Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin.     

Flow microfluorometric analysis of mouse thymus development in vivo and in vitro

The phenotypic properties of lymphoid cells in the developing embryonic thymus were characterized using monoclonal antibodies and flow microfluorometry. CBA/J-T6/T6 thymocytes stained with antibodies directed against Thy-1.2, Lyt-1, Lyt-2 or H-2K^k were simultaneously analyzed for fluorescence intensity and forward light scatter (FLS), a cell size-related parameter. Whereas Thy-1 and Lyt-1 antigens were already present on 15-day fetal thymocytes, Lyt-2 expression was first detectable on day 16 and increased rapidly thereafter to reach adult levels by day 19. Concomitant with these phenotypic changes, rapid changes in FLS occurred during this time period. The FLS distribution of Lyt-2⁺ cells was initially homogeneously high (day 16) but became biphasic at days 17–18. Thereafter, the lower FLS subpopulation predominated. FLS changes in Lyt-2^? cells could be dissociated kinetically from changes in the Lyt-2⁺ subpopulation. Thus high FLS Lyt-2^? cells were the predominant subpopulation throughout the entire fetal period and could still be detected after birth, when a population with lower FLS first appeared. The embryonic thymus developing in vivo was then compared with the 13-day embryonic thymus maintained for 14 days in an in vitro organ culture system. Based on a combination of fluorescence and FLS analysis, the organ-cultured thymus appeared to share certain phenotypic properties with the 18–19 day in vivo developing thymus.     

Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection

The fully automated IMx immunoassay analyzer was used to develop a system for the detection of IgG and IgM antibodies to rubella virus for immune status screening and diagnosis of primary infections. Reagents and assay protocol software were developed using rubella virus sensitized microparticles as the solid phase to capture specific antibodies from serum samples. Anti-human IgG or IgM antibody coupled to alkaline phosphatase enzyme followed by methylumbelliferyl phosphate substrate was used to detect the presence or absence of antibodies specific to the antigens on the solid phase. To evaluate the efficacy of the IMx rubella IgG assay, immune status screening was performed with a clinical patient population of 501 sera. When compared to an IgG specific enzyme immunoassay and passive hemagglutination assay the agreement was greater than 99%. The IMx rubella IgM assay was utilized to determine the presence of rubella specific IgM antibodies in 462 sera. These results were compared to IgM specific enzyme immunoassay results and also demonstrated greater than 99% agreement. Seroconversion following rubella vaccination of susceptible individuals was demonstrated by IgG and IgM antibody responses as early as two weeks postvaccination. In addition to automation, the IMx system offers rapid assay times and calibration curve storage without sacrificing clinical efficacy.     

Studies of the association of the A, B and Lewis blood group antigens with carcinoembryonic antigen (CEA)

Carcinoembryonic antigen (CEA) was purified from primary tumour or from hepatic metastases obtained from ten cases of carcinoma of the colon. In nine cases the blood group antigens A, B, Le^a or Le^b were detected in CEA preparations by the binding of ¹²⁵I-labelled CEA by blood group antibodies. The extent of binding appeared to preclude simple contamination of CEA preparations by blood group glycoprotein. In all cases the blood group antigens detected were consistent with the patients' known blood groups. Blood group I and i activities were not detected.

It is concluded that the determinants of A, B and Lewis antigens and of CEA share the same glycoprotein carrier molecules.

     

Effect of treadmill exercise on food intake and body weight in lean and obese rats

Body weight and food intake of lean and obese, male and female Osborne-Mendel rats following treadmill exercise were compared. Rats were assigned, separately by sex, to one of three diet groups; Group 1 was fed a low fat (10%) diet throughout the study, Group 2 was fed a high fat (55%) diet for 16 weeks and then switched to the low fat diet 1 week prior to exercise, and Group 3 was fed the high fat diet throughout the study. To control for differences in work output between the leanest and heaviest animals, exercise intensity was adjusted across groups such that all exercised rats had equivalent energy expenditure. After a 3 day training period, the exercise was successively increased over 8 days until a work output of 374.9J was reached. Relative to their respective controls, obese exercised males showed a reduction in body weight but no change in food intake. In contrast, exercised females showed no change in body weight or food intake, regardless of dietary condition.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Tissue-specific segregation of TCRgamma delta+ NKT cells according to phenotype TCR repertoire and activation status: parallels with TCR alphabeta+NKT cells

Whereas the majority of NKT cells in the mouse express an alpha beta TCR (NKTalpha beta cells), a small subset of NKT cells express a gamma delta TCR (NKTgamma delta). Here we have systematically analyzed the phenotype, TCR repertoire and activation status of NKTgamma delta cells in the thymus, liver, spleen and bone marrow of normal C57BL/6 mice. Our data indicate that NKTgamma delta cells segregate in a tissue-specific manner according to these parameters. While most NKTgamma delta cells in the thymus and liver have a recently activated CD62L(lo) phenotype and a TCR repertoire that is heavily biased to Vgamma1.1 and Vdelta6.3, the majority of NKTgamma delta cells in the spleen and bone marrow are CD62L(hi) and have a much less biased TCR repertoire. Moreover, expression of NK markers is high on NKTgamma delta cells in spleen and bone marrow but low in thymus and liver. Collectively our results reveal a tissue-specific segregation of NKTgamma delta cells that is strikingly similar to that recently described for CD1d-dependent and Cd1d-independent NKTalpha beta cells. We therefore propose that chronic TCR activation by tissue-specific endogenous ligands is a generic property of NKT cells of both the alpha beta and gamma delta lineages.     

The glioblastoma-derived T cell suppressor factor/transforming growth factor-beta 2 inhibits T cell growth without affecting the interaction of interleukin 2 with its receptor

Human glioblastoma cells secrete a peptide termed glioblastoma-derived T cell suppressor factor (G-TsF) which inhibits T cell activation. Recently, purification and cloning of G-TsF revealed that G-TsF is identical to transforming growth factor-beta 2. As shown here, G-TsF suppresses the growth of an ovalbumin-specific mouse T helper cell clone (OVA-7T) independently of the stimulus used being either (a) antigen in the presence of antigen-presenting cells, or (b) interleukin 2 (IL2) or (c) phorbol ester and calcium ionophore. Furthermore, in the presence of antibodies against IL2 receptors, G-TsF was able to suppress the residual proliferation still observed when OVA-7T were stimulated with phorbol ester/ionophore. G-TsF failed to inhibit the release of IL3 from OVA-7T activated with IL2. Taken together, the data provide evidence that G-TsF does not directly interfere with interactions of IL2 with its receptor but rather inhibits T cell activation by interfering with an as yet unidentified pathway used by both IL2 and phorbol ester/ionophore. When analyzing different monokines and lymphokines for its effect on G-TsF-induced suppression of T cell growth the only factor found to partially neutralize the effect of G-TsF was tumor necrosis factor-alpha.