The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.     

Percentages of cervical cytologic diagnoses as a quality assurance method

OBJECTIVE: To demonstrate empirically that the efficiency of rescreening to discover false negative cytologic diagnoses is greatly enhanced by prospectively stratifying accessions according to risk level. STUDY DESIGN: We stratified accessions from 11 clinical sources and established the rate of diagnoses according to three categories: (1) "within normal limits"/"benign cellular changes" (WNL/BCC), (2) "atypical squamous/glandular cells of undetermined significance" (ASCUS/AGCUS) and (3) "squamous intraepithelial lesion/invasive carcinoma" (SIL/CA). We then prospectively rescreened all negative smears from sources with rates of positive diagnoses (ASCUS/AGCUS and SIL/CA) in excess of 20% and 5% of negative smears from sources with rates of positive diagnoses < 20%. We compared the detection rates of false negatives on rescreening target groups with random rescreening of 10% of all negative smears. RESULTS: The rates of SIL/CA, ASCUS/AGCUS and WNL/BCC varied from 0 to 43%, 4% to 14% and 46% to 94%, respectively. Rescreening 10% of all negative smears revealed a false negative fraction of 3%; rescreening target groups revealed a false negative fraction of 5.9%. CONCLUSION: The yield of prospectively detected false negative diagnoses was significantly increased by targeting high-risk accession groups. When cytology laboratories serve diverse populations, stratifying accessions by risk to permit increased sampling from the proportionately higher risk categories is a simple and effective device to maximize the yield and benefit from rescreening.     

Staphylococcal enterotoxin B primes cytokine secretion and lytic activity in response to native bacterial antigens

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.     

Apolipoprotein E*3-Leiden transgenic mice as a test model for hypolipidaemic drugs

PURPOSE: We report an investigation into the distribution of proteoglycans (PGs) in normal, organ-cultured and dextran-treated human corneas. METHODS: Immunogold labeling was carried out at the electron microscope level to localize keratan sulphate (KS), chondroitin sulphate (CS), and heparan sulphate (HS) PGs. RESULTS: High levels of labeling for CS was found in the epithelium, endothelium, and keratocytes, with light labelling present in the basement membranes and the corneal stroma. Labeling for HS was present in the epithelium, endothelium, and keratocytes, with intense labeling present at the endothelium/Descemet's membrane interface and the epithelium/Bowman's layer interface. Large filaments were also observed in these regions in cuprolinic blue-stained specimens. Keratan sulphate was present at high levels in the stroma and the basement membranes with low levels present within the keratocytes, epithelium, and endothelium. The pattern of KS labeling along the collagen fibrils in the stroma sometimes showed evidence of periodicity. Organ-cultured corneas had extensive collagen-free "lakes," the interior of which immunolabeled positively for KS and showed staining with cuprolinic blue. The lakes were greatly reduced in the dextran-treated samples. CONCLUSION: This investigation determined the ultrastructural distribution of KS, CS, and HS PGs in human cornea and showed that organ culture is associated with a change in distribution of stromal PGs.     

Induced candidosis in mice with artificially established oral flora

The aim of this study was to gauge the effect of an artificially established flora, unnatural for mice, on the induction of oral candidosis in mice. Four groups of BALB/c mice were compared; conventional Candida albicans-free mice, "germ-free" mice which had been inoculated with Candida-free human saliva, germ-free mice which had been exposed to a cocktail of Streptococcus mitis, S. sobrinus and S. sanguis, and uncontaminated germ-free mice. After exposure to C. albicans via drinking water, the four groups of mice were killed and their oral cavities examined for candidal growth and oral lesions. Conventional mice yielded significantly less candidal growth and exhibited significantly fewer oral lesions than the other three groups. Candidal lesions in the two groups of contaminated germ-free mice were significantly fewer than in the uncontaminated germ-free mice. The latter exhibited extensive candidal lesions with little inflammatory infiltrate. It is concluded that mice with human oral micro-organisms have some resistance against candidal infection albeit at a reduced level, that mice with natural oral flora are highly resistant, and that germ-free mice are extremely susceptible to C. albicans infection.     

Effect of set-up error on the dose across the junction of matching cranial-spinal fields in the treatment of medulloblastoma

PURPOSE: The effect of systematic and stochastic setup error on the dose delivered to the gap region for the three field radiation treatment of medulloblastoma is studied. The consequences of such setup error is discussed. METHODS AND MATERIALS: The treatment of medulloblastoma is typically a 3 field technique, in which two lateral cranial fields are matched with a spine field. The x-ray dose delivered to the region between the matched fields depends upon the gap size. The choice of the gap width between the cranial and spinal fields is controversial. It is currently a compromise between minimizing the risk of dose hot spots to the spine, and the associated clinical complications, as well as the magnitude of cold spots (underdosing) across the gap, with the associated risk of disease recurrence. In this paper, we examine the effect of gap width with a moving junction, referred to as "field feathering", on the dose across the field junction for a 6MV photon beam. In addition, we have studied 129 portal films and 40 simulation films to assess the accuracy and precision of patient setup during treatment with a plan involving feathered fields. Selected landmarks observable on both portal and simulation films were identified and the variation in the distances to the field edges measured. The distribution of patient setup error was convoluted with the beam profiles for a 6MV linac. These convoluted field edges were used obtain dose profiles across the gap region as a function of gap separation. The consequences for therapy are discussed. In addition, analysis of patient setup error on an alternative treatment involving beam modifiers to broaden the beam penumbra is discussed. RESULTS: The magnitude of the spatial stochastic and systematic setup error was determined to be approximately three and two millimeters respectively. The dosimetric consequences of patient setup error lead to over and under dosing in the spinal gap region for the three field technique. The degree of under or over dose depends on the nature and magnitude of the patient setup error. CONCLUSIONS: The effect of patient setup error can lead to significant dosimetric errors in the dose to the gap region depending on the magnitude of the setup errors. The effective over and under dose can be compensated by the use beams modifiers such as a beam spoiler or vibrating jaws.     

Influence of Matrix Properties on Fragmentation Test

Physical aging was used to vary the mechanical properties of model single fiber composites without changing the chemistry at the interface in order to study how property changes affect the measurement of interfacial adhesion by the fragmentation test. The properties of epoxy matrix/AS4 single fiber composites driven to full cure (T_g = 166°C) are altered by annealing below T_g. Neat resin samples with identical thermal histories are tested. All aged panels show roughly the same embrittlement with aging characterized by an average 30% decrease in tensile failure strain and 7.3% increase in compressive yield relative to quenched samples. Fragmentation results indicated no change between aged and quenched samples. Results are discussed in terms of micromechanics models for the fragmentation test. Strain at fragmentation increased with aging. This was related to the residual stress state in the model composite and the possibility of the zero stress state of the single fiber composites increasing with thermal annealing.     

Fiber/Matrix load transfer in cyanate resin carbon fiber systems

Comparative single fiber fragmentation test measurements are used to charcterize cynate and epoxy resin interface load transfer with high modulus (HMS4) and high strength (AS4) carbon fibers. The HMS4 fiber forms a weak interface with a fiber controlled failure mode, and the AS4 fiber forms a strong interface with the resin properties, apparently determining the level of load transfer. The resin properties examined are critical surface energy for wetting, cure shrinkage, thermal shrinkage, and mechanical modulus and strength. The cynate and epoxy resins display no significant difference in critical surface energy. Cure shrinkage has a negligible effect on load transfer. The compressive force from thermal shrinkage is significant, but the larger Tg to testing temperature range of the cyanate resin is offset by the larger thermal expansion coefficient of the epoxy, resulting in a near equal compressive force for the two resins. There is little difference in modulus between the two resins but a significant difference in shear strength. This difference is reflected in the larger load transfer measurement for the cyanate resin. A comparison of simple one-dimensional elastic and plastic models for the fiber fragmentation experiment resulted in better conformity with the plastic model. This would indicate that interfacial failure occurs by plastic deformation of the resin for the systems of this study.