Minor histocompatibility antigen DDX3Y induces HLA-DQ5-restricted T cell responses with limited TCR-Vbeta usage both in vivo and in vitro.

In vitro stimulation of human female T cells with male HLA-identical dendritic cells resulted in the generation of HLA-DQB1*0501/0502-restricted minor histocompatibility H-Y antigen-specific CD4(+) T cell clones. Two clones generated from different HLA-identical pairs were analyzed. Use of HLA-DQ5-expressing female Epstein-Barr virus transformed B lymphoblastoid cell lines transfected with various H-Y genes and loaded with overlapping peptides demonstrated that both T cell clones are specific for a peptide encoded by DDX3Y. Previously, an HLA-DQ5-restricted T cell clone specific for the same peptide was isolated from a patient with graft-versus-host disease. Thus, we compared the T cell receptor (TCR) rearrangements of the 2 in vitro generated T cell clones and the ex vivo isolated T cell clone. All 3 clones shared the same TCRBV5-4* gene segment and 2 of 3 clones also used similar TCR-Valpha segments. Our results suggest that T cells recognizing the HLA-DQ5/DDX3Y T cell epitope might be characterized by a relatively limited TCR-beta repertoire. The differences in the junctional TCR-beta region had no effect on the antigen specificity, but altered the capacity of the TCR to distinguish the HLA-DQ5/DDX3Y complex from its allelic counterpart. The results also demonstrate that in vitro stimulation of T cells with allogeneic HLA-identical dendritic cells may facilitate the characterization of in vivo, potentially relevant HLA class II-restricted minor H epitopes.     

Detection of Staphylococcal Superantigenic Toxins by a CD69-Specific Cytofluorimetric Assay Measuring T-Cell Activation

The presence of staphylococcal superantigenic toxins in the supernatants of liquid cultures was detected by an easy and rapid method assessing the activation of T lymphocytes by cytofluorimetric measurement of CD69 expression. Staphylococcus aureus cells were grown in Eagle’s minimum essential medium supplemented with 5% heat-inactivated fetal calf serum. Supernatant fluids from all S. aureus strains producing superantigen-related toxins, including enterotoxins A to E, toxic shock syndrome toxin, and exfoliative toxins A and B, induced CD69 expression in a significantly higher number of T cells than a cutoff of 2%. This CD69 assay might be used for initial detection of superantigens from S. aureus strains isolated in the context of staphylococcal toxemia or related chronic human diseases such as atopic dermatitis or Kawasaki syndrome.     

Rapid diagnosis of smallpox infection and differentiation from its mimics.

The potential for a bioterrorism-induced smallpox outbreak has been much discussed of late. The literature of the late 1960s stressed that the distinction between smallpox and the other viral-induced vesicle-forming diseases, namely varicella zoster and disseminated herpes simplex, was difficult to make. Given that the cutaneous manifestations of smallpox would be among the initial symptoms, we reviewed 2 cases of smallpox diagnosed in South America in the 1970s in conjunction with 9 cases of multiple skin vesicles diagnosed as either disseminated herpes simplex or varicella-zoster. These were examined by routine hematoxylin and eosin stain (H&E) as well as by in situ hybridization. A blind review of the cases demonstrated that each showed striking intraepithelial vesicles containing multinucleated squamous cells exhibiting a ground glass appearance of the nuclear chromatin. Thus, as expected, routine H&E examination could not differentiate the 2 smallpox cases from the other 9 samples. In situ hybridization easily distinguished the 2 cases of smallpox from the other 9 samples, 5 of which contained varicella-zoster (two had been misdiagnosed as herpes) and the other 4 were disseminated herpes simplex. The in situ test, readily accomplished in any histology-based molecular laboratory in 4 hours, allows for the rapid and specific identification of smallpox infection and, importantly, its distinction from its mimics. Formalin fixation, which is optimal for in situ hybridization, guarantees the inactivation of the smallpox virus.     

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-α pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-α pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.     

Low elementary movement speed is associated with poor motor skill in Turner's syndrome

The article aims to discriminate between 2 features that in principle both may be characteristic of the frequently observed poor motor performance in girls with Turner's syndrome (TS). On the one hand, a reduced movement speed that is independent of variations in spatial accuracy demands and therefore suggests a problem in motor execution. On the other hand, a disproportional slowing down of movement speed under spatial-accuracy demands, indicating a more central problem in motor programming. To assess their motor performance problems, 15 girls with TS (age 9.6-13.0 years) and 14 female controls (age 9.1-13.0 years) were tested using the Movement Assessment Battery for Children (MABC). In additionally, an experimental procedure using a variant of Fitts' graphic aiming task was used to try and disentangle the role of spatial-accuracy demands in different motor task conditions. The results of the MABC reestablish that overall motor performance in girls with TS is poor. The data from the Fitts' task reveal that TS girls move with the same accuracy as their normal peers but show a significantly lower speed independent of task difficulty. We conclude that a problem in motor execution is the main factor determining performance differences between girls with TS and controls.     

Preserved contribution of nitric oxide to baseline vascular tone in deconditioned human skeletal muscle

Deconditioning is a risk factor for cardiovascular disease. Exercise reduces this risk, possibly by improving the vascular endothelial nitric oxide (NO) pathway. The effect of deconditioning on the NO pathway is largely unknown. This study was designed to assess baseline NO availability in the leg vascular bed after extreme, long-term deconditioning (spinal cord-injured individuals, SCI) as well as after moderate, short-term deconditioning (4 weeks of unilateral lower limb suspension, ULLS). For this purpose, seven SCI were compared with seven matched controls. Additionally, seven healthy subjects were studied pre- and post-ULLS. Leg blood flow was measured by venous occlusion plethysmography at baseline and during infusion of 5 incremental dosages of N ^G-monomethyl- l -arginine ( l -NMMA) into the femoral artery. Sodium nitroprusside (SNP) was infused to test vascular responsiveness to NO. Baseline leg vascular resistance tended to be higher in SCI compared with controls (37 ± 4 versus 31 ± 2 arbitrary units (AU), P = 0.06). Deconditioning altered neither the vasoconstrictor response to l -NMMA (increase in resistance in SCI versus controls: 102 ± 33% versus 69 ± 9%; pre- versus post-ULLS: 95 ± 18% versus 119 ± 15%), nor the vascular responsiveness to NO. In conclusion, two human in vivo models of deconditioning show a preserved baseline NO availability in the leg skeletal muscle vascular bed.     

Morphometric and immunohistochemical characterization of the intimal layer throughout the arterial system of elderly humans

The purpose of the present study was to obtain insight into the natural development of adaptive intimal thickening and atherosclerosis in the arterial tree of human species. The morphometry and composition of the intimal layer were studied in the arterial system of elderly individuals. Post mortem, a total of 703 arterial segments were dissected from 24 subjects (age 81.9 ± 9.9 years). From each subject, segments were dissected from 31 different arteries. Area stenosis [(plaque area/vessel area) × 100%] was determined in each segment. By (immuno)histochemistry, lipid content and the presence of inflammatory cells (macrophages) were assessed in the coronary, common carotid, brachial, radial and internal iliac arteries. Adaptive intimal thickening or advanced atherosclerosis was observed in all studied artery types. Area stenosis was highest in the coronary arteries (median, 30%) and lowest in the arteries supplying the brain (median, ≤ 7%). Plaques hiding a lipid‐rich core and plaques with macrophage infiltration were observed in all five selected artery types. In summary, the present observation demonstrates that intimal thickening is a systemic process involving most artery types. Within elderly humans, features of advanced atherosclerotic disease, a lipid‐rich core and macrophages, can be observed in the intimal layer of artery types that are recognised for their relation with clinical syndroms as well as artery types that remain clinically silent.     

Application of multidimensional scaling to ratings of foods for obese and normal weight individuals

Sixteen obese patients, aged 19–31, and 27 normal weight individuals, aged 18–22, tasted and smelled fresh, unseasoned blended foods while blindfolded. Then they rated the foods on 51 semantic differential adjective scales which related to stimulation of the gustatory, olfactory, and trigeminal systems. The stimuli included fruits, vegetables, meat, fish, nuts, dairy products, grains, and a set of four standards: sucrose (sweet), NaCl (salty), lemon (sour), and coffee (bitter) in a thin cornstarch base. Proximity measures among stimuli for each subject were developed from the ratings on the adjective scales. Two multidimensional scaling (MDS) procedures, SINDSCAL (a variation of INDSCAL) and ALSCAL, were applied to the proximity measures yielding two-dimensional food maps which were very similar. Both MDS procedures provided weights for each subject on the two dimensions of the space common for all subjects. The weights indicated that the obese subjects found the first dimension, which was related to the hedonic and flavorous aspects of the stimuli, relatively more important than normal weight individuals. Also, obese subjects were significantly better at identifying the blended foods when compared to normal weight subjects.