The role of CD4+ T-cell subsets in determining transplantation rejection or tolerance.

Peripheral tolerance to allogeneic organ grafts can be induced in rodents by treating with non-depleting CD4 and CD8 monoclonal antibodies. This tolerance is maintained by CD4+ T cells with a potent capacity to induce tolerance in further cohorts of T cells (i.e. infectious tolerance). We have cloned CD4+ T-cell subsets against the male transplantation antigen in vitro and find, in contrast to Th1 or Th2 clones that elicit rejection, that there is a distinct population of CD4+ T cells that suppress rejection by adoptive transfer (here called Treg). In order to identify molecular markers associated with tolerance and gain insights into the mechanisms of action of Treg cells, we carried out serial analysis of gene expression. We identified genes overexpressed in Treg compared to Th1 and Th2 cultures and found that some of these correlated in vivo with CD4-induced transplantation tolerance rather than rejection. The genes overexpressed in Treg cultures and within tolerated skin grafts were primarily expressed by mast cells (e.g. tryptophan hydroxylase and FcepsilonR1alpha), suggesting that regulatory cell activity and this form of tolerance may be associated with a localised but non-destructive form of Th2-like activation and a recruitment of mast cells.

Variations in the relative amounts of biotin-containing enzymes present in both tumorigenic and non-tumorigenic hybrid cells and other cell lines.

The observation that radioactively labelled streptavidin binds to several biotin-containing enzymes in mammalian cells has led to the finding that there is considerable variation in the proportion of these enzymes present (namely beta-methyl crotonyl CoA; propionyl CoA; pyruvate and acetyl CoA, carboxylases). This is particularly striking when certain tumorigenic and non-tumorigenic hybrid cells are compared. It is found that there is a consistently higher proportion of pyruvate carboxylase in the tumorigenic hybrid cells. However, not all tumorigenic cell lines show this same characteristic and reasons for this are discussed. It is also shown that whilst the proportions of the four enzymes are apparently constant for a given cell type, there is a substantial degree of clonal variation and this is particularly so in tumorigenic cells in vitro. However, the more tumorigenic cells in a given population do show a higher proportion of pyruvate carboxylase. Also a range of cells derived from lymphoid tissue has been compared with normal human lymphocytes and considerable differences are again observed. The significance of these findings is considered in relation to other phenotypic properties of hybrid cells.

Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells.

1. Maximal activities of some key enzymes of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and glutaminolysis were measured in homogenates from a variety of normal, neoplastic and suppressed cells. 2. The relative activities of hexokinase and 6-phosphofructokinase suggest that, particularly in neoplastic cells, in which the capacity for glucose transport is high, hexokinase could approach saturation in respect to intracellular glucose; consequently, hexokinase and phosphofructokinase could play an important role in the regulation of glycolytic flux in these cells. 3. The activity of pyruvate kinase is considerably higher in tumorigenic cells than in non-tumorigenic cells and higher in metastatic cells than in tumorigenic cells: for non-tumorigenic cells the activities range from 28.4 to 574, for tumorigenic cells from 899 to 1280, and for metastatic cells from 1590 to 1627 nmol/min per mg of protein. 4. The ratio of pyruvate kinase activity to 2 x phosphofructokinase activity is very high in neoplastic cells. The mean is 22.4 for neoplastic cells, whereas for muscle from 60 different animals it is only 3.8. 5. Both citrate synthase and isocitrate dehydrogenase activities are present in non-neoplastic and neoplastic cells, suggesting that the full complement of tricarboxylic-acid-cycle enzymes are present in these latter cells. 6. In neoplastic cells, the activity of glutaminase is similar to or greater than that of hexokinase, which suggests that glutamine may be as important as glucose for energy generation in these cells.

Immunoreactive determinants of CA 125 in women with endometriosis.

Among 10 patients with endometriosis CA 125 was increased (greater than 35 U/ml) in endometriotic cyst fluid in all the patients, but only two had increased serum concentrations. Gel electrophoresis of serum, endometriotic cyst fluid, and endometriotic tissue resolved the CA 125 immunoreactive fragments from the three sources into bands of similar electrophoretic mobilities. Electrophoresis under reducing and non-reducing conditions showed immunoreactive fragments of apparent masses of 55,000 and 140,000 daltons, respectively. Analysis under reducing conditions did not result in loss of activity. CA 125 antigen is thought to be a high molecular weight glycoprotein complex. As far as is known, this is the first report describing lower molecular weight immunoreactive determinants of CA 125.

Further characterization of the antigen defined by the monoclonal antibody M27.

Using immunoblotting techniques, the antigen that binds the monoclonal antibody M27 has been clearly defined in terms of apparent molecular mass and distribution. In reducing conditions it has an apparent mass of 178K (K = 10(3) Mr) and is present in the cytoplasm and membranes of all mammalian tissue culture cells so far examined. It is absent from lines derived from avian, piscine and amphibian sources. It is also absent from foetal liver of both rat and mouse, but subsequently appears after cultivation in vitro. Similarly, it can be detected on rat lymphocytes only after mitogenic stimulation. However, it is found on both hepatoma and lymphoma cells in vitro, and on in vivo tumours from murine sources. It thus appears to be associated with cell proliferation.

A modified adenosine polyphosphatase histochemical method to demonstrate Langerhans cells in fragile epidermis.

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate "plastic" slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 X 10(-4) M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.

The immunohistochemical demonstration of thyroglobulin in human thyroid tumour xenografts using a monoclonal antibody.

A monoclonal antibody (RBU/01) was raised against human thyroglobulin and its suitability for the immunohistochemical staining of thyroglobulin was determined on fixed, wax-embedded tissue, using the peroxidase anti-peroxidase (PAP) method. The antibody was then used to demonstrate the expression of human thyroglobulin in sections of a human follicular carcinoma of the thyroid which had been grown in immunodeficient mice. It is concluded that the immunohistochemical evaluation of the xenografts with the antibody provides useful information on this xenograft system as a potential model for thyroid carcinoma.

Changes with time in Langerhans cell number, ATPase reactivity and morphology in murine epidermis after exposure to UVB.

Effects on Langerhans cells of a single 200 mJ/cm2 dose of 284-316 nm UVB were studied in the dorsal epidermis of CBA/H mice. ATPase-positive cells were counted using light microscopy and compared with numbers of cells containing Birbeck granules. In interfollicular epidermis from untreated mice 633 +/- 20 (mean +/- S.E., n = 30) ATPase-positive cells/mm2 were found. By 3 days after UVB treatment, the number of ATPase-positive cells decreased by 90%, whilst the Birbeck granule-containing cell population was only reduced by 40% and very few cells had ultrastructural damage, suggesting that many Langerhans cells lost ATPase marker but remained intact. 7 d after irradiation there were 50% more cells with Birbeck granules than in controls, but the number of cells bearing ATPase remained below normal. ATPase-positive cell numbers approached normal only after a further 2-3 weeks. Langerhans cell responses after UVB differed from those previously found after X-irradiation.