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Experimental Models of Adjuvants

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Abstract

Many studies have been carried out to investigate the immune pathways stimulated by adjuvants, the role of adjuvants in enhancing pathogen-specific immune responses, and the potential noxious effects of adjuvants for the recipient, both in animal models and in humans. In order to prevent several costly infectious diseases, farmed salmon are intraperitoneally injected with vaccines containing adjuvant oil and a number of different antigens. The rabbit model is often used for vaccines intended for intramuscular injection. The adjuvants were inoculated in swine neck, quadriceps, and semitendinosus muscles. Several kinds of animal models have been employed to investigate adjuvant effects and midterm reactions in vivo, in an attempt to better unravel adverse events in humans following vaccinations or prosthesis implantations. Higher organisms, such as primates, are rarely used; rather, different mouse models have provided the bulk of the evidence for vaccine and adjuvants effects in living beings.

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Squalene, a hydrocarbon obtained for commercial purposes primarily from shark liver oil and other botanic sources, is increasingly used as an immunologic adjuvant in several vaccines, including seasonal and the novel influenza A (H1N1) 2009 pandemic flu vaccines. Nearly a decade ago, squalene was supposed to be the experimental anthrax vaccine ingredient that caused the onset of Persian Gulf War syndrome in many veterans, since antibodies to squalene were detected in the blood of most patients affected by this syndrome. This evidence has raised a widespread concern about the safety of squalene containing adjuvants (especially MF59) of influenza vaccines. Nevertheless, further clinical evidence clearly suggested that squalene is poorly immunogenic, that low titres of antibodies to squalene can be also detected in sera from healthy individuals, and that neither the presence of anti-squalene antibodies nor their titre is significantly increased by immunization with vaccines containing squalene (or MF59) as an adjuvant. This review summarizes the current scientific evidence about the relationship between squalene, anti-squalene antibodies and vaccination.
Article
Tetramethylpentadecane (TMPD, or commonly known as pristane)-induced lupus is a murine model of systemic lupus erythematosus (SLE). Renal disease and autoantibody production strictly depend on signaling through the interferon (IFN)-I receptor. The major source of IFN-I is immature monocytes bearing high levels of the surface marker Ly6C. Interferon production is mediated exclusively by signaling through TLR7 and the adapter protein MyD88. It is likely that endogenous TLR7 ligands such as components of small nuclear ribonucleoprotein complexes are involved in triggering disease. Lupus autoantibodies are produced in ectopic lymphoid tissue developing in response to TMPD. This model is well suited for examining links between dysregulated IFN-I production and the pathogenesis of human SLE, which like TMPD-lupus, is associated with high levels of IFN-I.
Article
Four inbred strains differed greatly in their response to a standard inoculation of rat thyroid extract with complete Freund's adjuvant and pertussis vaccine. Strains HO and AU, both Ag-B5, developed only minimal lesions, although AU rats had a high titer of circulating antibodies to thyroid antigens. AO rats (Ag-B2) and DA rats (Ag-B4) showed more severe thyroiditis. AO rats had high titers of antibody while the DA strain had only moderate titers.
Article
Adjuvant arthritis in rats is usually induced by injection of mycobacterium tubercle cell walls suspended in various adjuvant oils such as Freund's incomplete adjuvant (FIA) or pristane. We have recently shown that injection of adjuvant oils without inclusion of mycobacterium tubercle cell walls triggers arthritis [oil adjuvant-induced arthritis (OIA)] in the DA rat strain. The OIA is a genetically restricted disease since only DA rats are susceptible while Lewis, DA-fostered Lewis and F1 (Lew x DA) rats are relatively resistant. Activated alpha beta T cells infiltrate the affected joints of adjuvant oil-injected DA rats and treatment with monoclonal antibodies to the alpha beta T-cell receptor abrogates development of arthritis. These findings show that alpha beta T-cell activation is a critical event in the development of OIA.
Article
Injection of complete Freund's adjuvant into one paw of Holtzman rats induced an inflammatory response in the injected site (primary lesion) and from the 10th day on, severe polyarthritis developed (secondary lesions). In this susceptible strain, cyclophosphamide dose dependently reduced the intensity of the primary and secondary lesions. In contrast, in Buffalo rats, which are considered as resistant, cyclophosphamide in the dose of 25 mg/kg significantly potentiated the secondary lesions. Higher doses (200 and 300 mg/kg) also reduced the intensity of the lesions in this strain. Our data suggest that the development of adjuvant induced arthritis is controlled by a cyclophosphamide sensitive suppressor cell population.
Article
Detailed characterization of several aspects of visual function was made in two groups of monkeys exposed developmentally to methyl mercury. One group of monkeys (Macaca fascicularis) was dosed from birth onward with 50 micrograms/kg/day of mercury as methyl mercury. Another group was exposed in utero by dosing the mother with 10, 25, or 50 micrograms/kg/day of mercury as methyl mercury, and postnatally until 4.0-4.5 years of age with the same dose the mother had received. Spatial and temporal visual function was tested in both groups. Spatial visual deficits observed in the group dosed beginning postnatally were reported previously (Rice and Gilbert, 1982, Science, 216, 759-761). Monkeys exposed in utero plus postnatally exhibited impaired high- and low-luminance spatial vision. They also exhibited deficits in low-frequency high-luminance temporal vision, while low-luminance temporal vision was superior to that of control monkeys. Monkeys in which exposure began at birth displayed superior low-luminance temporal vision, while high-luminance temporal vision was not impaired. These effects were observed in the absence of constriction of visual fields. These data suggest that the pattern of visual deficits produced by developmental exposure to methyl mercury may be different from that in the adult, and that the developing visual system may be able to remodel as a result of early insult by a neurotoxic agent.
Article
Observations of the social behavior of Macaca fascicularis exposed in utero to methylmercury (MeHg) and nonexposed control infants were performed as part of a study of the toxic, reproductive and developmental effects of maternal MeHg intake. Infants were tested twice weekly from 2 weeks to 8 months of age. Data were summarized into 6 categories of social behavior and 7 categories of nonsocial behavior. Analysis of the most prevalent behavior indicated that MeHg-exposed offspring exhibited a decrease in social play behavior and a concomitant increase in nonsocial passive behavior. The MeHg effect on social play behavior tended to decrease with age, while the group differences in nonsocial passive behavior tended to increase. The results indicate that maternal intake of MeHg during pregnancy can affect the social development of infant primates by suppressing social interactions and increasing nonsocial behavior.
Article
Infant Macaca fascicularis exposed prenatally to maternal subclinical levels of methylmercury (MeHg) and their nonexposed controls were administered a test of visual recognition memory beginning at 210 days postconception (mean postnatal age = 51.88 days, SD = 5.30). The test consisted of a series of problems in which two identical 35 mm slides of a monkey's face were presented for a study period, followed by a test trial in which the previously exposed stimulus was paired with a novel one, and the looking time to each was recorded. The nonexposed group showed differential visual attention to the novel stimuli, indicating visual recognition abilities. The exposed group's visual attention to the novel stimuli was random. These results, in conjunction with earlier findings, suggest that prenatal MeHg exposure is associated with impaired visual recognition memory performance.
Article
Bile acid transport in female DA (dark Aguti) rats, a model for debrisoquine hydroxylation deficiency in man, was investigated. Compared to hydroxylation competent male DA and Sprague-Dawley rats of either sex, the female DA rat had a significantly lower taurocholate maximal secretory rate in vivo. Studies in the perfused liver showed this to be due to a decreased extraction efficiency during exogenous taurocholate loading. To characterize further the defect, taurocholate uptake velocity into isolated hepatocytes was studied. This showed a decreased maximal uptake velocity in the female DA rat (P less than 0.02). Whether this defect in bile acid uptake is related to the defective debrisoquine hydroxylation, remains to be established.
Article
THE induction of monoclonal, plasma cell tumours which produce immunoglobulin in the inbred BALB/c strain of mice is a unique form of carcinogenesis1,2. Mouse plasma cell tumours have been produced by intraperitoneal implantation of solid materials, such as plastic diffusion chambers, polymethylmethacrylate plastic shavings3,4 and circular disks4, and by intraperitoneal injection of various high and low viscosity white mineral oils1,2,5. White mineral oils comprise a poorly defined mixture of saturated hydrocarbons, including normal and iso-paraffins, and naphthenes6, and such oils contain many hundreds of different molecular species (C. Steenbergen, personal communication). Because of the chemical heterogeneity of the oils used, it has been impossible to identify or trace the particular chemical species involved in plasma cell tumour induction. Since the discovery that the immunoglobulin products of some human Waldenström macroglobulinaemias7, multiple myelomas8 and some mouse plasma cell tumours2,9,10 exhibit antibody activity, it has become important to elucidate more fully the factors involved in the carcinogenesis of the murine plasma cell tumours.
Article
Five monkeys were treated from birth with oral doses of mercury as methylmercury (50 micrograms per kilogram of body weight per day); concentrations in the blood peaked at 1.2 to 1.4 parts per million; and declined after weaning from infant formula to a steady level of 0.6 to 0.9 part per million. There were no overt signs of toxicity. When tested between 3 and 4 years of age under conditions of both high and low luminance, treated monkeys exhibited spatial vision that was impaired compared with that of control monkeys.
Article
The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.
Article
Collagen-Induced Arthritis (CIA) is an experimentally induced and genetically controlled animal model of chronic joint inflammation. In rats, there are informative strain differences in susceptibility to CIA. DA rats (RT1avl) develop severe CIA after immunization with bovine (BII), chick (CII), or homologous rat (RII) type II collagens. In contrast, the MHC-congenic DA. 1N(BN) and WF.1N(BN) rats (RT1n) are relatively resistant to CIA and develop moderate CIA in response to immunization with CII but not BII or RII. We previously found that simultaneous infection with rat cytomegalovirus (RCMV) greatly exacerbates the severity of arthritis that develops in BII-immunized DA rats. To examine the mechanism of RCMV amplification of CIA, the effect of simultaneous infection with RCMV on arthritis and autoimmunity to type II collagen was determined in WF.1N and DA.1N rats after immunization with BII, CII and RII. RCMV increased the incidence of CIA and the level of autoimmunity to type II collagen (skin-testing and IgG antibody titer) selectively in DA.1N and WF.1N rats immunized with CII, but not in littermates immunized with BII, although the transient reversal of CD4+/CD8+ mononuclear cell ratios in peripheral blood that is associated with RCMV infection occurred equally in both BII- and CII- immunized DA.1N rats. Likewise, RCMV infection moderately increased the levels of anti-RII autoimmunity and arthritis in DA rats sub-optimally immunized with RII but had no consistent effect on either anti-RII immunity or arthritis in RII-immunized DA.1N and WF.1n rats. The data show that RCMV augments arthritis only in rats that are genetically susceptible to CIA and that are appropriately immunized with a species of type II collagen that is arthritogenic for the MHC-haplotype being tested. Two possible mechanisms are suggested by these data: RCMV-associated increases in anti-RII autoimmunity in rats with CIA may result from amino acid sequence homologies between RCMV and type II collagen; alternatively, virus-induced pro-inflammatory cytokines may activate RII-reactive lymphocytes thereby potentiating autoimmunity and arthritis.
Article
Intraperitoneal injection of pristane (2,6,10,14 tetramethylpentadecane) is a standard technique for obtaining monoclonal antibody-enriched ascitic fluid. However, pristane also induces plasmacytomas and an erosive arthritis resembling rheumatoid arthritis in BALB/c mice, probably as a consequence of enhanced interleukin 6 production. We report here that the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) is a further consequence of injecting pristane in BALB/c mice. Anti-Su antibodies appeared as early as 1-2 mo after a single injection of 0.5 ml pristane, followed by anti-U1RNP and anti-Sm antibodies after 2-4 mo. Within 6 mo of pristane injection, 9 of 11 BALB/c mice had developed anti-Su, anti-U1RNP, anti-U2RNP, anti-Sm, and possibly anti-U5RNP antibodies. Autoantibodies were not produced by 20 BALB/c mice of the same age and sex that were not injected with pristane. Thus, autoantibodies characteristic of lupus were induced in mice that are not usually considered to be genetically susceptible to the disease. The induction of autoantibodies associated with SLE by pristane may be relevant to understanding the role of abnormal cytokine production in autoantibody production and the pathogenesis of autoimmune disease. Furthermore, the induction of high titer autoantibodies by pristane dictates caution in the use of ascitic fluid as a source of monoclonal antibodies, since the polyclonal antibodies induced by pristane may copurify with the monoclonal antibody secreted by an injected hybridoma.
Article
To review the literature examining the association of silicone gel filled implants and connective tissue disease. Computerized literature searches and manual review of bibliographies. Numerous concerns have arisen regarding the safety of silicone gel filled breast implants. The structure of these prostheses is reviewed. Silicones are not biologically inert. Injectable as well as implantable silicones have proven capable of eliciting inflammatory and fibroproliferative responses. Silicone leakage from silicone gel filled implants is well documented as is distant migration of silicone in the host. In the past decade, over 60 cases of connective tissue disease following mammoplasty with silicone gel filled implants have been reported. About half of these patients developed scleroderma or scleroderma-like illnesses. This reported overrepresentation of scleroderma compared to other rheumatic diseases mimics the Japanese experience with injectable silicones. Possible biological rationale for the association is presented. The physical and biological properties of silicone gel filled implants and their behavior in vivo is compatible with the hypothesis that they may contribute to the development of connective tissue disease. The association seems most likely with scleroderma; however, there is as yet inadequate epidemiological data to definitively establish causality.
Article
The arthritogenic properties of adjuvant oil upon percutaneous administration in DA rats was investigated. Groups of rats were administered single or repeated percutaneous applications of Freund's incomplete adjuvant (FIA) or olive oil on shaved skin with or without prior abrasion of the skin. Control rats were shaved and abrased only. A transient arthritis developed in 8/16 animals after repeated applications of FIA on abrased skin. The incidence of arthritis increased to 7/8 animals when FIA was repeatedly administered via filter paper on abrased skin and covered with a bandage. Histological examination of the arthritic joints showed proliferation of the synovial lining layer, infiltration of mononuclear cells and polymorphonuclear cells in the subsynovial tissue. Some bone and cartilage destruction was also seen. Repeated treatment with olive oil on abrased skin induced joint swelling in 3/15 animals, which did not, however, correspond to any microscopically observable signs of inflammation. Also, a single application of FIA on abrased skin or repeated applications of FIA without abrasion induced arthritis, although with low penetration, whereas control animals had no clinical signs of joint involvement. These findings demonstrate that percutaneous administration of adjuvant oil can cause arthritis in genetically susceptible animals.
Article
To define the arthritogenic effects of incomplete Freund's adjuvant (ICFA) injections in DA rats. ICFA was injected into DA rats and other inbred rat strains. The severity of arthritis was scored and associated changes in weight recorded. After ICFA injection a mild transient symmetric arthritis developed in 15/22 (68%) of the DA rats observed for greater than 4 weeks. Histologic examination of the observed arthritis showed edema and neutrophil infiltrates with some lymphocytes and plasma cells. No arthritis developed in DA rats given normal saline injections nor in Lewis, DA.1N and WF.1N rats given ICFA. Injection of DA rats with ICFA alone can produce arthritis.
Article
In the present study the neurotoxic effects of a low dosage (0.5 mg/kg per day) of methylmercury (MeHg) on the developing nervous system were investigated. Pregnant rats were treated with MeHg from day 7 of pregnancy to day 7 of lactation. Locomotor activity (locomotion, rearing, and motility) and spatial learning ability were tested in the offspring at 6 months of age. The expression of tyrosine hydroxylase (TH) was examined by immunohistochemistry and in situ hybridization. A significant decrease in spontaneous motility and rearing was observed only in the MeHg-treated male rats. After administration of a low dose of d-amphetamine (0.5 mg/kg) no differences could be observed between control and MeHg-treated male rats, suggesting that changes in dopaminergic transmission were involved. However, no change in TH messenger RNA expression was observed. No changes in spatial learning acquisition or memory were shown in MeHg-treated rats. Taken together, these findings show that during development a very low dosage of MeHg exerts neurotoxic effects detectable in adulthood, and that susceptibility is gender-dependent.
Article
Vaccine adjuvants help antigens elicit rapid, potent, and long-lasting immune responses. The lack of understanding of the immunological mechanism of action of adjuvants has limited the rational development of vaccines for human use. In particular, little is known about how the immune system processes adjuvants. The goal of the present study was to determine the fate of the vaccine adjuvant MF59, labeled with the fluorescent dye Dil, after injection with fluorescein-labeled gD2 antigen from type 2 herpes simplex virus. At 3 h after intramuscular injection into BALB/c mice, most of the MF59 was still in the form of extracellular droplets in the muscle, but a detectable fraction of the MF59 was in cells in the subcapsular sinus of draining inguinal lymph nodes. At 48 h, most of the MF59 at the site of injection was inside cells that were immunoreactive for the dendritic cell markers DEC-205 and MHC class II molecules, reflecting the interaction of MF59 with antigen presenting cells. At this time, intracellular MF59 was also abundant in the paracortical (T cell) region of lymph nodes. The gD2 antigen was also intracellular in muscle and colocalized MF59 at 48 h, and the presence of MF59 increased the amount of intracellular antigen. Similarly, serological antibody titers to gD2 were 207-fold higher after two injections when MF59 was administered with the antigen. These findings suggest that MF59 interacts with antigen presenting cells at the site of injection and then moves to the draining lymph nodes, where it increases the efficiency of antigen presentation to T cells.
Article
MF59, which is an adjuvant approved for human use, typically elicits higher antibody titers than alum when used in combination with a variety of recombinant and natural subunit antigens. The mechanisms responsible for the adjuvant action of MF59 are not fully understood. In particular, little is known about the in vivo distribution of MF59 and of antigen after intramuscular (i.m.) injection. The goal of the present study was to determine the distribution of MF59 injected with soluble antigen gD2 from type 2 herpes simplex virus (HSV) and to compare the distribution of gD2 injected with or without MF59. At 4 h, 36% of the injected dose of labeled MF59 was in the quadriceps muscle and about 50% was in the inguinal fat surrounding the muscle. Half of the initial amount of labeled MF59 in muscle was detected 42 h after injection. The amount of labeled MF59 in the draining lymph nodes was maximal 2 d after injection, which represented 0.1-0.3% of the injected dose. At 4 h, 12% of the injected dose of labeled gD2 was found in the muscle. The presence of MF59 did not significantly modify the distribution of gD2. The results indicate that MF59 and gD2 distribute and are cleared independently after i.m. injection. Importantly, MF59 is unlikely to have a repository effect, whereby it slowly releases the antigen.
Article
Gulf War Syndrome (GWS) is a multisystemic illness afflicting many Gulf War-era veterans. The molecular pathological basis for GWS has not been established. We sought to determine whether the presence of antibodies to squalene correlates with the presence of signs and symptoms of GWS. Participants in this blinded cohort study were individuals immunized for service in Desert Shield/Desert Storm during 1990-1991. They included 144 Gulf War-era veterans or military employees (58 in the blinded study), 48 blood donors, 40 systemic lupus erythematosus patients, 34 silicone breast implant recipients, and 30 chronic fatigue syndrome patients. Serum antibodies to squalene were measured. In our small cohort, the substantial majority (95%) of overtly ill deployed GWS patients had antibodies to squalene. All (100%) GWS patients immunized for service in Desert Shield/Desert Storm who did not deploy, but had the same signs and symptoms as those who did deploy, had antibodies to squalene. In contrast, none (0%) of the deployed Persian Gulf veterans not showing signs and symptoms of GWS have antibodies to squalene. Neither patients with idiopathic autoimmune disease nor healthy controls had detectable serum antibodies to squalene. The majority of symptomatic GWS patients had serum antibodies to squalene.
Article
Squalene is a cholesterol precursor, which stimulates the immune system nonspecifically. We demonstrate that one intradermal injection of this adjuvant lipid can induce joint-specific inflammation in arthritis-prone DA rats. Histopathological and immunohistochemical analyses revealed erosion of bone and cartilage, and that development of polyarthritis coincided with infiltration of alphabeta(+) T cells. Depletion of these cells with anti-alphabeta TcR monoclonal antibody (R73) resulted in complete recovery, whereas anti-CD8 and anti-gammadelta TcR injections were ineffective. The apparent dependence on CD4(+) T cells suggested a role for genes within the major histocompatibility complex (MHC), and this was concluded from comparative studies of MHC congenic rat strains, in which DA.1H rats were less susceptible than DA rats. Furthermore, LEW.1AV1 and PVG.1AV1 rats with MHC identical to DA rats were arthritis-resistant, demonstrating that non-MHC genes also determine susceptibility. Some of these genetic influences could be linked to previously described arthritis susceptibility loci in an F2 intercross between DA and LEW.1AV1 rats (ie, Cia3, Oia2 and Cia5). Interestingly, some F2 hybrid rats developed chronic arthritis, a phenotype not apparent in the parental inbred strains. Our demonstration that an autoadjuvant can trigger chronic, immune-mediated joint-specific inflammation may give clues to the pathogenesis of rheumatoid arthritis, and it raises new questions concerning the role of endogenous molecules with adjuvant properties in chronic inflammatory diseases.