The mechanisms of immune suppression by high-pressure stress in mice

The effects of high-pressure stress on the induction of anti-sheep red blood cells (SRBC) and of plaque-forming cells (PFC), and on thymus weight, were studied in BALB/c mice in-vivo and in-vitro. The efficacy of high-pressure stress in suppressing PFC and thymic involution was maximum when the stress was applied 1 h day(-1) for 2 days before immunization with SRBC. Both effects were blocked by administration of indomethacin, atropine, naloxone or phentolamine before the first application of stress, whereas hexamethonium and propranolol had no such effect. Hexamethonium, naloxone and propranolol administered before the second application of high-pressure stress blocked both effects. Prostaglandin and acetylcholine given 24 h before application of high-pressure stress caused a marked reduction in PFC count, but not in thymus weight. The reduced PFC count caused by acetylcholine was blocked by pretreatment with indomethacin. When adrenaline was injected 24 h after application of high-pressure stress a marked reduction in PFC was observed, but without thymic involution. When adrenaline was injected 24 h after prostaglandin injection the PFC count was also markedly reduced, but not thymus weight. The decrease in PFC caused by two exposures to stress or one exposure to stress plus injection of adrenaline was blocked by diethylcarbamazine before the second exposure to stress or the injection of adrenaline. In addition, normal spleen cells, were induced as suppressor cells when incubated with the serum of stressed mice, but not when supplemented with anti-leukotriene C4, D4 antibody. These data suggest that mice fall into a pre-stress condition via the release of prostaglandin after the first stress, and then immunosuppression is induced in these prestressed mice via the release of leukotriene C4, D4, caused by the activation of the autonomic nervous system by the second exposure to stress.     

Selective immunotoxic effects in mice treated with the adenosine deaminase inhibitor 2'-deoxycoformycin

Mice were administered the adenosine deaminase inhibitor 2'-deoxycoformycin by daily intraperitoneal injection for five days and evaluated 24 h, 72 h and 6 days after the final dose. Spleen weight was decreased in treated mice for up to 6 days after treatment whereas body weight was significantly affected only at 24 h in mice administered 4 micrograms 2'-deoxycoformycin/g of body weight. The number and relative percentage of circulating lymphocytes were decreased 24 and 72 h after the last injection. Lymphoproliferative responses to T cell mitogens were suppressed for at least 72 h post-treatment whereas the mixed lymphocyte response was normal at 24 h but was depressed at 72 h post-treatment. Conversely, natural killer cell activity was greater in treated mice than in controls for the entire observation period. Data from cell surface marker analysis provided indirect evidence that natural killer effector cells lack sensitivity to 2'-deoxycoformycin. The antibody responses of mice treated with 2 or 4 micrograms 2'-deoxycoformycin/g over four days prior to or after immunization with sheep erythrocytes were suppressed or enhanced, respectively, compared to controls. These results indicate selective effects of 2'-deoxycoformycin on immune function and suggest that subpopulations of lymphocytes differ in the degree of sensitivity to 2'-deoxycoformycin.     

The effects of 5-fluorouracil on immunocompetent cells depend on the genotype of experimental animals used

A detailed study was made of the effects of 5-fluorouracil (5-FU) on primary and secondary immune responses to a T-dependent antigen in mice. Subcutaneous, intraperitoneal and intravenous applications gave similar results, oral administration was less effective. The minimal effective dose was found to be 40 mg/kg. Inbred strains known to differ in terms of IgG immune responses were compared and found to respond quite differently to the drug. For example, when 5-FU was injected 24 h before or simultaneously with antigen, IgM plaque-forming cell (PFC) responses of C57BL/10 mice were not substantially affected. In contrast, responses of A/J and Balb/c mice were suppressed 50-90% under the same conditions. If injected 24 h after the antigen, 5-FU inhibited PFC responses completely in all three strains. IgM PFC recovery was fastest in the C57BL/10 strain and started on day 7. In secondary responses, IgM overstimulation was seen in C57BL/10 and Balb/c mice if 5-FU was injected once, i.e. 24 h after primary immunization. IgG PFC were completely suppressed in primary responses in all strains. In secondary responses, the decrease was influenced by the time of drug injection and by the genotype of the treated animal. Inhibition of isotype switching was greatest in low-IgG-responding C57BL/10 mice. These results demonstrate that genotypic variation can be substantial with respect to effects of 5-FU on the immune system.     

The effects of acute and chronic lithium treatment on pilocarpine-stimulated phosphoinositide hydrolysis in mouse brain in vivo.

1. Measurements were made of the in vivo formation of inositol phosphates in the brains of C57/B1/601a mice treated acutely or chronically with lithium chloride (LiCl). 2. A single injection of LiCl (10 mEquiv kg-1, s.c.) 18 h before death increased the accumulation of [3H]-inositol phosphates ([3H]-Ins P's) in the brains of mice injected i.c.v. with [3H]-myo-inositol 24 h previously. 3. Pilocarpine (200 mg kg-1, i.p.) injected 15 min before death further enhanced the formation of [3H]-Ins P's in the brains of LiCl-treated, but not saline-treated, mice. The enhancement due to pilocarpine was abolished by injection of atropine sulphate (10 mg kg-1, i.p.) 10 min earlier. 4. Chronic (14 days) LiCl feeding produced an accumulation of [3H]-Ins P's significantly less than that due to a single injection of LiCl, but the response to pilocarpine was markedly greater in mice chronically fed with LiCl when compared with mice acutely injected with LiCl. 5. Mass measurements of endogenous inositol 1,4,5 triphosphate revealed increases due to pilocarpine and chronic LiCl feeding alone. A combination of the two treatments produced levels greater than either alone. 6. These results demonstrate that LiCl treatment enhances both basal and pilocarpine-stimulated inositol phospholipid hydrolysis in vivo and this might be relevant to its therapeutic effects.     

Intramuscular absorption and regional lymphatic uptake of liposome-entrapped inulin

An investigation of the effects of the liposome variables (size, surface area, and amount of injected lipid) on the intramuscular absorption and subsequent lymphatic uptake of drug from the injection site (left muscle) and in the opposite (right) muscle and lymph node was done in mice. Two size ranges were studied (0.3-2.0 mu and 0.15-0.7 mu). Large multilamellar vesicles were studied at lipid doses of 20.7 and 9.0 mg/kg, whereas small multilamellar vesicles were investigated at a dose of 9.5 mg/kg. Liposomes were composed of phosphatidylcholine/cholesterol/phosphatidylserine/alpha-tocopherol/14C-dipalmitoylphosphatidylcholine (as a marker for the lipid phase), 4:5:1:0.01:0.01 (molar ratio). 3H-inulin was used to monitor the aqueous phase. At 24 hr after administration, large liposomes tended to result in the largest fraction of drug at the injection site, with the 3H/14C ratio indicating stability of the remaining vesicles. Small liposomes showed a greater uptake of inulin via the lymphatics. Decreasing the total amount of injected lipid resulted in a slower absorption of large liposomes and a greater uptake via the lymphatics.     

Substance P effects on intraspecific aggressive behaviour of isolated male mice: an ethopharmacological analysis

Adult male mice of the Swiss CD1 strain were used to evaluate the effects on isolation-induced aggressive behaviour of a single intravenous administration of substance P (SP; 0.25, 1.0 or 2.5mg/kg dose). All mice were injected 15min before testing (10min videotaped dyadic encounters with an isolated male untreated opponent). Control mice were injected with vehicle. All animals were tested again 24h later in a drug-free state. SP treatment produced a decrease in offensive scores (Attacks and Rattling behaviour), a longer latency to the first Attack episode, and enhanced defensive displays. These effects were reversed 24h later. In no case did SP treatment affect locomotor activity levels or freezing behaviour. A role of SP in the regulation of murine aggressive response is strongly suggested through a direct action of the drug on the central nervous system and specifically on the hypothalamus.     

Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains.

The immunosuppressive action of phosphatidylserine has been studied in mitogen-activated human peripheral blood mononuclear cells. The addition of phospholipid (10-60 nmol/10(6) cells) causes a dose-dependent inhibition of DNA synthesis induced by PHA, anti-CD3 mAb, allogeneic lymphocytes and tetradecanoylphorbol acetate plus ionomycin. In contrast, the interleukin-2-dependent DNA synthesis is less affected. Flow cytometric analysis and binding of radioiodinated interleukin-2 show that the phospholipid prevents the expression of interleukin-2 and transferrin receptors. Removal of monocytes by adherence does not change the action of phosphatidylserine. Furthermore, the phospholipid is equally effective in preparations depleted of CD4+ or CD8+ lymphocytes. Phosphatidylinositol partly reproduces the action of phosphatidylserine. Phosphatidic acid, phosphatidylglycerol and phosphatidylcholine are inactive. Also unsaturated phosphatidylserine analogues inhibit DNA synthesis whereas saturated phosphatidylserines do not. The data suggest that phosphatidylserine mainly affect the steps of T cell activation preceding the production of interleukin-2 and the expression of its receptor. The phosphorylserine headgroup and the unsaturated acyl chains contribute to this effect.     

Modification of acquired immunity in BALB/c mice by aztreonam

Recent studies have suggested that antibiotics may act as biological response modifiers. In this study we investigated the effect of aztreonam, a monobactam antibiotic, on different parameters of acquired immunity in BALB/c mice. Different dosages of aztreonam injected into mice induced an increase in the lymphoproliferative response to specific mitogens and in the production of interleukin-2 by splenic cells, as well as a decreased response of this immune population to sheep erythrocytes lower total blood cell counts and a lower percentage of monocytes than in untreated mice. These results show a modulatory action of aztreonam on different immune parameters, which is independent of its antimicrobial activity and that could be of interest in human therapy.     

Phosphatidylserine reverses reserpine-induced amnesia

The effects of phosphatidylserine (PS) were studied in rats treated with reserpine (1 mg/kg) immediately after training in the passive avoidance task. In experiment I, phosphatidylserine (25 mg/kg) was administered 30 min before or immediately after training. Acute pre- or post-treatment with phosphatidylserine was effective in reversing the amnestic effect of reserpine in test trials performed 24 h and 1 week after training. Experiment II was performed to determine if the long-term pretreatment with phosphatidylserine (25 mg/kg) for 7 days is able to protect the rats against the amnestic effects of reserpine in this task. The data show that phosphatidylserine reverses the impairment induced by reserpine in trials performed 24 h and 1 week after training. These results indicate that the memory deficits associated with catecholamine depletion caused by reserpine can be attenuated by acute pre- or post-training or by long-term pretreatment with this phospholipid.     

Loading of curcumin into macrophages using lipid-based nanoparticles

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, Cm) is a natural compound which possesses antioxidant, anti-inflammatory and anti-tumor ability. Here, phospholipid vesicles or lipid-nanospheres embedding Cm (CmVe or CmLn) were formulated to deliver Cm into tissue macrophages through intravenous injection. Cm could be solubilized in hydrophobic regions of these particles to form nanoparticle dispersions, and these formulations showed ability to scavenge reactive oxygen species as antioxidants in dispersions. At 6h after intravenous injection in rats via the tail vein (2mg Cm/kg bw), confocal microscopic observations of tissue sections showed that Cm was massively distributed in cells assumed as macrophages into the bone marrow and spleen. Taken together, these results indicate that the lipid-based nanoparticulates provide improved intravenous delivery of Cm to tissues macrophages, specifically bone marrow and splenic macrophages in present formulation, which has therapeutic potential as an antioxidant and anti-inflammatory.     

Agmatine increases proliferation of cultured hippocampal progenitor cells and hippocampal neurogenesis in chronically stressed mice

AIM: To explore the mechanism of agmatine's antidepressant action. METHODS: Male mice were subjected to a variety of unpredictable stressors on a daily basis over a 24-d period. The open-field behaviors of the mice were displayed and recorded using a Videomex-V image analytic system automatically. For bromodeoxyuridine (BrdU; thymidine analog as a marker for dividing cells) labeling, the mice were injected with BrdU (100 mg/kg, ip, twice per d for 2 d), and the hippocampal neurogenesis in stressed mice was measured by immunohistochemistry. The proliferation of cultured hippocampal progenitor cells from neonatal rats was determined by colorimetric assay (cell counting kit-8) and 3H-thymidine incorporation assay. RESULTS: After the onset of chronic stress, the locomotor activity of the mice in the open field significantly decreased, while coadministration of agmatine 10 mg/kg (po) blocked it. Furthermore, the number of BrdU-labeled cells in the hippocampal dentate gyrus significantly decreased in chronically stressed mice, which was also blocked by chronic coadministration with agmatine 10 mg/kg (po). Four weeks after the BrdU injection, some of the new born cells matured and became neurons, as determined by double labeling for BrdU and neuron specific enolase (NSE), a marker for mature neurons. In vitro treatment with agmatine 0.1-10 micromol/L for 3 d significantly increased the proliferation of the cultured hippocampal progenitor cells in a dose-dependent manner. CONCLUSION: We have found that agmatine increases proliferation of hippocampal progenitor cells in vitro and the hippocampal neurogenesis in vivo in chronically stressed mice. This may be one of the important mechanisms involved in agmatine's antidepressant action.     

Effect of 5-iodouracil and 2,6-diaminopurine on passive avoidance task

The effect of two antimetabolites, 2,6-diaminopurine and 5-iodouracil on passive avoidance learning was studied in male albino mice. The drugs were injected intracranially 48 h, 24 h or 2 h before and 1 h, 2 h or 24 h after the acquisition trial. The injection of 5-iodouracil 2 h before the acquistion trial or 1 h after it impairs the performance in the same experimental situation of the animals tested 48, 72 h or 1 week after. The same impairment of performance appears after the injection of 2,6-diaminopurine performed 24 and 2 h before or 1 h after the acquisition trial. The effect of these substances is probably caused by the interference with the metabolism of nucleic acids during learning.     

Biodegradable Microparticles Loaded with Doxorubicin and CpG ODN for In Situ Immunization Against Cancer

In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen-presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. We designed a comprehensive approach to in situ immunization using poly(lactic-co-glycolic acid) (PLGA)-biodegradable microparticles (MPs) loaded with doxorubicin (Dox) and CpG oligodeoxynucleotides (CpG) that deliver Dox (chemotherapy) and CpG (immunotherapy) in a sustained-release fashion when injected intratumorally. Dox induces immunogenic tumor cell death while CpG enhances tumor antigen presentation by DCs. PLGA MPs allow their safe co-delivery while evading the vesicant action of Dox. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs. In vivo, Dox/CpG MPs combined with antibody therapy to enhance and maintain the T cell response generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent an efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.KEY WORDS: cancer vaccine, CpG, doxorubicin, in situ immunization, microparticles, PLGA     

The effects of (-)-Δ9-tetrahydrocannabinol on reserpine-induced hypothermia in rats

1. An intravenous injection into rats of 1 mg/kg (-)-Delta(9)-tetrahydrocannabinol Delta(9)-THC) had no effect on rectal temperature and produced in the subcellular fractions of the brain a shift of 5-hydroxytryptamine (5-HT) from the particulate or ;bound' 5-HT to the supernatant or ;free' fraction, whereas the noradrenaline (NA) decreased in both fractions.2. Pretreatment of rats by an intravenous injection of 1 mg/kg Delta(9)-THC three times a week for four weeks, prevented the hypothermia and the reduction in brain 5-HT produced by an intraperitoneal injection of 15 mg/kg reserpine given 24 h after the last Delta(9)-THC injection.3. Pretreatment of rats by a single intravenous injection of 1 mg/kg Delta(9)-THC prevented the hypothermia and reduction in brain 5-HT produced by an intraperitoneal injection of reserpine given 1 h before. The reduction in brain NA was not prevented except at the 18 h time interval.4. An injection of 1 mg/kg Delta(9)-THC intravenously into rats 3 h after an intraperitoneal injection of reserpine accentuated the reserpine hypothermia as well as the reduction of 5-HT but not of NA in the brain.5. The reserpine hypothermia was not prevented by a single intravenous injection of 1 mg/kg Delta(9)-THC when cinanserin, a 5-HT inhibitor, was injected 30 min before the reserpine.     

Pharmacological effects of phosphatidylserine liposomes: the role of lysophosphatidylserine.

1. Unique among the phospholipids, phosphatidylserine depresses brain energy metabolism when injected intravenously into mice in the form of sonicated liposomes. The possibility that this effect results from a metabolic transformation of phosphatidylserine is examined in this paper. 2. A strong enhancement of the phosphatidylserine effect is induced by the incubation of liposomes with rat serum. Similar phosphatidylserine activation is observed after the incubation of the phospholipid with purified phospholipase A2 from pancreas. In both cases phosphatidylserine is split into the deacylated derivative, lysophosphatidylserine. 3. Lysophosphatidylserine reproduces with greater efficacy the effect of phosphatidylserine on brain energy metabolism. Other lysophospholipids are not effective. 4. It is concluded that the pharmacological effects of phosphatidylserine liposomes is due to the generation of lysophosphatidylserine.     

Long-lasting anti-inflammatory properties of Crotalus durissus terrificus snake venom in mice

In the present study, we investigated the effects of Crotalus durissus terrificus venom (CdtV) on vascular and cellular events of inflammation induced by carrageenan (cg) in mice. To evaluate edema, CdtV (75 μg kg⁻¹) was administered subcutaneously before (1 h, 7 or 14 days) or after (1, 4 or 48 h) subplantar injection of cg (15 mg kg⁻¹) into the mouse right hind paw; to analyze leukocyte influx, cg (200 μL) was injected i.p. in mice. The inhibitory action of CdtV on edema, either before or after cg injection, was prolonged, lasting even 72 h after administration. Besides, CdtV significantly inhibited migration of polymorphonuclear cells to peritoneal cavity when administered before or after cg. Such inhibitory effects of CdtV on edema and cell migration were also compared with well-known anti-inflammatory drugs. The results demonstrated that CdtV, when injected either 7 or 14 days or 1 h before cg, induced a more effective and long-lasting anti-inflammatory effect than that observed with classical anti-inflammatory drugs. The association of CdtV with different drugs did not potentialize their actions on cell migration. These results demonstrate that CdtV exhibits long-lasting anti-inflammatory effects.     

CYCLOSPORINE A IMMUNOSUPPRESSION IN SHEEP WITH RESPONSE ENHANCEMENT BY CONCOMITANT KETOCONAZOLE

1. The pharmacokinetics of a single dose of Cyclosporine A (CsA) administered to sheep by intravenous (i.v.) route were examined. 2. Concomitant administration of ketoconazole was found to increase the area under the blood CsA concentration-time curve (AUC) and was effective when adminstered by the oral or intraperitoneal route. 3. The effects of CsA and ketoconazole on the immune system of sheep were also assessed. 4. A single dose of CsA 5mg/kg resulted in abrogation of in vitro lymphocyte function manifest at 24h after injection of CsA. Normal responsiveness recovered in 48-72h. Numbers of T lymphocytes in peripheral blood were elevated transiently at 48h although no other significant alteration in lymphocyte subsets was observed with this treatment. 5. Concomitant ketoconazole administration enhanced the CsA-induced suppression of in vitro lymphocyte responses. Blood levels of CsA (AUC values to 24h) were significantly elevated with concomitant ketoconazole administration and depression of lymphocyte responses to mitogens were also significantly enhanced. An increase in the proportion of T4 positive cells in the blood was observed at 48h and at 7 days after administration of CsA with ketoconazole. 6. These findings indicate that CsA effectively abrogates immunocompetence in the sheep and this immunosuppressive effect is enhanced by concomitant administration of ketoconazole.     

Local thrombus formation in the site of intravenous injection of chlorpromazine: possible colloid-osmotic lysis of the local endothelial cells

Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artificial membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakage. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenously dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.     

Pharmacokinetics of liposome-encapsulated anti-tumor drugs: Studies with vinblastine,actinomycin D,cytosine arabinoside,and daunomycin

We have investigated the effects of encapsulation within liposomes (phospholipid vesicles) on the plasma clearance kinetics and tissue disposition of four anti-tumor drugs, namely vinblastine. cytosinc arabinoside, actinomycin-d and daunomycin. In each case, subsequent to intravenous administration, the liposome-encapsulated drugs were cleared from the plasma much more slowly than were the free drugs. For example, the major portion of daunomycin injected in free form had a plasma half-life of less than 5 min, while liposome-encapsulated daunomycin had a plasma half-life in excess of 150 min. Encapsulation also caused a marked alteration in the tissue disposition of the injected drugs. Thus, encapsulation within liposomes resulted in a large increase in the total amount of drug equivalents retained by the tissues at various times after injection. In the case of cytosine arabinoside, for example, the level of drug equivalents in the liver at 16 hr post injection was 68-fold greater for liposome-encapsulated drug than for free drug. Encapsulation also altered the relative distribution of drugs in the tissues, with tissues rich in reticuloendothelial cells, such as liver and spleen, being the favored sites of uptake.     

Effect of Kp,an antitumor protein-polysaccharide from mycelial culture ofPhellinus linteus on the humoral immune response of tumor-bearing ICR mice to sheep red blood cells

The immunomodulating activity of Kp, an antitumor protein-polysaccharide preparation from the shake-cultured mycelia ofPhellinus linteus, was investigated in ICR mice subcutaneously implanted with 1×10⁶ cells of sarcoma 180. The mice were intraperitoneally administered with Kp at a dose of 100 mg/kg once daily for five consecutive days starting from 24 hrs after the tumor implantation. Ten days after the last injection, the mice were immunized with 1×10⁷ or 4×10⁸ sheep red blood cells (SRBC) and five days later, the antibody-forming immune response were assessed by direct hemolytic plaque assay. To an immunization dose of 1×10⁷ SRBC, the Kp-treated mice elicited a successful humoral immune response despite the tumor-burden and produced 259×10³ plaque-forming cells (PFC)/spleen, while the corresponding tumor-bearing control mice showed virtually no response (2.0×10³ PFC/spleen) (the stimulation index=129.5). However, to an immunization dose of 4×10⁸ SRBC, both of the control mice and Kp-treated mice showed almost the same level of strong humoral immune response. From these data it is clear that Kp effectively restores the humoral immune response of the tumor-bearing ICR mice.