Effects of lysozyme dimer on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunosuppressed mice.

The effects of lysozyme dimer on humoral response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (200 mg/kg) were tested on mice. The effect of lysozyme dimer on the humoral response to SRBC in non-treated with cyclophosphamide mice was determined in relation to doses (0.2, 2, 20 or 200 micrograms/kg) and the time of the drug administration with respect to the antigen before or after SRBC immunization. Moreover, the effect of lysozyme dimer on the humoral response in cyclophosphamide-treated mice was studied depending on the dose applied and time of exposure to the drug in relation to SRBC. It has been found that lysozyme dimer potentiates the humoral response to SRBC in mice, resulting in an increased number of splenocytes producing haemolytic antibodies (PFC) and the total and 2-mercaptoethanol resistant level of anti-SRBC antibodies. A single exposure to lysozyme dimer gave the strongest stimulating action on SRBC when the doses of 2 or 20 micrograms/kg were administered 2 h prior to the antigen. The potentiating effect of the drug was reduced when it was administered 24 h before the antigen and also when single doses were as high as 200 micrograms/kg and as low as 2 micrograms/kg. Exposure to four doses of lysozyme dimer at 24 h intervals was more activating than a single injection. A strong potentiating effect on the specific response to SRBC was noted after four injections of lysozyme dimer at doses from 0.2 to 20 micrograms/kg. The effect of the drug did not depend on the time of exposure to the antigen. It has also been found that lysozyme dimer significantly reduces the suppressive effect of a high cyclophosphamide dose (200 mg/kg) on the humoral response of SRBC-immunized mice. The protective action of lysozyme dimer was dose- and time-dependent. The strongest protection was observed after three doses of 20 micrograms/kg administered prior to pharmacological immunosuppression. Reduction in the dose to 2 micrograms/kg and shorter treatment resulted in reduced protective effects. We have also found that the protective action of three doses of lysozyme dimer (2 or 20 micrograms/kg each) administered between cyclophosphamide injection and the antigen, or after antigen administration is weaker than such a treatment prior to cyclophosphamide immunosuppression.

Effects of levamisole, DTC and low-dose mechlorethamine on humoral response of SRBC-immunized rabbits exposed to cold stress.

The experiments were carried out on normothermal rabbits and rabbits exposed to cold stress (hypothermia). The animals of the latter group were submerged in ice-water for 20 s and then placed in a freezer at -15 degrees C for 8 min until their body temperature dropped by 3 degrees C. Both the normothermal and hypothermal rabbits were immunized i.p. with 3 ml of 10% sheep red blood cells (SRBC). Levamisole (2 mg/kg), DTC (sodium diethyldithiocarbamate, 20 mg/kg) or mechlorethamine (mustine; 5 micrograms/kg) were injected i.v. three times at 24-h intervals. The number of PFC, total (19S + 7S) and 2-mercapthoethanol resistant (7S) serum haemagglutination titres were determined. It was found that, in normothermal rabbits, all three agents potentiated the number of plaque-forming cells (PFC); the impact of DTC was the strongest, while the weakest influence was observed for mechlorethamine. Furthermore, DTC increased anti-SRBC haemagglutinin titre, whereas mechlorethamine did not. Levamisole, on the other hand, reduced total serum haemagglutinin titre. Cold stress reduced humoral response to SRBC, which was reflected in the decreased number of PFC and serum haemagglutination titres (19S + 7S and 7S). Each agent showed a different way of action. Pretreatment with DTC prevented the immunosuppression caused by cold stress, while levamisole and mechlorethamine only reduced the immunosuppressive effect.

The effect of Tolpa Peat Preparation on the phagocytic and metabolic activity of neutrophils in normothermic rabbits and with pyrogen-induced fever.

The studies on normothermic rabbits show that intravenous administration of Tolpa Peat Preparation (TPP) at a dose of 5 mg/kg for 3 or 6 consecutive days increases the percentage of phagocytizing cells and the number of bacteria phagocytized by a single neutrophil. The stimulated phagocytic activity of neutrophils coincides with increased nitroblue tetrazolium (NBT) reduction. In contrast, a single administration of TPP to rabbits with fever induced by E.coli lipopolysaccharide (LPS) has a modulating effect on the metabolic activity of neutrophils, depending on TPP dose. TPP administered at a dose of 0.5 mg/kg potentiates the stimulating effect of pyrogen on the percent of NBT-positive neutrophils. A tenfold increase of TPP dose (5 mg/kg) reduces the stimulating effect of LPS and a hundredfold increase (50 mg/kg) leads to total inhibition of LPS-induced changes.

The influence of long-term administration of Tolpa Peat Preparation on immune reactivity in mice. I. Morphological changes in the thymus.

In experiment I BALB/c 200 mice were given in drinking water the TPP (Tolpa Peat Preparation) in daily doses of 1, 10 and 50 mg/kg over the period of 3, 5, 7, 9 and 12 weeks. In experiment II before the administration of TPP (as in experiment I) mice were immunized with a single dose i.p. administration of 0.2 cm3 of a 10% suspension of sheep erythrocytes (SRBC), i.e. 4 x 10(8) cells. Histopathological and ultrastructural studies have shown that TPP in all three doses causes morphological changes characteristic of thymus activity stimulation. However, the doses of 10 and 50 mg/kg administered longer than 7 weeks caused retrograde changes that did not occur after administration of 1 mg/kg of TPP. Morphological changes in the thymus of immunized and non-immunized mice simultaneously receiving TPP were similar.

The influence of long-term administration of Tolpa Peat Preparation on immune reactivity in mice. II. The effect of intermittent TPP administration on the morphological picture of lymphatic organs.

The studies were conducted on 200 Balb/c mice (inbred strain), aged 8 weeks. The mice were administered Tolpa Peat Preparation (TPP) in drinking water (10 or 50 mg/kg/day) for seven weeks (experiment I). Two lobes of the thymus, the spleen and the axillary and mesenteric lymph nodes of mice were taken for histological examinations after 3, 5 and 7 weeks of continuous TPP treatment and also 12 and 16 weeks from the beginning of the treatment. In experiment II, TPP was administered daily at the same doses for 3 weeks, and intermittently for 9 consecutive weeks; a week interval followed each seven-day cycle. The lymphatic organs of mice were taken for histological examinations after 3, 5, 7, 9 and 12 weeks of TPP treatment. It was found that daily administration of TPP (as described above) to mice for 7 weeks (10 mg/kg) induced a marked stimulation of the lymphatic organs (in the first weeks of TPP treatment) and resulted in retrograde changes (depletion of thymocytes and lymphocytes) in the lymphatic structures when TPP administration was prolonged (4-7 weeks). The morphological changes due to TPP administered at the dose of 50 mg/kg were less pronounced in the organs examined. The retrograde changes in the lymphatic structures disappeared 9 weeks after TPP treatment had been stopped. Similarly, the changes showing stimulation of the lymphatic organs were maintained throughout the entire period of 9 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of long-term administration of Tolpa Peat Preparation on immune reactivity in mice. III. The effect on primary humoral response to sheep erythrocytes.

Tolpa Peat Preparation (TPP) was administered to mice in daily doses of 1, 10 and 50 mg.kg-1 for 3, 5, 7, 9 or 12 consecutive weeks. After each of the above mentioned periods the primary response to sheep erythrocytes (SRBC) was examined by determination of the percentage of splenocytes forming E rosettes, the number of splenocytes producing anti-SRBC haemolysins of 19S and 7S type, and the level of serum haemagglutinins to SRBC (19S + 7S and 7S types). The effect of enhanced humoral response by TPP was observed in mice immunized with SRBC. This effect depended, first of all on TPP does but also on the duration of time TPP administration. The stimulating effect on the humoral response occurred after the daily dose of 1 mg.kg-1 during 12 weeks. On the other hand, the daily doses of 10 or 50 times higher enhanced the humoral response only during the first 5-7 weeks of TPP administration. Longer administration of TPP in these doses caused disappearance of this effect or reduction of the response to SRBC.

Responses of neutrophils and lymphocytes in the cold stress: effects of nonsteroid anti-inflammatory drugs.

The cold stress induced in rabbits by lowering their body temperature by 3 degrees C resulted in neutrophilia and a decrease in number of phagocytes and phagocytized bacteria. The stress did not affect the number of lymphocytes and the ability of forming E rosettes by T lymphocytes, but depressed the formation of EAC rosettes by B lymphocytes. This inhibition of neutrophil activity was antagonized completely by acetylsalicylic acid, and substantially by mefenamic acid and indomethacin administered, in doses inhibiting pyrogen-induced fever, either 2.5 h before or 1.5 h after the hypothermia. The drugs did not antagonize the depression of the ability of formation of EAC rosettes.

The dose-dependent influence of mechloretamine on the response to sheep erythrocytes in mice. Comparison of immunostimulating properties of mechloretamine with levamisole.

The effect of the following doses of mechloretamine: 1, 5, 10, 25, 50, 100, 250 and 500 micrograms/kg on the immunological response in mice immunized with sheep red blood cells (SRBC) was investigated. The number of plaque forming cells (PFC) to SRBC, the serum hemagglutinins level and the number of lymphocytes forming E or EAC-rosettes were determined. Depending on mechloretamine dose the following effects on the tested parameters were obtained: (i) only stimulating--1 and 5 micrograms/kg, (ii) stimulating or suppressive according to the test--10-100 micrograms/kg, (iii) only suppressive--250 and 500 micrograms/kg. Mechloretamine (5 micrograms/kg) induced the increase in PFC in comparison with levamisole (2 mg/kg). The difference between the action of mechloretamine and levamisole used in immunostimulating doses on the increased anti-SRBC antibodies or on the E-rosette forming lymphocytes was revealed.

[Immunomodulating effects of low doses of nitrogen mustard derivatives]. / Immunomodulujace dzialanie malych dawek pochodnych iperytu azotowego.

Chlormethine (Nitrogen mustard) in small doses proved to have immunopotentiating and anti-inflammatory activities. The influence of two nitrogen mustard derivatives : chlorambucil (1 or 10 micrograms/kg p.o.) and cyclophosphamide (0.03 or 0.3 mg/kg i.v.) as well as busulphan (0.5 or 5 micrograms/kg p.o.)--the agent of ++alkylating cytostatic group were investigated in rabbits. Whole blood count, the number of T and B lymphocytes, serum IgG level, phagocytic and microbicidal activities of neutrophils and the plasma level of free glucocorticoids were estimated. The drugs were used in the doses 10-100 times lower than cytostatic ones. Moreover, the ability of alkylating drugs to enhance or to suppress the changes evoked by lipopolysaccharide of E. coli in examined parameters was assessed. The results were compared with chlormethine data obtained previously. None of two nitrogen mustard derivatives (chlorambucil or cyclophosphamide) in the doses many times lower than cytostatic ones, exhibited an immunostimulating and adjuvant properties characteristic of chlormethine. Such properties did not demonstrate small doses of busulphan, another compound of alkylating drugs.

Influence of nonsteroid anti-inflammatory drugs on lymphocyte subpopulation and on the primary humoral response in normothermic and feverish rabbits.

Rabbit peripheral blood was tested for the ratio of T and B lymphocytes and for the number of plaque forming cells (PFC) on day 5 and 6 after immunization with sheep erythrocytes. Fever was induced by intravenous injection of lipopolysaccharide (LPS) of E. coli. At higher doses, suppressing the fever, only indomethacin (40 mg/kg per day) inhibited the number of lymphocytes, subpopulation of B cells and their capability for hemolytic antibodies synthesis. This effect was not observed with indomethacin at five fold lower dose. Mefenamic acid (60 mg/kg per day) and acetylsalicylic acid (200-900 mg/kg per day) per se irrespectively of the dose applied, do not affect lymphocyte subpopulations and the primary humoral response. All the three drugs investigated counteracted with the changes in the number of B lymphocytes and PFC induced by LPS.

Pyrogenic fever and blood plasma glucocorticoids after nonsteroid anti-inflammatory drugs.

Rabbits were injected with the lipopolysaccharide from E. coli (LPS) and received orally nonsteroid anti-inflammatory drugs (NSAIDs): acetylsalicylic acid, indomethacin, mefenamic acid, ibuprofen, aminophenazone, metamizole sodium, and phenylbutazone. These NSAIDs exerted antipyretic action without inhibiting the increase in the level of plasma glucocorticoids induced by LPS. This finding indicates the lack of correlation between the pyrogenic action of bacterial pyrogen and pyrogenic increase in the plasma glucocorticoid level. The investigated NSAIDs when given alone to normothermic rabbits differently affected the plasma glucocorticoid level: acetylsalicylic acid, indomethacin and ibuprofen depressed the plasma level of these hormones, mefenamic acid and phenylbutazone elevated it, and aminophenazone and metamizole sodium did not alter it significantly.

Inhibition of postpyrogenic increase of phagocytic and killing activity of neutrophils by nonsteroid antiinflammatory drugs.

Acetylsalicylic acid (300 mg/kg), mefenamic acid (30 mg/kg) or indomethacin (20 mg/kg) given orally in the doses preventing the postpyrogenic fever, inhibited the stimulatory effect of LPS on phagocytic and killing activity of neutrophils. The dose of acetylsalicylic acid that did not eliminate fever in rabbits (100 mg/kg), had no suppressive effect upon fever-stimulated killing activity of neutrophils. The drugs administered twice a day to normothermic animals did not evoke any suppressive changes in the activity of neutrophils.

Chlormethine in small doses as immunostimulator--LPS synergism.

Normothermic rabbits and rabbits with LPS induced fever were treated with chlormethine (Nitrogranulogen, Ntg) in the doses of 1 microgram/kg and 10 micrograms/kg. The blood was collected 4, 24, 48 hrs and 4, 7, 10 days after Ntg injection. Following indices of immunity were studied: T and B cells number, number of IgM producing cells after immunization with SRBC, serum IgG level, killing activity of neutrophils and number of phagocytized bacteria. It was observed that both doses of Ntg injected intravenously to normothermic rabbits, significantly increased the number of T and B lymphocytes and of IgM producing lymphocytes a well as the level of IgG in the serum, number of phagocytized bacteria and killing activity of neutrophils. Ntg in combination with LPS shortened the period of fever, and through the synergistic effect, significantly increased T lymphocytes number in the blood, IgG level in the serum, number of phagocytized bacteria and killing activity of neutrophils.

Migration of isolated rabbit lymphocytes in the course of pharmacological inhibition of pyrogenic fever.

The studies concerned in vitro migration of peripheral blood lymphocytes of rabbits given intravenously one dose of E. coli lipopolysaccharide (LPS) and antipyretic doses of acetylsalicylic acid (ASA), indomethacin (IND) or mefenamic acid (MEFA). In normothermic animals, ASA appeared to inhibit in vitro the spontaneous migration of lymphocytes. Contrary to ASA, MEFA and IND did not produce any significant changes in lymphocytes migration. Four hours after pyrogen injection migration of lymphocytes was slightly enhanced and after 24 h it was significantly inhibited. All non-steroid anti-inflammatory drugs examined counteracted this effect.

The influence of copper-dextran complex (C-79), non-steroid anti-inflammatory drugs and bacterial pyrogen on stabilization of rabbit erythrocyte membrane.

We investigated the effect of copper-dextran complex (C-79), acetylsalicylic acid (ASA), mefenamic acid (MEFA) and indomethacin (IND), alone or combined with E. coli lipopolysaccharide (LPS) on osmotic fragility of rabbit erythrocytes. It has been found that LPS in combination with ASA, MEFA, and IND did not change the stabilizing effect of the antipyretics on rabbit erythrocyte membrane. C-79 in doses of 0.4-0.8 mg Cu/kg iv stabilized the erythrocyte membrane for several hours. In lower doses, the compound produced a weak stabilizing effect, and an opposite effect was induced by a dose of 1.6 mg Cu/kg. After administration of C-79 and LPS in combination, the duration of LPS-induced fever was shortened and the erythrocyte stabilization by C-79 was weaker. A combination of ASA with C-79 depressed the body temperature in normothermic animals, while the stabilizing effect of both compounds on the erythrocyte membrane was non-additive.

Plasma catecholamines and 11-hydroxycorticosteroids during interaction between hexamethonium and noradrenaline.

Intravenous infusion of noradrenaline leads to much higher elevation of concentrations of noradrenaline and adrenaline in the rabbit plasma in the presence than in the absence of hexamethonium. This effect is related to stimulation of the hypophyseal-adrenal axis, as adrenalectomy or hypophysectomy prevents it. The stimulation of the system takes place without involvement of circulatory reflexes. The 11-hydroxycorticosteroid levels in plasma increase also under the influence of the interaction in hypophysectomized animals, but this does not affect the level of noradrenaline. The causes of this phenomenon are discussed in the light of data on inhibitory effect of steroids on the extraneuronal uptake of catecholamines.

Participation of the aortal nerve in reflectory release of 11-hydroxycorticosteroids during noradrenalinemia.

Intravenous infusion of 30 micrograms/kg noradrenaline (NA) produced reflectory pituitary-dependent release of 11-hydroxycorticosteroids (11-OHCS) in rabbits and sheep. It has been demonstrated that the afferent tract carrying the impulses stimulating this process runs from the circulatory system in aortal nerves (nn. depressores). The study has revealed the relationship between the increase in the plasmatic levels of 11-OHCS and endogenous adrenaline (A) during pharmacologically induced noradrenalinemia.

The effect of organic nitrites and nitrates on glycogenolytic activity in the heart and skeletal muscle.

Organic nitrites (sodium nitrite, amyl nitrite) and nitrates (nitroglycerin, pentaerythritol, nitromannite) affect similarly phosphorolytic, amylolytic and total glycogenolytic activities in the heart and skeletal muscle. The action of these drugs on glycogen metabolism is due to liberation of adrenaline and concurrent inhibition of its effect on enzymatic activities. The drugs antagonize the stimulatory action of adrenaline on phospholytic activity and inhibitory action on amylolytic activity.

Endogenous catecholamines during noradrenaline and adrenaline hypertension.

Intravenous infusion of noradrenaline (NA) (30 micrograms/kg) results in reflectory increase of adrenaline (A), this has been established by spectrofluorometrical assay of the difference in the plasma catecholamine level during noradrenalinemia with and without elimination of circulatory reflexes. Analogically, intravenous infusion of A brings about reflectory increase of NA level. In the presence of hexamethonium a substantial amount of injected NA remains in the blood. After adrenalectomy the uptake of injected catecholamines is greatly enhanced.