Complete Freund's adjuvant (CFA) has historically been one of the most useful tools of immunologists. Essentially comprised of dead mycobacteria and mineral oil, we asked ourselves what is special about the mycobacterial part of this adjuvant, and could it be recapitulated synthetically? Here, we demonstrate the essentiality of N-glycolylated peptidoglycan plus trehalose dimycolate (both unique in mycobacteria) for the complete adjuvant effect using knockouts and chemical complementation. A combination of synthetic N-glycolyl muramyl dipeptide and minimal trehalose dimycolate motif GlcC14C18 was able to upregulate dendritic cell effectors, plus induce experimental autoimmunity qualitatively similar but quantitatively milder compared to CFA. This research outlines how to substitute CFA with a consistent, molecularly-defined adjuvant which may inform the design of immunotherapeutic agents and vaccines benefitting from cell-mediated immunity. We also anticipate using synthetic microbe-associated molecular patterns (MAMPs) to study mycobacterial immunity and immunopathogenesis.

It has recently been suggested that non-reflex behavioral readouts, such as burrowing, may be used to evaluate the efficacy of analgesics in rodent models of pain.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by joint infiltration and bone damage. The aim of the present study was to evaluate the beneficial effects of losartan in adjuvant-induced arthritis (AIA). Arthritis was induced in rats by subcutaneous injection of 0.2 mL of Complete Freund's adjuvant (CFA) in the planter surface of the hind paw. Arthritic rats were allocated into three groups (n = 10), the first group (arthritis control), received 1% of tween 80, the second and the third groups received prednisolone (10 mg/kg/day; p.o) and losartan (20 mg/kg/day; p.o) respectively for two weeks. A fourth group (vehicle control) received 1% tween 80. At the end of the experiment, blood samples were collected for biochemical, oxidative stress, and hematological analysis. Histopathological and macroscopical examinations on joints were also performed. Our results revealed that losartan significantly reduced serum levels of tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6) , rheumatoid factor (RF), and erythrocytes sedimentation rate (ESR).It significantly decreased serum malondialdehyde and increased blood glutathione .Losartan exhibited significant decrease in serum level of total cholesterol (TC), triglycerides (TG) and low density lipoprotein (LDL) coupled with marked increase in high density lipoprotein (HDL).Furthermore, losartan decreased white blood cell cells (WBC's) count and increased red blood cells (RBC's) , hemoglobin (Hb) , platelets, and hematocrit (Hct) counts. These findings were further supported by histopathological and macroscopical examinations. It could be concluded that losartan was able to repress biochemical, oxidative and hematological changes associated with AIA. These effects could be attributed to anti-arthritic, hypolipidemic, antioxidant and anti-anemic properties.

Apelin is the endogenous ligand for APJ, a G-protein-coupled receptor. Apelin gene and protein are widely distributed in the central nervous system and peripheral tissues. The role of apelin in chronic inflammatory pain is still unclear. In the present study, a mouse model of complete Freund's adjuvant (CFA)-induced inflammatory pain was utilized, and the paw withdrawal latency/threshold in response to thermal stimulation and Von Frey filament stimulation were recorded after intrathecal (i.t.) injection of apelin-13 (0.1, 1, and 10 nmol/mouse). The mRNA and protein expression, concentration of glutamic acid (Glu), and number of c-Fos immunol staining in lumbar spinal cord (L4/5) were determined. The results demonstrated that Apln gene expression in the lumbar spinal cord was down-regulated in the CFA pain model. Apelin-13 (10 nmol/mouse, i.t.) alleviated CFA-induced inflammatory pain, and it exhibited a more potent antinociceptive effect than apelin-36 and (pyr)apelin-13. The antinociception of apelin-13 could be blocked by APJ antagonist apelin-13(F13A). I.T. apelin-13 attenuated the increased levels of Aplnr, Grin2b, Camk2d, and c-Fos genes expression, Glu concentration, and NMDA receptor 2B (GluN2B) protein expression caused by CFA. Apelin-13 significantly reduced the number of Fos-positive cells in laminae III and IV/V of the dorsal horn. This study indicated that i.t. apelin-13 exerted an analgesic effect against inflammatory pain, which was mediated by activation of APJ, and inhibition of Glu/GluN2B function and neural activity of the spinal dorsal horn.

Rabbits injected with Freund's adjuvant, without any added antigen, developed antibodies against tubular brush border (BB) antigens of rat kidney, as demonstrated by the indirect immunofluorescence technique. After injection of the antibodies in rats, diffuse granular deposits of rabbit IgG, typical of passive Heymann nephritis, were detected along the glomerular capillary walls. The adjuvant-induced anti-BB antibodies thus resembled the immune anti-BB antibodies elicited in rabbits by immunization with isolated rat kidney BB membranes. Both the in vitro and in vivo binding of the adjuvant-induced antibodies could be inhibited by absorption with isolated rat kidney BB membranes. To characterize the BB antigens, radioactively labeled and solubilized BB membrane vesicles were precipitated with the adjuvant-induced and the immune anti-BB antibodies. Analysis of the immune precipitates by gel elecrophoresis showed polypeptides of more than 200, 130, and 80 kilodaltons as the major components of the precipitates. These antigens are major glycoproteins of the BB membranes and appear to be associated with the nephritogenic antigens of passive Heymann nephritis.

Anxiety disorder is highly prevalent worldwide and represents a chronic and functionally disabling condition, with high levels of psychological stress characterized by cognitive and physiological symptoms. Scopoletin (SP), a main active compound in Angelica dahurica, is traditionally used for the treatment of headache, rhinitis, pain, and other conditions. Here, we evaluated the effects of SP in a mouse model of complete Freund's adjuvant (CFA)-induced chronic inflammation anxiety. SP (2.0, 10.0, 50.0 mg/kg) administration for 2 weeks dose-dependently ameliorated CFA-induced anxiety-like behaviors in the open field test and elevated plus maze test. Moreover, we found that SP treatment inhibited microglia activation and decreased both peripheral and central IL-1β, IL-6, and TNF-α levels in a dose-dependent manner. Additionally, the imbalance in excitatory/inhibitory receptors and neurotransmitters in the basolateral nucleus after CFA injection was also modulated by SP administration. Our findings indicate that the inhibition of the nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways involving anti-inflammatory activities and regulation of the excitatory/inhibitory balance can be attributed to the anxiolytic effects of SP. Moreover, our molecular docking analyses show that SP also has good affinity for gamma-aminobutyric acid (GABA) transaminase and GABAA receptors. Therefore, these results suggest that SP could be a candidate compound for anxiolytic therapy and for use as a structural base for developing new drugs.

The present study aims to evaluate the antiarthritic activity of diacetylcurcumin (DAC), a synthetic derivative where the free phenolic groups of curcumin are derivatized by acetylation, thereby conferring greater lipophilicity to the parent molecule and partially overcoming the limited systemic bioavailability of curcumin. Antiarthritic activity was evaluated on a Freund's complete adjuvant (FCA)-induced murine model of arthritis. Oral administration of DAC (60 and 120 mg/kg) resulted in a significant inhibition of inflammation in the acute and chronic phases, respectively, demonstrating an improved and sustained anti-inflammatory effect, comparable to that of curcumin (150 mg/kg) in the chronic stage at a lower dose. Phenylbutazone (80 mg/kg) was used as a reference drug. The pharmacological consequence of DAC or curcumin treatment is the prevention of secondary lesions commonly associated with this biological model.

Vesicular glutamate transporter type 2 (VGLUT2) is known to play an important role in mediating heat hyperalgesia induced by inflammation. However, the underlying mechanism for this activity is poorly understood. Cyclin-dependent kinase 5 (Cdk5), serving as a key regulator in modulating release of glutamate, acted a key player in the formation of heat hyperalgesia of inflammatory pain. However, it remains unknown whether there is a bridge between Cdk5 and VGLUT2 for mediating inflammatory pain. Therefore, we designed the experiment to determine whether VGLUT2 signaling pathway is involved in inflammatory pain mediated by Cdk5 in the inflammatory pain model induced by complete Freund's adjuvant (CFA). Our results showed that the coexpression of Cdk5/VGLUT2 in small- and medium-sized neuronal cells of the dorsal root ganglion (DRG) and spinal cord between days 1 and 3 following subcutaneous injection of CFA was significantly increased. Moreover, our study revealed that the expression of VGLUT2 protein in the DRG and spinal cord was remarkably increased between days 1 and 3 following CFA injection and was significantly reduced by roscovitine, a selective antagonist of Cdk5. Additionally, p25 but not p35, an activator of Cdk5, protein was significantly increased by CFA and reduced by roscovitine. Our findings suggested that VGLUT2/Cdk5 signaling pathway contributes to inflammatory pain mediated by Cdk5/p25.

Sirtuin1 (SIRT1), which is regulated by microRNA-34a (miR-34a), can modulate pathophysiology processes, including nonalcoholic fatty liver disease and intestinal ischemia/reperfusion injury. We previously reported that SIRT1, an NAD+-dependent deacetylase, plays a vital role in the development of neuropathic pain. However, the role of miR-34a/SIRT1 in complete Freund's adjuvant (CFA)-induced inflammatory pain remains unclear. In the present study, we examined miR-34a and SIRT1 in CFA mice. MiR-34a levels increased, while SIRT1 decreased in the spinal cord. Inhibiting miR-34a by intrathecal injection of miR-34a antagomir attenuated CFA-induced pain behavior. Moreover, miR-34a antagomir inhibited the CFA-induced SIRT1 decrease in the spinal cord. Furthermore, the analgesic effect of miR-34a antagomir was abrogated by the SIRT1 inhibitor EX-527. Our data provide support that the underlying mechanisms of miR-34a in promoting inflammatory pain may involve negative regulation of SIRT1.

Current therapies for autoimmune diseases focus on treating the symptoms rather than the underlying disease cause. A major setback in improving current therapeutics for autoimmunity is the lack of antigen specificity. Successful antigen-specific immunotherapy (ASIT) would allow for improved treatment of autoimmune diseases. In this work, dexamethasone was co-delivered with autoantigen (PLP) in vivo to create effective ASIT for the treatment of experimental autoimmune encephalomyelitis (EAE). Using an emulsion of incomplete Freund's adjuvant (IFA) as a co-delivery vehicle, it was discovered that the controlled release of autoantigen was important for the suppression of clinical disease symptoms. Analysis of the immune response via cytokines revealed that dexamethasone was important for shifting the immune response away from inflammation. Co-delivery of both autoantigen and dexamethasone increased B-cell populations and antibody production, signifying an increased humoral immune response. Overall, this data indicated that the co-delivery of PLP and dexamethasone with a water-in-oil emulsion is effective in treating a murine autoimmune model.

Migraine is a neurovascular primary headache disorder, which causes significant socioeconomic problems worldwide. The pathomechanism of disease is enigmatic, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Migraine research of recent years has focused on neuropeptides, such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP1-38) as potential pathogenic factors and possible therapeutic offensives. The goal of present study was to investigate the simultaneous expression of CGRP and precursor of PACAP1-38 (preproPACAP) in the central region of the TS in a time-dependent manner following TS activation in rats.

Chronic inflammation pain is a debilitating disease, and its mechanism still remains poorly understood. This study attempted to illuminate the metabolic mechanism of chronic inflammation pain induced by complete Freund's adjuvant (CFA) injection, especially at spinal level. The chronic inflammation pain model was established by CFA administration. Behavioral testing including mechanical allodynia and thermal hyperalgesia was performed. Meanwhile, a liquid chromatography-mass spectrometry-based metabolomics approach was applied to analyze potential metabolic biomarkers. The orthogonal partial least squares discrimination analysis mode was employed for determining metabolic changes, and a western blot was performed to detect the protein expression change. The results showed that 27 metabolites showed obviously abnormal expression and seven metabolic pathways were significantly enriched, comprising aminoacyl-tRNA biosynthesis, arginine and proline metabolism, histidine metabolism, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glutathione metabolism, and phenylalanine metabolism. Meanwhile, the results showed that the expression of arginase I and nitric oxide levels were elevated in the CFA group compared with the control group, while the argininosuccinate synthetase and argininosuccinatelyase proteins were not significantly different between the groups. These findings demonstrate that metabolic changes of the spinal cord may be implicated in neurotransmitter release and pain conductivity following CFA administration.

Adjuvant formulations capable of inducing high titer and high affinity antibody responses would provide a major advance in the development of vaccines to viral infections such as HIV-1. Although oil-in-water emulsions, such as Freund's adjuvant (FCA/FIA), are known to be potent, their toxicity and reactogenicity make them unacceptable for human use. Here, we explored different adjuvants and compared their ability to elicit antibody responses to FCA/FIA. Recombinant soluble trimeric HIV-1 gp140 antigen was formulated in different adjuvants, including FCA/FIA, Carbopol-971P, Carbopol-974P and the licensed adjuvant MF59, or combinations of MF59 and Carbopol. The antigen-adjuvant formulation was administered in a prime-boost regimen into rabbits, and elicitation of antigen binding and neutralizing antibodies (nAbs) was evaluated. When used individually, only FCA/FIA elicited significantly higher titer of nAbs than the control group (gp140 in PBS (p<0.05)). Sequential prime-boost immunizations with different adjuvants did not offer improvements over the use of FCA/FIA or MF59. Remarkably however, the concurrent use of the combination of Carbopol-971P and MF59 induced potent adjuvant activity with significantly higher titer nAbs than FCA/FIA (p<0.05). This combination was not associated with any obvious local or systemic adverse effects. Antibody competition indicated that the majority of the neutralizing activities were directed to the CD4 binding site (CD4bs). Increased antibody titers to the gp41 membrane proximal external region (MPER) and gp120 V3 were detected when the more potent adjuvants were used. These data reveal that the combination of Carbopol-971P and MF59 is unusually potent for eliciting nAbs to a variety of HIV-1 nAb epitopes.

Xuelian injection (XI), a classic preparation extracted from Saussureae Involucratae Herba, has been clinically used to manage rheumatoid arthritis (RA) for nearly twenty years in China. However, the underlying anti-RA mechanism of XI remains unclear. In this study, complete Freund's adjuvant (CFA)-induced acute arthritic model was used to examine the anti-RA effects of XI in vivo. The molecular mechanisms of this action were further investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Various parts of Margaritaria discoidea find use in traditional medicine in the treatment of pain and oedema. This study evaluated the anti-allergic, anti-inflammatory and anti-arthritic effects of a 70% (v/v) aqueous ethanol extract of the stem bark of Margaritaria discoidea, MDE in rodents.

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE-/- ) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1β at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes-and not epithelial cells-and was dependent on NLRP3 inflammasome activation and IL1β-signalling. In females, CFA-induced TSLP was independent of IL1β and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR-/- mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE-/- and ApoE-/- /TSLPR-/- mice with either CFA/IFA or PBS. ApoE-/- mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE-/- /TSLPR-/- mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.

This study aimed to assess the effect of petroleum ether extract (PEE), ethyl acetate extract (EthE), and ethanol extract (EAE) of Trichilia monadelpha stem bark on bone histomorphology in arthritis.

Rheumatoid arthritis (RA) is characterized by inflammation of one or more joints, and affects ~1% of the adult population worldwide. Sulforaphane (SFN) is a natural compound that has been suggested as an antioxidant. Here, SFN’s effects were evaluated in a murine mono-arthritis model. Mono-arthritis was induced in mice by a single intra-articular injection of Complete Freund’s Adjuvant (CFA-10 µg/joint, in 10 µL) into the ipsilateral joint. The contralateral joint received an equal volume of PBS. On the 4th day post-joint inflammation induction, animals received either SFN (10 mg/kg) or vehicle (3% DMSO in saline), intraperitoneally (i.p.), twice a day for 3 days. Joint swelling and secondary mechanical allodynia and hyperalgesia were evaluated over 7 days post-CFA. After this period, animals were culled and their blood and synovial fluid samples were collected for analysis of cell populations, cytokine release and thioredoxin reductase (TrxR) activity. Knee joint samples were also collected for histology. SFN reduced joint swelling and damage whilst increasing the recruitment of Ly6C⁺ and Ly6G⁺ cells to CFA-injected joints. SFN-treated animals presented down-regulation of CD11b and CD62L on synovial fluid Ly6G⁺ cells. Synovial fluid samples obtained from CFA-injected joints and plasma samples of SFN-treated mice presented higher levels of IL-6 and increased activity of TrxR, in comparison with controls. These results indicate that SFN reduces knee joint damage by modulating cell activation/migration to the joints, cytokine production and increasing the activity of TrxR, and therefore, may represent an alternative treatment to joint inflammation.

Strychnos potatorum Linn (Loganiaceae) is a moderate sized tree found in southern and central parts of India, Sri Lanka and Burma. In traditional system of medicine, Strychnos potatorum Linn seeds were used for various ailments including inflammation, diabetes etc. To investigate the folkloric use of the seeds the present study was carried out on Freund's adjuvant induced arthritic rats.

Group B coxsackieviruses (CVBs) belonging to the genus, Enterovirus and contain six serotypes that induce various diseases, whose occurrence may involve the mediation of more than one serotype. We recently identified immunogenic epitopes within coxsackieviruses B3 (CVB3) viral protein 1 that induce anti-viral T cell responses in mouse models of CVB infections. In our investigations to determine the protective responses of the viral epitopes, we unexpectedly noted that animals immunized with complete Freund's adjuvant (CFA) alone and later challenged with CVB3 were completely protected against myocarditis. Similarly, the pancreatitis-inducing ability of CVB3 was remarkably reduced to only 10% in the CFA group as opposed to 73.3% in the control group that received no CFA. Additionally, no mortalities were noted in the CFA group, whereas 40% of control animals died during the course of 21 days post-infection with CVB3. Taken together, our data suggest that the adjuvant effects of CFA may be sufficient for protection against CVB infections. These observations may provide new insights into our understanding of the occurrence of viral infections.