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.     

Con A activates an Akt/PKB dependent survival mechanism to modulate TCR induced cell death in double positive thymocytes

While low avidity ligation of the T cell receptor (TCR) leads to positive selection and further maturation of developing thymocytes providing the immune system with mature CD4(+) and CD8(+) (single positive) T cells, high avidity ligation triggers negative selection by apoptotic cell death and therefore the TCR repertoire is purged of autoreactive T cells. On peripheral T cells, however, high avidity ligation of the TCR triggers activation and survival not death. In the present study we used concanavalin A (Con A) and alpha-CD3 epsilon antibody to investigate a possible survival mechanism in connection with TCR ligation. Con A and alpha-CD3 epsilon were used in the study for the following reasons: (1) they both mimic the effects of high avidity TCR ligation by activating peripheral T cells, and (2) they trigger distinctively different physiological changes in developing thymocytes. While Con A supports events associated with cellular survival, alpha-CD3 epsilon induces apoptotic cell death. In our experimental system the TCR was cross-linked by Con A and alpha-CD3 epsilon in thymocytes of major histocompatibility complex (MHC) deficient thymus organ cultures, where signals from the TCR can be triggered on zero background signal level. We have found that TCR cross-linking by Con A and not by alpha-CD3 epsilon decreases the gene and protein expression of the pro-apoptotic molecule, Bad; and that Con A is capable of the activation of the survival signalling pathway including protein kinase B (Akt/PKB) independently of phosphatidyl inositol kinase (PI3K).     

Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males

Adult male CD rats and CD-1 mice were given a single oral dose of ethylene glycol monomethyl ether (EGM) at 0, 500, 750, 1,000, or 1500 mg/kg. Groups of 10 were killed at weekly intervals after dosing for analysis of sperm counts and morphology or testicular histology; further groups of 10 were sequentially mated to pairs of virgin females to test for dominant lethality or gross foetal malformations in the F1 generation (F1 abnormalities). EGM was found to deplete the spermatocytes of both species severely, principally pachytene cells, but with other stages affected with increasing dose. A delay in the progression of spermatogenesis may account for a discrepancy between the apparent stage-specificity of damage deduced from lowered sperm counts and that observed histologically. In the rat, morphological abnormalities were observed in sperm that had been exposed as spermatocytes; in the mouse, however, the sensitive cells were the late spermatocytes and early spermatids. In all these parameters there was an indication of a dose-response relationship in both rats and mice. In the mating studies EGM induced a dose-related decrease in fertility 5 weeks after dosing in the rat, but complete sterility in all but the lowest dose after 6 weeks. In contrast, EGM had no effect on the reproductive capacity of the mouse. There was no statistically significant evidence for the induction of dominant lethal mutations or F1 abnormalities in either species. A single oral dose of cyclophosphamide (CTX) at 100 mg/kg induced a significant increase in dominant lethality in both species. CTX reduced the number of total implants in the rat and induced a nonsignificant increase in the number of abnormal offspring sired by treated male mice.     

Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions.

Candida albicans binds to several species of oral streptococci, in particular Streptococcus gordonii, through recognition of a streptococcal cell wall polysaccharide receptor (A. R. Holmes, P. K. Gopal, and H. F. Jenkinson, Infect. Immun. 63:1827-1834, 1995). We now show that isogenic cell surface protein mutants of S. gordonii DL1, unaltered in expression of cell wall polysaccharide, are reduced in ability to support adherence of C. albicans cells in a solid-phase assay. Inactivation of the S. gordonii cshA and cshB genes, encoding high-molecular-mass cell surface polypeptides, and inactivation of the sspA and sspB genes, encoding antigen I/II salivary adhesins, resulted in 40 and 79% reductions, respectively, in adherence of C. albicans cells. Inactivation of the S. gordonii scaA gene encoding a cell surface lipoprotein had no effect on C. albicans adherence. Polyclonal antiserum to streptococcal antigen I/II protein SpaP and antibodies specific to the amino-terminal nonrepetitive (NR) domain of CshA both inhibited adherence of C. albicans to S. gordonii cells. Conversely antibodies to the amino acid repeat block repetitive (R) domain of CshA, or to ScaA, did not inhibit C. albicans adherence. Immobilized recombinant polypeptide fragments of CshA comprising NR domain or R domain sequences both supported adherence of C. albicans cells. Expression of S. gordonii SspB protein on the surface of Enterococcus faecalis conferred on the enterococcal cells the ability to bind C. albicans, and this was ablated by antigen I/II antiserum. Collectively the results suggest that interaction of C. albicans with S. gordonii is mediated by a complement of adhesin-receptor interactions that involves two families of streptococcal multifunctional polypeptide adhesins, bacterial cell wall polysaccharide, and as yet unidentified yeast cell surface components.     

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.     

Adherence of Streptococcus sanguis

The presence of adhesins on the cell surface of S. sanguis enables the organism to grow and survive in the oral cavity. Many workers are now actively involved in attempting to characterise these adhesins and the molecular basis for adherence of various streptococci. The complexity of the adhesion processes is underlined by the many varied opinions as to the nature of the molecular components and biochemical interactions that are involved. Understanding the molecular mechanisms involved in bacterial adherence and in dental plaque formation will have significant impact on the prevention and treatment of oral diseases.