Simultaneous cross-linking of CD6 and CD28 induces cell proliferation in resting T cells

In the present study, we showed that simultaneous ligation of the monoclonal antibodies (mAb) against CD6 and CD28 induces T-cell proliferation in purified resting T lymphocytes in the absence of T-cell receptor (TCR) occupancy. No cell proliferation was observed when the mAb were cross-linked alone or used simultaneously in the soluble form. T-cell proliferation mediated through CD6/CD28 is accompanied by the up-regulation of interleukin-2 (IL-2) mRNA and expression of IL-2 receptors on the cell surface. In the presence of IL-2-neutralizing mAb the proliferative response of the T cell induced through CD6/CD28 was inhibited dose dependently. Cross-linking mAb to CD6 and CD28 alone or together did not down-regulate the CD3/TCR complex. T-cell proliferation mediated through CD6/CD28 was only partially blocked by the immunosuppressive drug, cyclosporin A (CsA), whereas anti-CD28-induced T-cell proliferation in the presence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was unaffected. In sharp contrast T-cell proliferation mediated by anti-CD6 in the presence of TPA was efficiently blocked by CsA. In addition, two protein kinase C (PKC) inhibitors, GF 109203X and H-7 dose-dependently inhibited T-cell proliferation mediated through CD6/CD28, suggesting that PKC activation may be involved. Furthermore, there was a marked differential dose-dependent inhibitory effect of the PKC inhibitors on T-cell proliferation mediated by the co-ligation of anti-CD6 or anti-CD28 in the presence of anti-CD3, with the former being more sensitive to PKC inhibition. Taken collectively, our results suggest that T-cell activation can occur through an antigen-independent pathway by cross-linking the accessory molecules, CD6 and CD28, and that these two cell surface antigens may have distinct signalling pathways.     

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits.

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1). The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.     

NMR structure and mutagenesis of the FADD (Mort1) death-effector domain

When activated, membrane-bound receptors for Fas and tumour-necrosis factor initiate programmed cell death by recruiting the death domain of the adaptor protein FADD to the membrane. FADD then activates caspase 8 (also known as FLICE or MACH) through an interaction between the death-effector domains of FADD and caspase 8. This ultimately leads to the apoptotic response. Death-effector domains and homologous protein modules known as caspase-recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. Here we describe the solution structure of a soluble, biologically active mutant of the FADD death-effector domain. The structure consists of six antiparallel, amphipathic alpha-helices and resembles the overall fold of the death domains of Fas and p75. Despite this structural similarity, mutations that inhibit protein-protein interactions involving the Fas death domain have no effect when introduced into the FADD death-effector domain. Instead, a hydrophobic region of the FADD death-effector domain that is not present in the death domains is vital for binding to FLICE and for apoptotic activity.     

Concentration-response relationships for adenosine agonists during preconditioning of rabbit cardiomyocytes

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 microM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 microM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 microM CCPA. The APNEA curve appeared moriophasic, the major slope occurring between 1-100 nM; DPCPX (1 microM) shifted the concentration-response curve approximately 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of approximately 5 nM; DPCPX shifted the BNECA concentration-protection relationship approximately 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.     

Priapism: etiology and management

Priapism is a urologic emergency. All patients should receive prompt urologic consultation. Management is based on prompt recognition, differentiation between low- and high-flow priapism, reversal of any potential precipitating factors, and the use of corporal aspiration/irrigation combined with intracavernosal alpha-agonist injection therapy. It cannot be over-emphasized that severely prolonged erections are associated with the development of irreversible problems with erectile function and, therefore, immediate and aggressive management is mandatory.     

Image compression in digital mammography: effects on computerized detection of subtle microcalcifications

Our previous receiver operating characteristic (ROC) study indicated that the detection accuracy of microcalcifications by radiologists is significantly reduced if mammograms are digitized at 0.1 mm x 0.1 mm. Our recent study also showed that detection accuracy by computer decreases as the pixel size increases from 0.035 mm x 0.035 mm. It is evident that very large matrix sizes have to be used for digitizing mammograms in order to preserve the information in the image. Efficient compression techniques will be needed to facilitate communication and archiving of digital mammograms. In this study, we evaluated two compression techniques: full frame discrete cosine transform (DCT) with entropy coding and Laplacian pyramid hierarchical coding (LPHC). The dependence of their efficiency on the compression parameters was investigated. The techniques were compared in terms of the trade-off between the bit rate and the detection accuracy of subtle microcalcifications by an automated detection algorithm. The mean-square errors in the reconstructed images were determined and the visual quality of the error images was examined. It was found that with the LPHC method, the highest compression ratio achieved without a significant degradation in the detectability was 3.6:1. The full frame DCT method with entropy coding provided a higher compression efficiency of 9.6:1 at comparable detection accuracy. The mean-square errors did not correlate with the detection accuracy of the microcalcifications. This study demonstrated the importance of determining the quality of the decompressed images by the specific requirements of the task for which the decompressed images are to be used. Further investigation is needed for selection of optimal compression technique for digital mammograms.     

Current concepts in dentin bonding: focusing on dentinal adhesion factors

With greater need for treating root surface lesions, dentinal adhesives are more in demand. For successful treatment, all factors--dentin, tooth, patient and materials--must be considered.