Nanodiamonds in Oil Emulsions as Effective Vaccine Adjuvants and Antitumor Therapeutic Agents

Background Composed of mineral oil and mycobacteria pathogens, complete Freund’s adjuvant (CFA) is one of the most commonly used adjuvants for antibody production and scientific research due to its high efficiency. However, the dead mycobacteria in CFA can cause many allergic reactions. We propose here a new formulation based on the use of nanodiamonds (NDs) as biocompatible non-allergic additives in incomplete Freund’s adjuvant (IFA) to avoid these adverse effects. Methods Chicken egg ovalbumin (OVA) was used as the antigens and 100-nm NDs after purification by air oxidation and strong oxidative acid washes were used as the additives. Levels of OVA-specific IgG antibody in mouse sera were measured by using enzyme-linked immunosorbent assays (ELISA) after the second and third immunizations of healthy mice with OVA and OVA/ND in IFA or CFA. Abilities of the OVA/ND/IFA vaccination to inhibit the tumor growth of mice inoculated with EL4 cells or OVA-expressing E.G7 cells were examined over 1 month. Results The new formulation worked well as a potent vaccine adjuvant, which could boost the immune responses and reduce the consumption of antigens in producing antibodies of interest in model animals like mice. Additionally, the composites showed distinct therapeutic activities, as proven by the OVA/ND/IFA treatment that effectively inhibited the tumor progression of E.G7-inoculated mice, allowing the animals to survive over 35 days post tumor-cell challenges. About 0.2% of the injected ND particles were found in mouse spleens on day 24 after vaccination of the E.G7-inoculated mice with OVA/ND/IFA. Conclusions The multiple functionality of ND makes it useful as an active and trackable component of a vaccine adjuvant not only to enhance antibody production but also to suppress tumor growth in vivo. The ND-based new formulation can be developed into single-dose vaccines with promising potential for real-world applications.

Antinociceptive Investigations of Niranthin in Complete Freund’s Adjuvant-induced Chronic Pain in Mice

The main objectives of the present work are to determine the clinical effect of niranthin on visceral or somatic inflammatory pain. The study was performed to determine the effects of niranthin on visceral or somatic inflammatory hypersensitivity of adult Swiss albino mice by using complete Freund’s adjuvant (CFA) induced pain model. The effect of CFA injection was determined after 24 hours of injection by using an aesthesiometer such as Von Frey filaments to evaluate tactile acetone-evoked cooling and thermal sensitivity. We used a digital Plethysmometer to measure paw edema. Single dose of niranthin intraperitoneal injection (5 & 10 mg/kg) was injected into mice having CFA-induced mechanical hypersensitivity and after 30 minutes of administration, reduced mechanical hypersensitivity was observed. In addition, niranthin also reduced acetone-evoked hypersensitivity within 4 hours. Compared to DMSO, niranthin was most highly active to reduce CFA-induced paw edema. To reduce mechanical hypersensitivity, multiple doses of niranthin (bis in die (b.i.d.)) from 1st - 5th day and b.i.d. day 9th and 10th) were given and remarkable results were observed such as did not cause tolerance in multiple dosing and significantly reduced in CFA induced hypersensitivity. This work reported niranthin having antinociceptive activity and indicated that niranthin is conventionally active in the management of persistent pain.

Appraisal of Anti-Arthritic Potential of Quercus Leucotrichophora Methanolic Extract in Complete Freund’s Adjuvant Induced Arthritic Rats; a Mechanistic Study

This research work was conducted to validate the folkloric use and therapeutic potential of Quercus leucotrichophora (QL) leaf methanolic and aqueous extracts against inflammation and arthritis and to determine the chemical composition by HPLC. The in-vitro anti-oxidant and anti-inflammatory activities were carried out along with in-vivo assays such as carrageenan induced paw edema, xylene induced ear edema and Complete Freund’s Adjuvant induced arthritis in Wistar rats. The CFA (0.1 ml) was inoculated to the left hind paw at day 1 to induce arthritis and oral dosing with QLME at 150, 300 and 600 mg/kg was begun at 8th day till the 28th day in all groups while methotrexate was given as standard treatment. There was a noteworthy (p<0.05-0.0001) restoration in body weight, paw edema, arthritic index, altered blood parameters and oxidative stress biomarkers in treated rats as compared to diseased group. Moreover, QLME considerably (p<0.0001) downregulated TNF-α, IL-6, IL-1β, COX-2, and NF-κB, while significantly (p<0.0001) upregulated IL-10, I-κB, IL-4 in relation to diseased group. The QLME exhibited no mortality in acute toxicity study. It was concluded that QLME possessed substantial anti-inflammatory and anti-arthritic potential at all dosage levels, mainly at 600 mg/kg might be due to presence of quercetin, sinapic acid and ferulic acid.

Effect of Human Mesenchymal Stem Cells on Freund's adjuvant-induced Rheumatoid Arthritis in Sprague Dawley Rats

Introduction: Mesenchymal stem cells (MSC) therapy is a new approach to treat RA. Studies evaluating anti-inflammatory effects of MSCs per RA severity are scarce. Our primary objective was to evaluate anti-inflammatory effects, change in cytokine levels and cartilage regeneration of two different MSC preparations delivered through two different routes of administration in three RA stages: mild, moderate and severe. Methods: Human-derived umbilical cord tissue MSCs (hUCT-MSCs) and human bone marrow-derived MSCs (hBM-MSCs) delivered via intra-plantar and intravenous routes were tested in Freund's adjuvant-induced arthritis in rats. Arthritis severity was based on the arthritis score (<3=mild, 3=moderate and 4=severe). Assessments included changes in arthritis scoring, paw swelling, haematology parameters, biomarkers (TNFα and IL-10) and histopathology analysis. Results: MSC treatment significantly reduced arthritis scores in all treatment groups. IL-10 levels increased 30 days after treatment with (IP)hUCT-MSCs (P=0.0241), (IV)hUCT-MSCs (P=0.0095) and (IP)hBM-MSCs (P=0.0002). TNF-α levels reduced compared to positive control at 30 days: (IP)hUCT-MSCs (P=0.0060), (IV)hUCT-MSCs (P=0.0003), (IP)hBM-MSCs (P=0.0005), (IV)hBM-MSCs (P<0.0001) and continued through 30-60 days. Microscopic examination showed regenerative changes in animal joints treated with both intra-plantar or intravenous MSCs. Arthritis scores reduced in all RA severity groups while benefits (changes in IL-10 and TNF-α) were more pronounced in moderate and severe RA. Haematology parameters remained similar among all animal groups at baseline, 30 days and 60 days indicating safety of MSCs. Conclusion: Treatment with hUCT MSCs and hBM MSCs were safe, well-tolerated and effectively reduced joint inflammation, synovial cellularity and pro-inflammatory cytokine levels in CFA-induced RA rat model.

Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis

The 68Gallium-labeled 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid conjugated radiolabelled arginine-glycine-aspartic acid peptide ([68Ga]Ga-NODAGA-RGD) is a positron emission tomography (PET) tracer binding to cell surface receptor αvβ3 integrin that is upregulated during angiogenesis and inflammation. We studied whether αvβ3 targeting PET imaging can detect myocardial inflammation in a rat model of autoimmune myocarditis. To induce myocarditis, rats (n = 8) were immunized with porcine cardiac myosin in complete Freund's adjuvant on days 0 and 7. Control rats (n = 8) received Freund's adjuvant alone. On day 21, in vivo PET/CT imaging with [68Ga]Ga-NODAGA-RGD followed by ex vivo autoradiography and immunohistochemistry were carried out. Inflammatory lesions were detected histologically in the myocardium of 7 out of 8 immunized rats. In vivo PET images showed higher [68Ga]Ga-NODAGA-RGD accumulation in the myocardium of rats with inflammation than the non-inflamed myocardium of control rats (SUVmean 0.4 ± 0.1 vs. 0.1 ± 0.02; P = 0.00006). Ex vivo autoradiography and histology confirmed that [68Ga]Ga-NODAGA-RGD uptake co-localized with inflammatory lesions containing αvβ3 integrin-positive capillary-like structures. A non-specific [68Ga]Ga-DOTA-(RGE)2 tracer showed 76% lower uptake than [68Ga]Ga-NODAGA-RGD in the inflamed myocardium. Our results indicate that αvβ3 integrin-targeting [68Ga]Ga-NODAGA-RGD is a potential PET tracer for the specific detection of active inflammatory lesions in autoimmune myocarditis.

Effects of different doses of complete Freund’s adjuvant on nociceptive behaviour and inflammatory parameters in polyarthritic rat model mimicking rheumatoid arthritis

Complete Freund’s adjuvant (CFA) has been used to develop the arthritic or inflammatory condition in the animal, but there is a lack of information concerning high CFA doses on nociceptive behaviour and inflammatory parameters. This study aimed to compare the effects of different high doses of CFA in rat to closely mimic nociceptive and inflammatory parameters of rheumatoid arthritis (RA) in humans. Twenty-four male Sprague-Dawley rats were randomly divided into four groups (n = 6): Control (C), CFA-induced polyarthritic groups at 5.0 mg/mL (CFA 5.0), 7.5 mg/mL (CFA 7.5) and 10.0mg/mL (CFA 10.0). The rats’ right hindpaw was inoculated with CFA intradermally and developed into a polyarthritic state within 20 days. Nociceptive behavioural assessments, including von Frey and hot plate tests and spontaneous activities, were conducted on day 0, 7, 15 and 20. Bilateral ankle joints diameter and circumference, full blood count, joints and paw histological examinations were also conducted throughout the study period. Based on the results, CFA 5.0 and CFA 7.5 groups showed a significant increase in spontaneous activities and development of thermal hyperalgesia but no change in body weight and food intake, no development of tactile allodynia and haematological indices, and no significant morphological changes of joints histology. Meanwhile, CFA 10.0 group demonstrated significant and constant changes in all nociceptive and inflammatory parameters investigated. In conclusion, CFA at the dose of 10mg/mL has the most potential and reliable dosage to develop polyarthritis in a rat model to mimic RA condition in humans.

Anti-Inflammatory Effects of BoNT/A Against Complete Freund’s Adjuvant-Induced Arthritis Pain in Rats: Transcriptome Analysis

Arthritis is the most common cause to lead to chronic pain. Botulinum toxin type A (BoNT/A) has been widely used to treat chronic pain. In our previous study, we confirmed the anti-inflammatory and antinociceptive effects of BoNT/A in the Complete Freund’s Adjuvant (CFA)-induced arthritis model, but the underlying anti-inflammatory mechanism was not fully elucidated. The purpose of this study was to investigate the anti-inflammatory effects and mechanisms of BoNT/A on arthritis using transcriptomic analysis. The BoNT/A was injected into the rat ankle joint on day 21 after CFA injection. The von Frey and hot plate tests were applied to assess the pain-related behaviors at different time points. Five days after BoNT/A treatment, gene expression profiling in dorsal root ganglion (DRG) was performed using RNA sequencing (RNA-seq). The differentially expressed genes (DEGs) were analyzed by various tools. The mechanical allodynia and thermal hyperalgesia were significantly reversed after BoNT/A injection. RNA-seq revealed 97 DEGs between the CFA group and Sham group; these DEGs were enriched inflammatory response, IL-17 signaling pathway, etc. There are 71 DEGs between the CFA+BoNT/A group and the CFA group; these DEGs related to response to peptide, PI3K-Akt signaling pathway, ECM–receptor interactions, etc. Three key genes were significantly decreased after CFA-induced arthritis pain, while BoNT/A increased the expression of these genes. The identification of S100A9, S100A8, and MMP8 genes can provide new therapeutic targets for arthritis pain and affect the signaling pathway to play an anti-inflammatory role after the treatment of BoNT/A.