Ovalbumin injected with complete Freund's adjuvant stimulates cytolytic responses

CD8⁺ cytotoxic T lymphocytes (CTL) recognize antigens (Ag) associated with class I major histocompatibility complex (MHC) molecules. Endogenously synthesized protein Ag are processed into peptides in the cytoplasm and transported to the endoplasmic reticulum where they are bound by class I proteins. Exogenous Ag do not enter the class I processing pathway of most cells and thus do not activate CD8⁺ CTL. Nevertheless, several investigators have reported that immunization with exogenous Ag can activate CD8⁺ T cells that have immunoregulatory activity. To determine how exogenous Ag entered the class I pathway in vivo and whether immunosuppressive CD8⁺ T cells were cytolytic, we have shown in this report that injection with OVA emulsified in the complete Freund's adjuvant (CFA) primed CD8⁺, class I MHC-restricted, OVA-specific CTL in mice. These CTL recognize the OVA_257–264 epitope, produce tumor necrosis factor-α and interferon-γ upon activation. Both oil and mycobacteria components in CFA were required for inducing CTL responses. Priming was not attributed to direct sensitization of class I-bearing cells by contaminating peptides. Rather, phagocytic cells, but not CD4⁺ helper T cells, were required for priming CD8⁺ CTL by OVA-CFA. Thus, OVA in CFA is taken up by antigen-presenting cells and processed into the class I pathway by phagocytic cells in vivo. In addition, CTL induced by OVA-CFA suppressed the antibody response to OVA in adoptive recipients. These results suggest that CD8⁺ CTL specific for exogenous proteins might be routinely stimulated by injecting proteins in conventional adjuvants and that such cells have the potential to regulate immune responses in vivo.     

In interleukin-4-deficient mice,alum not only generates T helper 1 responses equivalent to Freund's complete adjuvant,but continues to induce T helper 2 cytokine production

The role of interleukin (IL)-4 in the activity of two frequently used vaccine adjuvants, Freund's complete adjuvant (FCA) and the aluminum hydroxide gels (alum), was studied using the standard antigen ovalbumin (OVA) in IL-4 genedisrupted mice (IL-4 -/-). In the absence of adjuvant, there was an overall reduction in antibody production to OVA in IL-4 −/− mice and significantly greater amounts of interferon (IFN)-γ were produced following restimulation of splenocytes with antigen in vitro compared with immunocompetent controls (IL-4 +/+). FCA and alum boosted the immune response to OVA in both IL-4 −/− and IL-4 +/+ mice. In IL-4 +/+ mice, while FCA stimulated a wide-spectrum immunoglobulin response, including both Th1-associated IgG2a and Th2-associated IgG1, alum enhanced only Th2 antibody production and no OVA-specific IgG2a could be detected. In IL-4-deficient mice, however, not only was IgG2a production increased in all adjuvant-treated groups, but alum was as potent at stimulating this antibody subclass as FCA. Similarly, increased production in vitro by splenocytes of the Th1 cytokine IFN-γ, equivalent to that produced after inoculation with FCA/OVA, was only detected in IL-4 −/− mice inoculated with alum/OVA. There was no IgE production in IL-4 −/− mice and OVA-specific IgG1 production, although still at significant levels, was reduced compared with wild-type mice irrespective of the adjuvant used. However, although production of the Th2 cytokine IL-5 was totally inhibited in IL-4-deficient mice inoculated with FCA/OVA, there was no significant difference in IL-5 production between the two strains when alum was used as adjuvant.     

Effects of neurokinin-1 receptor agonism and antagonism in the rostral ventromedial medulla of rats with acute or persistent inflammatory nociception

The rostral ventromedial medulla (RVM), a central relay in the bulbospinal pathways that modulate nociception, contains high concentrations of substance P (Sub P) and neurokinin-1 (NK1) receptors. However, the function of Sub P in the RVM is poorly understood. This study characterized the actions of Sub P in the RVM in the absence of injury and then used two NK1 receptor antagonists, L-733,060 and L-703, 606, to probe the role of endogenously released Sub P in the development and maintenance of persistent inflammatory nociception of immune or neurogenic origin. In uninjured rats, microinjection of Sub P in the RVM produced a transient thermal antinociception that was attenuated by pretreatment with L-733,060 or L-703,606. It did not alter threshold to withdrawal from tactile stimulation with von Frey filaments. Microinjection of the antagonists alone did not alter paw withdrawal latency (PWL) or threshold suggesting that Sub P is not tonically released in the RVM in the absence of injury. However, microinjection of either antagonist in the RVM was sufficient to reverse heat hyperalgesia 4 h, 4 days or 2 weeks after intraplantar (ipl) injection of complete Freund's adjuvant (CFA). Antagonism of NK1 receptors in the RVM did not prevent or reverse tactile hypersensitivity induced by CFA, but did attenuate that produced by capsaicin. NK1 receptor antagonism did not prevent the development of thermal hyperalgesia, tactile hypersensitivity or spontaneous pain behaviors induced by mustard oil (MO). The results suggest that Sub P has bimodal actions in the RVM and that following inflammatory injury, it can play a critical role as a pronociceptive agent in the development and maintenance of hyperalgesia and tactile hypersensitivity. However, its actions are highly dependent on the stimulus modality and the type of injury, and this may be an additional basis for the poor efficacy of NK1 receptor antagonists in clinical trials.     

Granisetron and carvedilol can protect experimental rats againstadjuvant-induced arthritis

Context: Rheumatoid arthritis (RA), a disabling autoimmune disorder of the joints as well as other organs, affects about 1% of population. Unfortunately, all current treatments of RA cause severe gastrointestinal, renal and other complications.

Objective: We aimed to evaluate the possible antiarthritic effects of a serotonin 5-HT₃ receptor blocker, granisetron, and a nonselective adrenergic receptor blocker, carvedilol, on complete Freund's adjuvant-induced RA in adult female albino rats.

Materials and methods: Rats were allocated into a normal control group, an arthritis control group, two reference treatment groups receiving dexamethasone (1.5?mg/kg/day) and methotrexate (1?mg/kg/day), and two treatment groups receiving granisetron (2.5?mg/kg/day) and carvedilol (10?mg/kg/day). Serum-specific rheumatoid, immunological, inflammatory and oxidative stress biomarkers were assessed. A confirmatory histopathological study on joints and spleens was performed.

Results: Granisetron administration significantly improved all the measured biomarkers, with the values of rheumatoid factor, matrix metalloproteinase-3, cartilage oligomeric matrix protein, immunoglobulin G, antinuclear antibody and myeloperoxidase being restored back to normal levels. Carvedilol administration significantly improved all biomarkers, with serum MPO value restored back to normal levels.

Discussion and conclusions: Serotonin 5-HT₃ receptor blockers and adrenergic receptor blockers, represented by granisetron and carvedilol, may represent new promising protective strategies against RA, at least owing to immune-modulator, anti-inflammatory and antioxidant potentials.     

Assessing the effect of verbal working memory load on visuo-spatial exogenous orienting

Recent reports have suggested that variants in the sortilin-related receptor gene (SORL1) increase the risk of late onset Alzheimer's disease (AD) in Northern European, Hispanic, African-American and Isreali-Arab populations. SORL1 directs trafficking of amyloid precursor protein (APP) and under-expression of SORL1 may lead to over-expression of beta amyloid peptides. Adults with Down syndrome (DS) over-express APP and have early onset and high risk for AD. We investigated the relation of seven variants in the gene for SORL1 to age at onset and risk for AD among 208 adults with DS, 45-70 years of age at baseline. Participants were ascertained through the New York State developmental disability service system and followed at 18-month intervals. Information from cognitive assessments, caregiver interviews, medical record review and neurological examination was used to establish the diagnosis of dementia. Homozygosity for the minor T allele in rs556349 and for the minor C allele in rs536360 was associated with later age at onset and reduced risk of AD (HR=0.26, 95% CI: 0.08-0.86; and HR=0.40, 95% CI: 0.16-0.98, respectively). Mean age at onset was approximately four years later in individuals who were homozygous for those alleles compared with those who had at least one major allele. These findings indicate a modest association of variants in SORL1 with AD. In addition, we did not observe the same alleles to be associated with AD compared with earlier studies, suggesting that these SNPs are in linkage disequilibrium (LD) with the putative functional variants or that expression of the SORL1 gene and hence its interaction with APP might be modified by the extremely high levels of APP characteristic of Down syndrome. Thus, further studies are needed to identify functional variants that influence risk for AD in this uniquely vulnerable population.     

Serotonin immunoreactivity in spinal cord axons and terminals of rodents with experimental allergic encephalomyelitis

Spinal cord axons and terminals stained for serotonin-like immunoreactivity were examined in rats and guinea-pigs with experimental allergic encephalomyelitis, an animal disease model for multiple sclerosis. During the paraplegic stage of experimental allergic encephalomyelitis, many serotonin-positive axons in the ventral and lateral funiculi of both rats and guinea-pigs were found to be grossly distorted, often appearing to end in bulbous enlargements. Serotonin-immunoreactive terminal varicosities in the gray matter were swollen and diminished in number in paraplegic rats with experimental allergic encephalomyelitis. In addition, the intervaricose segments which were observed in control rats appeared to be missing in rats with experimental allergic encephalomyelitis. Depletion of serotonin-positive terminals was much more pronounced in paraplegic guinea-pigs with experimental allergic encephalomyelitis than in rats with experimental allergic encephalomyelitis, but those terminals which remained in the guinea-pigs were morphologically similar to those of control guinea-pigs. The greater depletion of serotonin-positive terminals in guinea-pigs may reflect a more severe disease state in this species, as none of the guinea-pigs survived the acute stage of paralysis. As the time period between recovery from paralysis and sacrifice increased in rats with experimental allergic encephalomyelitis, serotonin-positive terminals in the gray matter became increasingly more normal in appearance. Even by the first day of recovery, some intervaricose segments could again be observed, and after two weeks of recovery, the terminals were much more numerous and less swollen than during the paraplegic stage of disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type II collagen-induced arthritis in the same rat strain are different diseases.

The DA rat is highly susceptible to induction of arthritis after immunization with homologous type II collagen (CII) emulsified in Freund's incomplete adjuvant (FIA), resulting in collagen-induced arthritis (CIA). The DA rat also develops arthritis after injection of FIA alone (oil-induced arthritis (OIA)). This finding allows a direct comparison of two different models for rheumatoid arthritis; one induced with a defined auto-immunogen and one with a pure adjuvant. Both CIA and OIA develop approximately 2 weeks after induction but OIA is a self-limited acute disease whereas CIA induced with homologous CII follows a chronic disease course. Immunization with CII leads to a strong autoantibody response to CII while injection of FIA leads to no or very limited anti-CII antibody response. The Lewis rat develops neither CIA nor OIA while F1 (DA x Lewis) rats develop CIA but not OIA. Olive oil or CII emulsified in olive oil does not induce arthritis in DA rats. Pretreatment with CII in olive oil vaccinates against CIA but not OIA whereas pretreatment with FIA vaccinates against OIA but not CIA. These findings demonstrate that inclusion of CII in the adjuvant leads to a disease distinct from OIA which is characterized by a CII autoimmune response and chronicity of the disease course.     

Anti-Inflammatory and Anti-Nociceptive Activities of Methanolic Extract of the Leaves of Fraxinus Floribunda Wallich

Fraxinus floribunda Wallich (Family-Oleaceae) is a wide green tree in the sub-alpine region of Sikkim, India. The methanolic extract of the leaves of Fraxinus floribunda (MEFF) at 100, 200 and 400mg/kg/p.o was screened in rats for anti-inflammatory activity by acute-carrageenan induced paw edema, sub-acute cotton pellet induced granuloma and chronic Freund''s adjuvant induced arthritis models. In all the three models of anti- inflammatory studies 200 and 400mg/kg/p.o doses of the extract showed significant effect (P<0.001). Antinociceptive evaluation was performed by writhing and tail-immersion tests in mice. Anti-nociceptive evaluation revealed that MEFF at the dose of 400mg/kg/p.o had significant activity against the control. The relieving effect was through the peripheral and central mechanism of action of the extract. This study rationalized the ethno medicinal use of the plant for relieving pain in inflammatory pathological conditions like fracture and dislocation.     

Human colonic intraepithelial lymphocytes from patients with inflammatory bowel disease fail to down-regulate proliferative responses of primed allogeneic peripheral blood mononuclear cells after rechallenge with antigens.

Human colonic intraepithelial lymphocytes from control subjects down-regulate the proliferative responses of primed allogeneic peripheral blood mononuclear cells on rechallenge with antigens or phytohaemagglutinin (PHA). In contrast, human colonic intraepithelial lymphocytes from patients with inflammatory bowel disease fail to down-regulate the proliferative responses of primed allogeneic peripheral blood mononuclear cells on rechallenge with antigens. These findings may be important in the development and maintenance of the mucosal immunological activation of inflammatory bowel disease.     

Toll or Toll-Free Adjuvant Path Toward the Optimal Vaccine Development

Successful vaccines contain an adjuvant component that activates the innate immune system, thereby eliciting antigen-specific immune responses. Many adjuvants appear to be ligands for toll-like receptors (TLR), which are thus promising targets for the development of novel adjuvants to elicit vaccine immunogenicity. However, recent evidence suggests that some adjuvants activate the innate immune system in a TLR-independent manner possibly through other pattern recognition receptors and signaling machinery. In particular, newly identified intracellular retinoic-acid-inducible gene (RIG)-like receptors, NOD-like receptors, or even as yet unknown recognition machinery for the adjuvant may regulate TLR-independent vaccine immunogenicity. To develop optimal vaccines, it will be critical to understand how TLR-dependent and TLR-independent innate immune activation, by various adjuvants, control the consequent adaptive immune responses to vaccine.     

Inhibition of the neutrophilic infiltrate of experimental tubulointerstitial nephritis in the Brown-Norway rat by decomplementation

Anti-tubular basement membrane (TBM) antibody-associated tubulointerstitial nephritis (TIN) in Brown-Norway rats is induced by immunization with bovine TBM antigens and adjuvants. The lesion is characterized by linear deposition of IgG and C3 along the TBM with sequential neutrophil (Days 8-9)- and mononuclear (Day 10 and after)-dominated inflammatory infiltrates. To study the complement dependence of the infiltrative process, immunized rats were decomplemented with cobra venom factor (CVF). The CVF treatment did not affect the production or renal deposition of anti-TBM antibodies. CVF markedly reduced the neutrophilic inflammatory infiltrate. In rats immunized with suboptimal doses of soluble bovine TBM antigens to produce a mild lesion, decomplementation also decreased the mononuclear inflammatory infiltrates on Days 10-13. In rats immunized optimally with particulate TBM to induce maximally severe TIN, decomplementation did not affect the mild mononuclear cell infiltrate on Days 8 and 9 but did somewhat reduce the subsequent mononuclear infiltrate on Days 10 and 12. These results demonstrate that the anti-TBM antibody- and C3-associated neutrophilic inflammatory infiltrate is largely complement dependent. The early mononuclear cell infiltrate that was unmodified by CVF treatment may be dependent on complement-independent humoral events or related to cell-mediated immune events. A portion of the later mononuclear inflammatory infiltrate could be dependent on the preceding neutrophilic inflammatory phase.     

Raised anti-endothelial cell autoantibodies (AECA), but not anti-neutrophil cytoplasmic autoantibodies (ANCA), in recurrent oral ulceration: modulation of AECA binding by tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)

Recurrent oral ulceration (ROU) is a common oral mucosal condition of unknown etiology. However, there is evidence to suggest that vasculitis may play a role. Here we investigate the presence in ROU of two autoantibodies associated with vasculitis, AECA and ANCA. AECA target as yet unidentified antigens on the endothelial cell surface and have been identified in patients with vasculitic disorders and inflammatory conditions with a vasculitic component. ANCA target specific neutrophil-associated proteins and are detected in specific vasculitic and chronic inflammatory disorders. AECA and ANCA levels were studied in 20 ROU patients and 20 controls. IgG AECA to the endothelial cell line ECV 304 were detected in 19 ROU patients and four controls. Levels were significantly raised in ROU both to ECV 304 (P < 0.000 05) and to human umbilical vein endothelial cells (HUVEC) (P < 0.005). Although levels were highest during episodes of ulceration, they were also raised between episodes. Stimulation of endothelial cells with TNF-α significantly increased AECA binding of both ROU (P < 0.005) and control samples (P < 0.0001), while IFN-γ decreased binding (ROU P < 0.0001; controls P < 0.05). In contrast, ANCA were detected in only one patient and none of the controls. The presence of raised levels of AECA lends support to the hypothesis that a vasculitic process may underlie ROU. Moreover, these findings suggest that endothelial cell expression of AECA target antigens is increased by TNF-α and decreased by IFN-γ stimulation.     

Immunochemical partial identity between two independently identified and isolated major allergens from Alternaria alternata (Alt-I and Ag 1)

Partially purified preparations of Alt-I, the main allergenic fraction of Alternaria alternata isolated by Yunginger, and of Ag 1, shown in crossed radioimmunoelectrophoresis (CRIE) to be the dominating major allergen of A. alternata (Løwenstein, Nyholm), were compared by tandem crossed immunoelectrophoresis (CIE), RAST inhibition, and the CRIE-related technique, single radial radioimmunodiffusion (SRRID). The two allergen preparations showed reaction of identity in tandem-CIE and indistinguishable specific IgE binding in CRIE and SRRID, regardless of antibodies and serum pools used. In RAST inhibition, the relative potencies of the allergen preparations and of the crude extracts correlated well with their Alt-I/Ag 1 content as estimated by rocket immunoelectrophoresis. Moreover, all inhibition curves were parallel, confirming identical IgE binding by Alt-I and Ag 1 with the serum pools used. A second preparation of Alt-I, isolated from another strain of Alternaria, showed reaction of partial identity with Ag 1 in tandem-CIE, indicating that different variants of Alt-I (Ag 1) may exist in different strains of A. alternata.     

Characterization of a dominant anti-Ia idiotype using the IA mutant mouse strain B6.C-H-2bm12

Initial studies of antibody recognition of Ia molecules using the IA mutant mouse strain bm12 suggested that two anti-Ia monoclonal antibodies (mAbs), 25-9-17 and 34-5-3, share several features: (1) indistinguishable serologic specificity including a lack of reactivity with Iabm12, (2) binding of the same spatial epitope (cluster), and (3) definition of a cross-reactive idiotype (CRI) as defined by xenogeneic antisera. In the present study we characterize a rabbit anti-idiotype (anti-Id) to 25-9-17 by affinity chromatography, and demonstrate that it detects at least two distinct idiotopes, one shared by 25-9-17 and 34-5-3 designated CRI (25-9-17) and one unique for 25-9-17 molecules. Experiments were also undertaken to determine whether CRI (25-9-17) represents a measurable component of allogeneic humoral responses to Iab antigens. By both absorption analyses of a polyspecific antiserum and production of antigenically-restricted antisera using bm12 mice, CRI (25-9-17) was found to represent a significant proportion of the antibodies to Iab. By several criteria it was shown that the CRI (25-9-17)+ molecules were among the antibodies defining the serologic lesion of bm12 mice. In preparation for future studies to alter in vivo T-cell responses involving recognition of Ia (e.g. graft vs host disease and allogeneic transplant rejection), various immunization protocols and mouse strains were tested for induction of Id (25-9-17) following in vivo administration of various anti-idiotypic reagents. Rabbit anti-Id (25-9-17) successfully induced CRI (25-9-17) positive molecules in all strains tested regardless of IA or Ig genotype. Moreover, some of these treated mice produced antibodies to an Ia determinant missing on bm12 cells, suggesting that they recognize the same serologic determinant as mAb 25-9-17.     

Maintenance of granuloma macrophages in serum-free medium

Granulomas were induced by injecting polyacrylamide beads into subcutaneous pouches created by divulsion of the dorsal skin of mice. More than 10(7) phagocytic cells (60% macrophages, 40% polymorphonuclear cells) could be recovered from this granuloma. The separation of the phagocytic cells can be achieved either following sedimentation in Percoll or following the incubation of cells on plastic petri dishes. Phagocytosis of zymosan was observed in macrophages maintained in vitro for 1 month in Eagle's serum-free medium.     

MPTP administration increases plasma levels of acute phase proteins in non-human primates (Macaca fascicularis)

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc). Parkinsonian patients and animal models of PD show inflammatory phenomena such as microglial activation and cytokine production that could modulate the progression of the disease, since they play a crucial role in the degenerative process. Since acute phase proteins (APPs) are involved in a number of homeostatic alterations and inflammatory processes, we analyzed the levels of APPs in primates before and after treatment with MPTP. A significant increase in C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (HP) levels after MPTP treatment. These results demonstrate that MPTP induces a systemic generalized inflammatory reaction after specific dopaminergic neurotoxicity insult, suggesting that the inflammatory process in Parkinsonism may affect other immune-inflammatory responses outside the brain.     

Use of dynamic weight bearing as a novel end-point for the assessment of Freund's Complete Adjuvant induced hypersensitivity in mice

Animal models are an integral part of pain research. However, current models tend to rely on evoked responses and there is a belief that non-evoked responses may be a more relevant behavioural readout as the animal responds in a more natural manner. Here, dynamic weight bearing (DWB), a novel method for assessing mechanical hypersensitivity, was evaluated using the Freund's Complete Adjuvant (FCA) model of inflammatory pain in mice. DWB enables the measurement of weight placed through all four paws of a freely moving animal. The data obtained from DWB was compared with data acquired using the standard static weight bearing (incapacitance) test. In both tests reversal of FCA induced mechanical hypersensitivity was investigated using the selective COX2 inhibitor celecoxib. Mice treated with FCA placed less weight through the ipsilateral hindpaw compared to vehicle controls. This reduction was reversed by celecoxib (30 mg/kg p.o.) in the dynamic and static weight bearing tests. The data presented here suggests that dynamic weight bearing may provide a novel end point for the development of new analgesics.     

Polymeric microparticles for sustained and local delivery of antiCD40 and antiCTLA-4 in immunotherapy of cancer

This study investigated the feasibility of the use of polymeric microparticles for sustained and local delivery of immunomodulatory antibodies in immunotherapy of cancer. Local delivery of potent immunomodulatory antibodies avoids unwanted systemic side effects while retaining their anti-tumor effects. Microparticles based on poly(lactic-co-hydroxymethyl-glycolic acid) (pLHMGA) and loaded with two distinct types of immunomodulatory antibodies (CTLA-4 antibody blocking inhibitory receptors on T cells or CD40 agonistic antibody stimulating dendritic cells) were prepared by double emulsion solvent evaporation technique. The obtained particles had a diameter of 12–15 μm to avoid engulfment by phagocytes and were slightly porous as shown by SEM analysis. The loading efficiency of the antibodies in the microparticles was >85%. The in vitro release profile of antiCD40 and antiCTLA-4 from microparticles showed a burst release of about 20% followed by a sustained release of the content up to 80% of the loading in around 30 days. The therapeutic efficacy of the microparticulate formulations was studied in colon carcinoma tumor model (MC-38). Mice bearing subcutaneous MC-38 tumors were treated with the same dose of immunomodulatory antibodies formulated either in incomplete Freund's adjuvant (IFA) or in microparticles. The antibody-loaded microparticles showed comparable therapeutic efficacy to the IFA formulation with no local adverse effects. The biodegradable microparticles were fully resorbed in vivo and no remnants of inflammatory depots as observed with IFA were present in the cured mice. Moreover the microparticles exhibited lower antibody serum levels in comparison with IFA formulations which lowers the probability of systemic adverse effects. In conclusion, pLHMGA microparticles are excellent delivery systems in providing long-lasting and non-toxic antibody therapy for immunotherapy of cancer.