Repeatability of diffusion-based stiffness prediction - A healthy volunteer study.

INTRODUCTION: The study investigated the repeatability of brain diffusion-based stiffness prediction (DWIstiff) in healthy volunteers. METHODS: Thirty-one healthy volunteers were examined with DWIstiff using two different sets of b-values: b200-1500 s/mm2 (DWIstiff, 1500) and b200-1000 s/mm2 (DWIstiff, 1000). Each b-value set was scanned twice per imaging session without repositioning the participants. DWIstiff images were reconstructed from each set. Two observers delineated regions of interest (ROIs) on each DWIstiff image. The repeatability coefficient (RC), coefficient of variation (CV), inter- and intraobserver agreement were calculated. RESULTS: After excluding three participants due to image artifacts, the study included twenty-eight volunteers (mean age (range)) 37 years (24-62), 10 males, 18 females). For DWIstiff, 1500, the lowest and the highest RCs were in the parietal lobe (0.52) and respectively the brain stem (1.17). The lowest RC for DWIstiff, 1000 was in the frontal lobe (0.42) and the highest in the brain stem (1.58). The CV for whole brain measurements was 3.83 % for DWIstiff, 1500 and 4.93 % for DWIstiff, 1000. The Bland‒Altman (BA) limits of agreement (LoA) for the intraobserver agreement of DWIstiff, 1500 were -0.90 to 1.06 and respectively -0.78 to 0.88 for DWIstiff, 1000. Regarding interobserver agreement, the LoA were -0.85 to 0.94 for DWIstiff, 1500 and -0.61 to 0.66 for DWIstiff, 1000. CONCLUSION: DWIstiff is a precise technique with some observer dependence. Repeatability is higher for DWIstiff, 1000 s/mm2 than for DWIstiff 1500 s/mm2. IMPLICATIONS FOR PRACTICE: Our findings suggest that DWIstiff can reliably detect stiffness changes larger than 4.93 % in healthy volunteers. Further studies should investigate whether the repeatability of DWIstiff may be affected by the presence of pathology such as a brain tumor.

Community analysis of biting midges (Culicoides Latr.) on livestock farms in Denmark.

This study presents descriptive statistics and community analysis of adult biting midges trapped at 16 livestock farms by means of light traps on Zealand and Lolland-Falster, Denmark. A total of 9,047 male and female Culicoides divided into 24 species, were caught. Biotic and abiotic factors ranging from presence of different host species (cattle or sheep/goats), presence of small woody areas or wetlands in the surrounding landscape, and agricultural practice (organic or conventional) were included in the community analysis. Only differences in the Culicoides communities between conventional and organic practices were tested significantly different. Total numbers of Culicoides individuals were higher on the organic farms than on the conventional farms. The larger loads of biting midges on the organic farms may be due to free-ranging animals that attracted the midges on pastures and carried them to the stable environment (the cattle of the conventional farms were held inside the stables). Presence of deciduous trees within 500 m of the farms resulted in higher numbers of Culicoides obsoletus s.s., while presence of wetlands increased the numbers of Culicoides punctatus and Culicoides pulicaris. Furthermore, Culicoides riethi and Culicoides puncticollis (subgenus Monoculicoides) were recorded in high numbers on individual farms. C. puncticollis was found for the first time in Denmark and so far only recorded from Zealand.

Comparison of quantitative ultrasound and dual X-ray absorptiometry in estrogen-treated early postmenopausal women.

Identifying individuals at risk of developing osteoporosis is important in order to initiate early treatment. Many new techniques have been proposed as alternatives for DXA-scanning. Some of these alternatives certainly have advantages, but none have so far been demonstrated to predict fractures better, or even to identify individuals at risk of osteoporosis as well as with the standard method. In this study, comprising a group of women from the Danish Osteoporosis Prevention Study, we wished to investigate whether a technique based on quantitative ultrasound (QUS) could identify individuals with low BMC/BMD as measured by dual X-ray absorptiometry (DXA). Furthermore, we wished to test whether the method could detect differences between untreated individuals and those treated with hormone replacement therapy. We found that QUS could detect differences between the treated and untreated groups, but it was unable to identify women with low BMD, although it might be able to identify persons not at risk of osteoporosis. Low QUS values should be followed by a regular DXA measurement to confirm the presence of osteoporosis.

The nature of the subset of MHC class II molecules carrying the CDw78 epitopes.

A CDw78 mAb FN1 was shown to recognize DP and/or DR molecules under the conditions of Western blotting. DP molecules were specifically retarded on a column of the FN1 immunosorbent; binding of FITC-labeled FN1 to B cell lines was completely blocked by excess of mAb to DR/DP beta chains, partially by several mAb to DP and weakly by some mAb to DR. The binding of two other CDw78 mAb, FN4 and MR11, to the B cell surface was most strongly inhibited by excess of different mAb to DR. Kinetics of stable binding of the CDw78 mAb indicated that their monovalent binding is of low affinity and that the stable binding to the surface is due to bivalent binding to two spatially close MHC class II molecules. FN1-based immunosorbent effectively immunoisolated complexes of MHC class II proteins with several tetraspanin molecules from a mild detergent lysate of a B cell line. It is concluded that FN1 and most likely also the other two CDw78 mAb recognize with low affinity determinants on MHC class II molecules (DP or DR) and preferentially bind in a stable fashion to dimerized or aggregated MHC class II molecules. Such dimers or aggregates may either exist as preformed on the cell surface or may be gradually formed and stabilized by bivalent interaction with mAb. These structures may be related to the previously described 'superdimers' of MHC class II and/or 'MHC-tetraspanin complexes'. CDw78 mAb may be valuable tools targeting such aggregated fraction of MHC class II molecules which can exhibit important signaling and antigen-presenting properties.

CDw78--a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton.

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10% of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90% of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10-20% of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (Ka = 1.5 x 10(8) M(-1)) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-mu suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation.

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.

Cross-linking of CD53 promotes activation of resting human B lymphocytes.

The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cell membrane proteins. The structure and functional characteristics of these molecules indicate that they may play important roles in transmembrane signaling in different cells. Recently, it was reported that cross-linking of CD53 on human B cells led to an increase in cytoplasmic calcium fluxes. In the present study, we wished to further explore the possible role of CD53 in functional B cell responses. Cross-linking of CD53 with the use of the mAb MEM-53 and a polyclonal sheep anti-mouse Ig promoted activation of resting B cells into the G1 phase of the cell cycle as judged by increased expression of the early activation Ag CD69, increases in cellular volume, RNA synthesis, and c-myc protein levels, and enhanced binding of 7-aminoactinomycin D. In contrast, MEM-53 alone had no detectable effects. Cross-linking of anti-CD53 induced negligible S phase entry in the absence of other stimuli. However, cytokines, in particular IL-2 and IL-4, potentiated the DNA synthesis induced by cross-linking of CD53. Furthermore, cross-linking of the CD53 Ag induced Ig production in the presence of T cell supernatant. Taken together, the data suggest that CD53 plays an important functional role in B cell activation and differentiation.

Cyclic AMP sensitive signalling by the CD28 marker requires concomitant stimulation by the T-cell antigen receptor (TCR/CD3) complex.

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RI alpha 2-C beta 2) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anti-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP).

Immobilization and recovery of fusion proteins and B-lymphocyte cells using magnetic separation.

A new approach to facilitate immobilization and affinity purification of recombinant proteins and selected human B lymphocytes has been developed. Using magnetic beads with attached DNA containing the Escherichia coli lac operator, fusion proteins comprising the DNA-binding lac repressor could be affinity-purified and recovered by gentle elution conditions, such as with a lactose analogue or by enzymatic means using either deoxyribonuclease (DNase) or restriction endonucleases. The results show for the first time that a DNA-binding protein can be used for affinity purification of fusion proteins as exemplified by the specific and gentle recovery of beta-galactosidase and alkaline phosphatase from bacterial lysates using immunomagnetic separation. The approach was further extended to cell separation by the efficient recovery and elution of human CD37 B lymphocytes from peripheral blood.

Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells.

In the present paper we demonstrate that retinol-retinol-binding protein and chylomicron remnant retinyl esters in concentrations normally found in human plasma inhibit growth of normal human B lymphocytes. Physiological concentrations of retinoic acid (about 30 nM) were less active than physiological concentrations of retinol (about 3 microM). Pharmacological concentrations of retinol and retinoic acid were more active than the concentrations normally found in plasma. Retinol (3 microM) inhibited anti-IgM-mediated DNA synthesis as measured by [3H]thymidine uptake at 72 h by 78%. Furthermore, we found that the cells were blocked in the mid-G1 phase of the cell cycle. Thus, neither MYC up-regulation measured at 3 h nor the expression of the early activation antigen 4F2 was reduced by retinol, whereas the late activation markers (transferrin receptor expression and actinomycin D staining at 48 h of stimulation) were markedly inhibited. Retinol reduced the interleukin 6 production induced by anti-IgM and interleukin 4 after 48 h, whereas the induction of interleukin 6 and tumor necrosis factor by O-tetradecanoylphorbol-13-acetate and ionomycin was less affected. We also noted that the retinoids reduced the formation of plaque-forming cells (i.e. Ig synthesis). These data imply that vitamin A present in human plasma is a normal modulator of B cell function.

Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.

A new method for detachment of Dynabeads from positively selected B lymphocytes.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.

[Total intravenous anesthesia with propofol-fentanyl. Is it necessary to supply nitrous oxide?]. / Total intravenøs anaestesi med propofol-fentanyl. Er det nødvendigt at supplere med kvaelstofforilte?

Forty patients admitted for elective hemilaminectomy were randomly allocated in two groups: Group I: intravenous anaesthesia with propofol-fentanyl and Group II: Propofol-fentanyl anaesthesia with a supplement of nitrous oxide. The purpose of the study was to investigate whether this supplement involved any advantages or disadvantages for the patients. Mean arterial pressures, awakening times, complications immediately postoperatively and total doses of fentanyl showed no statistically significant differences. In group I (-N2O), the median dose of supplementation with fentanyl was significantly greater, 0.89 micrograms/kg versus 0 in group II (p less than 0.05). We conclude that total intravenous anaesthesia is preferable, because pollution of the air in the operating theatre is avoided.

Secondary poisoning hazards in stone martens (Martes foina) fed bromadiolone-poisoned mice.

Stone martens (Martes foina) are particularly exposed to secondary poisoning by feeding on anti-coagulant-loaded rats and mice in premises. This study indicates that the risk can be considered relatively small as up to 31 bromadiolone-loaded mice were consumed during four days by a single stone marten without symptoms of poisoning.