Modification of vitamin A metabolism in rats fed a copper-deficient diet.

The liver is the main storage site of vitamin A and copper. Inverse relationships between copper and vitamin A liver concentrations have been suggested. We have investigated the consequences of a copper-deficient diet on liver and blood vitamin A storage in Wistar rats. Animals were fed either a copper-deficient diet for 45 days from weaning, or an identical diet containing adequate amounts of copper. Concentrations of vitamin A were determined by isocratic high performance liquid chromatography using UV detection. We have observed in the liver of the rats fed a copper-deficient diet a significantly higher mean level of retinyl esters (148 +/- 37 micrograms/g of liver) and retinol (3.3 +/- 1.4 micrograms/g of liver) compared to the mean concentration of the retinyl esters (53 +/- 8.5 micrograms/g of liver) (p less than 0.01) and retinol (1.4 +/- 0.5 micrograms/g of liver) (p less than 0.01) in controls. Opposite results were observed in the serum of the group fed a copper-deficient diet as these rats had a significantly lower level of retinol (22 +/- 4 micrograms/100 ml) compared to the mean concentration in the controls (64 +/- 20 micrograms/100 ml) (p less than 0.01). These findings suggest that a copper-deficient diet may cause defective transport of vitamin A from liver to blood. This experimental model may be useful to further investigate unusual liver vitamin A and copper concentrations observed in children during various hepatobiliary diseases.

Cyclosporine administration decreases liver vitamin A stores in normal and vitamin A-deficient rats.

Thirty-four Wistar rats were fed a marginal or normal vitamin A diet and received daily for 14 days an intragastric intubation of oil supplemented with 0, 20, or 60 mg X kg-1 of cyclosporine A. The hepatic content and concentration of vitamin A were significantly decreased by cyclosporine treatment, whereas no modification occurred in kidney or serum vitamin A levels. No induction of hepatic cytochrome P-450 was observed in treated animals. These results suggest that cyclosporine interferes with vitamin A stores; thus, vitamin A supplementation may be useful in patients receiving cyclosporine therapy. Drug-metabolizing enzymes, which are cytochrome P-450 dependent, did not seem to be involved in the hepatic vitamin A decrease observed.

Influence of the embryo on the distribution of maternal immunoglobulins in the mouse uterus.

The effect of the embryo on the distribution of IgA, IgG and IgM was studied by an immunoperoxidase technique on mouse uterine sections, (1) during the first part of pregnancy and pseudopregnancy, and (2) in delayed implantation combined with different progesterone-oestradiol treatments designed to extend the delay or induce implantation, and in nonpregnant ovariectomized mice similarly treated. The number of glandular lumina containing IgA increased particularly from the implantation period, but in pseudopregnancy this number decreased from the morning of Day 4, and afterwards continued to decline. In delayed implantation, the number of glandular lumina containing IgA also rose considerably when implantation was induced by oestradiol, whereas under the same progesterone-oestradiol treatment, nonpregnant ovariectomized animals displayed no such increase. Significant staining for IgG in the stroma was observed on Day 4 of pregnancy and pseudopregnancy but prolonged staining for IgG was observed only during pregnancy. In addition, significant numbers of IgA-plasma cells in the stroma were observed mostly in uteri containing embryos. These results indicate that embryos might affect the process by which ovarian hormones regulate IgA and IgG distribution.

Maternal immunoglobulins G, A and M in mouse uterus and embryo during the postimplantation period.

The distribution of maternal IgG, IgA and IgM was studied by an immunoperoxidase technique on mouse uteri and embryo sections from 4.5 to 10 days of gestation. In the embryo, from day 5, IgG was found in the endoderm, and later in the trophoblast, distal and extraembryonic proximal endoderm, embryo cavity and early vitelline vessels. From day 6, IgA was found in the same areas but staining was less intense and was not seen in vitelline vessels. From day 8, small amounts of IgM were found in the same areas, but not in vitelline vessels. Between implantation sites, IgG and plasma cells containing IgA remained present in the stroma; most glands contained IgA, and the uterine lumen stained for IgA and IgG. IgG granules were found in the apical region of luminal epithelial cells in the implantation sites at 5 and 5.5 days; IgA granules were seen in the apical region of luminal epithelial cells between implantation sites from 4.5 to 6.5 days. These results suggest that IgA and some IgM are transmitted along with IgG to the early mouse embryo but do not reach the embryonic circulation, and that during the implantation period reabsorption of IgG and IgA from the uterine lumen into the epithelium differs, depending on the position of this epithelium in relation to the embryo. The persistent IgA and IgG secretion in the uterus after implantation suggests that during this period the mechanisms regulating immunoglobulin secretion may differ from those during the oestrous cycle.

Immunoglobulins in the mouse uterus during the oestrous cycle.

The distribution of IgA, IgG and IgM was studied by an immunoperoxidase technique on sections of mouse uteri at each stage of the oestrous cycle. Staining for IgG and IgA was highest at pro-oestrus, declined at oestrus and was very low during the other stages. At pro-oestrus IgG was found throughout the stroma, in the uterine lumen, and in 10% of glandular lumina; very few IgG-containing plasma cells were present. At pro-oestrus, IgA was found in the uterine lumen, and in most of the uterine glands, both in the lumen and in the epithelium; little IgA was present in the stroma. IgA-plasma cells were detected at each stage of the cycle and were particularly numerous at pro-oestrus and oestrus. These results suggest that IgA is secreted locally from plasma cells into the uterine gland through the glandular epithelium, but that IgG enters the stroma from the local capillaries. The obvious increase in IgG and IgA secretion at pro-oestrus, when plasma oestradiol levels are highest, supports the hypothesis that, during the oestrous cycle, the humoral immune response is regulated in the uterus by ovarian hormones.

Immunoglobulins in the mouse uterus before implantation.

An indirect immunoperoxidase technique was used to study the distribution of IgA, IgG, IgM and albumin in the uterus of primigravid mice. As pregnancy proceeds from Days 2 to 6, IgA-containing plasma cells concentrate around the uterine glands, IgA is found in an increasing number of glands and then in the uterine lumen. At the same time the stroma is progressively invested by IgG, but IgG plasma cells are not present in significant numbers and IgG is very rarely found in glands. IgM remains in blood vessels until Day 5 when it is present in small amounts in the stroma. Albumin tends to follow a pattern similar to that of IgG but in addition is present in the lumen and in a few cells in the luminal epithelium. The growing decidua does not contain immunoglobulin. These results suggest that, as the embryo reaches the uterine lumen, IgA, produced locally by plasma cells, is secreted into the uterine lumen via uterine glands while IgG infiltrates the stroma as a result of increased permeability of the uterine capillaries at the time of implantation.

Immunological studies of mouse decidual cells. II. Studies of cells in artificially induced decidua.

Cells from artificially induced decidual tissue (deciduoma) in the mouse were examined for Thy-1 surface antigen and receptors for the Fc portion of immunoglobulin G (FcR) and compared with cells of the normal decidua from 6 to day 9 of pregnancy. It was shown that (1) Thy-1 antigen is present on the same proportion of cells in decidua and deciduoma on day 6 and day 7, (2) FcR-bearing cells can be detected in similar numbers on day 6 and day 7 but this does not increase on day 8 in deciduoma as it does in decidua, and (3) progesterone treatment after induction of decidualization allowed further increase of FcR-bearing cells in deciduoma. These results present further evidence of the similarity between deciduoma and decidua in the mouse. They indicate that these two membrane markers are present in the early decidua, regardless of the presence of an embryo, and suggest that progesterone may play a part in the increase of FcR-bearing cells in the decidua during pregnancy.

Preparation of single cell suspension from rat liver. A simple method designed for immunologic studies.

Single cell suspensions from rat liver were prepared by mean of a 10 min. in vivo liver perfusion with an isotonic solution. This solution was devoid of calcium but contained 27 mM of sodium citrate and 1% of bovine serum albumin. The liver cells were dissociated over chromium nickel screen in presence of 0.8 mM of calcium and 0.25 M of saccharose; they were further separated by gravity sedimentation for 10 min. The dissociated cells, when incubated in Waymouth's medium enriched with 15% of heat-inactivated horse serum and 1% of BSA, actively synthetized urea and proteins. They also incorporated [3H] thymidine. Isolated hepatocytes, obtained without resorting to extraneous enzymes, conform to the conditions required for the study of cell surface receptors involved in immunologic recognition mechanisms.

[Culture of hepatitis virus B]. / Culture du virus B de l'hepatite

For the last 25 years, numerous attempts have been made to isolate the HBV agent responsible for hepatitis B by means of cultures 'in vitro'. We have undertaken longterm cultures of children's hepatic tissue (C.H.), conjunctive tissue (human adult H.A.F. and human embryonic fibroblasts H.E.F.) and KB cells; these were put in the presence of 7 sera HB + rich in Dane particles. These cells were trypsinized twice a week for almost 3 months and did not present any cytopathogenic effects. Electromicroscopy revealed, 15 days after infection, the presence of icosahedral particles (25 to 27 nm in diameter), free or in dense clusters, but more often empty (20 nm in diameter). These structures seemed to be made up of an assembly of capsomers approximately 5 nm in diameter, joined together in fours to form a ridge. Older cultures revealed clusters of icosahedrons some of which degenerated spontaneously; others were surrounded by proteinic structure having a fringed aspect. Certain rare particles of 35 to 45 nm in diameter are similar to full Dane particles. EID immunological results were positive in the case of sera of patients convalescent from hepatitis B, containing anti-HBc antibodies, on C.H. cells the 27th and 40th days, and negative with anti-HBs antibodies. By immunofluorescence we observed 12 to 20 days after infection of the cells, a clear fluorescence at first nuclear, then essentially cytoplasmic, by means of fluorescent anti-HBc sera of human or animal origin. With the fluorescent anti-HBs antibodies, the reaction is weak and solely cytoplasmic although in DRI, with H.E.F. and KB cells, we obtained from time to time weakly positive results in HBs. The relations between the morphological structures and the immunological results observed are discussed.