Liposomes in Immunology: Further Evidence for the Adjuvant Activity of Liposomes

The immune response to HSA-phosphatidylcholine complexes administered to rabbits was not markedly enhanced when compared with the response to unmodified ESA.

It was found in earlier work that HSA entrapped in liposomes (mainly composed of phosphatidylcholine) evoked a strong immune response under conditions where no response was detected to free HSA.

The present results exclude the possibility that HSA- phosphatidylcholine complexes which may arise from liposome- encapsulated HSA may be responsible for the adjuvant activity of the liposomes.

The adjuvant activity of liposomes could also be established after administration of a liposome-associated strong antigen (BGG).     

Liposomes in Immunology: The Immune Response Against Antigen-Containing Liposomes

Three ways of antigen administration to rabbits were compared with respect to the resulting immune responses.

Antigen was administered free in solution, entrapped in liposomes or free in solution but together with empty liposomes.

Liposomal material showed adjuvant activity when injected together with free antigen, but a much stronger adjuvant effect was found when antigen was injected entrapped in liposomes.     

Liposomes in Immunology: Impairment of the Adjuvant Effect of Liposomes by Incorporation of the Adjuvant Lysolecithin and the Role of Macrophages

The immune response against HSA (human serum albumin) was studied in rabbits after intravenous injection of various HSA preparations. When HSA was injected one day after, together with or coupled to lysolecithin, a late response was found in twelve out of thirteen rabbits, whereas a minority of the rabbits responded when lysolecithin was omitted.

These results confirm the adjuvant activity of lysolecithin. A rapid response starting on day 6 was found in rabbits injected with HSA entrapped in liposomes which had been composed of lecithin, phosphatidic acid and cholesterol (PPC liposomes). The response against liposome entrapped HSA was delayed for about one day when the phospholipid adjuvant lysolecithin was incorporated in the liposomes (LPPC liposomes).

Results lend support to the hypothesis that the adjuvant activity of lysolecithin and its opposite inhibition of the adjuvant activity of liposomes are mediated by the same mechanism, i.e. inhibition of enzymatic digestion in lysosomes of macrophages.     

The Secondary Immune Response Against Liposome Associated Antigens

In the present experiments, the secondary immune response against antigens is studied after priming with liposome associated antigens and booster injections with the antigen alone, in order to study the effect of liposomes on the generation of immunological memory against the associated antigens.

Liposomes show adjuvant activity with respect to both the primary and secondary immune response against associated human serum albumin (HSA). When the injected dose of liposome associated HSA was too low to elicit a primary immune response, generation of immunological memory against the antigen could not be detected. Horse radish peroxidase (HRP) associated with liposomes did not elicit a primary immune response, but immunological memory against the antigen was established.     

Selective Platelet Immune Retention

Inbred rats were immunized with Freund's Adjuvant containing proteins of Mycobacterium tuberculosis, Mycobacterium butyrium, or dinitrochlorobenzene (DNCB). Native arterial blood was then passed over glass beads coated with those antigens. The platelet retention on the glass beads was measured.

Rats immunized with Complete Freund's Adjuvant developed accelerated platelet retention on beads coated with protein derivatives of tuberculosis (EkD) after just 18 seconds of blood flow. Rats Immunized twice maintained selective retention longer than those immnunized once. The test animals developed no cutaneous hypersensitivity nor precipitin lines on Ouchter-long gels against PPD. Rats sensitized to DNCB had accelerated platelet retention on MCB-coated beads. Results were temperature and complement dependent, and antigen-specific. Heparin caused a dose-dependent inhibition of the accelerated retention.

PYD potentiated the UP-induced aggregation of platelet-rich plasma (YW) from immunized rats.

These experiments suggest that platelets react selectively to antigens in the intravascular spaces in immune reactions.     

Liposomes coated with chemically modified dextran interact with human endothelial cells.

Some liposomal formulations are now in clinical use. New applications in biology and medicine using targeted liposomes remain an intensive research area. In this context, liposomes constituted of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (70/10/20 mol %) were prepared by detergent dialysis and coated with dextran (Dx) or functionalized dextran (FDx), both hydrophobized by a cholesterol anchor which penetrates the lipid bilayer during the vesicle formation. The coating of liposomes with these polysaccharides was performed because chemically modified dextran but not native Dx interacted with vascular cells. The liposome uptake by human endothelial cells was followed using uncoated and coated liposomes radiolabeled with a neutral lipid (3H-cholesterol) and a polar phospholipid (14C-PC). The results indicated for both radiolabels a preferential uptake by endothelial cells of FDx-coated liposomes compared to uncoated or Dx-coated liposomes. Addition to the culture medium of calcium up to 10 mM further enhanced the level and rate of incorporation of FDx-coated liposomes, whereas interaction of endothelial cells with uncoated liposomes or liposomes coated with Dx was poorly affected. Liposome membranes were then labeled with N-(lissamine rhodamine B sulfonyl)diacyl-PE and liposome uptake by endothelial cells was observed by fluorescence microscopy. The punctate intracellular fluorescence of cells incubated at 37 degrees C with fluorolabeled liposomes is indicative of the liposome localization within the endocytotic pathway of the cells. Altogether, these data demonstrate that coating of liposomes with FDx enable specific interactions with human endothelial cells in culture. Consequently, these liposomes coated with bioactive polymers represent an attractive approach as materials for use as drug delivery vehicles targeting vascular cells.     

Damage of Liposomes in Antibody-Dependent Cell-Mediated Cytotoxicity

Lymphoid cells bearing Fc receptors are able to lyse antibody-coated animal target Cells in an antibody-dependent cell-mediated cytotoxicity reaction. The results presented here show that liposomes consisting of sphingomyelin, cholesterol, and cardiolipid and coated by cardiolipidic antibodies could be destroyed by spleen or thymus cells. No alteration of liposomes was observed when normal rabbit serum was used or when the effector cell population was depleted of cells bearing Fc receptors. The lysis of antibody-coated liposomes by effector cells could be carried out in two steps. In the first step, the fixation of antibodies on the cardiolipidic antigens could lead to a reorganization of the liposomal membrane. In the second step, the effector Fc-receptor-bearing cells might amplify this alteration of the liposomes.     

Monocyte Locomotion in Anergic Chronic Brucellosis Patients: The In Vivo Effect of Ascorbic Acid

In 14 patients suffering from relapsing chronic brucellosis who were anergic to brucella antigens, we have studied peripheral blood monocyte random migration and chemotaxis against non-specific and specific leukoattractants, as well as plasma and monocyte ascorbic acid levels.

We found that all parameters studied, were significantly beneath normal, when compared to normal subjects.

After the oral administration of ascorbic acid at a daily dose of 1gr for 15 conseguetive days, random and directed migration against a non-specific stimulus (casein)returned to normal. Directed migration against disease associated leukoattractants (brucella melitensis and brucella abortus) antigens improved significantly, without reaching normal values.

We concluded that ascorbic acid supplementation might partially restore peripheral, monocyte function and help the monocyte-macrophage system to mount an effective immune response against chronicity of brucella infection.     

In Vitro Effect of Inosiplex on T Lymphocytes. I Influence on T Cells with Receptors for IgG (T γ)

Inosiplex, a complex of inosine and 2-hydroxypropyldimethyl ammonium-4-(acetylamino) benzoate, 1:3 molar ratio, originally developed for antiviral use, is now under wider investigation because of its immunopotentiating properties.

This compound can have some actions on T cells at various stages of differentiation, thus promoting an enhancement of their blastogenic responses to varied mitogenic agents (PHA, Con A, PWM, MLC, tetanus toxoid, and viral antigens).

Our studies demonstrate that under the influence of inosiplex human peripheral blood T lymphocytes bearing Fc IgG receptors have an augmented receptor avidity for SRBC which result in an increased E active rosette formation, and that T cells preincubated with the drug at the appropriate concentrations express more Fc IgG receptors.

Even though Tγ cells exert “in vitro” immunoregulatory properties, the increase in percentage of Tγ lymphocytes do not correlate with a potentiation of the Con A-induced suppressor activity of T cells.

Moreover, the lymphocytes treated with the substance in the absence of Con A exert helper functions, increasing the mitogenic responses of the second culture PHA - treated lymphocytes.

These data appear to suggest a pro-proliferative inosiplex-induced effect which could mask a concomitant suppressor cell induction.     

Liposomes in immunology: further evidence for the adjuvant activity of liposomes.

The immune response to HSA-phosphatidylcholine complexes administered to rabbits was not markedly enhanced when compared with the response to unmodified HSA. It was found in earlier work that HSA entrapped in liposomes (mainly composed of phosphatidylcholine) evoked a strong immune response under conditions where no response was detected to free HSA. The present results exclude the possibility that HSA-phosphatidylcholine complexes which may arise from liposome-encapsulated HSA may be responsible for the adjuvant activity of the liposome. The adjuvant activity of liposomes could also be established after administration of a liposome-associated strong antigen (BGG).     

Modulation of Nonisduced and Phorbol Ester-Induced Generation of Superoxide Anion by Free Liposomes and Liposomes Containing Dexamethasone.

We tested the relative efficacy of free dexamethasone, dexamethasone containing liposomes and free liposomes in preventing superoxide anion, O^-₂ generation by neutrophils. O^-₂ production by 5 ± 10⁵ neutrophils, whether primed or not with lipopolysacchaiide, was stimulated by phorbol 12-myristate 13-acetate (PMA) to 13.4 ± 1.3 nmoles after 15 minutes compared to 1.2 ± 0.3 nmoles with nonstimulated cells. Free liposomes but not dexamethasone (dexa) decreased non-stimulated as well as PMA-induced O^-₂ generation. Dexa-containing phosphatidylcholine from egg yolk: phosphatidylserine from bovine brain (POPS 7:3) liposomes, unlike free dexa, diminished PMA-stimulated O^-₂ production in a dose-dependent manner with a maximal effect at 37.5 μg/ml phospholipid (6.6 ± 1.6 nmoles). The kinetics of cytochrome-c reduction revealed that decreased O^-₂ production resulted from an extended lag-time of release to almost 8 minutes with PMA induction and consequently led to the conclusion that liposomes modified the activity of NADPH oxidase as well as that of protein kinase C. Liposomes prepared with PC and PS of natural origin had a greater inhibitory effect on O^-₂ generation by neutrophils than dipal-mitoylphosphatidylcholine (DPPC) and phosphatidylethanolamine from egg yolk (PE):PC (3:1) liposomes. When 100 μM of Ca²⁺ was added to the medium, the inhibitory action of liposomes prepared with egg yolk PC and DPPC was increased by 30 and 60% respectively, while that of PS and PE:PC was prevented. We also verified that liposomes by themselves, even if phagocytized, did not induce O^-₂ generation or its concentration was too low to be detected by this technique. From the clinical point of view, some formulations delayed non-induced and PMA-induced O^-₂ generation, thus adding to the anti-inflammatory effect of the glucocorticoid they transported.     

Regulation of Murine T-Lymphocyte Function by Spleen Cell-Derived and Exogenous Serotonin

The modulatory effects of serotonin on T-cell activity were investigated. T-cell blastogenesis of normal spleen cells was slightly stimulated by the addition of low doses (1 and 10 ng/ml) of the inducer of serotonin release, fenfluramine. In contrast to the stimulatory effects of low doses of fenfluramine, high doses of fenfluramine (1 and 10 ug/ml) or of exogenously added serotonin (≥0.1 ug/ml) inhibited T-cell activation. Both the stimulation by low dose fenfluramine and the inhibition by high dose fenfluramine were accentuated by pretreating mice with tryptophan to heighten intracellular stores of serotonin and then inducing serotonin release. Pretreatment of mice with the serotonin inhibitor p-cholorphenylalanine (PCPA) abolished the fenfluramine inhibition of T-cell activation indicating that the fenfluramine inhibitory effect was mediated via endogenous spleen cell-derived serotonin. However, the PCPA treatment diminished T-cell activation. These results suggest that endogenous serotonin causes a biphasic dose-response effect on T-cell activity with serotonin being required for optimal T-cell function, low doses being immune stimulatory and higher doses being suppressive.

Con-A, concanavain A

5-HT, 5-hydroxytryptamine

LAK, lymphokine-activated killer

NK, natural killer

PCPA, p-cholorphenylalanine

PHA, phytohemagglutinin     

The Lack of Antigenicity of Tuftsin: A Naturally Occurring Phagocytosis Stimulating Tetrapsptide

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide that stimulates all known functions of the polymorphomaclear. leukocyte and macrophage cell lines. Tuftsin is located in the F_c region of IgG between the 289 and 292 amino acid sequence of the CH₂ domain. We describe unsuccessful attempts to generate antituftsin antibodies. In separate experiments tuftsin was chemically conjugated to methylated bovine serum albumin (CH₃ BSA), BSA, keyhole limpet hemocyanin (KLH) and purified protein derivative (PPD). Tuftsin was also polymerized with glutaraldehyde. Animals used for immunization were rabbits, roosters, and dogs. All experiments failed to produce antituftsin antibody. Probable reasons for the lack of antigenicity include:

I) Lack of “foreignness” of tuftsin in mammalian species.

II) The small size of the tetrapeptide.

III) Tuftsin may be exerting an adjuvant effect when coupled to foreign antigens and is therefore not recognized by the host immune system.     

Immunological Consequences of Zidovudine Treatment in Control and Morphine or Methadone Treated Mice

The effects of acute and chronic zidovudine (AZT) administration on immunologic test responses of mice were studied. The effects of AZT administration combined with morphine or methadone treatment, were also studied separately comparing the effects of each drug.

We noted that AZT-treatment did not modify the T-lymphocyte subsets (L₃T₄/LyT₂ rate), whereas morphine-treatment and AZT plus morphine treatment decreased the percentage of T helper cells.

Acute and chronic AZT-treatment increased Natural Killer cell (NK) activity and also recovered the decreased NK cell activity produced by morphine-treatment.

AZT-treatment, morphine-treatment, AZT plus morphine treatment and AZT plus methadone treatment strongly depressed the phagocytic physiological activity of Polymorphonuclear leukocytes (PMNs).

Another evidence of immunologic responsiveness against AZT was the reduction of the mitogenic and antigenic response of lymphocytes.

These results suggest a negative role of AZT-treatment especially on phagocytic activity and confirms a depressive effect of morphine-treatment on several immune functions studied. Furthermore, there is no indication of additive or synergistic toxic effects of AZT, morphine and methadone on the immune functions above that seen with each of these drugs when tested alone.     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.     

Lupus anticoagulant activities of murine monoclonal antibodies to liposomal phosphatidylinositol phosphate.

Four murine monoclonal antibodies having high levels of activity against phosphatidyl-inositol phosphate (PIP) were tested for lupus anticoagulant activity. The antibodies showed different degrees of potency in a modified partial thromboplastin time test (APTT) that used dilutions of either bovine brain extract (Thrombofax) or liposomes consisting of phosphatidylcholine/phosphatidylserine (PC/PS) as the phospholipid source. The same relative order of anticoagulant potency that was observed in the APTT that used the PC/PS liposomes was maintained when the anti-PIP antibodies were tested for cross-reactivity either by induction of complement-dependent immune damage to liposomes containing PS, or in enzyme-linked immunosorbent assays that used PS, cardiolipin (CL), or phosphatidylinositol (PI) as antigens. The data indicate that monoclonal antibodies to PIP can express anticoagulant activity in a modified APTT that correlates with their different degrees of cross-reactivity against the negatively-charged phospholipids PS, CL, and PI.     

Liposomes as carriers of antigens and adjuvants.

Liposomes have been widely used as carriers of protein or peptide antigens. Antigenic materials can be attached to the outer surface, encapsulated within the internal aqueous spaces, or reconstituted within the lipid bilayers of the liposomes. The natural tendency of liposomes to interact with macrophages has served as the primary rationale for utilizing liposomes as carriers of antigens. Liposomes also serve as carriers of a variety of adjuvants and mediators, including lipid A, muramyl dipeptide and its derivatives, interleukin-1, and interleukin-2. Research utilizing in vitro cell culture models has demonstrated that liposomes containing both appropriate antigens and major histocompatibility gene complex molecules can induce antigen-specific genetically restricted cytotoxic T lymphocytes. Liposomes induce immune reactions through classical interactions with antigen presenting cells. However, modelling experiments have also demonstrated that liposomes can even substitute for antigen presenting cells, and cell-free genetically restricted and nonrestricted presentation of antigens by liposomes to helper T lymphocytes has been demonstrated. Liposomes are successful for inducing potent immunity in vivo and they are now being employed in numerous immunization procedures and as vehicles for candidate vaccines.     

Benzodiazepines Inhibit in Vitro Free Radical Formation from Human Neutrophils Induced by Fmlp and A23187.

Diazepam (DZP) inhibited in vitro in a concentration-dependent manner superoxide anion generation and chemiluminescence from human neutrophils stimulated by the formylated oligopeptide FMLP and by the calcium ionophore A23187.

The dose-dependent inhibitory effect of DZP on A23187-dependent superoxide generation in the presence of Ca⁺⁺ 0.6 mM was highly antagonized by increasing extracellular Ca⁺⁺ concentration to 1.5 mM and to 2.0 mM.

Ro 5-4864, a specific ligand for peripheral type benzodiazepine (BZ) binding site, inhibited superoxide generation induced by FMLP, while clonazepam (CNZ), which is selective for brain sites, did not possess any activity. Ro 15-1788, a central type BZ receptor antagonist, did not show any antagonistic activity on DZP-dependent inhibition.

A new physiological property for substances presenting an affinity for peripheral type BZ binding sites is supposed. The inhibitory effect of BZ on neutrophil functions seemed to be associated with a Ca⁺⁺-involving mechanism.     

In Vitro Effects of Tp5 Analogs on E-Rosette Formation and Cell Division

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests.

Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides.

Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED₇₅) inhibited E-receptor. Treatment of azathioprine (ED₇₅-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC.

Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED₃₀. These “suppressive” peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours.

Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.     

Therapeutic Effect of Ok-432 Induced Endogenous Tnf on Tumor Bearing Mice and Cancer Patients

The therapeutic effect of OK-432 induced endogenous TNF on tumor bearing mice and cancer patients was investigated.

OK-432 (10 KE/mouse) was administered intraperitoneally to Balb/c mice 7 days prior to the transplantation of Meth A cells (1×10⁶/mouse) into the abdominal cavity. And at day 1 of tumor inoculation, 1 KE/mouse of OK-432 was administered intraperitoneally.

The significant prolongation of life span was observed in these mice.

On the basis of these observation, therapeutic effect of endogenous TNF on cancer patients was clinically evaluated. OK-432 was administered intraperitoneally or intrapleurally to cancer patients with peritonitis carcinomatosa or pleuritis carcinomatosa 4 times (10KE each) every other day and 50KE of OK-432 was readministered with the interval of 7 days.

An appreciable activity of TNF was detected in peritoneal fluids or pleural effusion, and the significant decreasing of these fluids was observed. It is therefore concluded that these therapeutic approach may well be taken into account in treatment of cancer.