Effects of a protein-free diet on the changes in cyclic AMP and cyclic GMP levels induced by immunization in splenic T and B lymphocytes in the rat.

The levels of cyclic nucleotides, cAMP and cGMP, were determined in splenic lymphocytes of normally fed (N) and protein deprived (PD) rats before and at different time intervals after a single injection of sheep erythrocytes. Assays were performed with protein binding methods on unseparated as well as on T and B cells fractionated by filtration through nylon wool. The cAMP levels increased in unfractionated cells and in T and B lymphocytes 2 hours following immunization of N rats. Another rise in cAMP levels occurred after 3 days in B lymphocytes, but there was also a simultaneous increase of the cGMP levels in preparations of unfractionated cells and in B lymphocytes. The PD diet suppressed or delayed most of the aforementioned changes. Thus, the immunodepressive effect of such a diet may be ascribed to the inhibition of both the early signal (increase of cAMP levels and of cAMP/cGMP ratio) leading to T and B differentiation and the later signal (increase of cGMP levels) which initiates antibody production.

Comparative effects of protracted protein deprivation on the DNA synthetic and mitogenic actions of phytohemagglutinin and concanavalin A in vivo on the lymphocytes of the rat.

The increase rate of DNA synthesis and mitotic activity in popliteal lymph nodes following subplantar injection of PHA was significantly reduced in rats subjected to a protein-free diet given for 9 weeks. On the contrary, the lymph node responses to Con A were higher with latter diet than with a balanced 18% casein diet. On the other hand, PHA reduced the weight and proliferative activity of the thymus, and probably also increased the emigration of thymocytes in protein deprived rats, while Con A was devoid of any effects. Thus, mainly the activity of the PHA-responsive subset of T lymphocytes and thymocytes appeared to be disturbed after protein deprivation. According to previous work, this subset was formed of high theta, cortisone-sensitive celles.

Effects of a protein deprived diet on the hepatotoxicity, and the DNA synthetic, mitogenic, and immunological actions of microbial lipopolysaccharides in the rat.

Rats fed an 18% casein (Cs) or a protein deprived diet (PD) for 8 weeks received injections of Escherichia coli lipopolysaccharides (LPS) in both hind foot pads. While these injections were tolerated in Cs rats, about 50% of PD rats died after 1 or 2 days as a result of a massive necrosis of the liver. To a large extent these lesions were prevented by cortisone. Three days after injection of LPS, Cs rats exhibited a hypertrophy of the popliteal lymph nodes (PLN) and spleen, as well as a drastic increase in DNA synthesis in DNA synthesis in the PLN. Mitotic indices did not increase. The DNA synthetic responses to PLN in the surviving PD rats were much lower than in Cs animals, but a sharp rise in DNA synthesis and mitotic activity occurred in the spleen. The comparison with the effects of LPS in cortisone-treated rats showed that both cortisone-sensitive and -resistant cells participated in PLN activation in rats fed both diets, but that only cortisone-resistant lymphocytes entered mitosis in the spleens of PD rats. LPS also provoked a sharp drop in both DNA synthesis and mitosis in the thymus, probably due to a stress effect, since only cortisone-sensitive thymocytes were involved. In a second experimental series, immunological tests (Rosette-forming cells, Plaque-forming cells, serum hemagglutinin titers) were performed 7 days after intraperitoneal injection of LPS. The responses were not significantly different in Cs and PD rats. This is in contrast with the protein deficiency-induced depression of thymus-dependent humoral immunity.

Comparative effects of a protein deprived diet on the weight, lymphocyte content and the number of DNA-synthesizing and dividing cells in the lymphoid organs of the rat. Influence of the diet on the stimulating action of phytohemagglutinin and the inhibitory action of cortisone.

The changes induced in adult male rats by a protein deprived (PD) diet lasting 7 weeks were investigated from the viewpoint of the cell contents of the popliteal lymph nodes (PLN), spleen and thymus; the levels of DNA-synthesizing lymphocytes counted on cell smear autoradiographs, and the levels of mitoses. The stimulating effect of a subplantar injection of phytohemagglutinin (PHA) and the inhibitory action of cortisone (3 mg/100 g per day for 5 days) were also determined in normal and PD rats. The PD diet significantly reduced the cell contents, primarily in the thymus, as well as the number of labeled cells and mitoses per mg of tissue and per entire organ. When expressed on the basis of 10(3) cells, the levels of both cell groups were also reduced in the spleen but not in the PLN's where they were already very low with a balanced diet. In the thymus, the sharp drop in the mitotic index (M.I.) contrasted with unchanged levels of labeled cells and an important increase in the labeling density per cell. PHA increased the M.I. to a great extent in the PLN's and spleen in normal rats but not in PD rats. In contrast, the labeling index (p. 10(3) cells) in the spleen, as well as the number of labeled cells per mg and per organ, increased in PLN's and the spleen after PHA to a much greater extent with a PD diet than with a normal diet. These results suggest a premitotic block of the cell cycle after protein deprivation. Also, the mitotic index appears to be a more reliable test, than the labeling index, of the lymphostimulating action of PHA and of its inhibition following protein removal from the diet. The effects of cortisone in normal rats resembled to a large degree those of a protein-free diet. If the hormone was administered to PD rats, the residual lymphocytes of the atrophic thymus were still more cortisone-sensitive than those of normal rats. On the contrary, DNA synthesizing cells in the PLN's and spleen, and mitoses in the spleen resisted cortisone in PD rats, while they were partly destroyed by the hormone in the controls. This suggests that primarily the cortisone-resistant lymphocytes of these organs are capable of synthesizing DNA after prolonged protein deprivation. The results are in agreement with the hypothesis of an intervention of endogenous glucocorticoid hormones in the protein lack-induced lymphoid involution.

Hematological and immunological effects of excess dietary leucine in the young rat.

Young rats were subjected to a dietary leucine overload for several weeks. Although no significant changes in growth, food consumption, and hematological and immune responsiveness occurred when the basic diet was balanced (18% casein), rats which were both overloaded with leucine and subjected to a protein-poor diet (4% casein) displayed a strong impairment of immunological reactions to sheep red cells (rosette and plaque-forming cells in the spleen, serum hemagglutinins, and hemolysins). The effect was significantly more pronounced than in rats on a protein-poor nonsupplemented diet or in pair-fed controls on a balanced diet. The immunodepression was as profound as after complete protein deprivation. It is suggested that a secondary defect in valine and isoleucine utilization may play a role in the harmful effects of excess leucine, because isoleucine and especially valine have been shown to be highly deleterious to lymphopoiesis.

Cyclic AMP and GMP levels in thymocytes and splenic T and B lymphocytes in protein deprived rats.

Cyclic nucleotide levels were determined in thymocytes and in splenic T and B lymphocytes separated by nylon wool filtration. In control rats, thymocytes contained less cAMP and cGMP than splenic T cells. The level of cGMP was lower, and consequently cAMP:cGMP ratio was higher in splenic B than in T cells. Protein deprivation increased the nucleotide ratios in both T and B splenic lymphocytes; this effects was due to a decrease in the cGMP levels in the case of the T cells and to an increase in the cAMP levels in the B cells. These data could be related to the reduced mitotic activity of the splenic lymphocytes. However, the nylon-nonadherent thmyocytes of protein deprived rats paradoxically displayed increased cGMP contents without changes in the cAMP levels, despite the drop in their proliferative potential. The impairment of cyclic nucleotide metabolism may intervene in the immunological disturbances due to protein malnutrition.

Graft-versus-host reaction and lymphoid organs in normally fed and protein-deprived rats.

Lymph node graft-versus-host reaction (GVHR) induced by parental splenic lymphocytes inoculated into hind foot pads of F-1 hybrid rats is correlated with the state of the thymus and the spleen of the recipients. This may explain the depression of the reaction after protracted protein deprivation. Furthermore, GVHR provokes mainly in normal rats a reduction of thymus and spleen possibly due to a T-cell transfer to the grafted area.

The effects of late thymectomy on the immunosuppressive action of cortisone and testosterone in rats immunized to sheep red blood cells.

The immunological effects of cortisone and testosterone were studied in intact and lately thymectomized rats immunized to sheep red cells. The results showed that the effects of low doses of cortisone as well as those of high doses of testosterone (drop in the total number of rosette- and plaque-forming cells: RFC and PFC) were conditioned by the involution of the thymic cortex provoked by such hormonal doses, since these changes did not occur in thymectomized rats. The effects of high doses of cortisone were partly not influenced by thymectomy (drop in the RFC and PFC levels) but partly thymus-dependent (drop in the serum antibody titers). The latter were apparently related to advanced lesions of the thymic medulla.

[Comparative studies of the cyclic AMP content and renewal of this nucleotide in thymic, splenic and lymph-node lymphocytes in adult rats]. / Etude comparée des teneurs en AMP cyclique et du renouvellement de ce nucléotide dans les lymphocytes thymiques, spléniques et ganglionnaires chez ie rat adulte

Cyclic AMP (c-AMP) content and turnover were measured in pure preparations of lymphocytes obtained from thymus, spleen and lymph nodes in the Rat. The c-AMP content was determined by combining the methods of Krishna and of Thomson and Appleman, and its turnover was estimated from the activities of adenylcyclase and phosphodiesterase using 3H-adenine. The values, espressed per 10(8) cells, were the lowest for the thymus and the highest for the lymph nodes, while they were intermediary for the spleen. The differences in the c-AMP turnover between the three organs may be correlated with the extent of the mitotic activity of the corresponding lymphocytes, this activity being related inversely to the turnover of c-AMP.

[Immunosuppressive effect of ethionine in rats. Resistance of this effect to methionine, tryptophan, ATP, adenosine and uridine]. / Effet immunodépresseur de l'éthionine chez le rat. Résistance de cet effet a l'administration de méthionine, de tryptophane, d'ATP, d'adénosine et d'uridine

One hundred eleven male adult rats of a pathogen-free Sherman or Charles River strain were divided into 3 series. Each of them contained non-treated controls and rats treated with intraperitoneal (i.p.) injections of DL-ethionine (E) [75 mg/d p. 100 g] for 6 weeks (series 1) or for only 2 weeks (series 2 and 3). Series 1 and 2 included also rats injected simultaneously with E and DL-methionine (68.4 mg/d p. 100 g). In the series 2 there were also groups receiving at the same time E, ME and tryptophan (5 mg/d p. 100 g) or E, Me and ATP (1.65 mg/d), and in the series 3, groups were injected with E + adenosine (31.2 mg/d p. 100 g) and (or) uridine (284 mg/d p. 100 g). All animals were immunized i.p. 1 week before killing with sheep red blood cells (SRBC): either 2.4 X 10(9) SRBC p. 100 g without adjuvant (series 1 and 2) or 1 X 10(9) SRBC with Freund's adjuvant added (series 3). Rosette-and plaque-forming cells (RFC and PFC) were counted in the spleen, and titers of serum hemagglutinins and hemolysins were determined with separation of 2-mercaptoethanol-resistant IgG from non-resistant immunoglobulins. E provoked within 2 weeks a drastic inhibition of the immune responses: drop in the RFC and PFC levels p. 10(6) (approximately 2900 leads to approximately 320 and approximately 415 leads to 40 respectively, in the study 3), and significant decrease in the serum antibody titer, especially IgG. Addition of Me still amplified the immunosuppression. Supplementation with tryptophan, ATP, adenosine and (or) uridine was also ineffective. Though not displaying any immunoprotective potency, Me partly neutralized the non-immunological effects of E: arrest of the body growth and involution of lymphoid organs and male genital organs, particularly apparent after 6 weeks of treatment. In conclusion, the ethionine-induced immunosuppression does not result from a metabolic exclusion of labile methyl groups or from an acute ATP deficiency due to an excess trapping of adenine as S-adenosyl-ethionine. Lack of pyrimidines or tryptophan must also be discarded. On the other hand, the possibility of maintaining the immunosuppressive activity of E, while reducing the toxicity of this compound, by addition of Me deserves to be emphasized.