Interactions of glucocorticoids and heparin on the humoral immune response of mice.

Interactions of glucocorticoids and heparin on the humoral immune response of mice to SRBC have been studied. Heparin injected subcutaneously in a depot-form 2 hours before immunization restores the antibody-forming ability of mice immuno-suppressed by hydrocortisone, through pretreatment of mice only with heparin 3 days before immunization decreases the PFC-count in the spleen. Cortisone (ip) and heparin (sc) per se injected at about the time of immunization with suboptimal doses of SRBC resulted only in a slight stimulating or no effect on spleen PFC count and serum antibody level. Cortisone and heparin applied in combination, however, exerted a stimulatory effect on the humoral immune response to SRBC. The route of steroid administration pointed to the significance of macrophages in this phenomenon. As the cooperative effect of heparin + cortisone on the humoral immune response shows a good correlation with their cooperative effect on in vivo (3H)dThd incorporation and thymidine kinase activity of spleen, it was hypothesized that an in vivo mitogenic activity for the spleen cells was responsible for the adjuvant effect observed.

A kinetic study of glucocorticoid-heparin interaction on the in vivo DNA-synthesis of mouse lymphatic organs.

The effect of glucocorticoids and heparin (in depot form) on the DNA-synthesis of thymus and spleen of mice was investigated by measuring (3H)dThd incorporation in a four day period. The kinetics of glucocorticoid action was similar whether the hormones were applied in 11 beta-hydroxy-, or keto-form and was characterized by a suppressive effect with a maximum on the first two days after administration. The suppressive effect of glucocorticoids proved to be more marked in the thymus than in the spleen. Heparin per se did not change thymidine incorporation into the lymphoid organs, but injected 3 hours prior steroid administration inhibited the suppressive effect of cortisone, but not that of hydrocortisone. In addition, heparin even reversed the suppressing effect of cortisone on splenic (3H)dThd uptake. The effects of heparin on glucocorticoid action could be only observed when the steroid was applied intraperitoneally. This observation and other data discussed suggest the involvement of peritoneal cells (macrophages) in glucocorticoid-heparin interactions on DNA-synthesis of lymphoid organs.

Enhanced take of spontaneous murine tumors in mice treated with inhibitors of macrophage and/or NK cell function.

Both macrophages and NK cells have been suggested to play a role in recognizing and eliminating early, in situ neoplasms. Therefore we studied the effect of inhibitors of macrophage and/or NK cell function on the take of transplantable spontaneous murine tumors in syngeneic mice. The treatment of animals with trypan blue, a selective inhibitor of macrophage function, decreased considerably the period of latency of BSP3 adenocarcinoma; however, it did not increase the take of SP4, SP82 and SP84 adenocarcinomas. The treatment of recipients with neutral red, a selective inhibitor of NK cell function, enhanced the take of SP4 adenocarcinoma. The treatment of mice with agents depressing both macrophage and NK cell function (silica or carrageenan) decreased the both macrophage and NK cell function (silica or carrageenan) decreased the period of latency and/or increased the take of SP4, SP82 and SP84 adenocarcinomas. Carrageenan or a combined treatment with both trypan blue and neutral red also enhanced the take of BaF1, a benzo(a)pyrene-induced fibrosarcoma. We concluded that both macrophages and NK cells may function as effector cells of an antitumoral surveillance system.

Immunodiffusion analysis of the antigenic structure of the transplantable MC 29 tumor.

By the use of specific anti-chicken liver sera it could be proved with immunodiffusion techniques that the transplantable MC 29 tumor line contains liver-specific antigens. Production of chicken serum-proteins by the tumor could also be demonstrated. Thus, the transplantable MC 29 tumor line can be regarded as a hepatoma.

Thymidine kinase activity in murine lymphoid organs following glucocorticoid and heparin administration.

Thymidine kinase (TK) activity was studied in thymus and spleen of mice after glucocorticoid and heparin administration. Glucocorticoids (cortisone and hydrocortisone) injected intraperitoneally caused a prolonged 80--90% decrease in TK activity of both lymphoid organs. Heparin per se administered in depot-form subcutaneously did not alter the enzyme activity in the lymphoid organs significantly. By contrast, heparin injected 6 hours before glucocorticoid treatment inhibited the decreasing effect of cortisone but not that of hydrocortisone on TK activity in the thymus and fully inhibited the effect of both hormones on the enzyme activity of spleen. In addition the combined use of heparin and cortisone increased the splenic TK activity above the control value on the 2nd day after treatment. The possible mode of action of heparin is discussed.

Effect of heparin on the priming with a protein antigen in mice.

Heparin has been found to stimulate or suppress the priming activity of a protein antigen (cock muscle phosphorylase-b) in mice depending on the various parameters (the dose of antigen, timing of administration, etc.). The adjuvant effect of endotoxin on priming was potentiated by a single dose of heparin injected 10-12 hours after immunization. Optimal doses of heparin incorporated into Freund's incomplete adjuvant also enhanced the humoral immune response first of all the development of 7 S immunologic memory. The possible mechanisms are discussed.

Effect of endotoxin on the peritoneal mast cells in mice.

Endotoxin administered intraperitoneally has been found to decrease the absolute and relative number of the peritoneal mast cells in mice. The number of peritoneal mast cells showed a dose-dependent biphasic response to endotoxin. Maximal and more consistent decrease in the peritoneal mast cell count was observed at about 24 h and lasted for at least 4 days. Mast cell damage also occurred. The possible mechanism of this phenomenon is discussed.

Cellular- and permeability-changes in the peritoneal cavity of mice after injection of endotoxon.

Injection of endotoxin into the peritoneal cavity of mice causes an early emigration of PMN-leucocytes and therafter a decrease in macrophage count. The number of lymphocytes shows only a transient increase. One and 2 injections of endotoxin rise the fibrinogen and protein level of the peritoneal fluid. A simple test for measuring the change in permeability (and lymph circulation) was described ("congo red elimination's test"). Changes in permeability occur after a latent period of 2 hours following endotoxin administration, culminate during the 1st day and last about 3-4 days. The possible mechanisms of the above phenomena are discussed.

Antagonistic effect of heparin and a water-soluble glucocorticoid on the peripheral lymphocyte count in mice.

The effect of heparin and prednisolone succinate on the peripheral white blood cell count of mice has been studied 3 hours after injecting the drugs. Heparin in low doses did not influence the cell count but in higher doses (20 to 400 U) elicited marked lymphocytosis. When prednisolone and heparin were administered together, heparin (5 to 10 U) markedly inhibited the prednisolone-induced lymphopenia. The effect on the lymphocyte count of prednisolone combined with a large dose of heparin depends on the doses of the drugs. The antagonism seems to be non-competitive in nature. The PMN count shows only minor changes and the reaction of eosinophils to heparin is less pronounced. The antagonistic effect was manifest on the spleen weight.

Comparison of the effects of peritoneal and spleen cells of syngeneic or allogeneic origin on the take of transplantable murine tumours.

We compared the effects of various potential effector cells of syngeneic or allogeneic origin on the take of a spontaneous adenocarcinoma (SP4) and Lewis lung (LL) carcinoma. As reported earlier, syngeneic resident (non-activated) peritoneal cells (PC) did not inhibit the take of these tumours. On the contrary, transfer of resident PC from allogeneic donors suppressed the tumour take. Syngeneic and allogeneic PC activated by poly I:C or by a combination of indomethacin, poly I:C and Syncumar ("combined treatment") inhibited the tumour take to a similar extent. Syngeneic spleen cells (from untreated mice or from donors underwent "combined treatment") did not inhibit the take of Lewis lung tumour. Transfer of activated allogeneic spleen cells resulted in a stronger inhibition of tumour take than the transfer of resident allogeneic spleen cells.

Characterization of activated peritoneal cells inhibiting the take of transplantable murine tumours.

We studied the properties of activated peritoneal cells (PC) inhibiting the take of SP4 spontaneous adenocarcinoma and Lewis lung carcinoma in syngeneic mice. Treatment of the poly I:C activated PC from Balb/c mice suppressing the take of SP4 tumour with anti-asialo GM1 antibody and complement before transfer did not affect their tumour-inhibitory potential. PC from Balb/c nude mice treated with poly I:C also inhibited the take of SP4 tumour. Spleen cells from untreated or poly I:C treated Balb/c and Balb/c nude mice, however, did not inhibit the take of SP4 adenocarcinoma. Treatment of peritoneal cells activated by a combination of poly I:C, indomethacin and Syncumar (referred to as "combined treatment") with anti-asialo GM1 antibody and complement could not, or could only partly abolish their tumour-inhibitory potential. The cells mediating the suppression of the take of Lewis lung tumour proved to be Thy-1,2+/-, Lyt-1-, Lyt 2.2- cells. We conclude that the activated peritoneal cells inhibiting the take of SP4 adenocarcinoma and Lewis lung tumour are different from NK cells, NC cells and LAK cells and represent a distinct antitumoural effector cell population.

Effect of poly I:C-activated peritoneal cells on the take of transplantable murine tumours.

The effect of syngeneic mouse peritoneal cells (PC) on the growth of four different transplantable tumours was studied in adoptive transfer experiments (Winn's test). PC from unstimulated mice did not influence the growth of a benzpyrene induced fibrosarcoma (BaF1) and a methylcholantrene induced mastocytoma (P815), but significantly enhanced the growth of a spontaneous adenocarcinoma (Sp4) and Lewis lung carcinoma (LL). PC induced by a single injection of thioglycollate did not influence, whereas PC elicited by proteose peptone markedly enhanced the growth of BaF1 fibrosarcoma. The enhancing effect of peptone induced PC was diminished by a single intraperitoneal dose (100 micrograms/mouse) of polyinosinic-polycytidylic acid (poly I:C) given after peptone injection. Transferring PC obtained after a single injection of poly I:C (100 micrograms intraperitoneally) resulted in retardation of growth of BaF1 fibrosarcoma and Sp4 adenocarcinoma or in a marked decrease in their take depending on the PC/tumour cell ratio. The effector cells involved in the protective effect proved to be different, using these two tumour models. Lewis lung carcinoma and P815 mastocytoma proved to be insensitive to poly I:C-stimulated PC.

Mouse peritoneal cells activated with a combination of indomethacin, poly I:C and Syncumar inhibit the take of Lewis lung carcinoma in adoptive transfer assay.

Effect of peritoneal cells (PC) from mice treated with a combination of drugs (indomethacin, poly I:C and Syncumar) on the take of Lewis lung (LL) carcinoma was studied in Winn-type adoptive transfer experiments. Transfer of PC from mice given a single intraperitoneal injection of polyinosinic-polycytidylic acid (poly I:C) or indomethacine or Syncumar (100 micrograms of each) per se did not suppress the take of Lewis lung carcinoma in the recipient mice. PC obtained from mice treated with a combination of indomethacin and poly I:C or poly I:C and Syncumar also failed to inhibit the take of the tumour. In contrast, PC collected from mice after a combined treatment with the three drugs (indomethacin + poly I:C + Syncumar) resulted in a 30-60% decrease in tumour take depending on the tumour cell/PC ratio. This effect could not be observed when a single intraperitoneal dose of cyclophosphamide was administered three days before starting of the combined treatment of the donor mice. The effector cells contributing to the tumour inhibitory effect proved to be nonadherent cells, probably large granular lymphocytes (LGL), as their suppressive effect was abrogated after treatment with the lysosomotrop vital dye neutral red.

Are mast cells involved in adjuvant action?

A variety of adjuvant substances have been found to reduce the absolute and relative number of peritoneal mast cells in mice. This observation is discussed in the light of literary data suggesting a potential role of mast cells in the regulation (modification) of the immune response.

Inhibition of cortisone action in mice by heparin.

A single dose of heparin applied in a depot-form (Freund's incomplete adjuvant or Ca-phosphate gel) inhibits the effects of intraperitoneally injected cortisone on the lymphoid organs (thymus and spleen), on the peritoneal and peripheral lymphoid cell count and serum gamma globulin level as well as on the liver glycogen deposition in mice. The same dose of heparin did not influence the action of hydrocortisone measured on the thymic and spleen involution and liver glycogen content. The route of cortisone administration seems to be critical, as heparin shows no or only minor effects when cortisone is applied subcutaneously; moreover, the action of cortisone per se is more marked by subcutaneous than by intraperitoneal administration. The results suggest the hypothesis that heparin inhibits cortisone-cortisol conversion and this inhibition is mediated by macrophages.

Mycobacterium simiae and related mycobacteria.

Fifty mycobacterial strains were isolated from freshly imported tuberculin-negative Macacus rhesus and Cercopithecus ethiops monkeys. Of these strains, 14 were identified as Mycobacterium simiae and 4, as Mycobacterium asiaticum. These two species are slow growing with a delayed photochromogenicity. M. Simiae is niacin-positive. Both species are resistant to the antituberculous chemotherapeutic compounds streptomycin, isoniazid, p-aminosalicylate, and rifampin but are sensitive to cycloserine. The two species are virulent for white mice. Infection is contagious; 25% of noninfected cage mates become infected during 12--60 days of exposure to infected animals. Intrauterine transmission of infection also occurs. Utilizing the gel-precipitation method, we have observed up to 16 antigens in each species. Four to six antigens are shared with Mycobacterium tuberculosis. Mycobacterium species strain 52 is antigenically distinct. The 14 strains of M. simiae belong to two serotypes. Mycobacterium habana belongs to M. simiae serotype 1.

Interferon-inducibility is diminished in tumour-bearing mice.

Inducibility of interferons was studied in tumour-bearing mice. Using five different transplantable tumour-models, diminishing of interferon-inducibility was revealed during tumour progression, irrespectively of the tumour-model used. Diminishing of interferon-inducibility runs parallel with development of the tumours.