Comparative evaluation of commonly used laboratory tests for post-mortem diagnosis of rabies

Animal brain samples received at WHO Collaborating Centre laboratory at National Institute of Communicable Diseases (NICD) during the years 1991-2002 were tested by Seller's stain, Fluorescent Antibody Test (FAT) and Mouse Innoculation Test (MIT) as methods of rabies diagnosis. Negri bodies on Seller's staining could be detected in 52.5% of MIT positive brains, the concordance of this test with MIT was found to be 77.8%. FAT was positive in 91.5% of MIT positive brains, though it showed concordance of 95.7% with MIT results in the total samples. 12.2% of the samples were found positive by FAT of which 1/3rd also showed the presence of Negri bodies when MIT was negative i.e. showing that the virus is present in inactivated form. Thus emphasizing the need for timely and proper collection and transportation of specimens for testing. Seller's stain and FAT give reliable diagnosis of rabies in the brain samples in majority of the cases. MIT being time-intensive test, is of academic value only in decision making as regards initiation of Post Exposure Treatment (PET), it is recommended that in cases where Seller's stain and FAT have yielded negative results the decision to initiate PET should give due consideration to the nature and circumstances of the animal bite and other epidemiological features.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities

An RNA-binding motif (RBM) gene family has been identified on the human Y chromosome that maps to the same deletion interval as the 'azoospermia factor' (AZF). We have identified the homologous gene family (Rbm) on the mouse Y with a view to investigating the proposal that this gene family plays a role in spermatogenesis. At least 25 and probably >50 copies of Rbm are present on the mouse Y chromosome short arm located between Sry and the centromere. As in the human, a role in spermatogenesis is indicated by a germ cell-specific pattern of expression in the testis, but there are distinct differences in the pattern of expression between the two species. Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are female due to a position effect resulting in non-expression of Sry ; sex-reversing such mice with an Sry transgene produces males with a high incidence of abnormal sperm, making this the third deletion interval on the mouse Y that affects some aspect of spermatogenesis. Most of the copies of Rbm map to this deletion interval, and the Yd1males have markedly reduced Rbm expression, suggesting that RBM deficiency may be responsible for, or contribute to, the abnormal sperm development. In man, deletion of the functional copies of RBM is associated with meiotic arrest rather than sperm anomalies; however, the different effects of deletion are consistent with the differences in expression between the two species.      

Ultrastructural determinants of murine achilles tendon strength during healing

The mechanisms by which tendon strength is established during growth and development and restored following injury are not completely understood and are likely to be complex, multifactorial processes. Several studies examining the relationship between mechanical behavior and ultrastructural characteristics of tendons and ligaments during growth and maturation suggest that collagen fibril diameter is strongly correlated with tendon strength. Because of the similarities between development and repair processes of musculoskeletal tissues, increases in tendon strength during healing may be related to increases in fibril ultrastructural parameters such as fibril size, numerical density, and area fraction. In this study, we compared murine Achilles tendons at various time points after tenotomy with sham-operated controls in tensile tests to failure and examined tendons using electron microscopy to assess collagen fibril ultrastructure. We found that in the 6-week period following Achilles tenotomy, fibril mean diameter remained significantly smaller than sham-side diameter by a factor of 2-3. Despite the persistently small fibril size, increasing numerical density resulted in a gradual increase in fibril area fraction. Biomechanical strength did not reach that of intact tendons until some time between 5 and 7 weeks, approximately the same time period when fibril area fraction began to approach sham values. These data suggest that parameters other than collagen fibril size are most responsible for increased tendon strength during healing.     

Effects of Vitamin E on airway responses and biochemical parameters in guinea pigs sensitized to ovalbumin

The effect of dietary supplementation with Vitamin E was studied in sensitized guinea pigs. After measurement of baseline airway reactivity and sensitization with ovalbumin, the animals were randomized into two groups: Group A, on a commercial feed and Group B, on dietary supplementation with oral Vitamin E (0.7 IU/kg). These were challenged with inhaled ovalbumin after 4 weeks. The following outcomes were studied: airway responses to ovalbumin inhalation, airway reactivity, sodium and calcium ion influx in isolated tracheal cells, Na+ K+ ATPase and Ca2+ ATPase activity in tracheal homogenate and plasma malonaldehyde. Sensitization increased airway reactivity in Group A but not in Group B. The tracheal cells of animals in Group B showed significantly lower rates of 45Ca and 22Na influx and lower activities of tracheal Na+ K+ ATPase and Ca2+ ATPase as compared to Group A. Plasma malonaldehyde was similar between two groups. We concluded that Vitamin E suppresses the increase in airway reactivity following sensitization and has membrane stabilizing actions.