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Characterization of suppressor cells in adjuvant arthritis was performed by using highly susceptible DA strain rats. The results showed that suppressor cells were induced after a single inoculation of subarthritogenic dose of mycobacterial adjuvant. Relatively long incubation period was required for the induction of suppressor cells. Such cells were predominated in the draining lymph node and, after fractionation, only sIg- population was effective in conferring unresponsiveness. In vivo irradiation or hydrocortisone treatment suggested that low dose induced suppressor cells were resistant against such immunosuppressive treatments. In addition, by using alkyldiamine as a non-mycobacterial arthritogenic adjuvant, it was suggested that unresponsiveness induced by low dose priming with mycobacterial adjuvant was antigen specific.

Rats of inbred DA strain were given 20 intraperitoneal injections of typhoid-paratyphoid bacilli at 10 day intervals. Thymic lymph follicles with germinal centres were formed in females but not in males. However, orchidectomy permitted the formation of large numbers of thymic lymph follicles with germinal centres in males. The formation of lymph follicles with germinal centres in the thymus was not affected by ovariectomy. These results would appear to suggest an inhibitory role of androgens on the formation of thymic lymph follicles with germinal centres. In addition, large numbers of plasma cells, mast cells, and reticular cells, or macrophages with haemosiderin in their cytoplasm, appeared in the parenchma of the thymus in close association with the formation of thymic germinal centres.

A single oral administration of natural principal estrogens, estrone (E1), 17 beta-estradiol (E2) and estriol (E3), caused active proliferation of hepatocytes of adult mouse liver. Steroid hormones tested (testosterone, progesterone and cortisone) other than estrogens were not stimulants of proliferation of hepatocytes. Among these three natural estrogens, E3 was found to be the most potent stimulant of hepatocyte proliferation and E1 was the weakest. The possible mechanism of the hepatocyte-proliferating potency of estrogens was discussed in close relationship to their stimulating effect on the reticuloendothelial system.

The spleen of (PvG/c X DA)F1 rats, intravenously injected with carbon, was investigated. Large heavily carbon-laden (LHC) macrophages, which were found only in the red pulp at 30 min, appeared along marginal zone bridging channels (MZBC) from the red pulp towards the white pulp side successively during 1-6 h after carbon injection. After this time, they appeared in the periarterial lymphatic sheaths (PALS) near MZBC and then in the deeper PALS along the arteries by 5-10 days. Frequently, they were found in rows from MZBC into the white pulp. These findings suggest migration of LHC macrophages from the red towards the white pulp trough MZBC. Possible migration of LHC macrophages through MZBC was observed for a long period--at least 3 months examined. LHC macrophages came together preferentially in PALS and in and around the germinal centers consisting of large pyroninophilic lymphoblastoid cells. Occasionally, possible migration of LHC macrophages from regions around sinuses crossing the marginal zone vertically (vertical sinus) was also observed. Sinuses accompanied by LHC macrophages often ran parallel in close association with MZBC, particularly at sites of MZBC near the red pulp.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.

In an attempt to study the functional relation between pituitary cyclic AMP and TSH secretion in response to thyrotropin releasing hormone (TRH) or thyroid hormone administration, pituitary concentration of cyclic AMP was measured by protein binding assay after in vivo and in vitro administration of test materials (TRH, thyroxine, triiodothyronine, actinomycin D, puromycin and cycloheximide singly or in combination). Small dose of TRH apparently augmented TSH secretion as evidenced by a marked increase of intrathyroidal colloid droplet, but failed to elevate the pituitary concentration of cyclic AMP. Triiodothyronine (T3) and thyroxine (T4) blocked an increase of TSH secretion produced by TRH, but they elevated pituitary concentration of cyclic AMP in vivo and in vitro. Actinomycin D (Act D), puromycin and cycloheximide elevated pituitary cyclic AMP concentration without stimulating TSH secretion. From the data accumulated, it appears that the measurement of total anterior pituitary concentration of cyclic AMP is not useful to evaluate the activity of TSH cells in response to thyroid hormone, TRH and TRH plus thyroid hormone.

In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immunohistological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2'-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.

The splenic macrophages of rats and mice were studied by light and fluorescence microscopy to determine their phagocytotic uptake of carbon and neutral polysaccharide (Fic-F), and their lysosomal enzyme activities. In rats, the large macrophages of the marginal zone (MZ) showed a moderate to strong acid phosphatase activity, and took up most of the Fic-F, even though they showed a weak phagocytotic activity to carbon particles. Red-pulp macrophages, however, ingested a large quantity of carbon particles, and are considered to be the major scavengers in the rat spleen. In contrast, the MZ macrophages in the mouse spleen were the major scavengers and showed a vigorous uptake of both carbon and Fic-F. In rats, the marginal metallophils (MM), located at the outer border of the periarterial lymphatic sheath and boundary between the MZ bridging channel and surrounding tissue, ingested Fic-F, whereas those located around the follicular area did not. In mice, on the other hand, the MM never ingested Fic-F. Lightly carbon-ladened small cells were constantly seen in the MZ of both rats and mice. They showed little acid phosphatase activity and did not ingest Fic-F. They were also present in the blood circulation.

Diazepam (10(-5)-3 X 10(-4) M) selectively enhanced the negative inotropic responses of guinea-pig atria and the relaxation of guinea-pig taenia coli caused by adenosine and ATP. In the atria, the effect of 2-chloroadenosine, a stable analog of adenosine, was not affected by diazepam. Segments of guinea-pig atria or taenia coli took up 3H-activity during incubation with [3H]adenosine but did not take up 32P-activity from [32P]ATP. Diazepam at concentrations sufficient to enhance the in vitro responses reduced by half the uptake of 3H-activity into the preparations. Adenosine (10(-6) M) and ATP (10(-6) M) were degraded to inactive inosine during incubation with atrial segments and their degradation was inhibited by diazepam. In contrast, in rat atria, diazepam did not enhance the negative inotropic effects of adenosine and ATP, and did not prevent the uptake of adenosine. These results suggest that in guinea-pig atria and taenia coli, diazepam like dipyridamole, acts as an adenosine potentiator by preventing the uptake and degradation of adenosine.

A single intraperitoneal injection with a 10 mg estriol (E3) in aqueous suspension induced a large and prolonged elevation of serum alpha-fetoprotein (AFP) in adult mice. E3 also raised the mitotic activity of hepatocytes in the absence of liver injury. Although both the AFP concentration and hepatocyte proliferation reached the peak on day 5 after E3 administration, a high level (about 12,500 ng/ml) of serum AFP persisted for a long period after hepatocyte proliferation declined. Five mg E3 showed a remarkable threshold effect on AFP elevation and 3 mg E3 on hepatocyte proliferation. Immunohistochemical studies indicated AFP production by hepatocytes in adult mice after the E3 administration.

After a single pharmacologic dose of estriol (E3) treatment, an increase in number of focal areas of hepatic hemopoiesis was observed in adult mice. When syngeneic bone marrow cells were transfused into E3-treated mice, focal hepatic hemopoiesis was increased further. In a single experiment, using a single cell suspension, the concentration of CFUs in the liver was increased 5 days after E3-treatment, while that in the blood decreased. In addition, 51Cr-labeled bone marrow cells selectively accumulated in the E3-treated mouse liver. These results suggest, but in not sense prove, that circulating hemopoietic stem cells are trapped in the E3-treated mouse liver, and that E3-activated Kupffer cells may play a central role in focal hemopoiesis in the liver.

In a middle-aged woman with virilizing adenoma, 2 mg dexamethasone increased urinary excretion of 17-ketosteroids (17-KS) and 17-hydroxycorticosteroids, whereas 8 mg dexamethasone increased urinary excretion only of 17-KS. With discontinuation of dexamethasone, 17-KS excretion returned to the predexamethasone level. Dexamethasone depressed the basal level of cAMP synthesis and basal testosterone production by the normal adrenal tissue in vitro. Dexamethasone also depressed the increase of cAMP produced by ACTH in the normal tissue. In contrast, dexamethasone increased basal cAMP synthesis and stimulated testosterone secretion in the tumor tissue. ACTH and dexamethasone were additive in their effects on cAMP and testosterone in the tumor tissue. It is suggested that dexamethasone acted directly on the adrenal tumor to stimulate steroid secretion in this patients.

A single intraperitoneal injection of 10 mg estriol (E3) rapidly induced a transient decrease in the serum albumin (ALB) level in 3 to 5 days in adult mice. At a dose of 5 mg, E3 showed a remarkable threshold effect on the decline of serum ALB. By immunohistochemistry, two types of ALB-positive cells were found in the liver. Type 1 cells were stained intensely and almost homogeneously throughout all or nearly all their entire cytoplasm, and type 2 cells showed only granular or vesicular cytoplasmic deposits. After administration of 10 mg E3, the type 1 cells diminished rapidly and markedly in direct proportion to the fall in serum ALB from the centro- and mediolobular zones, where they had been normally localized either singly or in small groups. Simultaneously, the granular or vesicular deposits that had been normally stained very weakly in the type 2 cells in the centro- and mediolobular zones became intensely stained along with cells in the perilobular zone. Alpha-fetoprotein (AFP)-containing cells in the liver appeared in maximal numbers at 5 to 7 days after E3 administration. These serological and cytological changes after E3 administration are here discussed on the basis of a possible inverse relationship between ALB and AFP gene expression.

Various doses (1 microgram to 10 mg) of oestriol (E3) were intraperitoneally injected into mice immediately after subcutaneous inoculation of an oestrogen receptor-negative lymphoma cell line (KE-5) established from a spontaneously developed AKR thymic lymphoma. The growth of KE-5 cells was markedly promoted by E3 at the early stage of tumour growth. At this stage, 1 microgram E3 enhanced tumour growth significantly and the maximum effect was obtained with 1 mg E3. Normal female mice showed a higher incidence and shorter latency than males. However, once tumours became palpable, the tumour growth rate appeared to be unaffected. Histological observations using Alcian blue and colloidal iron revealed a marked increase of hyaluronic acid in the subcutaneous connective tissue of the tumour-injection site within 3-5 days after intraperitoneal administration of 1 mg E3. Biochemical analyses showed a rapid and marked increase in skin hyaluronic acid content to over 3 times the control levels (0.25 +/- 0.10 mg g-1 skin) within 3 days of E3 administration. Subcutaneous inoculation of KE-5 cells together with hyaluronic acid (0.2 mg) resulted in markedly enhanced tumour growth, particularly at the early stage. These results suggest that an increase in stromal hyaluronic acid content is the most likely mechanism responsible for the promoting effect of E3 on KE-5 cells.

Histological observations of the mouse spleen were carried out at different times after intravenous carbon injection. Large carbon-laden macrophages appeared in great numbers in the marginal zone soon after injection. They came together favorably around the germinal centers. Possible migration of these cells toward the germinal centers diffusely from the periphery of the white pulp or through the periarterial lymphoid sheath was suggested. These macrophages entered the germinal centers on a large scale and clustered for a long period--at least 180 days. Since the same type of macrophages were observed persistently in the marginal zone, it was thought that some of them might arise from the blood stream. Possible migration of these cells from the marginal zone toward the germinal centers was also persistently observed. A second type of much smaller carbon-laden macrophages was seen in the white pulp. However, they never showed any favorable localization in the germinal centers as did large carbon-laden macrophages.

When the serum insulin valves during a 50-gm oral glucose tolerance test were expressed as the function of glucose concentrations, a good linear relationship was found in normal subjects and in patients with diabetes, hyperthroidism, hypothroidism or chronic hepatitis. The insulin index calculated from the line representing this relationship was less than 100 in all 19 diabetic patients and most of the 13 hyperthyroid patients with oxyhyperglycemia, whereas it was more than 100 in the 14 normal subjects, the 4 hypothyroid patients, and the 8 patients with oxyhyperglycemia due to chronic hepatitis. The insulin index decreased with increasing maximal blood glucose concentration during the glucose tolerance test in diabetic patients. Furthermore, improvement in the insulin index was correlated with the effectiveness of sulfornylurea drugs in the diabetic group.

Lymph flow and the composition of lymph lipids from the hepatic and thoracic ducts of rabbits with cirrhosis of the liver (induced by 46-51 intramuscular injections of a mixture of carbon tetrachloride and olive oil at 4-day intervals) have been compared with those of control animals injected with olive oil only. In cirrhotic animals, the concentration of lymph lipids was not greatly altered, but lymph flow, and consequently the hourly transport of lipids by lymph were greatly increased; the increase in transport of cholesteryl esters, free cholesterol, and phospholipids by way of the thoracic and hepatic duct lymph was particularly striking. The concentration of these lipid fractions in serum from the cirrhotic rabbits was also increased. The differences normally observed between lipid fatty acid compositions of serum and lymph disappeared in cirrhotic animals; this is interpreted as due to increased hepatic permeability to lipoproteins.