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Bleeding due to platelet dysfunction can result from quantitative or qualitative disorders. Qualitative disorders may affect any of the many steps involved in platelet adhesion, activation, and aggregation. We present an adolescent with a qualitative platelet disorder storage pool defect, which results in abnormalities of secondary platelet aggregation and the potential for excessive bleeding following minor trauma or a surgical procedure. The normal function of platelets and their role in coagulation is reviewed, qualitative platelet disorders discussed, and the perioperative treatment of such patients presented. [ABSTRACT FROM AUTHOR]

We have evaluated the immunological abnormalities present in a 6 year old patient with primary intestinal and generalized lymphangiectasia confirmed by intestinal, lung and lymph node biopsies. Lymphocyte loss through the gut was confirmed by the detection of lymphocytes in her stool. An increased enteric protein loss was suggested by hypopro-teinaemia. peripheral oedema, and a very short half-life for i.v. immune serum globulin (3 days). Lymphocyte subpopulation analysis revealed a selective loss of T lymphocytes. with a proportionally increased loss of the OKT4 positive helper/inducer subpopulation. Functionally, there was a decrease in proliferative responses to some mitogens and to allogeneic cells, and a lack of T cell help for in vitro B lymphocyte differentiation into immunoglobulin secreting cells. Natural killer function was normal. In this patient, a concomitant thymic deficiency was documented by failure to identify thymic tissue on a thymus biopsy and by an absence or decrease of the serum thymic factor (thymulin) and thymosin α1. No compensatory lymphopoiesis was detected in the bone marrow. In an attempt to increase T lymphocyte development, the patient was treated with thymosin fraction 5. Daily treatment with this preparation resulted in a transient clinical improvement which could not be sustained on a weekly thymosin treatment schedule. However, lymphocyte numbers did not increase during this treatment. The findings in this patient support the notion that T lymphocytes are needed to stimulate thymic epithelium. In situations of excessive loss of long lived T lymphocytes a secondary thymic atrophy may occur and further contribute to the development of a deficiency in cell-mediated immunity. [ABSTRACT FROM AUTHOR]

Anadromous fishes such as steelhead trout, Oncorhynchus mykiss , are exposed to a suite of infectious agents and migratory challenges during their freshwater migrations. We assessed infectious agent load and richness and immune system gene expression in gill tissue of Bulkley River (British Columbia, CA) steelhead captured at and upstream of a migratory barrier to evaluate whether infectious burdens impacted migration success. We further considered the potential influences of water temperature, sex and fish size on host infectious agents and transcription profiles. There were eight infectious agents detected in steelhead gill tissue, with high prevalence of the bacteria Candidatus Branchiomonas cysticola (80%) and Flavobacterium psychrophilum (95%) and the microparasite Sphaerothecum destruens (53%). Fish sampled at the falls had significantly greater relative loads of Ca. B. cysticola and F. psychrophilum , higher infectious agent richness and differential gene expression compared to fish captured upstream. Flavobacterium psychrophilum was only associated with immune gene expression (particularly humoral immunity) of fish sampled at the falls, while water temperature was positively correlated with genes involved in the complement system, metabolic stress and oxidative stress for fish captured upstream. This work highlights interesting differences in agent-host interactions across fisheries and suggests that hydraulic barriers may reduce the passage of fish with the heaviest infectious agent burdens, emphasizing the selective role of areas of difficult passage. Further, this work expands our knowledge of infectious agent prevalence in wild salmonids and provides insight into the relationships between infectious agents and host physiology.

Purpose: To determine the rate of progression of eyes with subclinical diabetic macular edema (DME) to clinically apparent DME or DME necessitating treatment during a 2-year period., Methods: In all, 43 eyes from 39 study participants with subclinical DME, defined as absence of foveal center edema as determined with slit lamp biomicroscopy but a center point thickness (CPT) between 225 and 299 μm on time domain (Stratus, Carl Zeiss Meditec) optical coherence tomography (OCT) scan, were enrolled from 891 eyes of 582 subjects screened. Eyes were evaluated annually for up to 2 years for the primary outcome, which was an increase in OCT CPT of at least 50 μm from baseline and a CPT of at least 300 μm, or treatment for DME (performed at the discretion of the investigator)., Results: The cumulative probability of meeting an increase in OCT CPT of at least 50 μm from baseline and a CPT of at least 300 μm, or treatment for DME was 27% (95% confidence interval (CI): 14%, 38%) by 1 year and 38% (95% CI: 23%, 50%) by 2 years., Conclusions: Although subclinical DME may be uncommon, this study suggests that between approximately one-quarter and one-half of eyes with subclinical DME will progress to more definite thickening or be judged to need treatment for DME within 2 years after its identification.

The v-Cbl oncogene induces myeloid and B-cell leukemia; however, the mechanism by which transformation occurs is not understood. An oncogenic form of c-Cbl (Cbl-DeltaY371) was expressed in the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to determine whether it was able to induce growth factor-independent proliferation. We were unable to isolate clones of transfected 32Dcl3 cells expressing Cbl-DeltaY371 that proliferated in the absence of IL-3. In contrast, 32Dcl3/Cbl-DeltaY371 cells did not undergo apoptosis like parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/CblDeltaY371 cells arrested in G(1) when cultured in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured in the absence of IL-3 for 24 h were present in a sub-G(1) fraction, while only 4% of the 32D/Cbl-DeltaY371 and 2% of the 32D/Bcl-2 cells were found in a sub-G(1) fraction. There was no difference in the pattern of tyrosine-phosphorylated proteins observed following stimulation of either cell type with IL-3. The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical in both cell types. No differences were detected in the activation of Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-DeltaY371 cells compared with parental 32Dcl3 cells. Likewise, there was no difference in the pattern of phosphorylation of JAK2, STAT5, ERK1, ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from medium containing IL-3. The protein levels of various Bcl-2 family members were examined in cells grown in the absence or presence of IL-3. We observed a consistent increased amount of Bcl-2 protein in five different clones of 32D/Cbl-DeltaY317 cells. These data suggest that the Cbl-DeltaY371 mutant may suppress apoptosis by a mechanism that involves the overexpression of Bcl-2. Consistent with this result, activation of caspase-3 was suppressed in 32D/Cbl-DeltaY371 cells cultured in the absence of IL-3 compared with 32Dcl3 cells cultured under the same conditions.

Purpose: We have previously reported increased levels of Osteonectin/SPARC transcript in age-related cataractous compared to normal human lenses. The purpose of the present study was to evaluate the corresponding levels of osteonectin/SPARC protein in age-related cataractous relative to normal lenses and to evaluate the levels of osteonectin/SPARC transcript in specific types of age-related human cataracts. The spatial expression of osteonectin/SPARC was also evaluated in normal human lenses., Methods: Specific types of age-related cataracts were collected and graded. Normal human lenses were microdissected into epithelia and fibers. Osteonectin/SPARC protein levels were monitored by Western immunoblotting, and transcript levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Osteonectin/SPARC expression patterns were examined by RT-PCR and by immunostaining., Results: Higher levels of osteonectin/SPARC protein were detected in age-related cataractous relative to normal human lenses. Increased levels of osteonectin/SPARC transcript were also detected in posterior-subcapsular and nuclear cataractous lenses relative to normal lenses. Osteonectin/SPARC transcripts were detected in the lens epithelium but not fibers. Osteonectin/SPARC protein levels were highest in the peripheral lens epithelium., Conclusions: Consistent with our previous studies on osteonectin/SPARC mRNA levels, osteonectin/SPARC protein levels were also elevated in cataractous compared to normal human lenses. Increased levels of osteonectin/SPARC mRNA were also found in nuclear and posterior-subcapsular cataracts relative to normal lenses. Osteonectin/SPARC expression is confined to the lens epithelium, and osteonectin/SPARC levels are highest in the peripheral lens epithelium.

We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most single-base substitutions when carried out on products amplified with a primer to which a GC clamp has been added. Although DGGE has been primarily used for the detection of limited numbers of single-base mutations in disease studies, it offers great potential for use in population analysis of genetic markers with greater levels of sequence variation. The methodology described was developed to identify the number and distribution of MHC class I alpha 1 alleles among chinook salmon (Oncorhynchus tshawytscha) populations. DGGE detects 28 of 31 identified alpha 1 sequences, which differ by between 1 and 16 nucleotides and a two-codon indel. By creating a network of control alleles, 22-23 of the MHC alleles can be resolved rapidly and accurately by a single gel run condition, and 27 alleles can be resolved by two gel run conditions. This techniques has been used in surveys scoring alleles from two MHC markers (class I alpha 1 and alpha 2) in 20,000 individuals of chinook and coho (O. kisutch) salmon. A single person in our laboratory now analyzes 160 salmon from one MHC locus per day with DGGE.

Unlabelled: In this study, we evaluated the effects of viewing an educational videotape about pediatric anesthesia on measures of parental knowledge of anesthesia and preoperative anxiety using a randomized, controlled design. During their routine preoperative visit, 85 parents of children scheduled to undergo ambulatory surgical procedures under general anesthesia were randomized to view either the experimental videotape about pediatric anesthesia or a control videotape with no medical content. Before and immediately after viewing the assigned videotape, parents completed measures of situational anxiety (State-Trait Anxiety Inventory-State), preoperative anxiety and need for information (Amsterdam Preoperative Anxiety and Information Scale), and anesthesia knowledge (Standard Anesthesia Learning Test). Repeated-measures analyses of variance showed that parents who viewed the experimental videotape showed a significant increase in anesthesia knowledge (P < 0.022) and a significant reduction in their state of anxiety (P < 0.031), anesthesia-specific anxiety, and need for information (P < 0.0001) compared with the control group. These results demonstrated that viewing a preoperative educational videotape about pediatric anesthesia can provide immediate educational and anxiolytic benefits for parents of children undergoing ambulatory surgery. The duration of these benefits remains to be determined., Implications: In this study, we demonstrated the benefits of viewing an educational videotape about pediatric anesthesia on measures of parental knowledge of anesthesia and preoperative anxiety using a randomized, controlled design. We found that videotape viewing facilitated preoperative preparation and lessened preoperative anxiety.

Unlabelled: Parents' anxiety about their children's anesthesia may adversely affect the children's outcomes and compromise the quality of informed consent. Studies of these issues have been limited by the lack of validated measures of parental anxiety and knowledge surrounding anesthesia. In the present study, we evaluated psychometric properties of the Amsterdam Preoperative Anxiety and Information Scale (APAIS) and the Standard Anesthesia Learning Test (SALT) among 85 parents who participated in an evaluation of the effects of a videotape about pediatric anesthesia. The results supported the internal consistency, test-retest reliability, and concurrent validity of both instruments and documented the equivalence of two forms of the SALT. Factor analysis supported the previously demonstrated factor structure of the APAIS, further confirming its construct validity. We conclude that the APAIS and SALT are reliable and valid measures of parental anxiety and knowledge of pediatric anesthesia that can be used for clinical and research purposes., Implications: This study verified the reliability and validity of two questionnaires for measuring parents' knowledge and anxiety about pediatric anesthesia. These questionnaires can be used in further research on factors affecting parental anxiety and knowledge before their children's surgery.

Matrix metalloproteinases (MMPs) are a family of endopeptidases capable of enzymatic digestion of subendothelial basement membrane and other components of the extracellular matrix. Expression of MMP-2, -3, -7 and -9 is increased around multiple sclerosis plaques and in brain tissue in experimental allergic encephalomyelitis. To measure quantitatively the expression of these MMPs and their endogenous inhibitors (TIMP-1 and -2), we analysed samples from 52 patients with relapsing-remitting and primary progressive multiple sclerosis by ELISA (enzyme-linked immunosorbent assay) and substrate-gel electrophoresis (zymography). MMP-9 was increased over controls in 100% of relapsing-remitting multiple sclerosis cases, with similar levels detected in relapses and clinically stable phases of disease. In primary progressive multiple sclerosis, MMP-9 was increased in 57% of CSF samples, but concentrations were below those encountered in the relapsing-remitting form. The selective upregulation of MMP-9 suggests that T-cells and macrophages invading the brain parenchyma and the CSF space are the predominant source of MMP-9 in multiple sclerosis. TIMPs and other MMPs (MMP-2 and -3) were not upregulated or not detectable (MMP-7) in CSF of patients with relapsing-remitting and primary progressive multiple sclerosis. The sustained increase of MMP-9 in clinically stable multiple sclerosis supports the concept that multiple sclerosis is associated with ongoing proteolysis that may result in progressive tissue damage. The selective inhibition of MMP-9 could be a useful approach for the prevention of disease progression in multiple sclerosis.

Experimental autoimmune neuritis (EAN) is an animal model of Guillain-Barré syndrome. We have shown recently that BB-1101, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, prevents development of EAN when given from the day of immunization and, more important clinically, reduces disease severity when given from symptom onset. This suggests the involvement of MMP activity in the pathogenesis of EAN. However, the exact function and expression patterns of MMPs in acute inflammation of the PNS have not been investigated. MMP-like enzymes are also involved in the processing of tumour necrosis factor-alpha (TNF-alpha), which has been implicated previously in the pathology associated with EAN. In the present study we investigated the profile of MMP and TNF-alpha expression and their localization in sciatic nerve tissue during EAN, using a semiquantitative competitive reverse transcriptase-coupled polymerase chain reaction and immunohistochemistry. In the normal rat PNS, four of the 10 MMPs studied were constitutively expressed and four MMPs were differentially regulated during EAN. Expression of TNF-alpha was elevated at peak disease severity and localized to Schwann cells, macrophages and endoneurial blood vessels. Expression levels of 92 kDa gelatinase and stromelysin-1 were significantly increased early in the development of EAN and continued to rise, peaking at day 15 coincident with maximum disease severity. Schwann cells and endothelial cells were the main cellular source of these enzymes. Prominent infiltration of inflammatory cells into the sciatic nerve was concordant with a significant increase in the expression levels of matrilysin and macrophage metalloelastase. Both matrilysin and macrophage metalloelastase were detected in invading macrophages, T lymphocytes and resident Schwann cells. The selective upregulation of specific MMPs during EAN and their varied cellular localization suggests that MMPs play a multifactorial role in the aetiology of EAN. Activity of MMPs could participate in the disruption of the blood-nerve barrier, breakdown of the myelin sheath, the release of TNF-alpha, and facilitate leukocyte invasion into the PNS. These observations highlight MMPs as potential targets for therapeutic intervention in acute peripheral neuropathies, such as Guillain-Barré syndrome.

The GM2 activator deficiency (also known as the AB variant), Tay-Sachs disease, and Sandhoff disease are the major forms of the GM2 gangliosidoses, disorders caused by defective degradation of GM2 ganglioside. Tay-Sachs and Sandhoff diseases are caused by mutations in the genes (HEXA and HEXB) encoding the subunits of beta-hexosaminidase A. The GM2 activator deficiency is caused by mutations in the GM2A gene encoding the GM2 activator protein. For degradation of GM2 ganglioside by beta-hexosamindase A, the GM2 activator protein must participate by forming a soluble complex with the ganglioside. In each of the disorders, GM2 ganglioside and related lipids accumulate to pathologic levels in neuronal lysosomes, resulting in clinically similar disorders with an onset in the first year of life, progressive neurodegeneration, and death by early childhood. We previously have described mouse models of Tay-Sachs (Hexa -/-) and Sandhoff (Hexb -/-) diseases with vastly different clinical phenotypes. The Hexa -/- mice were asymptomatic whereas the Hexb -/- mice were severely affected. Through gene disruption in embryonic stem cells we now have established a mouse model of the GM2 activator deficiency that manifests an intermediate phenotype. The Gm2a -/- mice demonstrated neuronal storage but only in restricted regions of the brain (piriform, entorhinal cortex, amygdala, and hypothalamic nuclei) reminiscent of the asymptomatic Tay-Sachs model mice. However, unlike the Tay-Sachs mice, the Gm2a -/- mice displayed significant storage in the cerebellum and defects in balance and coordination. The abnormal ganglioside storage in the Gm2a -/- mice consisted of GM2 with a low amount of GA2. The results demonstrate that the activator protein is required for GM2 degradation and also may indicate a role for the GM2 activator in GA2 degradation.

Geographic variation at an Mhc class I A1 exon was surveyed in 14 populations of coho salmon (Oncorhynchus kisutch) and 15 populations of chinook salmon (O. tshawytscha) inhabiting rivers of British Columbia, Canada. A total of 2,504 fish were sampled using denaturing gradient gel electrophoresis (DGGE), which distinguished 17 alleles in coho salmon and 20 alleles in chinook salmon. Heterozygosity at the A1 locus was moderately high for both coho (0.7) and chinook (0.6) salmon, but sequence divergence was low, with mean inter- and intraspecific nucleotide similarities of approximately 0.96. In a maximum parsimony tree, all of the observed alleles clustered into two trans-specific lineages. Within each lineage, coho and chinook alleles tended to fall into species-specific subclusters. Much of the intraspecific allelic variation within each lineage could be accounted for by nonsynonymous point mutation, indicative of balancing selection. The FST values for both coho (0.11) and chinook (0.13) salmon indicated that much of the allelic diversity was partitioned among populations. Neighbor-joining analyses of A1 allelic frequencies among coho and chinook salmon populations showed strong patterns of geographic differentiation similar to those based on neutral genetic markers such as microsatellite loci. Both natural selection and the salmonid zoogeographic history of frequent population bottlenecks have shaped the patterns of diversity observed at this and other Mhc exons in Pacific salmonids.

Three adult, female chickens, previously trained to detect pure tones using a positive reinforcement paradigm, were exposed to pure tones at 525 Hz at 120-dB sound-pressure level for 48 h. Immediately after exposure, pure-tone thresholds were elevated between 10 and 56 dB compared to pre-exposure values. The configuration of the initial hearing loss was essentially flat between 500 and 2000 Hz, with slightly less loss at lower and higher frequencies. Over the course of 100 to 600 h, depending on subject and test frequency, thresholds returned to preexposure levels. The slopes of the threshold-duration functions measured shortly after exposure were shallower than normal, but returned to the normal slope of approximately 3 dB per doubling of duration as hearing loss resolved to less than 15 to 20 dB. The cochleas from additional chickens examined by scanning electron microscopy (SEM) and fluorescence microscopy between 0 and 1512 h post-exposure showed extensive damage to the tectorial membrane over the middle two-thirds of the cochlea, with mild to moderate hair cell loss in the region of the cochlea associated with the exposure frequency.

Child-to-parent transmission of cytomegalovirus may be reduced by increasing protective behaviors (handwashing and glove use) and decreasing risky behaviors (intimate contact between child and parent). This study showed that an educational intervention resulted in increases in reported and objective measures of protective behaviors and decreases in reported risky behaviors. Further study must determine if changes in protective and risky behavior are maintained and prevent cytomegalovirus transmission.

We examined the latex surgical gloves used by 56 primary surgeons in 454 ophthalmic surgical procedures performed over a 7-month period. Of five techniques used to detect pinholes, air inflation with water submersion and compression was found to be the most sensitive, yielding a 6.80% prevalence in control glove pairs and a 21.8% prevalence in postoperative study glove pairs, for a 15.0% incidence of surgically induced perforations (P = 0.000459). The lowest postoperative perforation rate was 11.4% for cataract and intraocular lens surgery, and the highest was 41.7% for oculoplastic procedures. Factors that correlated significantly with the presence of glove perforations as determined by multiple logistic regression analysis were oculoplastic and pediatric ophthalmology and strabismus surgical procedures, surgeon's status as a fellow in training, operating time, and glove size. The thumb and index finger of the nondominant hand contained the largest numbers of pinholes. These data suggest strategies for reducing the risk of cross-infection during ophthalmic surgery.