Liposomes in Immunology: Multilamellar Phosphatidylcholine Liposomes as a Simple, Biodegradable and Harmless Adjuvant Without any Immunogenic Activity of its own

Multilamellar phosphatidylcholine liposomes were coated with the antigens human serum albumin (HSA) or bovine gamma globulin (BGG) simply by suspending the liposomes in a solution of the antigens.

Antigen coated phosphatidylcholine liposomes showed nearly the same adjuvant activity after intravenous injection as liposomes of a more complex phospholipid composition.

Since phosphatidylcholine liposomes are biodegradable, harmless, easily obtainable, have no immunogenic activity of their own and may be administered intravenously, they seem to be a promising immunoadjuvant.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

In Vitro Regulation of the Anamnestic Antibody Response Upon Addition of Heterologous Antigens to Primed Lymphoid Cells

Alum-precipitated keyhole limpet hemocyanin (KLH) and polymerized human serum albumin (HSA) were injected into the hind foot pads of rabbits. Many months later the popliteal lymph nodes were removed and cell cultures prepared and incubated in media containing different concentrations of KLH or native HSA. These primed cells responded to some concentrations of KLH with the synthesis not only of homologous antibody but of heterologous IgG antibody to HSA. They also responded to some concentrations of HSA with the production of heterologous antibody to KLH as well as homologous antibody. In some experiments the addition of KLH induced the cells to synthesize as much antibody to HSA as did the addition of HSA itself. The nonspecific stimulation of the anamnestic antibody response to HSA required the cultures to contain KLH, HSA, KLH-reactive memory cells and, presumably memory cells reactive with HSA.

The suspension of KLH-and HSA-primed cells responded to other concentrations of KLH or egg albumin (EA) with inhibition of the antibody response to HSA. This non-specific inhibition did not require lymph node cells which had been primed by the previous injection of KLH or EA.

KLH and HSA did not cross-react with respect to immunogenicity in vitro or in vivo or reactivity with antibody in vitro.

According to a model which was proposed, the KLH induced KLH-reactive T lymphocytes to produce soluble factor(s) which regulated the response of HSA-reactive B lymphocytes to HSA-specific determinants which persisted from the original injection.     

Impairment of Non-Specific Immunity in Patients in Persistent Vegetative State

In fourtheen patients in persistent vegetative state (PVS) immune responsivenes was investigated. In particular, we studied the relationship between brain lesions following traumatic injury and immune system. In this respect, phagocytosis and killing of Candida albicans by polymorphonuclear cells (PMN) and monocytes were tested. In addition serum levels of Interferon-γ (IFN-γ) were evaluated.

The patients come out fiom PVS by 3-4 month were used as control group.

Data shown a profound impairement of phagocytosis and killing of monocytes and low serum levels of IFNγ when compared with normal values.

Taken together, these findings suggest that brain lesions, may affect non-specific immune response.     

Spatial Visualization of Junctional Complexes by Backscattered Electron Imaging and Silver Staining

A method for the observation of junctional complexes by backscattered electron imaging in scanning electron microscopy, in tissue blocks, is presented.

The junctional complexes are revealed by a modified silver staining method (originally devised for nucleolar organizer regions), used “en bloc” after formalin or glutaraldehyde fixation.

Backscattered electron imaging allows, after this staining, the observation of junctional complexes through the surface of intact superficial cells, in the three tissues studied (liver, jejunum and urinary bladder)

The interest of this approach is to offer the possibility of observing junctional complexes “by transparence,” in nondissociated and nonsectioned tissues.     

Enhancement of Immune Response of Murine Peyer's Patches by a Diet Supplemented With Yogurt

The first line of defense against pathogens that enter the host by the oral route involves the Peyer's Patches (PP). For centuries many populations of the mediterranean basin have empirically administered soured milk (yogurt. to prevent and treat diarrhoea and entero-colitis. Recent reports have offered evidence in favour of a possible influence of yogurt on the host's immunocompetence.

Scope of the present study was to evaluate the influence of a diet supplemented with yogurt on the PP from BALB/c mice.

The results reported here suggest that yogurt feeding potentiates the host's cell-mediated immune response by increasing the percentage of B lymphocytes and the PHA and LPS-induced proliferative responses of PP cell suspensions.     

The Adjuvant Effect of Liposomes in Eliciting Anti-Galactosyl Antibodies

Negatively-charged, multilamellar liposomes, covalently coupled with p-aminophenyl-β-D-galactopyranoside, elicited in rabbits a nearly equivalent anti-galactosyl immune response in both the presence and the absence of complete Freund's-adjuvant (CFA). The antibody response obtained using liposomes both as the carrier and adjuvant was found to be better than that obtained through the conventional way of using protein carrier in CFA. The results suggest an effective role of liposomes as adjuvant in the production of sugar-specific antibodies.     

The Adjuvant Effect of Liposomes in Eliciting Anti-Galactosyl Antibodies

Negatively-charged, multilamellar liposomes, covalently coupled with p-aminophenyl-β-D-galactopyranoside, elicited in rabbits a nearly equivalent anti-galactosyl immune response in both the presence and the absence of complete Freund's-adjuvant (CFA). The antibody response obtained using liposomes both as the carrier and adjuvant was found to be better than that obtained through the conventional way of using protein carrier in CFA. The results suggest an effective role of liposomes as adjuvant in the production of sugar-specific antibodies.     

In vitro effects of naloxone on T-lymphocyte-dependent antibacterial activity in hepatitis C virus (HCV) infected patients and in inflammatory bowel disease (IBD) patients

Naloxone acts as an opioid antagonist, displacing opioid drugs from cellular receptors. Among opioid substances, β-endorphins are able to bind to several cell receptors, even including those expressed by immune cells. In this respect, evidence has been provided that in the course of viral infections, as well as in patients with ulcerative colitis high levels ofβ-endorphins are detectable. Here, peripheral blood lymphocytes (PBL) from 21 HCV infected patients and 14 patients with IBD, respectively, were incubated with Naloxone and Naloxone + Ca²⁺ in order to evaluate a putative modulation of PBL-mediated antibacterial activity. In fact, previous studies have demonstrated a reduction of this T-cell activity in HCV and IBD patients.

In general terms, the above treatment led to a recovery of the depressed antibacterial activity. In some cases, increase in T lymphocyte function was obtained with Naloxone alone, while in other cases the combination Naloxone + Ca²⁺ gave rise to a restorative effect. Of note, in some instances, lymphocytes were unresponsive to pharmacological modulation.

The overall results suggest that β-endorphins may down modulate T-cell antibacterial response in HCV and in IBD patients by saturating peripheral receptors on immune cells. Therefore, it is likely that Naloxone and/or Naloxone + Ca²⁺ may displace opioid drugs, thus antagonizing their effects.     

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.     

Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo . This study aimed to evaluate the immunostimulatory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes. BALB/c mice were immunized intramuscularly with different formulations of liposomes,CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation. High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.     

Liposomes as adjuvants with immunopurified tetanus toxoid: the immune response

Immune responses of BALB/c mice to immunopurified tetanus toxoid entrapped in dehydration-rehydration vesicles composed of equimolar egg phosphatidylcholine and cholesterol were compared to those of free toxoid. Animals were injected intramuscularly with the free or liposomal toxoid and identical injections were repeated 4 weeks and, in some experiments, 24 weeks later. Analysis of IgG1, IgG2a, IgG2b, IgG3 and IgM in the sera by an enzyme-linked immunosorbent assay suggested that adjuvanticity of liposomes is reflected in most antibody subclasses and that there is no shift in subclasses compared to the response obtained with the free antigen, thus establishing liposomes as a type I adjuvant. In other, appropriately designed experiments, the relative importance of events following the first and second injections in determining the adjuvant effect of liposomes was investigated. It was found that liposome adjuvanticity is the outcome of events following primary immunization.     

Cryoproteins in Acute Serum Sickness: Correlations Between Anticxn Cryoprecipitation, Immune Complexes and Immune Elimination in Acute Experimental Serum Sickness

To further define the relationship between serum cryoprecipitates and immune complexes, experimental serum sickness was produced in rabbits using radiolabelled bovine serum albumln (BSA) aei antigen.

BSA cryoprecipitation was not detected befors immune complexes appeared in the circulation. It reached peak quantities while immune complexes were present and BSA was undergoing immune elinination. The time come of BSA cryoprecipitation suggests that the BSA which appears in the cryopreclpitates may be in the form of immune couplexea.     

Eliciting Antibodies in Chickens to Human Dimeric IgA Removal of Factors from Human Colostrum Depressing Anti IgA Antibody Production

Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or “1st fraction” consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or “2nd fraction” consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the “1st fraction” antibodies to dimeric IgA were produced. When the “1st and 2nd fractions” of the column were remixed and used for immunization of chickens, the immune response was poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The immuno-depressing agent is therefore not universal. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The “activity” is therefore not specific for IgA and the remaining four antigens in human colostrum.

The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose.

The molecular weight (Mr) of the purified component was found to be 72 000 by sedimentation and diffusion and 80 000 by SDS page using Mr reference standards.

The properties of the immuno-suppressor strongly suggest that it is the secretory piece of dimeric IgA.