing meningoencephalitis of Pug dogs associates with dog leukocyte antigen class II and resembles acute variant forms of multiple sclerosis.

Necrotizing meningoencephalitis (NME) is a disorder of Pug Dogs that appears to have an immune etiology and high heritability based on population studies. The present study was undertaken to identify a genetic basis for the disease. A genome-wide association scan with single tandem repeat (STR) markers showed a single strong association near the dog leukocyte antigen (DLA) complex on CFA12. Fine resolution mapping with 27 STR markers on CFA12 further narrowed association to the region containing DLA-DRB1, -DQA1 and, -DQB1 genes. Sequencing confirmed that affected dogs were more likely to be homozygous for specific alleles at each locus and that these alleles were linked, forming a single high risk haplotype. The strong DLA class II association of NME in Pug Dogs resembles that of human multiple sclerosis (MS). Like MS, NME appears to have an autoimmune basis, involves genetic and nongenetic factors, has a relatively low incidence, is more frequent in females than males, and is associated with a vascularly orientated nonsuppurative inflammation. However, NME of Pug Dogs is more aggressive in disease course than classical human MS, appears to be relatively earlier in onset, and involves necrosis rather than demyelination as the central pathobiologic feature. Thus, Pug Dog encephalitis (PDE) shares clinical features with the less common acute variant forms of MS. Accordingly, NME of Pug Dogs may represent a naturally occurring canine model of certain idiopathic inflammatory disorders of the human central nervous system.

Development of an in vitro model of excess intracellular reactive oxygen species.

These investigations characterize an in vitro model for generating excess intracellular reactive oxygen species (ROS). This novel model may be useful in the identification and delineation of cellular mechanisms associated with aging due to the link between age and excess oxidative events. The human cell line, MCF7, was stably transfected using the pSV3.neo plasmid housing a gene encoding the Aequorea victoria green fluorescent protein (GFP). Transfected cells were analyzed for maintenance of GFP over time, showing stability of the GFP gene. These studies demonstrate that the presence of fluorescing GFP significantly increases intracellular ROS, creating oxidative stress in these cells. Antioxidant supplementation was evaluated to determine the effectiveness of intracellular H2O2 reduction. The results demonstrate that supplementation with a potent antioxidant, such as reduced glutathione, protects cells from oxidative damage by decreasing intracellular concentrations of H2O2. This model for intracellular generation of excess ROS establishes a clear method by which the utility of antioxidant supplementation to protect against intracellularly generated reactive oxygen species may be evaluated.

Digging up the canine genome--a tale to wag about.

There is incredible morphological and behavioral diversity among the hundreds of breeds of the domestic dog, CANIS FAMILIARIS. Many of these breeds have come into existence within the last few hundred years. While there are obvious phenotypic differences among breeds, there is marked interbreed genetic homogeneity. Thus, study of canine genetics and genomics is of importance to comparative genomics, evolutionary biology and study of human hereditary diseases. The most recent version of the map of the canine genome is comprised of 3,270 markers mapped to 3,021 unique positions with an average intermarker distance of approximately 1 Mb. The markers include approximately 1,600 microsatellite markers, about 1,000 gene-based markers, and almost 700 bacterial artificial chromosome-end markers. Importantly, integration of radiation hybrid and linkage maps has greatly enhanced the utility of the map. Additionally, mapping the genome has led directly to characterization of microsatellite markers ideal for whole genome linkage scans. Thus, workers are now able to exploit the canine genome for a wide variety of genetic studies. Finally, the decision to sequence the canine genome highlights the dog's evolutionary and physiologic position between the mouse and human and its importance as a model for study of mammalian genetics and human hereditary diseases.

The role of folate transport and metabolism in neural tube defect risk.

Neural tube defects (NTDs) are common congenital malformations in humans. While etiologically heterogeneous, for the most part they are multifactorial in their pathogenesis, having both genetic and environmental factors contributing to their development. In recent years, there has been a great deal of epidemiologic evidence demonstrating that women who received multivitamins containing folic acid periconceptionally had significantly reduced occurrence and recurrence risks for producing infants with such malformations. Unfortunately, the mechanism(s) underlying the beneficial effects of folic acid is not well understood. In this article, we review the fundamental embryological processes involved in closing the neural tube, the relevant epidemiologic data on folic acid supplementation and relative NTD risk, as well as several recent studies of candidate genes for NTD sensitivity that are involved in folate transport and metabolism.

Expression patterns of folate binding proteins one and two in the developing mouse embryo.

Expression patterns of mRNAs coding for the murine folate binding proteins one and two (FBP1 and FBP2) were determined by ribonuclease protection assay (RPA) in highly inbred SWV/Fnn mouse embryos. Tissue samples for RPA were collected from the anterior neural tube throughout the period of embryonic development, as well as from maternal- and fetal-derived term placenta. The peak in expression of FBP1 occurred in term placental tissue compared to neural tissue from any time point. This relative increase in FBP1 expression occurred in placental tissue of embryonic, as opposed to maternal, origin. The expression of FBP2 did not differ statistically between any timepoints or tissues examined. Expression of both FBP1 and FBP2 was slightly elevated throughout the period of neural tube closure (Gestational Days 8 through 10), although not significantly. These data fit the anticipated expression patterns of the homologues of human folate receptors alpha and beta, thus helping to resolve some of the confusion secondary to the nomenclature associated with this gene family. Furthermore, the expression of these two genes in the neural tube closure stage of embryological development supports their involvement in regulatory events related to normal neural tube morphogenesis.

Power output of pericardium-lined skeletal muscle ventricles, left ventricular apex to aorta configuration: up to eight months in circulation.

OBJECTIVE: The purpose of this experiment was to evaluate the potential for a skeletal muscle ventricle connected to the circulation between the left ventricle and the aorta to provide effective, long-term cardiac assist. METHODS: Skeletal muscle ventricles were constructed from the latissimus muscle in 10 dogs. After conditioning, the skeletal muscle ventricles were connected to the left ventricle and the aorta with 2 valved conduits. The skeletal muscle ventricle was programmed to contract during diastole. RESULTS: At time of implantation, skeletal muscle ventricles stimulated at 33 Hz and in a 1:2 ratio with the heart significantly decreased left ventricular work by 56% (P <.01) and at 50 Hz by 65% (P <.01). At a 1:2 ratio, the power output of the skeletal muscle ventricles was 59% of left ventricular power output at 33 Hz (P <. 01) and 93% at 50 Hz (P <.01). Animals survived 7, 11, 16, 17, 72, 99, 115, 214, and 249 days. Three deaths were directly related to the skeletal muscle ventricle. One animal is alive at 228 days. In the animal that survived 249 days, skeletal muscle ventricle power output at 8 months with a 33 Hz stimulation frequency and a 1:2 contraction ratio was 57% of left ventricular power output and 82% at 50 Hz. At a 1:1 ratio, skeletal muscle ventricle power output was 97% and 173% of the left ventricle at 33 and 50 Hz, respectively. CONCLUSIONS: Left ventricular assist with a skeletal muscle ventricle connected between the left ventricle and the aorta is the most hemodynamically effective configuration we have tested and can maintain significant power output up to 8 months.

Lack of association between mutations in the folate receptor-alpha gene and spina bifida.

Defects of neural tube closure are among the most common of all human malformations. Epidemiological and genetic studies indicate that most of these defects are multifactorial in origin with genetic and environmental causes. Although periconceptional supplementation of the maternal diet with folic acid has been shown to reduce the recurrence and occurrence of neural tube defects (NTDs) by up to 70%, the underlying mechanism remains unknown. Folic acid enters cells of certain tissues via a receptor-mediated process known as potocytosis. The folate receptor alpha (FR-alpha) gene codes for the protein responsible for binding folate, which is the first, and only, folate-dependent step in folate transport. The FR-alpha exons, which code for mature protein and the intron-exon boundaries, were examined for mutations in three separate studies. Initial screening was performed by single-stranded conformational polymorphism (SSCP) analysis in a subset of 1,688 samples obtained from a population-based case-control study of NTDs in California. In the second study, the DNA sequence of exons 5 and 6 was determined in a group of 50 NTD affected individuals. The final experiment involved using dideoxy fingerprinting (ddF) to screen a population-based case-control sample of 219 individuals who were stratified into four sub-groups on the basis of folate intake and pregnancy outcome. No polymorphism was detected in any of the four exons examined. It is unlikely that the beneficial effects of maternal folate supplementation in preventing NTDs acts through a mechanism involving pharmacological correction of a variant form of folate receptor alpha.

Neural tube and craniofacial defects with special emphasis on folate pathway genes.

Neural tube and orofacial defects are common congenital malformations in humans. While etiologically heterogeneous, they are for the most part multifactorial in their pathogenesis, having both genetic and environmental components in their development. In recent years, there has been a great deal of epidemiologic evidence demonstrating that women who received multivitamins containing folic acid periconceptionally had significantly reduced occurrence and recurrence risks for producing infants with such malformations. This risk reduction is not observed in all populations, further suggestive of a genetic regulation of this phenomenon. Unfortunately, the mechanisms underlying the beneficial effects of folic acid are not well-understood. In this article, we review the relevant epidemiologic data on both neural tube defects and orofacial malformations, the fundamental embryological processes involved in closing the neural tube, and the development of the craniofacies, and propose a working hypothesis for susceptibility to these malformations. This hypothesis is based on the interworkings of cellular folate transport, focusing on the key elements involved in potocytosis. We propose that infants with mutations in the folate receptor alpha gene might be at increased risk for congenital anomalies due to a reduced binding affinity for 5-methyltetrahydrofolate, the physiologic form of folic acid. Various experimental approaches to test the working hypothesis are considered.

Skeletal muscle ventricles, left ventricular apex-to-aorta configuration. 1 to 11 weeks in circulation.

BACKGROUND: Skeletal muscle ventricles (SMVs) have been used in animals in a variety of configurations to provide circulatory assistance. Long-term survival and function have been demonstrated. Our laboratory recently obtained promising short-term hemodynamic data in a left ventricular apex-to-aorta model. METHODS AND RESULTS: SMVs were constructed from the left latissimus dorsi muscle in five adult mongrel dogs. After a 3-week period of vascular delay and 5 to 7 weeks of electrical conditioning, valved conduits were used to connect the left ventricular apex to the SMV and the SMV to the descending aorta. The SMV was then stimulated to contract during cardiac diastole. Initial measurements showed a significant increase in the mean femoral diastolic pressure (62 +/- 6 versus 51 +/- 5 mm Hg, P < .05). There was also a decrease in the left ventricular tension-time index (11.5 +/- 2.5 versus 14.6 +/- 2.1 mm Hg.s, P < .05), indicating a decrease in the work requirement of the left ventricle. During SMV stimulation, the majority of flow (65%) was through the SMV circuit and was associated with reversal of flow in the proximal descending thoracic aorta. The longest-surviving animal survived 76 days, at which time pressure augmentation was still seen (mean femoral diastolic pressure, 63 +/- 0.9 versus 50 +/- 1.2 mm Hg, P < .05). CONCLUSIONS: Survival beyond the acute setting is possible with this model. Diastolic pressure augmentation can be effectively maintained over time.

Skeletal muscle as a myocardial substitute.

During the last several years, there has been intense worldwide interest concerning the use of skeletal muscle as a form of cardiac assistance. For over 10,000 people in the United States diagnosed each year with irreversible heart failure, the 1-year mortality approaches 50%. This comes despite recent advances in medical therapy, heart transplantation, and the artificial heart program. Because of the limitations of these treatments in terms of effectiveness, cost, and availability, we have used a different approach for cardiac augmentation. Skeletal muscle is shaped into the form of a pumping chamber and then used to aid the function of the failing myocardium. Another approach is cardiomyoplasty, where the latissimus dorsi muscle is wrapped around the heart and stimulated to contract in synchrony with the patient's failing myocardium. More than 500 patients have undergone cardiomyoplasty worldwide. These two areas of investigation represent the principle methods for skeletal muscle cardiac assistance.

Preliminary evidence of phenytoin-induced alterations in embryonic gene expression in a mouse model.

SWV mouse embryos collected on gestational days (GD) 9:12 and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both Wnt-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:12 embryos, including the growth factors TGF-beta and NT3, the proto-oncogene Wnt-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.

Specific pattern burn in a psychiatric patient.

Position at the time of burning can be precisely determined by analysis of the depth, configuration, distribution of the burn, flexion creases and spared areas. This information is very useful in determining non-accidental injuries in children. Although specific pattern injuries are rare in adults, we report an example of a self-inflicted burn in a schizophrenic which resembles a 'forced immersion burn'.

Controlled comparison of radio wave regional hyperthermia and peritoneal lavage rewarming after immersion hypothermia.

Anesthetized random source dogs were cooled by ice-water immersion to a stable core temperature of 25 degrees C and subsequently rewarmed with normal saline peritoneal lavage (43 degrees C, 175 ml/kg/hr) or radio frequency electromagnetic-induced regional hyperthermia (4-6 watts/kg). The mean time required for core rewarming to 30 degrees C was 183 +/- 79 minutes for lavage and 58 +/- 13 minutes for radio wave therapy (p less than 0.01). There was no evidence of tissue damage with either modality. These data suggest radio wave regional hyperthermia is superior to peritoneal lavage for core rewarming of rapidly induced immersion hypothermia.

Comparison of rewarming by radio wave regional hyperthermia and warm humidified inhalation.

Anesthetized random source dogs were cooled by ice water immersion to a stable core temperature of 25 degrees C and subsequently rewarmed with warm humidified inhalation (43 degrees C, 450 cc of min ventilation X kg-1) or radio-frequency induction hyperthermia (4-6 watts X kg-1). The mean time required for core rewarming to 30 degrees C was 280 +/- 114 min for ventilation and 58 +/- 13 min for radio wave therapy (p less than 0.001). There was no evidence of tissue damage with either modality. These data suggest radio wave heating is superior to warm humidified inhalation therapy for core rewarming of rapidly induced immersion hypothermia.