Automated histochemical analysis of cell populations in the intact follicle-associated epithelium of the mouse Peyer's patch

Summary A new technique of quantitative histochemistry has been developed to study the cellular composition of the follicle-associated epithelium of the mouse Peyer's patch. This technique involves applying naphthol AS-BI phosphate to the surface of intact tissue where it is hydrolysed by alkaline phosphatase present in the luminal membrane of the epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. M cells present in the epithelium contain little alkaline phosphatase activity and, therefore, remain white. Treatment with Alcian Blue is finally used to label goblet cells. Subsequent quantitative analysis of alkaline phosphatase-rich cells is carried out by scanning microdensitometry. Using this technique it is possible to detect two populations of alkaline phosphatase-containing cells in mice reared in a normal animal house environment.These results are discussed in relation to possible interactions taking place between enteric antigens and the gut-associated lymphoid tissue which could reduce the ability of follicle-associated enterocytes to express alkaline phosphatase.     

Adrenocorticotropin-dependent regulation of glutathione transferase subunit 4 in cultured rat adrenal cells.

After hypophysectomy, the level of glutathione transferase subunit 4 increases in the adrenal, as well as in the liver, as do those of several other forms of glutathione transferase. This increase in subunit 4 can subsequently be down-regulated by administration of adrenocorticotropin. The present investigation demonstrates that also in primary cultures of female rat adrenal cells an increase in the level of glutathione transferase subunit 4 (as shown by immunoblotting) occurs in the absence of adrenocorticotropin. When adrenocorticotropin or dibutyryladenosine 3',5'-phosphate was administered to these cells, a down-regulation of this enzyme level was observed, in agreement with the in vivo situation. This down-regulation was not affected by aminoglutethimide, an inhibitor of the cholesterol-side-chain-cleavage enzyme (cytochrome P-450scc) which is the rate-limiting step in the biosynthesis of steroids. Hence adrenal steroid production is not involved in the down-regulation of glutathione transferase subunit 4 by adrenocorticotropin.     

HLA 1990

The HLA system encompasses approximately one thousandth of the human genome and contains a series of closely linked loci coding for molecules which provide the context for the recognition of antigens by T lymphocytes. Within the HLA system, several phenotypically expressed and genomic polymorphisms can be distinguished. These polymorphisms are described and the main objectives for the future research are discussed.     

Lack of chondrogenic expression in mouse limb bud micromass cultures exposed to exogenous beta galactosidase or N-acetyl-beta-glucosaminidase

The effect of two exoglycosidases, beta-galactosidase and N-acetyl-beta-glucosaminidase (GlcNAc-ase) on chondrogenic expression of stage 19 mouse limb bud micromass cultures was investigated. Chondrogenic expression was monitored by Alcian blue staining and immunofluorescent localization of cartilage-specific proteoglycan and type II collagen. Chondrogenesis was inhibited by exposure to 0.1 U/ml beta-galactosidase or 0.025 U/ml GlcNAc-ase for 24 h or longer in culture. The effect of both enzymes was concentration and time dependent. Exoglycosidic hydrolysis of galactose or N-acetylglucosamine was substantiated by treatment with HRP-conjugated peanut agglutinin and succinylated wheat germ agglutinin, respectively. Cells treated with beta-galactosidase appeared to be flattened with a stellate morphology, whereas GlcNAc-ase-treated cells were bipolar forming ridge-like mounds that had a directional orientation. The antichondrogenic effect was not alleviated when the cells were induced to assume a spherical shape upon treatment with cytochalasin D. DNA measurements indicated that the lack of chondrogenic expression was not related to cell attachment or cell proliferation. These data support the hypothesis that the expression of specific terminal sugars on cell surface glycoconjugates of limb bud cells represents an important component of the chondrogenic process.     

The 'nothing dehydrogenase' reaction and the detection of ischaemic damage

The 'nothing dehyrogenase' reaction is defined as the reduction of tetrazolium salts in media lacking specific substrates for dehydrogenases. In this investigation, the kinetics of the 'nothing dehydrogenase' reaction were studied in cryostat sections of rat heart and liver with the use of various polyvinyl alcohol-containing incubation media. Formazan production was measured at 585 nm with a cytophotometer. The 'nothing dehydrogenase' reaction was substantially lower in the heart than in the liver which was due to low levels of endogenous lactate and the absence of proteins containing thiol groups, such as albumin, in the heart. In vitro ischaemia resulted in a reduced 'nothing dehydrogenase' reaction due to loss of NAD+, possibly as a consequence of its breakdown by glycohydrolase activity. One hour reperfusion following one hour ischaemia caused a decreased 'nothing dehydrogenase' reaction in certain areas of the liver. This reduction was a result of leakage of lactate dehydrogenase and thiol-containing molecules. It appeared at the ultrastructural level that parenchymal and endothelial cells were heavily damaged in the areas containing a low 'nothing dehydrogenase' activity. In conclusion, early ischaemic damage in liver can be detected with the 'nothing dehydrogenase' reaction.     

Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.