Micellar solubilization enhances the anti-inflammatory effect of xanthohumol

BackgroundXanthohumol is known to exert anti-inflammatory properties but has poor oral bioavailability. Using advanced micellization technology, it has been possible to markedly enhance its bioavailability.PurposeIn the present study, we compared the chronic anti-inflammatory activities of native and micellar xanthohumol in the rat adjuvant arthritis model, using diclofenac as a reference drug.MethodsAdjuvant arthritis was induced by injecting Freund's complete adjuvant into the right hind paw of rats and monitoring paw volume over 3 weeks. The drugs were given daily for 3 weeks, starting from the day of adjuvant inoculation. Serum was collected at the end of the experiment to measure inflammatory and oxidative stress parameters. Statistical comparisons between different groups were carried out by one-way analysis of variance followed by Tukey-Kramer multiple comparison test.ResultsMicellar solubilized xanthohumol showed a better anti-inflammatory activity than its native form. The reduction in paw volume was reflected in corresponding changes in relevant mediators of inflammation like tumor necrosis factor-α, interleukin-6 and C-reactive protein, myloperoxidase and lipid peroxidation markers.ConclusionThe findings confirm that micellar solubilization of xanthohumol enhances its anti-inflammatory activity, probably as a result of improving its bioavailabilty. The solubilized xanthohumol may prove to be a promising adjuvant tool for anti-inflammatory treatment and a potential anti-inflammatory alternative to synthetic drugs.     

Protective effect of betulinic acid on Freund's complete adjuvant‐induced arthritis in rats

The purpose of the experiment was to study the effects of betulinic acid (BA) on adjuvant‐induced arthritis in rats. The rat model of rheumatoid arthritis (AA) was established by Freund's complete adjuvant. Arthritis index, joint pathology, toe swelling, hemorheology, related cytokines and ROCK/NF‐κB signaling pathway were measured in rats. BA can significantly inhibit the arthritis index, improve joint pathology, reduce toe swelling, improve blood rheology, improve synovial cell apoptosis, and restore related cytokine negative regulation of ROCK/NF‐κB signaling pathways. BA has an obvious therapeutic effect on joint inflammation of toes in AA model rats, which may be due to the regulation of ROCK/NF‐κB signaling pathway.     

CXCL10 Controls Inflammatory Pain via Opioid Peptide-Containing Macrophages in Electroacupuncture

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund''s adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3⁺ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3⁺ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture.     

Investigation of the role of endosomal Toll-like receptors in murine collagen-induced arthritis reveals a potential role for TLR7 in disease maintenance

Introduction

Endosomal toll-like receptors (TLRs) have recently emerged as potential contributors to the inflammation observed in human and rodent models of rheumatoid arthritis (RA). This study aims to evaluate the role of endosomal TLRs and in particular TLR7 in the murine collagen induced arthritis (CIA) model.

Methods

CIA was induced by injection of collagen in complete Freund''s adjuvant. To investigate the effect of endosomal TLRs in the CIA model, mianserin was administered daily from the day of disease onset. The specific role of TLR7 was examined by inducing CIA in TLR7-deficient mice. Disease progression was assessed by measuring clinical score, paw swelling, serum anti-collagen antibodies histological parameters, cytokine production and the percentage of T regulatory (T_reg) cells.

Results

Therapeutic administration of mianserin to arthritic animals demonstrated a highly protective effect on paw swelling and joint destruction. TLR7^-/- mice developed a mild arthritis, where the clinical score and paw swelling were significantly compromised in comparison to the control group. The amelioration of arthritis by mianserin and TLR7 deficiency both corresponded with a reduction in IL-17 responses, histological and clinical scores, and paw swelling.

Conclusions

These data highlight the potential role for endosomal TLRs in the maintenance of inflammation in RA and support the concept of a role for TLR7 in experimental arthritis models. This study also illustrates the potential benefit that may be afforded by therapeutically inhibiting the endosomal TLRs in RA.     

Effect of ricinoleic acid in acute and subchronic experimental models of inflammation

Observational studies indicate that topical application of ricinoleic acid (RA), the main component of castor oil, exerts remarkable analgesic and anti-inflammatory effects. Pharmacological characterization has shown similarities between the effects of RA and those of capsaicin, suggesting a potential interaction of this drug on sensory neuropeptide-mediated neurogenic inflammation. The aim of this study was to assess RA anti-inflammatory activities in comparison with capsaicin in several models of acute and subchronic inflammation. The acute inflammation was induced by intradermal injection of carrageenan in the mouse or by histamine in the guinea-pig eyelid. In either experiment, the extent of the oedema thickness was measured. Subchronic oedema was induced by complete Freund's adjuvant injection in the ventral right paw of mice. Tissue substance P (SP) was measured in the carrageenan experiments by radioimmunoassay (RIA). It was found that the acute topical application of RA (0.9 mg/mouse) or capsaicin (0.09 mg/mouse) significantly increased the mouse paw oedema induced by carrageenan, while an 8-day repeated topical treatment with the same doses of both compounds resulted in a marked inhibition of carrageenan-induced paw oedema matched by a reduction in SP tissue levels. Similar effects were found against histamine-induced eyelid oedema in guinea-pigs after acute or repeated application of RA or capsaicin. RA and capsaicin given for 1-3 weeks reduced the established oedema induced by Freund's adjuvant, a subchronic model of inflammation, particularly if given by the intradermal route. Either in mouse paw or in guinea-pig eyelid, capsaicin but not RA by itself produced a slight hyperemia and activation of a behavioural response (e.g. scratching of the eyelids). On the basis of the present results, RA may be seen as a new capsaicin-like, non-pungent anti-inflammatory agent suitable for peripheral application.     

Therapeutic effects of standardized Vitex negundo seeds extract on complete Freund's adjuvant induced arthritis in rats

The seeds of Vitex negundo L. (Verbenaceae) have been commonly used as a folk remedy for the treatment of rheumatism and joint inflammation in Traditional Chinese Medicine. This study aimed to evaluate the anti-arthritic activity of the extract of V. negundo seeds (EVNS) using Freund's complete adjuvant (CFA) induced arthritis (AA) in rat model. As a result, EVNS, with abundant phenylnaphthalene-type lignans, significantly inhibited the paw edema, decreased the arthritis score and spleen index, and reversed the weight loss of CFA-injected rats. Histopathological studies showed a marked decrease of synovial inflammatory infiltration and synovial lining hyperplasia in the joints of EVNS-treated animals. The remarkable decrement of serum inflammatory factors (TNF-α, IL-1β and IL-6) were observed in EVNS-treated rats, whereas, IL-10, an anti-inflammatory cytokine, was found to be significantly increased by EVNS. The expressions of COX-2 and 5-LOX in PBMC were also inhibited by administration of EVNS. Our results demonstrated that V. negundo seeds possessed potential therapeutic effect on adjuvant induced arthritis in rats by decreasing the levels of TNF-α, IL-1β and IL-6 and increasing that of IL-10 in serum as well as down-regulating the levels of COX-2 and 5-LOX, and therefore may be an effective cure for the treatment of human rheumatoid arthritis.     

Hypothermia tolerance in guinea pigs with experimental myasthenia gravis

Hypothermia tolerance was studied in guinea pigs with experimental myasthenia gravis induced by immunization with homologous thymus extract and Freund's complete adjuvant. Untreated guinea pigs and guinea pigs immunized with liver extract and Freund's complete adjuvant served as controls. Thymus-immunized guinea pigs survived to a significantly lower temperature at asystole than did the liver-immunized controls. This finding is consistent with a slight diminution in temperature maintenance mechanisms in guinea pigs with experimental autoimmune thymitis. The observed increase in hypothermia tolerance may be due to impaired shivering as a consequence of the partial neuromuscular block demonstrable in the myasthenia gravislike state.     

Association between arthritis score at the onset of the disease and long-term locomotor outcome in adjuvant-induced arthritis in rats

IntroductionTo investigate the connection between the intensity of initial symptoms of inflammation and locomotor outcome in rheumatoid arthritis, we examined the relationship between long-term locomotor abnormalities and signs of inflammation at the onset of the disease in adjuvant-induced arthritis (AIA) in rats.MethodsThe arthritis score and hind-paw diameter were followed from immunization to day 195 (~7 months). At this time, locomotion was recorded during forced treadmill walking using 3D motion technology before radiographic scoring of hind limb joint damage. Many locomotor parameters were analyzed including time and length parameters, limbs kinematics, lateral paw position at toe off, maximal hind-paw elevation and posture. Ankle mobility was assessed from range of motion (ROM) of the joint during locomotion. Experiments were run in AIA (n = 18) and age-matched non-AIA rats (n = 8).ResultsAll AIA rats exhibited signs of inflammation at day 14 with a peak of inflammatory symptoms at day 22 post-immunization. After the first episode of inflammation, 83 % of AIA rats demonstrated recurrent disease (from week 6 to week 23). The frequency of inflammatory episodes (1 to 5) was not linked to the arthritis score at day 22. At day 195 post-immunization, AIA rats showed significantly impaired locomotion and radiographic lesions as compared to control rats. Significant relationships were observed between most locomotion-related parameters and concurrent ROM of ankle, which correlated negatively with the radiographic score. ROM of ankle at day 195 correlated negatively with both the arthritis score and hind-paw diameter measured at day 14, 22 and 30 post-immunization.ConclusionDecreased ankle mobility can be considered a driver of locomotion impairment in AIA. In this model, the severity of the initial inflammatory symptoms had a good prognostic value for long-term locomotor outcome.     

Mannan-Binding Lectin Deficiency Limits Inflammation-induced Myeloid-Derived Suppressor Cells Expansion via Modulating Tumor Necrosis Factor Alpha-triggered Apoptosis

Background: Mannan-binding lectin (MBL), a soluble pattern recognition molecule in the innate immune system, is reported to be associated with the function of immune cells. Myeloid-derived suppressor cells (MDSCs) are mainly characterized by immunosuppressive activities involving several inflammatory diseases such as cancer, infection, and arthritis. Some of the factors inducing their apoptosis are known, however, mechanisms have not been identified. The underlying impact of MBL on the MDSCs especially under inflammatory conditions remains unknown. This study was designed to investigate whether MBL affects MDSCs survival during inflammation conditions.Methods: WT and MBL-deficient (MBL^-/-) mice were induced on day 0 of the experiment by subcutaneous injection of complete Freund''s adjuvant and then injected with incomplete Freund''s adjuvant into the knee joint space under general anesthesia on day 14 to induce inflammatory arthritis. The proportions of MDSCs in the spleen and blood and the serum level of the inflammatory cytokines were measured. In vitro study, MDSCs were isolated from the bone marrow of WT and MBL^-/- mice and cultured in the presence of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days with or without tumor necrosis factor-alpha (TNF-α).Results: After adjuvant treatment, MBL^-/- mice had a significantly lower frequency of MDSCs as well as elevated serum inflammatory cytokines levels compared to WT mice. MBL deficiency markedly inhibited the MDSCs frequency from mice bone marrow induced by IL-6 and GM-CSF in the presence of TNF-α in vitro. Mechanistic studies established that MBL inhibited MDSCs apoptosis via down-regulation of TNF-α/tumor necrosis factor-alpha receptor 1 (TNFR1) signaling pathway and subsequent caspase 3-dependent manner.Conclusion: Mannan-binding lectin deficiency inhibits myeloid-derived suppressor cells expansion via modulating TNF-α triggered apoptosis.     

Twenty-four-Hour Rhythms of Serum Acth,Prolactin, Growth Hormone,and Thyroid-Stimulating Hormone,and of Median-Eminence Norepinephrine,Dopamine, and Serotonin,in Rats Injected with Freund's Adjuvant

The effect of Freund's adjuvant injection on 24-h variation of circulating ACTH, prolactin, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels, and of norepinephrine (NE) content, and dopamine (DA) and serotonin (5HT) turnover in median eminence, was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of 6–10 animals Freund's complete adjuvant or its vehicle at 1 lOOh 3 days before sacrifice and were killed by decapitation at six different time intervals throughout a 24-h cycle. In rats injected with adjuvant's vehicle, serum ACTH and prolactin exhibited peak values around the light-dark transition (p < 0.0001 and < 0.04, respectively), while the maximum in TSH was found in the late afternoon (p < 0.0001, one-way ANOVA). GH levels did not vary on a 24-h basis. In Freund's adjuvant-injected rats, 24-h variations of TSH levels became blunted, while 24-h variations of prolactin and ACTH persisted. Freund's adjuvant augmented serum ACTH and prolactin levels, and decreased GH and TSH levels (p < 0.0007, factorial ANOVA). Median-eminence NE content, and turnover of DA, assessed by measuring dihydroxyphenylacetic acid, DOPAC/DA ratio, and of 5HT, assessed by measuring 5-hydroxyindoleacetic acid, HIAA/5HT ratio, varied on a 24-h basis in rats receiving adjuvant's vehicle (p < 0.02). Median-eminence NE content attained its maximum at 1600–2000 h, while maxima in DOPA/DA and HIAA/5HT ratios occurred at 0400 h. Injection with Freund's adjuvant reduced the amplitude of the daily variation of NE content, shifted the maximum of DOPAC/DA ratio toward the light-dark transition, and blunted the daily variation in HIAA/5HT ratio in median eminence. The administration at 1200 of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) restored the augmented ACTH and prolactin levels (p < 0.0001, factorial ANOVA) and depressed GH and TSH levels (p < 0.02) found in Freund's adjuvant-injected rats. Cyclosporine was also effective in restoring 24-h rhythmicity of serum ACTH and TSH, but not of prolactin, levels. Cyclosporine did not modify the effect of Freund's adjuvant on time-of-day changes of median-eminence NE content, but it was effective in counteracting the changes of DA and 5HT turnover found after immunization. The results are compatible with a significant effect of immune-mediated inflammatory response at an early phase after Freund's adjuvant injection on ACTH, GH, prolactin, and TSH release, which is partially sensitive to immunosuppression by cyclosporine. (Chronobiology International, 14(3), 253–265, 1997)     

Histamine mediates the pro-inflammatory effect of latex of Calotropis procera in rats

INTRODUCTiON: Calotropis procera is known to produce contact dermatitis and the latex of this plant produces intense inflammation when injected locally. However, the precise mode of its pro-inflammatory effect is not known. In present study we have pharmacologically characterized the inflammation induced by latex of C. procera in a rat paw edema model and determined the role of histamine in latex-induced inflammation. METHODS: Inflammation was induced in the hind paw of rats by injecting different doses of dried latex (DL) of C. procera. The inhibitory effect of phenylbutazone, dexamethasone, celecoxib, cyproheptadine, chlorpheniramine and compound 48/80 on edema volume was evaluated and compared with that against carrageenan. The histamine content of DL was measured fluorometrically. RESULTS: DL produced dose-dependent inflammation of the rat paw. Cyproheptadine and chlorpheniramine effectively inhibited DL-induced inflammation (90%; p < 0.01), while anti-inflammatory drugs phenylbutazone, dexamethasone and celecoxib were more effective against carrageenan-induced inflammation. Depletion of mast cell histamine by compound 48/80 produced a significant decrease in DL-induced inflammation as compared with carrageenan (500% versus 25%). DL was also found to contain about 6 microg/g of histamine. CONCLUSIONS: Thus, our study shows that the biogenic amines play a significant role in C. procera latex-induced inflammation and antihistaminic drugs could be effectively used to inhibit inflammatory response elicited by exposure to latex.     

Limb kinematics during locomotion in the two-toed sloth (Choloepus didactylus,Xenarthra) and its implications for the evolution of the sloth locomotor apparatus

In order to gain insight into the function of the extant sloth locomotion and its evolution, we conducted a detailed videoradiographic analysis of two-toed sloth locomotion (Xenarthra: Choloepus didactylus). Both unrestrained as well as steady-state locomotion was analyzed. Spatio-temporal gait parameters, data on interlimb coordination, and limb kinematics are reported. Two-toed sloths displayed great variability in spatio-temporal gait parameters over the observed range of speeds. They increase speed by decreasing the durations of contact and swing phases, as well as by increasing step length. Gait utilization also varies with no strict gait sequence or interlimb timing evident in slow movements, but a tendency to employ diagonal sequence, diagonal couplet gaits in fast movements. In contrast, limb kinematics were highly conserved with respect to ‘normal’ pronograde locomotion. Limb element and joint angles at touch down and lift off, element and joint excursions, and contribution to body progression of individual elements are similar to those reported for non-cursorial mammals of small to medium size. Hands and feet are specialized to maintain firm connection to supports, and do not contribute to step length or progression. In so doing, the tarsometatarsus lost its role as an individual propulsive element during the evolution of suspensory locomotion. Conservative kinematic behavior of the remaining limb elements does not preclude that muscle recruitment and neuromuscular control for limb pro- and retraction are also conserved. The observed kinematic patterns of two-toed sloths improve our understanding of the convergent evolution of quadrupedal suspensory posture and locomotion in the two extant sloth lineages.     

Different forms of glycine- and GABAA-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons

Background

Inflammation-mediated hyperalgesia involves tissue acidosis and sensitization of nociceptors. Many studies have reported increased expression of acid-sensing ion channel 3 (ASIC3) in inflammation and enhanced ASIC3 channel activity with pro-inflammatory mediators. However, the role of ASIC3 in inflammation remains inconclusive because of conflicting results generated from studies of ASIC3 knockout (ASIC3 ^-/-) or dominant-negative mutant mice, which have shown normal, decreased or increased hyperalgesia during inflammation.

Results

Here, we tested whether ASIC3 plays an important role in inflammation of subcutaneous tissue of paw and muscle in ASIC3 ^-/- mice induced by complete Freund's adjuvant (CFA) or carrageenan by investigating behavioral and pathological responses, as well as the expression profile of ion channels. Compared with the ASIC3 ^+/+ controls, ASIC3 ^-/- mice showed normal thermal and mechanical hyperalgesia with acute (4-h) intraplantar CFA- or carrageenan-induced inflammation, but the hyperalgesic effects in the sub-acute phase (1–2 days) were milder in all paradigms except for thermal hyperalgesia with CFA-induced inflammation. Interestingly, carrageenan-induced primary hyperalgesia was accompanied by an ASIC3-dependent Nav1.9 up-regulation and increase of tetrodotoxin (TTX)-resistant sodium currents. CFA-inflamed muscle did not evoke hyperalgesia in ASIC3 ^-/- or ASIC3 ^+/+ mice, whereas carrageenan-induced inflammation in muscle abolished mechanical hyperalgesia in ASIC3 ^-/- mice, as previously described. However, ASIC3 ^-/- mice showed attenuated pathological features such as less CFA-induced granulomas and milder carrageenan-evoked vasculitis as compared with ASIC3 ^+/+ mice.

Conclusion

We provide a novel finding that ASIC3 participates in the maintenance of sub-acute-phase primary hyperalgesia in subcutaneous inflammation and mediates the process of granuloma formation and vasculitis in intramuscular inflammation.     

Sickle cell mice exhibit mechanical allodynia and enhanced responsiveness in light touch cutaneous mechanoreceptors

Background

Interleukin-8 (IL-8) is known for its roles in inflammation and plays critical roles in the development of pain. Its expression increases in the brain after peripheral inflammation. Prefrontal cortex, including the anterior cingulate cortex (ACC), is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission, however, little is known about the expression of IL-8 and its role in the enhanced ACC synaptic transmission in animals with persistent inflammatory pain.

Findings

In the present study, we examined IL-8 expression in the ACC, somatosensory cortex (SSC), and the dorsal horn of lumbar spinal cord following hind-paw administration of complete Freund's adjuvant (CFA) in mice and its effects on the ACC synaptic transmission. Quantification of IL-8 at protein level (by ELISA) revealed enhanced expression in the ACC and spinal cord during the chronic phases of CFA-induced peripheral inflammation. In vitro whole-cell patch-clamp recordings revealed that IL-8 significantly enhanced synaptic transmission through increased probability of neurotransmitter release in the ACC slice. ACC local infusion of repertaxin, a non-competitive allosteric blocker of IL-8 receptors, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in mice.

Conclusions

Our findings suggest that up-regulation of IL-8 in the ACC partly attributable to the enhanced prefrontal synaptic transmission in the mice with persistent inflammatory pain.     

Contralateral but not systemic administration of bupivacaine reduces acute inflammation in the rat hindpaw

The effects of contralateral treatment with local anesthetics following acute hindpaw inflammation were investigated in rats. Inflammation was induced by unilateral injection of either 50 or 100 &#119 l of 1% carrageenan into the right paw. Contralateral injection of either bupivacaine or saline was given immediately before the carrageenan. Hindpaw edema and withdrawal responses to thermal and mechanical stimulation were evaluated after 3, 6 and 24 h. The results showed that the proinflammatory effects of carrageenan were strongest at 6 h after the injection of 100 mul carrageenan with bilaterally decreased withdrawal latencies and ipsilateral edema formation. Contralateral treatment with bupivacaine (1.25, 2.5 or 5 mg/ml) dosedependently reduced nociceptive behavior for 3-24 h. The edema was also reduced at 6 h. No effects on pain-related behavior were observed following systemic administration of bupivacaine. Sciatic nerve ligation on the contralateral side or intrathecal administration of saline significantly reduced the effects of bupivacaine when respectively compared with shamoperation and subcutaneous saline injection. Contralateral treatment with bupivacaine into the knee joint induced the same anti-nociceptive effect as administered into the paw. Our findings indicate that contralateral administration of bupivacaine induces long-lasting anti-nociceptive effects and may serve as a new or complementary treatment approach in acute inflammatory pain conditions.     

Anti-rheumatoid arthritic activity of flavonoids from Daphne genkwa

The aim of the study was to investigate the anti-rheumatoid arthritic activity of four flavonoids from Daphne genkwa (FFD) in vivo and in vitro. Flavonoids of D. genkwa were extracted by refluxing with ethanol and purified by polyamide resin. An in vivo carrageenan-induced paw edema model, tampon-granuloma model and Freund's complete adjuvant (FCA)-induced arthritis mouse model were used to evaluate the anti-rheumatoid arthritic activities of FFD. Moreover, nitric oxide (NO) release and neutral red uptake (NRU) in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells were used to evaluate the anti-inflammatory effect in vitro. In addition, antioxidant effect of FFD was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A high dose of FFD significantly reduced the degree of acute inflammatory paw edema in mice as a response to carrageenan administration (p < 0.01). FFD displayed a dose-dependent inhibition of granuloma formation in mice (p < 0.05). FFD also inhibited chronic inflammation in adjuvant-induced arthritis rats when administered orally at the dose of 50 mg/kg/day (p < 0.001). In addition, FFD suppressed the production of NO and exhibited immunoregulatory function in LPS-activated RAW264.7 cells in a dose-related manner. Simultaneously, FFD revealed conspicuous antioxidant activity with IC₅₀ values of 18.20 μg/ml. FFD possesses significant anti-inflammatory and antioxidant activity, which could be a potential therapeutic agent for chronic inflammatory disorders such as rheumatoid arthritis.     

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant

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.     

Antibodies Specific for Carbamylated Proteins Precede the Onset of Clinical Symptoms in Mice with Collagen Induced Arthritis

Objective

The immune response to post-translationally modified antigens is a key characteristic of rheumatoid arthritis. Carbamylation is such a posttranslational modification. Recently, we demonstrated that autoantibodies recognizing carbamylated proteins are present in sera of rheumatoid arthritis. The molecular mechanisms underlying the break of tolerance and hence the induction of anti-CarP antibody responses are unknown as well as their appearance in mouse models for systemic arthritis. Therefore we analyzed their appearance in the mouse collagen-induced arthritis model.

Methods

collagen induced arthritis was induced by immunization with type II collagen in complete Freund''s adjuvant. Arthritis severity was monitored by clinical scoring and anti-CarP antibody levels were determined by ELISA.

Results

Anti-CarP antibodies were detectable in mice with collagen induced arthritis. We did not detect ACPA in mice with collagen induced arthritis. The specificity of the antibodies for carbamylated proteins was confirmed by inhibition assays and immunoblotting. Injection with complete Freund''s adjuvant without type II collagen could also induce anti-CarP antibodies, however, in mice with arthritis, the anti-CarP antibody response was stronger and developed more rapidly. The onset of collagen induced arthritis was preceded by an increase of anti-CarP IgG2a levels in the serum.

Conclusion

In mice with collagen induced arthritis we did not observe an immune response against citrullinated antigens, but we did observe an immune response against carbamylated antigens. This anti-CarP response already appeared before disease onset, indicating that collagen induced arthritis can be used as an in vivo model to study anti-CarP antibodies. Our data also indicate that the tolerance to carbamylated proteins, in contrast to the response to citrullinated proteins, is easily broken and that arthritis boosts the immune response against these proteins. The anti-CarP response in mice with CIA can be used as a model for immune responses to post-translationally modified proteins.