Multicenter evaluation of new double-antigen sandwich enzyme immunoassay for measurement of anti-human immunodeficiency virus type 1 and type 2 antibodies.

A new enzyme immunoassay (EIA), the Cobas Core Anti-HIV-1/HIV-2 EIA DAGS (also referred to as Roche DAGS), for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 was evaluated in four centers. The assay is based on the double-antigen sandwich (DAGS) format, which enables the detection of all classes of antibodies. The antigens consist of recombinant proteins in their native conformation and of synthetic peptides. Of a total of 5,836 negative serum samples, including 95 samples likely to produce false reactivities, 6 were false positive, resulting in a specificity of 99.9%. None of 35 sera that were from noninfected individuals but contained p24-cross-reacting antibodies as revealed by Western blot (immunoblot) analysis were reactive by the Roche DAGS assay. In samples from individuals infected with HIV-1 group M (n = 499) and HIV-2 (n = 200), the sensitivity of the assay was 100%. Although containing antigens with sequences from subtype B only, the assay was also able to correctly identify with high optical density/cutoff ratios samples from subjects infected with HIV-1 subtype O (n = 10). In 17 of 19 seroconversion panels tested, the assay detected the presence of HIV-1 antibodies as early as another sandwich EIA. Eight of these panels were also analyzed by an indirect second-generation assay, which detected antibodies 2 to 10 days later than did the DAGS assay under evaluation. The excellent specificity and sensitivity of the new Cobas Core Anti-HIV-1/HIV-2 EIA DAGS are the result of the DAGS format as well as of the native, naturally folded form of the recombinant protein used as the gag antigen.

Epitopes of the T19 lymphocyte surface antigen are extensively conserved in ruminants.

The reactivity of five monoclonal antibodies (mAbs SBU-T19, 197, IL-A29, CC-15 and CC-39) specific for the T19 molecule on sheep and cattle CD4-CD8- T cells was compared. MAbs SBU-T19 and 197 were shown to recognise separate epitopes on T19. All mAbs reacted with lymphocytes from several different ruminant species and the tissue distribution and frequency of positive cells was similar in each case. None of the mAbs reacted with horse, pig or camel lymphocytes. The extensive conservation of T19 epitopes in ruminants during the mammalian radiation could indicate an important role for this molecule in the ruminant immune system.

T cell receptor gene expression in sheep: differential usage of TcR1 in the periphery and thymus.

We have isolated cDNA clones for each chain of the sheep T cell receptor (TcR) and used these to analyze TcR gene expression in thymocyte and peripheral T cell subsets. Outer cortical thymocytes expressed a low level of mature message for all TcR chains suggesting that intrathymic precursors for the alpha/beta and gamma/delta lineages occur in this population. Inner cortical and medullary thymocytes expressed high levels of mature alpha/beta transcripts, low levels of mature delta transcripts but no detectable gamma message. Mature alpha/beta transcripts were detected in peripheral CD4+ lymphocytes and these as well as CD8+ cells expressed a surface heterodimer of 85 kDa which resolved into 40- and 50-kDa subunits after reduction. Peripheral CD4-CD8-lymphocytes, which in sheep are marked by the T19 antigen and may account for up to 60% of T cells in blood, expressed a surface heterodimer of 75 kDa. The T19+ cells had high levels of mature delta and truncated beta transcripts in their cytoplasm but did not express the C gamma gene detected in DN thymocytes, although they seem to share V gamma and/or J gamma elements. Both forms of the sheep TcR are associated with CD3 molecules on the cell surface. These results show that (a) in contrast to the situation in rodents and humans, a large proportion of peripheral sheep lymphocytes use TcR gamma/delta; (b) the proportion of T cells in the periphery which use TcR gamma/delta is greater than in thymus; and (c) CD4-CD8- cells in thymus and periphery (T19+) use the same C delta gene but appear to use different C gamma genes, suggesting that in sheep there may be more than one lineage of lymphocytes expressing TcR gamma/delta.

Evidence that the B5 molecule on sheep lymphocytes is a homologue of mouse Ly 6 and human CD59.

When analysed by SDS-PAGE, the B5 molecule immunoprecipitated from sheep thymocytes, T cells and B cells migrated as multiple bands within a size range of 11,000-17,000 MW. After digestion with endoglycosidase-F, the precipitate from all cell types yielded a 6000 MW band. T-cell lysates gave an additional band at 6300 MW. All forms of the B5 molecule were anchored to the cell membrane via phosphatidylinositol. These results strengthen earlier evidence that the B5 molecule is a sheep homologue of the mouse Ly 6 antigen. The additional protein unit found on T cells may be of functional importance since the B5 molecule is known to be involved in an activation pathway of T cells but not of thymocytes or B cells.

Gamma/delta T cells express a unique surface molecule appearing late during thymic development.

The vast majority of T cells in man and mouse use the alpha/beta form of T cell receptor (TcR), and express either CD4 or CD8, whereas the small subset of gamma/delta T cells are usually CD4-CD8-. In contrast to man and mouse, the gamma/delta subset in sheep, defined here using an anti-gamma/delta monoclonal antibody (mAb), comprises 30%-60% of T cells. We show that gamma/delta T cells in sheep express a unique surface molecule termed T19 which is 215 kDa in size and unrelated to either CD45 or the TcR. The T19 molecule was expressed at a distinct stage during gamma/delta T cell ontogeny within the thymus, since gamma/delta thymocytes which appeared early in fetal ontogeny were T19- and also major histocompatibility complex (MHC) class I- and localized almost exclusively to the outer cortex and cortex of the thymus. "Mature-type" gamma/delta thymocytes which emerged late in thymic development were T19+ and MHC class I+ and localized predominantly to the thymic medulla. The sequence of events indicated that these cells were most likely derived from the early gamma/delta thymocytes. These medullary gamma/delta thymocytes showed a very distinctive association with Hassall's corpuscles, suggesting a role for these structures in gamma/delta thymocyte maturation. In the periphery, T19 was expressed exclusively within the gamma/delta T cell subset, however some gamma/delta T cells were T19-. In particular, a large proportion of gamma/delta T cells within intestinal epithelium lacked T19, indicating a correlation between T19 expression and either function or homing patterns of gamma/delta T cells. Both T19+ and T19- gamma/delta T cells were CD2-, and expressed low levels of LFA-1 and CD5. In addition, gamma/delta T cells recirculated differently from other T cells, and appeared not to enter mesenteric lymph nodes at all from the blood. We propose that T19 is a maturation marker for gamma/delta T cells. In addition, the exclusive expression of T19 by gamma/delta T cells indicates that this molecule most likely serves a fundamental role in the interactions and function of gamma/delta T cells.

Ontogeny, morphology and tissue distribution of a unique subset of CD4-CD8- sheep T lymphocytes.

The morphology, distribution and ontogeny of the 197+ T lymphocyte subpopulation is described. Cells were identified by immunofluorescent and immunoenzymic staining of cell suspensions and tissue sections. In the circulation, 197+ cells are small and regular, indistinguishable in size from peripheral CD4+ and CD8+ cells, with little cytoplasm and a large amount of condensed chromatin. In the post-natal animal they are unevenly distributed, being most common in the circulatory pathways, particularly in the blood. In the thymus they chiefly localize in the regions around Hassal's corpuscles and medullary blood vessels. They are totally absent from B cell areas in all tissues. They are the last of the T cells to appear in ontogeny, and are first detected in the thymus. Their frequency in blood is age-dependent with peak levels occurring perinatally. There is a post-natal decline in the frequency of 197+ cells in ileocaecal but not in prescapular lymph nodes. We conclude that these T cells differ from the more commonly described T cells, not only in their surface expression of the CD4, CD8 and T19 antigens, but also in the time of their first appearance, their age-related prevalence and their distribution between and within tissues.

A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway.

We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.

A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population.

A mouse monoclonal antibody (mAb), ST-8, reactive with a subpopulation of sheep T lymphocytes was investigated by tissue distribution analysis and functional studies of the antigen-bearing (ST-8+) cells. Two other mouse mAb, ST-1 and T-80 that recognize all sheep T cells and a T-cell subset, respectively, were also examined for comparison. The ST-8+ cells represented 61-69% of thymocytes and about 10-30% of peripheral T lymphocytes. Histologically, ST-8+ cells were found mainly in the thymic cortex, T-dependent areas of the peripheral lymphoid tissues and the splenic red pulp and marginal zone, but not in B-dependent areas such as germinal centers of lymph follicles of the Peyer's patches. ST-8 mAb plus complement treatment completely abolished both the induction phase and the effector phase of alloreactive cytotoxic T lymphocytes but did not affect proliferative responses to T-dependent mitogens and allogeneic antigens. ST-8 mAb also blocked the cytotoxic T lymphocyte function of lysing specific targets in the absence of complement. ST-8 mAb immunoprecipitated an antigen from the surface of sheep T lymphocytes under reducing conditions with an apparent molecular weight of 33-35 kilodaltons. Therefore the antigen recognized by the ST-8 mAb showed striking similarities to human T8/Leu-2a and mouse Lyt-2 antigens not only in the tissue distribution and function of antigen-bearing cells but also the molecular weight. We conclude that the ST-8 antigen is the ovine homologue of the human T8 and mouse Lyt-2 antigens.

Studies on the differentiation of T lymphocytes in sheep. III. Preliminary characterization of an antigen recognized by two anti-pan T-cell monoclonal antibodies.

ST-1a and ST-1b are monoclonal antibodies that selectively react with cells in the T-cell lineage of the sheep. Preliminary structural studies using radioimmunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that both ST-1a and ST-1b recognize an antigen of apparent molecular weight 67,000 and 60,000-65,000, under either reducing or nonreducing conditions. Differential expression of these 67,000 and 60,000-65,000 MW proteins was observed in T cells obtained from various anatomical compartments. Cortical thymocytes and efferent lymph lymphocytes expressed predominantly the 67,000 molecule, whereas a thymus cell fraction enriched for medullary thymocytes expressed mainly the 60,000-65,000 MW molecule. Both proteins were found in equivalent amounts in immunoprecipitates obtained from peripheral blood lymphocytes and lymph node lymphocytes. Peptide mapping studies indicated that 67,000 proteins of both ST-1a and ST-1b immunoprecipitates have significant homology in peptide composition. Sequential immunoprecipitation experiments clearly demonstrated that the antigens recognized by ST-1a and ST-1b antibodies are the same. On the basis of the size, tissue distribution and trypsin sensitivity of the antigen, together with cytofluorometry data, we suggest that the target antigen of ST-1a and ST-1b monoclonal antibodies is the ovine homologue of mouse Lyt-1 and human Leu-1.

Studies on the differentiation of T lymphocytes in sheep. II. Two monoclonal antibodies that recognize all ovine T lymphocytes.

Two mouse monoclonal cytotoxic antibodies (ST-1a and ST-1b) recognize an antigen present on the large majority of thymocytes and all T cells in the periphery, but not B cells or other haemopoietic cells in sheep. Examination of frozen sections of various fetal tissues revealed that the cells expressing this antigen first appeared in the thymus, and these cells markedly increase in numbers in the peripheral lymphoid tissues after mid-gestation. Large accumulations of positive cells were located in the paracortex of lymph nodes, the periarteriolar lymphoid sheath of the spleen, and interfollicular areas of jejunal Peyer's patches, all of which are known to be T-dependent areas. Treatment of lymphocytes with ST-1a and complement resulted in the abrogation of T-proliferative responses, but the response to a B-cell mitogen, lipopolysaccharide, was not reduced. Neither ST-1a nor ST-1b cross-reacted to lymphocytes obtained from other species of animals (man, monkey, mouse, rat, guinea-pig, chicken, frog, pig, horse, goat and cattle). Based on these findings, it was concluded that the expression of the antigen recognized by ST-1a and ST-1b is restricted to the T-cell lineage of sheep, and that all ovine T cells express this antigen. Furthermore, ST-1a and ST-1b were determined to recognize the same antigen by reciprocal blocking experiments.

Skin polyamine levels in psoriasis: the effect of dithranol therapy.

Concentrations of putrescine, spermidine and spermine were determined in biopsies of skin of eight control subjects and of uninvolved skin and skin lesions of eight psoriatic patients before and after topical therapy with dithranol. Mean spermidine and spermine concentrations in uninvolved skin of psoriatic patients were normal, but putrescine concentration was elevated. In psoriatic skin lesions all three amines were significantly elevated. Chemotherapy effectively reduced polyamine concentrations in involved skin touards normal. This decrease in polyamine levels correlated with reduction of the proliferative activity of the epidermis and with clinical improvement of the patients.