Article

Effects of Cannabidiol on the In Vitro Lymphocyte Pro-Inflammatory Cytokine Production of Senior Horses

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Abstract

Cannabis sativa L. contains cannabidiol (CBD), a compound that has many anti-inflammatory properties. In this study, 99.9% CBD powder was used to determine its in vitro efficacy as an anti-inflammatory agent. Heparinized blood was collected via jugular venipuncture from senior horses. PBMCs were isolated then incubated for 24 hours with increasing dilutions of CBD dissolved in DMSO. PBMCs were stimulated the last 4 hours of incubation with PMA/IO and Brefeldin A. A Vicell counter was used to evaluate viability after incubation. PBMCs were stained intracellularly for IFNγ and TNFα then analyzed via flow cytometry. RT-PCR was used to analyze samples for gene expression. Five equine-specific intron-spanning primers/probes used are: CB1, CB2, TNFα, IFNγ, IL-10, and Beta-glucuronidase. Data was analyzed using RM One-way ANOVA (significance P< 0.05). Viability of PBMCs with CBD was completed to determine cytotoxicity. The dilution of CBD that did not affect cell viability was 4 µg/mL (P<0.05). CBD at 4 µg/mL significantly reduced production of IFN-γ and TNF-α (P<0.05). RT-PCR results for TNFα and IFNγ at 4 µg/mL showed a reduction compared with the positive control and IL-10 showed a similar reduction at 2 µg/mL and 4 µg/mL. RT-PCR gene expression results showed significance for 10 μg/mL CBD in CB1 and CB2. CBD at 4 µg/mL reduced in vitro production of inflammatory cytokines from senior horses. This in vitro study supports further investigation of CBD to determine if it may be effective as an anti-inflammatory treatment for chronic inflammation in the horse.

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... In fact, we have recently shown that CBD has an effect on inflammaging in vitro. 24 In that study, peripheral blood mononuclear cells collected from senior horses were incubated in vitro with various concentrations of pure CBD. We showed that at increasing concentrations of CBD there were reduced concentrations of proinflammatory cytokines TNFα and interferon-γ (IFNγ), and results indicated that CBD has the potential to modulate inflammation in horses 24 ; however, in vivo studies are warranted. ...
... Blood samples were collected via aseptic jugular venipuncture into EDTA-prepared tubes (Covetrus). Collection time points for both treatments were immediately before administration of CBC (time 0) and then 0.5, 1,4,8,24,48,72,96,120,144,168,192,216,240, and 264 hours after CBD administration. All blood samples were placed on ice immediately and within an hour of collection and centrifuged at 800 X g for 10 minutes, and then plasma was aliquoted into 1.5-mL plastic microcentrifuge tubes and stored at -20 °C until analyzed. ...
... The y-axes are logarithmically scaled. A and C-Results for evaluations at 0.5,4,8,24, 48, 72, 96, 120, 144, 168, 192, 216, 240, and 264 hours after treatment. B and D-Expansion of the x-axes allows depictions of the results for just the first 24 hours in greater detail. ...
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Objective: To determine the pharmacokinetics, bioavailability, and pharmacological effects of cannabidiol (CBD) in senior horses. Animals: 8 university-owned senior horses. Procedures: In this randomized, crossover study, horses were assigned to receive either a single oral dose of 2 mg/kg CBD in oil or a single IV dose of 0.1 mg/kg CBD in DMSO between August 10 and September 4, 2020. Blood samples were collected before and then 0.5, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, and 264 hours after CBD administration. Serum biochemical analyses and CBCs were performed. Plasma concentrations of CBD and its metabolites were determined with the use of liquid chromatography-tandem mass spectrometry. Results: Concentrations of CBD and metabolites (7-COH CBD and 7-COOH CBD) were detected in all plasma samples up to 8 hours after dosing (oral and IV), with 7-COOH CBD being the most predominant metabolite. Pharmacokinetic results for CBD oral dosing at 2 mg/kg were mean ± SD half-life of 7.22 ± 2.86 hours, maximum concentration of 18.54 ± 9.80 ng/mL, and time to maximum concentration of 2.46 ± 1.62 hours. For both oral and IV administrations, 7-COOH CBD did not fall below the limit of quantification for the times reported. Oral bioavailability for CBD was 7.92%. There was no meaningful effect of CBD on results for CBC, serum biochemical analyses, or vital signs for any horse. Clinical relevance: Pharmacokinetics and bioavailability of CBD in senior horses were determined, and there were no adverse effects of administering either the oral or IV dose of CBD evaluated.
... [18][19][20] Other studies in equine regarding the location of cannabinoids receptors and in vitro effects of cannabinoids on cytokine production also have been reported. [21][22][23] As a consequence, for a prudent use of CBD, it is mandatory to know the PK parameters derived after IV administration, such as volume of distribution (V ss ), clearance (Cl) and terminal half-life (t 1/2 ). Subsequently the bioavailability after oral route should be determined as has been recommended in the horse. ...
... [34][35][36] As inflammation and pain are very common in horses, there is great clinical interest in the development of specific formulations of CBD. 17,23,36 Nonlinear mixed effects models are not frequent for PK analysis in veterinary medicine although its use is increasing in recent years, mainly due to its ability to analyse unbalanced data, especially to cross-over designs very common in veterinary pharmacology. 31 On the other hand, it allows more robust simulations than with the classic two-stage methods and more efficient analysis of simultaneous data with different doses and routes, as have been recommended. ...
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Background Intravenous pharmacokinetics and oral bioavailability of cannabidiol (CBD) with different formulations have not been investigated in horses and may represent a starting point for clinical studies. Objectives To describe pharmacokinetics after intravenous and oral administrations with oil and micellar formulations and simulate different treatments. Study design Single intravenous experiment and two‐way randomised oral experiments, Latin‐square design. Methods Eight healthy horses received intravenous CBD at 1.00 mg/kg dose, oral CBD in sesame oil and in micellar formulation, both at 10.00 mg/kg. Concentrations were measured using LC–MS/MS and fitted by nonlinear mixed effect modelling. Parameters obtained were used to simulate single and multiple treatments at steady state. Results Intravenous and oral concentrations were simultaneously fitted using a three‐compartment model. Final estimates indicate that CBD has a volume of distribution of 36 L/kg associated with a systemic clearance of 1.46 L/h/kg and half‐lives ranged between 24 and 34 h. Oral bioavailability was close to 14% for both oral administrations. Simulated dose regimen of CBD every 12 and 24 h predicted similar percentages to reach effective plasma concentration with both oral formulation at 10.00 mg/kg. Main limitations A small horse population was used (8 horses per trial). Conclusions and clinical importance Oral bioavailability was low at the doses studied but fell within the range described for horse and other species. CBD had a high steady‐state volume of distribution, a high clearance and long half‐lives. No adverse reactions were detected at any dose or route. The micellar formulation showed a faster absorption and higher concentration peak, while the oil formulation presented lower levels, but more maintained over time. Simulations predicted that both could be useful in multiple oral dose treatments. These results indicated that CBD could be of interest, but further studies are needed to evaluate its clinical use in horses.
... In a preclinical study using rats with autism-like behavior, created through prenatal valproic acid exposure, CBDV proved to ameliorate behavioral abnormalities, restore hippocampal endocannabinoid signaling, and decrease neuroinflammation, indicating that CBDV could be a potential therapeutic agent for autism spectrum disorders [149]. ...
... In healthy people, ∆9-THC can cause acute psychotic reactions, together with a temporary decline in both cognitive function [9] and psychomotor control [164]. In patients with schizophrenia, ∆9-THC may intensify memory impairments, psychotic symptoms, and anxiety [150]; additionally, several studies have indicated a relationship between systematic Cannabis use and an increased risk of developing this condition [149]. Currently, there is growing preclinical evidence that ∆9-THC can suppress inflammation by activating CB2 or CB1/CB2 through multiple pathways, including: shifting the balance of human T helper 1 (Th1)/T helper 2 (Th2) cells [95], T reg differentiation [140], myeloid-derived suppressor cell induction (MDSC) [151], generation of apoptosis in dendritic and activated T cells [156,157], or induction of immunosuppressive MDSCs and T regs [165]. ...
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The most important discoveries in pharmacology, such as certain classes of analgesics or chemotherapeutics, started from natural extracts which have been found to have effects in traditional medicine. Cannabis, traditionally used in Asia for the treatment of pain, nausea, spasms, sleep, depression, and low appetite, is still a good candidate for the development of new compounds. If initially all attention was directed to the endocannabinoid system, recent studies suggest that many of the clinically proven effects are based on an intrinsic chain of mechanisms that do not necessarily involve only cannabinoid receptors. Recent research has shown that major phytocannabinoids and their derivatives also interact with non-cannabinoid receptors such as vanilloid receptor 1, transient receptor ankyrin 1 potential, peroxisome proliferator-activated receptor-gamma or glitazone receptor, G55 protein-coupled receptor, and nuclear receptor, producing pharmacological effects in diseases such as Alzheimer’s, epilepsy, depression, neuropathic pain, cancer, and diabetes. Nonetheless, further studies are needed to elucidate the precise mechanisms of these compounds. Structure modulation of phytocannabinoids, in order to improve pharmacological effects, should not be limited to the exploration of cannabinoid receptors, and it should target other courses of action discovered through recent research.
... For instance, CBD at concentrations ranging from 2.5 to 10 µg/mL was found to markedly inhibit the proliferation of canine cancer cells [34]. Another study on the toxicity of CBD on PBMC cells in horses demonstrated that CBD affected cell viability at a concentration range of 6-10 µg/mL [35]. However, these concentrations, including those in the present study, are often considered to exceed normal physiological ranges. ...
Article
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Cannabidiol, the primary non-psychoactive phytocannabinoid found in cannabis, has generated significant research interest due to its potential for biological effects, such as anti-inflammatory, analgesic, immunomodulatory, and anticonvulsant properties. Several studies have demonstrated the potential of CBD to alter inflammatory cytokines; however, data on CBD’s effects on cell viability and pro-inflammatory cytokines in target animals, such as dogs, are limited. Therefore, in this study, we investigated the effects of CBD on the cell viability and modulation of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), in canine PBMCs stimulated with LPS. To evaluate the effect of CBD on neuroinflammation in epilepsy pathology, an independent study of five refractory epileptic dogs co-treated with CBD for 30 days was conducted. The current findings revealed that CBD concentrations of 16 µg/mL had a statistically significant effect on the viability of canine PBMCs with a calculated IC50 of 15.54 µg/mL. The effect of CBD on inflammatory cytokines in LPS-stimulated PBMCs tended to be dose-dependent, with CBD concentrations of 5–30 μg/mL resulting in decreased production of the tested pro-inflammatory cytokines. Considering the effect of CBD on cytokine production by PBMCs from epileptic dogs, CBD has the potential to modulate immune responses and provide benefits when used in combination with antiepileptic drugs. The findings provided evidence of CBD cytotoxicity and its effect on the alteration of pro-inflammatory cytokines in canine PBMCs.
... 5 An in vitro experiment reported that CBD can reduce inflammatory cytokine production. 6 Abnormal mitochondrial function leads to the development of a wide variety of neurodegenerative, diabetes mellitus, and cardiovascular disorders. CBD modulates mitochondrial activities through voltage-dependent anion channel-I, which functions to exchange important metabolites involved in energy metabolism (ie, ADP and ATP) across the outer mitochondrial membrane. ...
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Background: Cannabidiol (CBD) is a non-psychoactive cannabinoid derived from Cannabis sativa L. with very low toxicity for human and a wide variety of therapeutic uses as medicine. The study objective is to evaluate the impact of CBD on vitamin D 3 receptor (VDR) protein expressions, tissue elasticity, anti-inflammatory, and anti-senescence activity in human and rodent cell lines. Methods: Cell viability was estimated by MTT assay. Relative quantification (RQ) of VDR protein expression was measured by RT-PCR. Tissue elasticity was measured by atomic force microscopy (AFM). Cellular senescence and ATP were performed using trypan blue exclusion test and colorimetric assay, respectively. Results: Cell viability assay data showed CBD was safe and nontoxic upto 7.5 µM. The VDR protein expression was significantly increased by 109.71% (p = 0.013), 236.96% (p ≤ 0.001), 170% (p ≤ 0.001), 100% (p = 0.019), 80% (p = 0.021), 427.27% (p ≤ 0.001), 366.67% (p ≤ 0.001), 56.31% (p = 0.016), and 63.84% (p ≤ 0.001) in MG-63, MDA-MB-231, SH-SY5Y, HEK-293, HT-29, EaHy-926, HepG2, A-549, and C2C12 cells, respectively compared to the normal control group. CBD treatment significantly reduced the levels of TNF-α (46.58%; p ≤ 0.049) and IL-6 (43.61%; p ≤ 0.001) at CBD-5 µM compared to the vehicle control group. Tissue elasticity was significantly (p ≤ 0.001) increased by 37.09% and 49.49% in CBD-2.5 and CBD-5 µM, respectively, compared to the vehicle control group. Significantly (p ≤ 0.001) reduced senescence cells by 39.22% in CBD-5 µM than the vehicle control group. The level of ATP was significantly (p ≤ 0.001) increased by 90.55, 117.06, and 153.54% in CBD-1, CBD-2.5, and CBD-5 µM, respectively, compared to the vehicle control group. Conclusion: Overall, data suggest that CBD considerably improved VDR protein expression, inflammation, cell growth, tissue elasticity, and enhanced mitochondrial bioenergetics in multiple cell lines. In this study, for the first time, we showed the evidence suggesting that the VDR plays a critical and multifaceted role in various types of human cells.
... Previous research has indicated that cannabinoids found in cannabis can inhibit inflammation in-vitro. For instance, CBD has been shown to significantly reduce the production of IFN-γ and TNF-α (Turner et al., 2021), while Δ 9 -THC has been found to decrease the level of IL-1β by inhibiting NLRP3 inflammasome activation (Suryavanshi et al., 2022). Similarly, turmeric rhizomes, particularly curcuminoids, have demonstrated anti-inflammatory activity, with curcumin being the most potent in suppressing TNF-induced nuclear factor kappa B activation, followed by DMC and BDMC (Sandur et al., 2007). ...
... In addition to the core pathology, patients with MS commonly face a myriad of symptoms, including pain, spasticity, and motor dysfunction, which significantly impact their daily lives. CBG exhibits anti-inflammatory properties and interacts with receptors involved in pain modulation, suggesting its potential use to relieve the challenges faced by MS patients [56,118]. Furthermore, CBG has neuroprotective properties and may promote remyelination and mitigate the progression of MS [119]. ...
Article
Neurodegenerative disorders pose significant societal and individual burdens, marked by progressive degeneration of the nervous system. The limited efficacies of current treatments underscore the pressing need for innovative therapeutic approaches. Cannabigerol (CBG) is a cannabinoid derivative found in the plant genus Cannabis that is viewed as a molecule of therapeutic interest due to its unique pharmacological profile and interactions with the endocannabinoid system (ECS). This comprehensive review explores the therapeutic potential of CBG for the treatment of disorders of the nervous system, such as Alzheimer’s and Parkinson’s disease. Preclinical investigations have revealed that the neuroprotective properties of CBG involve ECS modulation, promoting neuronal survival, and mitigating neuroinflammation and oxidative stress. However, it should be acknowledged that much of the research on the therapeutic potential of CBG is still in its early stages and primarily consists of in vitro and animal studies. Further clinical trials and research are needed to determine the efficacy and safety of CBG and the optimal dosing strategies required to treat neurodegenerative diseases. This review also explores the transformative potential of nanocarrier systems in CBG-centered therapies targeting neurodegenerative disorders and discusses the merits and demerits of various nanocarrier systems. This dual focus on the therapeutic potential of CBG and innovative distribution systems underscores the extent of the investigative work required to advance neurodegenerative disease management.
... It has been recognized to effectively reduce seizure severity and frequency in children and young adults (3,4) and appears promising for similar use in anticonvulsant-resistant epileptic animals (5). Furthermore, CBD has been demonstrated to have anti-inflammatory and immunomodulating properties in models using canine and equine whole blood (6,7). ...
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Objective To determine the pharmacokinetics (PK) of two oral doses of a Cannabis herbal extract (CHE) containing 1:20 THC:CBD in 12 healthy Domestic Shorthair cats. Methods Single-dose PK were assessed after oral administration of CHE at low or high dose (2 mg CBD + 0.1 mg THC, or 5 mg CBD + 0.25 mg THC per kg bw, respectively; n = 6 per group) in fasting cats. Blood samples were drawn up to 48 h following CHE administration. Plasma samples were analyzed for CBD, THC, and metabolites 6-OH-CBD, 7-OH-CBD, 11-OH-THC, and THC-COOH using a previously validated LC–MS/MS method. Results CBD and THC were quickly absorbed (mean Tmax of 2.4–2.9 h). Maximum plasma concentrations (Cmax) ranged from 36–511 ng/mL and 6.8–61 ng/mL for CBD and THC, respectively. Elimination was initially rapid for both CBD and THC, though a prolonged elimination phase was noted for CBD in some cats (T1/2 λ up to 26 h). Dose-adjusted Cmax and AUC0-last values were not statistically significantly different (p > 0.05) between dose groups indicating CBD and THC concentrations increased in a manner proportional (linear) to the dose. Dose-adjusted THC Cmax and AUC0-last were significantly higher than the corresponding dose-adjusted CBD parameters (p < 0.01). Low concentrations of the metabolite 6-OH-CBD were quantified but metabolites 7-OH-CBD, 11-OH-THC, and THC-COOH were not detected in any plasma samples. Inter-individual variance was notable. Salivation shortly after dosing was observed in two cats in the high dose group; these animals had substantially lower cannabinoid concentrations than other cats in this group. No adverse clinical signs (including vomiting, change in mentation or other neurological signs) were noted. Clinical significance Although cats did not display adverse effects after administration of a single oral dose of 1:20 THC:CBD CHE formulation at 2 or 5 mg CBD/kg bw, observed plasma concentrations were highly variable but generally lower than in dogs receiving the same dose and formulation. Administration of CHE in the fasting state may not optimize CBD absorption, and oral dosing may be challenging when administering an oil-based CHE in some cats.
... Similar results could be obtained in horses, which are also sensitive to stress as well. Studies in mice, dogs, and horses have shown that CBD reduces the production of inflammatory cytokines such as tumor necrosis factor alpha (TNFα), which may help to reduce inflammation in various tissues and promote a calming effect and reduced anxiety in horses (24,46,47). The OTM route has shown good absorption and efficacy of CBD 15% and therefore represents an interesting alternative to other routes of administration as it is a non-invasive and painless technique that allows the use of standardized concentrations and doses, requires minimal constriction and does not cause discomfort to the patient (3,25,48,49). ...
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The aim of this study was to evaluate the effect of oral cannabidiol (CBD) administration in addition to a conventional analgesic protocol on the clinical signs of 20 horses with mild joint osteoarthritis. The horses were randomly assigned to either the control group (C group) or the cannabidiol group (CBD group). Both groups were treated with phenylbutazone for 5 days. The CBD group received 0.03 mg/kg cannabidiol in hemp oil orally once daily for 14 days in addition to phenylbutazone treatment. All subjects were monitored for clinical parameters, oxidative status and blood counts. Pain and quality of life were also assessed using the Horse Chronic Pain Scale (HCPS). The CBD group showed a significant reduction in heart rate, respiratory rate, white blood cell count and oxidative stress (malondialdehyde lipid peroxidation). A significant reduction in HCPS scores was seen in both groups. Lower scores were recorded in the CBD group (3 med; range: 2/4) than in the C group (7 med; range: 4/10). The addition of a cannabidiol-based product to an analgesic protocol was well tolerated and showed positive effects on the treated subjects, improving their quality of life and pain relief.
... Produkcja cytokin zapalnych in vitro u koni starszych zmniejszyła się przy stężeniu CBD 4 µg/mL. Świadczy to o przeciwzapalnym działaniu kannabidiolu i daje podstawy do przeprowadzania dalszych badań poprzez suplementację CBD (21). ...
Article
Cannabidiol (CBD) is one of the cannabinoids found in Cannabis sativa L. The action of CBD in the body is similar to that of neurotransmitters, thanks to which it regulates most of the biochemical processes taking place in the body. Research on CBD properties in equine veterinary medicine has begun relatively recently. It has recently been proven to reduce inflammation, relieve pain, stabilize the nervous system, and have a calming effect. Human studies suggest that, due to its effects on the endocannabinoid system, CBD will have a number of positive effects on equine homeostasis. Current research is focused on determining appropriate CBD doses for desirable therapeutic or heeling effects. The results of studies carried out so far have confirmed the effectiveness of CBD, which may lead to further research on the use of CBD in the treatment and health prevention of horses.
... The use of cannabidiol (CBD), one of the non-psychoactive compounds found in hemp, is gaining in popularity as a potential alternative to conventional pharmaceutical treatments for a variety of conditions, including but not limited to arthritis and anxiety. Studies conducted in mice and horses have shown that CBD reduces the production of inflammatory cytokines such as tumor necrosis factor-α (TNFα), which may help reduce inflammation in various body tissues or may have a calming effect and lessen anxiety in horses [1,2]. Horses receiving 40 g of a pelleted CBD supplement containing 100 mg of CBD over 6 weeks were less reactive to a novel stimulus compared to a control group; however, heart rates were not different and blood serum was not evaluated [3]. ...
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Thirty stock type geldings (15 ± 3 years; 556 ± 63 kg BW) were used in a randomized complete design over 28 days to determine the influence of cannabidiol (CBD) oil supplementation levels on body weight, body condition, and blood chemistry. Horses were randomly assigned to one of three dietary treatments (n = 10 per treatment) formulated with canola oil to provide 1.50 mg CBD/kg BW (TRTA), 0.75 mg CBD/kg BW (TRTB), or 0.00 mg CBD/kg BW (canola oil; CTRL). Treatments were top-dressed onto concentrate and individually administered twice daily. Horses were maintained in adjacent dry lots and received coastal bermudagrass hay ad libitum. Body weight and body condition scores (BCS) were obtained every 14 days. On day 0 and 28, blood was collected via jugular venipuncture and serum was harvested to perform a blood chemistry panel and drugs of abuse screening at the Texas Veterinary Medical Diagnostic Laboratory. Data were analyzed using PROC MIXED of SAS (v9.4), and the model included treatment, time, and the treatment × time interaction, and linear and quadratic orthogonal polynomial contrasts to partition sum of squares. Analysis of composited treatment samples revealed lower CBD concentrations than indicated from initial testing by the manufacturer (0.13 mg CBD/kg in TRTA; 0.12 mg CBD/kg in TRTB). At this level of supplementation, canola-based CBD oil was well-accepted by mature horses, banned substances were not detectable in blood, and blood chemistry parameters were not adversely affected as a result of supplementation. More research is warranted to describe the discrepancy between formulated levels compared to tested levels of CBD in the canola-based supplement.
... 37 Cannabinoid's anti-inflammatory effects on equine lymphocytes in vitro have also been measured for the first time. 38 However, thus far, only one case report that identified a response of mechanical allodynia to therapy in a horse has been published. 39 The principal objective of the current study was to characterise the cellular expression of CB1 and CB2 in healthy synovial tissue to advance the understanding of the potential role of cannabinoid receptors in equine joint pain. ...
Article
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Background Osteoarthritis (OA) is a major cause of equine lameness. Cannabinoid (CB) receptors are now considered to be promising therapeutic targets in human rheumatology for pain and inflammation, however, little is known about the equine endocannabinoid system. Objectives The primary goal was to assess the presence and expression pattern of CB1 and CB2 in the synovium of healthy joints. A secondary goal was to explore the relationship between the CB expression, degree of synovitis and OA pathology. Study design Ex vivo experimental study. Methods Metacarpophalangeal joints (n = 25) from a tissue bank were studied. The joints were dissected, and the articular cartilage lesions were scored. Synovial membrane specimens (n = 45) were harvested, fixed and the degree of synovitis was graded on histological sections. Colocalised synovial sections were also immunostained with antibodies to CB1 and CB2. Five regions of interest were randomly selected from digital images of manually segmented synovial intima and scored blindly for positive cellular immunoreactive staining by two independent observers. Interobserver agreement was calculated with an intraclass correlation coefficient (ICC). Relationships between CB1 and CB2 immunoreactive scores and synovitis or joint OA grade were explored with mixed linear models. Results CB1 was expressed in synovial intimal cells in all specimens studied whereas CB2 expression was identified in 94%. Both receptors were also expressed in the subintimal blood vessel walls. ICCs were 84.6% (CB1) and 92.9% (CB2) for the immunoreactivity scores. Both CB1 and CB2 expression were significantly upregulated (p = 0.04 and p = 0.03, respectively) with increasing degree of synovitis. Conversely, CB1 expression significantly decreased (p = 0.03) with increasing severity of OA. Main limitations The type of synovial cell expressing CB1 or CB2 was not investigated. Conclusion Equine synovial intimal cells constitutively express both CB1 and CB2 receptors that are upregulated with synovitis and may have a role in joint pain. They are potential targets for therapy with cannabinoid molecules or their derivatives.
... 37 Cannabinoid's anti-inflammatory effects on equine lymphocytes in vitro have also been measured for the first time. 38 However, thus far, only one case report that identified a response of mechanical allodynia to therapy in a horse has been published. 39 The principal objective of the current study was to characterise the cellular expression of CB1 and CB2 in healthy synovial tissue to advance the understanding of the potential role of cannabinoid receptors in equine joint pain. ...
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Cannabidiol (CBD) is a non-psychotropic cannabinoid obtained from hemp (Cannabis sativa L.) used for pain management in companion animals including horses. The present study aimed to evaluate the efficacy of cannabidiolic acid (CBDA) and cannabigerol/cannabidiol oil (CBG/CBD) oral administration in alleviating pain in adult horses affected by chronic osteoarthritis (OA). Twenty-four horses (10 geldings and 14 mares), aged between 11 and 18 years old, were equally divided into two groups. One group received CBDA 15% oil and the other group received CBG/CBD oil (CBG20%-CBD10%) for 14 consecutive days. A standard dose of 0.07 mg/kg was chosen based on the mean body weight of 450 ± 28 kg. Horse Chronic Pain Scale (HCPS) and physiological parameters monitoring heart rate (HR), respiratory rate (RR), arterial blood pressure (systolic arterial pressure- SAP, diastolic arterial pressure- DAP) were assessed before (T0) and every day for the entire administration (T1-T14). Blood samples were collected for the evaluation of complete hemogram, Leukocyte subpopulation identification and counting and leukocyte differentiation antigens CD4 and CD8 at the day before the administration (T0) and every 7 days (T7 and T14). A reduction of HCPS pain scale scores and the number of WBC, monocytes and neutrophils and CD8 was observed with both CBDA and CBG/CBD treatment. No statistical differences were found in the physiological parameters. No subject required rescue analgesia or showed any adverse effects. The results of this study showed that oral administration of both CBDA and CBG/CBD oil may promote pain reduction in adult horses affected by chronic OA.
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Introduction: CD19-chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy for cancer treatment that has shown remarkable clinical responses, leading to approval by the FDA for relapsed and refractory B cell hematological malignancy treatment. Cannabidiol (CBD) is a nonpsychoactive cannabinoid compound that has been utilized as a palliative treatment in cancer patients due to its immunosuppressive properties. Currently, studies on using CBD during immunotherapy have gained increasing attention. However, the possible interaction between CBD and CAR T cell therapy has not been studied. Therefore, in this study, we aimed to examine the direct effects of CBD on CD19-CAR T cell function against hematologic malignancies. Materials and Methods: The cytotoxic effect of CBD was determined by a cell proliferation reagent water-soluble tatrazolium salt (WST-1) assay. CAR T cells were generated by retroviral transduction and treated with CBD at a nontoxic dose. The effect of CBD on immune characteristics, including transgene expression, T cell subset, and memory phenotype, was analyzed by flow cytometry. Proliferation, apoptosis, and cell cycle distribution were analyzed with standard methods. The effect on cytotoxic function was evaluated using degranulation assays, and antitumor activity was evaluated using flow cytometry. Results: The half-maximum inhibitory concentration (IC50) of CBD on NALM6, Raji, and T cells ranged from 16 to 22 μM. The maximum nontoxic dose of CBD that maintained cell viability at ∼100% was 8 μM. For the generation of CD19-CAR T cells, primary T cells were activated and transduced with a retroviral vector encoding CD19-CAR. CBD did not alter the surface expression or immune characteristics, including the T cell subset and memory phenotype, of CD19-CAR T cells. However, CBD suppressed CD19-CAR T cell proliferation by inducing apoptosis, as evidenced by an increase in the proportion of cells in the Sub-G1 phase in cell cycle arrest. However, the antitumor activity and cytokine secretion of CD19-CAR T cells were not altered by exposure to CBD in this study. Conclusions: In this study, a nontoxic dose of CBD affected CD19-CAR T cell proliferation but not its immune characteristics or cytotoxic function.
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Cannabidiol (CBD) has potential to reduce pain and inflammation in humans leading to the interest of use in equine. The purpose of this study was to determine the effects of CBD on immune function by measuring inflammatory cytokines and antibody responses to vaccination, as well as other health parameters in senior horses. Horses were orally-dosed with CBD (2 mg/kg: 13 horses) or control (soy oil: 14 horses) daily for 90 days, from July 2021 to November 2021. Peripheral blood samples were collected on days 0, 30, 60, and 90 before administering treatments. On day 90 all horses were kept on treatment and vaccinated with an equine influenza vaccine and blood samples were collected post-vaccination on days 14 and 21. For all time points, plasma samples were analyzed for determination of CBD and metabolites, 7-OH CBD and 7-COOH CBD, using tandem mass spectrometry. For time points 0, 30, 60 and 90, blood samples were analyzed for CBC and chemistry. Additionally, peripheral blood mononuclear cells (PBMC) were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, stained intracellularly for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) then analyzed via flow cytometry. Real-time PCR (RT-PCR) analyzed both stimulated PBMCs and whole blood for cytokine gene expression. Inflammatory proteins C-reactive protein, interleukin 1 receptor agonist, and prostaglandin E2 were measured with equine-specific enzyme linked immunosorbent assays. Thyrotropin-releasing hormone stimulation test and oral sugar test were performed on all horses before and after the study to analyze metabolic function. Hemagglutination inhibition (HI) titers were measured for immune responses pre- and post-vaccination. All data were analyzed using either a paired t-test or a two-way repeated measures analysis of variance (significance P < 0.05). Plasma concentrations of CBD and metabolites were determined with 7-COOH CBD, the most significant metabolite, in CBD treated horses compared to control treated horses. A significant decrease was determined for whole blood inflammatory cytokine expression of IFN-γ at day 60, and for IL6 at day 60 and 90 for CBD-treated horses when compared to control horses. CBD did not significantly affect any other immune factors, HI titers, or health parameters. This study demonstrated that treatment with CBD reduced some inflammatory cytokine production with no negative side effects as measured by CBC or chemistry profiles. This study reveals the initial understanding of CBD in the horse, however more in-depth research is needed to fully understand its efficacy on the health of the horse.
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Supplements containing Cannabidiol (CBD) are available for horses, however, few studies have been published on their effects on behavior and health parameters. The purpose of this study was to determine if a daily oral supplement containing CBD would cause sedation, ataxia or alterations in other health parameters during administration for 56 days. Twenty clinically healthy adult Thoroughbred horses were housed in stalls. Before treatment was initiated, a complete physical examination, complete blood count (CBC) and biochemical panel were evaluated. In addition, horses were examined for sedation and ataxia using standard scoring systems. Horses were randomly divided into two treatment groups, treated (supplement pellets containing CBD as Hemp Extract, 150 mg) or control (supplement pellets without CBD). Horses were treated daily and sedation and ataxia scores were assigned by two masked observers once weekly for 56 days. Horses were monitored daily for clinical signs or adverse events and body weights were recorded weekly. A CBC and biochemical panel were repeated on days 28 and 56, two hours after administration of the supplement. The supplement was readily consumed by the horses and no adverse effects were seen over the treatment period. Sedation and ataxia scores ranged from 0 to 2 for all horses during the weekly examinations and there was no statistical difference between treatment groups. There were no treatment effects on blood values, including indicators of anemia and blood proteins, liver enzymes, kidney values, electrolytes or calcium. Body weight significantly increased in all horses, by Day 56 compared to Day 0 but no treatment by day effect was noted. The CBD supplement (150 mg) was readily consumed and safe and did not result in changes in mentation, gait, or other health parameters, and no adverse clinical signs were observed during 56 days of oral administration.
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Cannabidiol (CBD) products have gained popularity among horse owners despite limited evidence regarding pharmacokinetics. The purpose of this study was to describe the pharmacokinetic profile of multiple doses of an orally administered cannabidiol product formulated specifically for horses. A randomized 2-way crossover design was used. Seven horses received 0.35 or 2.0 mg/kg CBD per os every 24h for 7 total doses, separated by a 2-week washout. Plasma CBD and delta-9-tetrahydrocannabinol (THC) were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) daily through day 10, then on day 14 after beginning CBD administration. On the final day of CDB administration, plasma CBD and THC were quantified at multiple times. After administration of 0.35 mg/kg of CDB, the Cmax of CBD was 6.6 ± 2.1 ng/mL while Tmax was 1.8 ± 1.2 h, whereas the Cmax for THC was 0.7 ± 0.6 ng/mL with a Tmax of 2.5 ± 1 h. After administration of 2.0 mg/kg of CBD, the Cmax of CBD was 51 ± 14 ng/mL with a mean Tmax of 2.4 ± 1.1 h and terminal phase half-life of 10.4 ± 6 h, whereas the Cmax of THC was 7.5 ± 2.2 ng/mL with a Tmax of 2.9 ± 1.1 h. Oral administration of a cannabidiol product at 0.35 mg/kg or 2.0 mg/kg once daily for 7 days was well-tolerated. Based on plasma CBD levels obtained, dose escalation trials in the horse evaluating clinical efficacy at higher mg/kg dose rates are indicated.
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This article retraces the story of cannabis from the earliest contacts of humans with the plant to its subsequent global expansion, its medicinal uses, and the discovery of the endocannabinoid system in the 20th century. Cannabis was attested to around 12 000 years ago near the Altai Mountains in Central Asia, and since then, cannabis seeds have accompanied the migration of nomadic peoples. Records of the medicinal use of cannabis appear before the Common Era in China, Egypt, and Greece (Herodotus), and later in the Roman empire (Pliny the Elder, Dioscorides, Galen). In the 19th century, orientalists like Silvestre de Sacy, and Western physicians coming into contact with Muslim and Indian cultures, like O'Shaughnessy and Moreau de Tours, introduced the medicinal use of cannabis into Europe. The structure of the main psychoactive phytocannabinoid, tetrahydrocannabinol (THC), was determined in Israel by Mechoulam and Gaoni in 1964. This discovery opened the gate for many of the subsequent developments in the field of endocannabinoid system (ECS) research. The advances in the scientific knowledge of the ECS place the debate on cannabis liberalization in a new context. .
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The use of CBD-rich hemp products is becoming popular among pet owners with no long-term safety data related to consumption in adult dogs and cats. The purpose of this study was to determine the single-dose oral pharmacokinetics of CBD, and to provide a preliminary assessment of safety and adverse effects during 12-week administration using a hemp-based product in healthy dogs and cats. Eight of each species were provided a 2 mg/kg total CBD concentration orally twice daily for 12 weeks with screening of single-dose pharmacokinetics in six of each species. Pharmacokinetics revealed a mean maximum concentration (Cmax) of 301 ng/mL and 43 ng/mL, area under the curve (AUC) of 1297 ng-h/mL and 164 ng-h/mL, and time to maximal concentration (Tmax) of 1.4 h and 2 h, for dogs and cats, respectively. Serum chemistry and CBC results showed no clinically significant alterations, however one cat showed a persistent rise in alanine aminotransferase (ALT) above the reference range for the duration of the trial. In healthy dogs and cats, an oral CBD-rich hemp supplement administered every 12 h was not detrimental based on CBC or biochemistry values. Cats do appear to absorb or eliminate CBD differently than dogs, showing lower serum concentrations and adverse effects of excessive licking and head-shaking during oil administration.
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There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.
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Various preparations and extracts of the plant Cannabis sativa (family cannabaceae) are used as herbal medicinal drugs against a series of disorders but the plant contains a wide series of pharmacologically active components which may confound evaluation of drug effects. In order to differentiate specific effects of the individual constituents on specific functions in the organism we advocate for controlled studies on specified constituents and their impact on the vertebrate organism. One of the dominating Cannabis constituents, delta(9)-tetrahydrocannabinol (THC), has previously been studied in depth whereas information on another main ingredient cannabidiol (CBD) is limited. We have performed a controlled study on CBD and its effect using an experimental zebrafish model. CDB treatment of zebrafish for 30 min affected mobility of the fish by decreasing swimming speed and swimming distance. In addition, out of 23 immune related genes studied it was shown that expression of two genes il1b and il17a/f2 were up-regulated and four genes, tgfba, ighm, cd4-1, and s100a10b were significantly down-regulated following CBD treatment. The study indicated that CBD affects motility and immunity of the vertebrate host.
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Objectives: The objectives of this study were to determine basic oral pharmacokinetics, and assess safety and analgesic efficacy of a cannabidiol (CBD) based oil in dogs with osteoarthritis (OA). Methods: Single-dose pharmacokinetics was performed using two different doses of CBD enriched (2 and 8 mg/kg) oil. Thereafter, a randomized placebo-controlled, veterinarian, and owner blinded, cross-over study was conducted. Dogs received each of two treatments: CBD oil (2 mg/kg) or placebo oil every 12 h. Each treatment lasted for 4 weeks with a 2-week washout period. Baseline veterinary assessment and owner questionnaires were completed before initiating each treatment and at weeks 2 and 4. Hematology, serum chemistry and physical examinations were performed at each visit. A mixed model analysis, analyzing the change from enrollment baseline for all other time points was utilized for all variables of interest, with a p ≤ 0.05 defined as significant. Results: Pharmacokinetics revealed an elimination half-life of 4.2 h at both doses and no observable side effects. Clinically, canine brief pain inventory and Hudson activity scores showed a significant decrease in pain and increase in activity (p < 0.01) with CBD oil. Veterinary assessment showed decreased pain during CBD treatment (p < 0.02). No side effects were reported by owners, however, serum chemistry showed an increase in alkaline phosphatase during CBD treatment (p < 0.01). Clinical significance: This pharmacokinetic and clinical study suggests that 2 mg/kg of CBD twice daily can help increase comfort and activity in dogs with OA.
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It is well known that certain active ingredients of the plants of Cannabis genus, i.e., the “phytocannabinoids” [pCBs; e.g., (−)-trans-Δ⁹-tetrahydrocannabinol (THC), (−)-cannabidiol, etc.] can influence a wide array of biological processes, and the human body is able to produce endogenous analogs of these substances [“endocannabinoids” (eCB), e.g., arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol (2-AG), etc.]. These ligands, together with multiple receptors (e.g., CB1 and CB2 cannabinoid receptors, etc.), and a complex enzyme and transporter apparatus involved in the synthesis and degradation of the ligands constitute the endocannabinoid system (ECS), a recently emerging regulator of several physiological processes. The ECS is widely expressed in the human body, including several members of the innate and adaptive immune system, where eCBs, as well as several pCBs were shown to deeply influence immune functions thereby regulating inflammation, autoimmunity, antitumor, as well as antipathogen immune responses, etc. Based on this knowledge, many in vitro and in vivo studies aimed at exploiting the putative therapeutic potential of cannabinoid signaling in inflammation-accompanied diseases (e.g., multiple sclerosis) or in organ transplantation, and to dissect the complex immunological effects of medical and “recreational” marijuana consumption. Thus, the objective of the current article is (i) to summarize the most recent findings of the field; (ii) to highlight the putative therapeutic potential of targeting cannabinoid signaling; (iii) to identify open questions and key challenges; and (iv) to suggest promising future directions for cannabinoid-based drug development.
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Osteoarthritis (OA) is a multifactorial joint disease, which includes joint degeneration, intermittent inflammation, and peripheral neuropathy. Cannabidiol (CBD) is a noneuphoria producing constituent of cannabis that has the potential to relieve pain. The aim of this study was to determine whether CBD is anti-nociceptive in OA, and whether inhibition of inflammation by CBD could prevent the development of OA pain and joint neuropathy. Osteoarthritis was induced in male Wistar rats (150-175 g) by intra-articular injection of sodium monoiodoacetate (MIA; 3 mg). On day 14 (end-stage OA), joint afferent mechanosensitivity was assessed using in vivo electrophysiology, whereas pain behaviour was measured by von Frey hair algesiometry and dynamic incapacitance. To investigate acute joint inflammation, blood flow and leukocyte trafficking were measured on day 1 after MIA. Joint nerve myelination was calculated by G-ratio analysis. The therapeutic and prophylactic effects of peripheral CBD (100-300 μg) were assessed. In end-stage OA, CBD dose-dependently decreased joint afferent firing rate, and increased withdrawal threshold and weight bearing (P < 0.0001; n = 8). Acute, transient joint inflammation was reduced by local CBD treatment (P < 0.0001; n = 6). Prophylactic administration of CBD prevented the development of MIA-induced joint pain at later time points (P < 0.0001; n = 8), and was also found to be neuroprotective (P < 0.05; n = 6-8). The data presented here indicate that local administration of CBD blocked OA pain. Prophylactic CBD treatment prevented the later development of pain and nerve damage in these OA joints. These findings suggest that CBD may be a safe, useful therapeutic for treating OA joint neuropathic pain.
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Many effects of the non-psychoactive cannabinoid, cannabidiol (CBD), have been described in immune responses induced by strong immunological stimuli. It has also been shown that CBD enhances IL-2 production in response to low-level T cell stimulation. Since IL-2, in combination with TGF-β1, are critical for Treg induction, we hypothesized that CBD would induce CD4⁺CD25⁺FOXP3⁺ Tregs in response to low-level stimulation. Low-level T cell stimulation conditions were established based on minimal CD25 expression in CD4⁺ cells using suboptimal PMA/Io (4 nM/0.05 μM, S/o), ultrasuboptimal PMA/Io (1 nM/0.0125 μM, Us/o) or soluble anti-CD3/28 (400-800 ng each, s3/28). CBD increased CD25⁺FOXP3⁺ cells from CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25⁻ T cells, as well as in CD4⁺ T cells derived from FOXP3-GFP mice. Most importantly, the Us/o + CBD-induced CD4⁺CD25⁺ Tregs robustly suppressed responder T cell proliferation, demonstrating that the mechanism by which CBD is immunosuppressive under low-level T cell stimulation involves induction of functional Tregs.
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Background Our previous studies showed that the non-psychoactive cannabinoid, cannabidiol (CBD), ameliorates the clinical symptoms in mouse myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis model of multiple sclerosis (MS) as well as decreases the memory MOG35-55-specific T cell (TMOG) proliferation and cytokine secretion including IL-17, a key autoimmune factor. The mechanisms of these activities are currently poorly understood. Methods Herein, using microarray-based gene expression profiling, we describe gene networks and intracellular pathways involved in CBD-induced suppression of these activated memory TMOG cells. Encephalitogenic TMOG cells were stimulated with MOG35-55 in the presence of spleen-derived antigen presenting cells (APC) with or without CBD. mRNA of purified TMOG was then subjected to Illumina microarray analysis followed by ingenuity pathway analysis (IPA), weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) elucidation of gene interactions. Results were validated using qPCR and ELISA assays. ResultsGene profiling showed that the CBD treatment suppresses the transcription of a large number of proinflammatory genes in activated TMOG. These include cytokines (Xcl1, Il3, Il12a, Il1b), cytokine receptors (Cxcr1, Ifngr1), transcription factors (Ier3, Atf3, Nr4a3, Crem), and TNF superfamily signaling molecules (Tnfsf11, Tnfsf14, Tnfrsf9, Tnfrsf18). “IL-17 differentiation” and “IL-6 and IL-10-signaling” were identified among the top processes affected by CBD. CBD increases a number of IFN-dependent transcripts (Rgs16, Mx2, Rsad2, Irf4, Ifit2, Ephx1, Ets2) known to execute anti-proliferative activities in T cells. Interestingly, certain MOG35-55 up-regulated transcripts were maintained at high levels in the presence of CBD, including transcription factors (Egr2, Egr1, Tbx21), cytokines (Csf2, Tnf, Ifng), and chemokines (Ccl3, Ccl4, Cxcl10) suggesting that CBD may promote exhaustion of memory TMOG cells. In addition, CBD enhanced the transcription of T cell co-inhibitory molecules (Btla, Lag3, Trat1, and CD69) known to interfere with T/APC interactions. Furthermore, CBD enhanced the transcription of oxidative stress modulators with potent anti-inflammatory activity that are controlled by Nfe2l2/Nrf2 (Mt1, Mt2a, Slc30a1, Hmox1). Conclusions Microarray-based gene expression profiling demonstrated that CBD exerts its immunoregulatory effects in activated memory TMOG cells via (a) suppressing proinflammatory Th17-related transcription, (b) by promoting T cell exhaustion/tolerance, (c) enhancing IFN-dependent anti-proliferative program, (d) hampering antigen presentation, and (d) inducing antioxidant milieu resolving inflammation. These findings put forward mechanism by which CBD exerts its anti-inflammatory effects as well as explain the beneficial role of CBD in pathological memory T cells and in autoimmune diseases.
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Abstract We have previously shown that the prophylactic treatment with cannabidiol (CBD) reduces inflammation in a model of acute lung injury (ALI). In this work we analyzed the effects of the therapeutic treatment with CBD in mice subjected to the model of lipopolysaccharide (LPS)-induced ALI on pulmonary mechanics and inflammation. CBD (20 and 80 mg/kg) was administered (i.p.) to mice 6 h after LPS-induced lung inflammation. One day (24 h) after the induction of inflammation the assessment of pulmonary mechanics and inflammation were analyzed. The results show that CBD decreased total lung resistance and elastance, leukocyte migration into the lungs, myeloperoxidase activity in the lung tissue, protein concentration and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) in the bronchoalveolar lavage supernatant. Thus, we conclude that CBD administered therapeutically, i.e. during an ongoing inflammatory process, has a potent anti-inflammatory effect and also improves the lung function in mice submitted to LPS-induced ALI. Therefore the present and previous data suggest that in the future cannabidiol might become a useful therapeutic tool for the attenuation and treatment of inflammatory lung diseases.
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Cannabinoids, the Cannabis constituents, are known to possess anti-inflammatory properties but the mechanisms involved are not understood. Here we show that the main psychoactive cannabinoid, Δ-9-tetrahydrocannabinol (THC), and the main nonpsychoactive cannabinoid, cannabidiol (CBD), markedly reduce the Th17 phenotype which is known to be increased in inflammatory autoimmune pathologies such as Multiple Sclerosis. We found that reactivation by MOG35-55 of MOG35-55-specific encephalitogenic T cells (cells that induce Experimental Autoimmune Encephalitis when injected to mice) in the presence of spleen derived antigen presenting cells led to a large increase in IL-17 production and secretion. In addition, we found that the cannabinoids CBD and THC dose-dependently (at 0.1-5 μM) suppressed the production and secretion of this cytokine. Moreover, the mRNA and protein of IL-6, a key factor in Th17 induction, were also decreased. Pretreatment with CBD also resulted in increased levels of the anti-inflammatory cytokine IL-10. Interestingly, CBD and THC did not affect the levels of TNFα and IFNγ. The downregulation of IL-17 secretion by these cannabinoids does not seem to involve the CB1, CB2, PPARγ, 5-HT1A or TRPV1 receptors. In conclusion, the results show a unique cannabinoid modulation of the autoimmune cytokine milieu combining suppression of the pathogenic IL-17 and IL-6 cytokines along with boosting the expression of the anti-inflammatory cytokine IL-10.
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Background and purpose: Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro. Experimental approach: Confluent Caco-2 cell monolayers were treated for 24 h with IFNγ and TNFα (10 ng·mL(-1) ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB(1) , CB(2) , TRPV1, PPARγ and PPARα. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids. Key results: Δ(9) -Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB(1) receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB(1) antagonism. No role for the CB(2) receptor was identified in these studies. Co-application of THC, cannabidiol or a CB(1) antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation. Conclusions and implications: These findings suggest that locally produced endocannabinoids, acting via CB(1) receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation. Linked articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
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Equine gastric ulcer syndrome (EGUS) is common in horses. A history of mild intermitted recurrent colic signs after eating is noted in many horses. Management of horses with abdominal pain caused by gastric ulcers is especially difficult, because non-steroidal anti-inflammatory agents, typically used to control abdominal pain, may exacerbate this condition. Effective pharmacologic agents are available to treat EGUS and eliminate abdominal pain, but more comprehensive measures of environmental and dietary management are needed to manage horses with EGUS and prevent recurrence. This article focuses on the history, clinical signs, diagnosis, and management of horses with abdominal pain associated with gastric ulcers. The primary goal is to provide an understanding of EGUS and to review effective pain management and specific antiulcer treatments and management strategies in horses with EGUS.
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This review covers reports published in the last 5 years on the anti-inflammatory activities of all classes of cannabinoids, including phytocannabinoids such as tetrahydrocannabinol and cannabidiol, synthetic analogs such as ajulemic acid and nabilone, the endogenous cannabinoids anandamide and related compounds, namely, the elmiric acids, and finally, noncannabinoid components of Cannabis that show anti-inflammatory action. It is intended to be an update on the topic of the involvement of cannabinoids in the process of inflammation. A possible mechanism for these actions is suggested involving increased production of eicosanoids that promote the resolution of inflammation. This differentiates these cannabinoids from cyclooxygenase-2 inhibitors that suppress the synthesis of eicosanoids that promote the induction of the inflammatory process.
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Cannabidiol (CBD) is one of the major nonpsychoactive cannabinoids produced by Cannabis sativa L. Recent studies have shown that CBD has a high protective index, comparable to that of phenobarbital and phenytoin. Because CBD has been reported to possess both anticonvulsant and antiepileptic activity, its pharmacokinetics were studied in dogs after the administration of two iv doses (45 and 90 mg) and one oral dose (180 mg) to dogs. After iv administration, CBD was rapidly distributed, followed by a prolonged elimination. It has a terminal half-life of 9 hr. CBD plasma levels declined in a triphasic fashion. The total body clearance of CBD was 17 liters/hr (after the 45-mg dose) and 16 liters/hr (after the 90-mg dose). This clearance value, after its normalization to blood clearance using mathematical equations, approaches the value of the hepatic blood flow; the extraction ratio in the liver is 0.74. CBD was observed to have a large volume of distribution, approximately 100 liters. In the dose range of 45 to 90 mg, the increase in the AUC was proportional to the dose, a fact that indicates that the pharmacokinetic profile of CBD in this dose range was not dose dependent. In three of the six dogs studied, CBD could not be detected in the plasma after oral administration. In the other three, the oral bioavailability ranged from 13 to 19%. The results of this study show that CBD is barely absorbed after oral administration to dogs. This low bioavailability may be due to a first pass effect.
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In this paper we extend the "network theory of aging," and we argue that a global reduction in the capacity to cope with a variety of stressors and a concomitant progressive increase in proinflammatory status are major characteristics of the aging process. This phenomenon, which we will refer to as "inflamm-aging," is provoked by a continuous antigenic load and stress. On the basis of evolutionary studies, we also argue that the immune and the stress responses are equivalent and that antigens are nothing other than particular types of stressors. We also propose to return macrophage to its rightful place as central actor not only in the inflammatory response and immunity, but also in the stress response. The rate of reaching the threshold of proinflammatory status over which diseases/disabilities ensue and the individual capacity to cope with and adapt to stressors are assumed to be complex traits with a genetic component. Finally, we argue that the persistence of inflammatory stimuli over time represents the biologic background (first hit) favoring the susceptibility to age-related diseases/disabilities. A second hit (absence of robust gene variants and/or presence of frail gene variants) is likely necessary to develop overt organ-specific age-related diseases having an inflammatory pathogenesis, such as atherosclerosis, Alzheimer's disease, osteoporosis, and diabetes. Following this perspective, several paradoxes of healthy centenarians (increase of plasma levels of inflammatory cytokines, acute phase proteins, and coagulation factors) are illustrated and explained. In conclusion, the beneficial effects of inflammation devoted to the neutralization of dangerous/harmful agents early in life and in adulthood become detrimental late in life in a period largely not foreseen by evolution, according to the antagonistic pleiotropy theory of aging.
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The objectives of this study were to evaluate the natural age-related variation and compare the level of pro-inflammatory cytokines in the peripheral blood and lower airways of horses. The mRNA expression of IL-1β, IL-6, IL-8 TNF-α TLR-4 in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMC) were studied by quantitative real time polymerase chain reaction (PCR) and differential cell count cytology from 44 horses of different ages. A significant age-related increase was found for the mRNA expression of IL-6, IL-8, TLR-4 and TNF-α in stimulated BAL cells and for TNF-α in stimulated PBMC. Furthermore, a significant decrease was found in the mRNA expression of IL-1β and TNF-α in stimulated BAL cells compared to stimulated PBMC. In conclusion, continued low antigen exposure of horses in the pasture environment could lead to a tight regulation of mRNA gene expression in the airway space compared to the peripheral blood and favour an age-related accumulation of mRNA genes.
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The chronic use of drugs that reduce the dopaminergic neurotransmission can cause a hyperkinetic movement disorder called tardive dyskinesia (TD). The pathophysiology of this disorder is not entirely understood but could involve oxidative and neuroinflammatory mechanisms. Cannabidiol (CBD), the major non-psychotomimetic compound present in Cannabis sativa plant, could be a possible therapeutic alternative for TD. This phytocannabinoid shows antioxidant, anti-inflammatory and antipsychotic properties and decreases the acute motor effects of classical antipsychotics. The present study investigated if CBD would attenuate orofacial dyskinesia, oxidative stress and inflammatory changes induced by chronic administration of haloperidol in mice. Furthermore, we verified in vivo and in vitro (in primary microglial culture) whether these effects would be mediated by PPARγ receptors. The results showed that the male Swiss mice treated daily for 21 days with haloperidol develop orofacial dyskinesia. Daily CBD administration before each haloperidol injection prevented this effect. Mice treated with haloperidol showed an increase in microglial activation and inflammatory mediators in the striatum. These changes were also reduced by CBD. On the other hand, the levels of the anti-inflammatory cytokine IL-10 increased in the striatum of animals that received CBD and haloperidol. Regarding oxidative stress, haloperidol induced lipid peroxidation and reduced catalase activity. This latter effect was attenuated by CBD. The combination of CBD and haloperidol also increased PGC-1α mRNA expression, a co-activator of PPARγ receptors. Pretreatment with the PPARγ antagonist, GW9662, blocked the behavioural effect of CBD in our TD model. CBD also prevented LPS-stimulated microglial activation, an effect that was also antagonized by GW9662. In conclusion, our results suggest that CBD could prevent haloperidol-induced orofacial dyskinesia by activating PPARγ receptors and attenuating neuroinflammatory changes in the striatum.
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Cannabidiol (CBD) has been reported to induce apoptosis in immune cells through oxidative stress-related mechanisms. The objective of the present study was to investigate the cellular mechanisms for CBD-induced apoptosis and oxidative stress in human monocytes. Exposure of freshly isolated human monocytes to CBD induced apoptosis in a time- and concentration-dependent manner. Time-course analyses revealed the induction of intracellular reactive oxygen species (ROS) at 1-2h post CBD (16 μM) exposure. By comparison, the CBD treatment rapidly elicited the depolarization of mitochondrial membrane potential (MMP) within 5min, and the oxidation of cardiolipin, a major lipid component of the mitochondrial inner membrane, within 15min. Moreover, CBD induced the release of cytochrome c (Cyt c) from mitochondria. Mechanistic studies revealed that CBD-induced ROS production and apoptosis were not associated with the alteration of mitochondrial superoxide dismutase activity, the electron leakage through mitochondrial respiratory chain, and Fe2+- and Ca2+-mediated mechanisms. In contrast, CBD-induced apoptosis and MMP depolarization were markedly attenuated by the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporin A (CsA), but not the calcineurin inhibitor FK506. Furthermore, CsA prevented cardiolipin oxidation and the MPTP opening induced by CBD. The present study suggests that CBD acts on the mitochondria to elicit ROS generation and apoptosis through MPTP opening and provides critical insights into the cellular mechanisms for CBD-induced oxidative stress in apoptotic monocytes.
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Plants have been the predominant source of medicines throughout the vast majority of human history, and remain so today outside of industrialized societies. One of the most versatile in terms of its phytochemistry is cannabis, whose investigation has led directly to the discovery of a unique and widespread homeostatic physiological regulator, the endocannabinoid system. While it had been the conventional wisdom until recently that only cannabis harbored active agents affecting the endocannabinoid system, in recent decades the search has widened and identified numerous additional plants whose components stimulate, antagonize, or modulate different aspects of this system. These include common foodstuffs, herbs, spices, and more exotic ingredients: kava, chocolate, black pepper, and many others that are examined in this review.
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Senior horses (aged ≥20 years) exhibit increased chronic, low-grade inflammation systemically, termed inflamm-aging. Inflammation is associated with many afflictions common to the horse, including laminitis and osteoarthritis, which are commonly treated with the non-steroidal anti-inflammatory drugs (NSAIDs) flunixin meglumine and phenylbutazone. Although these NSAIDs are effective in treating acute inflammatory problems, long-term treatment with NSAIDs can result in negative side effects. Thus, bioactive polyphenols including curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene were investigated to determine their effectiveness as anti-inflammatory agents in vitro. Heparinized blood was collected via jugular venipuncture from senior horses (n = 6; mean age = 26 ± 2 years), and peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll density gradient. PBMC were then incubated 22 h at 37 °C, 5% CO2 with multiple concentrations (320, 160, 80, 40, 20, 10 μM) of all five polyphenols (curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene), dissolved in DMSO to achieve the aforementioned concentrations. PBMC were stimulated the last 4 h of the incubation period with phorbol 12-myristate 13-acetate (PMA)/ionomycin and Brefeldin A (BFA). A Vicell-XR counter evaluated cell viability following incubation. PBMC were stained intracellularly for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) and analyzed via flow cytometry. Data was analyzed by one-way analysis of variance (ANOVA). Viability of PBMC incubated with various compound concentrations were compared with PBMC incubated with DMSO alone (positive control) to determine at what concentration each compound caused cytotoxicity. The highest concentration at which cell viability did not significantly differ from the positive control was: 20 μM for curcuminoids, 40 μM for hydroxypterostilbene, 80 μM for pterostilbene, and 160 μM for quercetin and resveratrol. Flunixin meglumine and phenylbutazone were then evaluated within this range of optimal concentrations for the polyphenol compounds (160, 80, 40, 20 μM) to compare the polyphenols to NSAIDs at equivalent concentrations. The highest concentration at which viability did not significantly differ from the positive control was: 40 μM for flunixin meglumine and 160 μM for phenylbutazone. All five polyphenols and flunixin meglumine significantly decreased lymphocyte production of IFN-γ, while only hydroxypterostilbene, pterostilbene, quercetin, and resveratrol significantly reduced lymphocyte production of TNF-α compared to the positive control (p < 0.05). Polyphenols performed similarly to or more effectively than common NSAIDs in reducing lymphocyte production of inflammatory cytokines of the senior horse in vitro. This study therefore supports the further investigation of polyphenols to determine whether they may be effective anti-inflammatory treatments for chronic inflammation in the horse.
Article
It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72 h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-L-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.
Article
Cannabidiol (CBD) is a cannabinoid compound derived from Cannabis Sativa that does not possess high affinity for either the CB1 or CB2 cannabinoid receptors. Similar to other cannabinoids, we demonstrated previously that CBD suppressed interleukin-2 (IL-2) production from phorbol ester plus calcium ionophore (PMA/Io)-activated murine splenocytes. Thus, the focus of the present studies was to further characterize the effect of CBD on immune function. CBD also suppressed IL-2 and interferon-gamma (IFN-gamma) mRNA expression, proliferation, and cell surface expression of the IL-2 receptor alpha chain, CD25. While all of these observations support the fact that CBD suppresses T cell function, we now demonstrate that CBD suppressed IL-2 and IFN-gamma production in purified splenic T cells. CBD also suppressed activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) transcriptional activity, which are critical regulators of IL-2 and IFN-gamma. Furthermore, CBD suppressed the T cell-dependent anti-sheep red blood cell immunoglobulin M antibody forming cell (anti-sRBC IgM AFC) response. Finally, using splenocytes derived from CB1(-/-)/CB2(-/-) mice, it was determined that suppression of IL-2 and IFN-gamma and suppression of the in vitro anti-sRBC IgM AFC response occurred independently of both CB1 and CB2. However, the magnitude of the immune response to sRBC was significantly depressed in CB1(-/-)/CB2(-/-) mice. Taken together, these data suggest that CBD suppresses T cell function and that CB1 and/or CB2 play a critical role in the magnitude of the in vitro anti-sRBC IgM AFC response.
Article
Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB1/2 receptors.
Article
Cannabinoids can influence the immune network. Data on the impact of exogenous cannabinoid ligands on immune function serve not only to understand how the endocannabinoid system modulates immune phenomena associated with infection or inflammation, but also to identify therapeutic targets for immune diseases. Cannabinoids can modulate immune reactions in the periphery but also in the brain, influence T cell subset balance and cytokine expression and play a role in the balance between neuroinflammation and neurodegeneration. Immune cells can synthesize endocannabinoids and also be influenced by cannabinoid analogues. Cannabinoid receptors show different expression on immune cells depending on activation status and stimuli. The complexity of relation between cannabinoid ligands of various classes and cannabinoid receptors brought the need to refine the simple conceptual frame of agonist-antagonists and offered potential implications for understanding interactions in pathological conditions. The immune influence of cannabinoid ligands is not fully elucidated. However, aspects of their immunomodulatory effects provide the basis for a context-dependent targeted therapeutic approach, thus leading to the possibility for the use of cannabinoids in the treatment of inflammatory disease.
Article
Nanomolar concentrations of Delta9-tetrahydrocannabinol or cannabidiol are demonstrated to enhance mitogen-induced degradation of tryptophan in human peripheral blood mononuclear cells in dependence of CB1- or CB2-receptor activation. In contrast, suppression of this pathway by cannabinoids in the micromolar concentration range was achieved independent of cannabinoid receptor activation. Both cannabinoids also suppressed tryptophan degradation in myelomonocytic THP-1 cells stimulated with lipopolysaccharide. We conclude, that suppression of tryptophan degradation by cannabinoids via indoleamine-2,3-dioxygenase, which is independent of cannabinoid receptor activation, might enhance the availability of tryptophan for serotonin biosynthesis and consequently can be important in the action of cannabinoids to improve mood disturbances.
Article
Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.
Article
A number of model systems have been employed to investigate age-associated changes in immune function. The purpose of the current study was to characterize senescent T cells and to investigate the inflamm-aging phenomenon both in vitro and in vivo using the old horse as a model. We examined whether decreased T cell proliferation induced by Con A is caused by increased apoptosis. We also utilized intracellular CFSE to analyze changes within each round of cell proliferation, in particular cytokine production. Intracellular staining with flow cytometry, RT-PCR, and ELISA were used to measure pro-inflammatory cytokines both in vitro and in vivo. While lymphocytes from old horses exhibit decreased proliferation, this is not the result of increased apoptosis. Instead, a larger percentage of the T cells remain in the parent generation and produce significant amounts of IFNgamma. Likewise, old horses have increased frequency of CD8-IFNgamma+ T cells and TNFalpha producing cells. We also show that old horses have elevated levels of IL-1beta, IL-15, IL-18 and TNFalpha gene expression in peripheral blood and significant levels of TNFalpha protein in serum, all characteristics of inflamm-aging.
Article
The in vivo metabolism of cannabidiol (CBD) was investigated in mice. Following the ip administration of CBD to mice, livers were removed and metabolites were extracted with ethyl acetate prior to partial purification on Sephadex LH-20 columns. Fractions from the columns were converted into trimethylsilyl, d9-trimethylsilyl, and methylester-trimethylsilyl derivatives for analysis by gas-liquid chromatography-mass spectrometry. In addition, metabolites containing carboxylic acid and ketone functional groups were reduced to alcohols with lithium aluminum deuteride before trimethylsilation. A total of 22 metabolites were characterized, 14 of which had not been reported previously. The metabolites could be categorized as follows: monohydroxylated (N=2), dihydroxylated (N=3), CBD-7-oic acid, side chain hydroxy-GBD-7-oic acids (N=3), side-chain acids (N=3), 7-hydroxy-side-chain acids (N=4), 6-oxo-side-chain acids (N=3) and glucuronide conjugates (N=3). The most significant biotransformations were glucuronide conjugation and, to a lesser extent, formation of CBD-7-oic acid.
Article
We investigated the in vitro effects of both psychoactive and nonpsychoactive marijuana components on leukocyte secretion of the immunoregulatory cytokines interleukin-1 alpha (IL-1), tumor necrosis factor alpha (TNF), interferon-gamma (IFN) and interleukin-2 (IL-2). Psychoactive delta-9-tetrahydrocannabinol (THC) and nonpsychoactive cannabidiol (CBD) were added to cultures of mitogen-activated human peripheral blood mononuclear cells (PBMC) and the concentrations of IL-1, TNF, IFN and IL-2 in culture supernatants were measured by ELISA systems. Concentrations of THC and CBD, comparable to plasma levels found after smoking marijuana (10-100 ng/ml), increased the concentration of measurable IFN (139 and 68%), while high concentrations of both cannabinoids (5-20 micrograms/ml) completely blocked synthesis and/or release of this cytokine. CBD was also shown to decrease the measurable quantity of both IL-1 and TNF. In contrast to the effects on IFN, IL-1 and TNF, both cannabinoids, had no effect on IL-2 secretion. This report suggests that both psychoactive and nonpsychoactive components of marijuana are immunomodulating and can potentially alter cytokine secretion of human PBMC.
Article
Marijuana, a widely abused drug in the US, and its derivatives (cannabinoids) have been used in AIDS and cancer patients for treatment of intractable nausea and cachexia. Yet, objective investigations of the effect of cannabinoids on the human immune system are few. We investigated the effect of delta9 tetrahydrocannabinol (THC) and cannabidiol (CBD) on cytokine production in vitro by human leukemic T, B, eosinophilic and CD8+ NK cell lines as models. THC decreased constitutive production of IL-8, MIP-1alpha, MIP-1beta, and RANTES and phorbol ester stimulated production of TNF-alpha, GM-CSF and IFN-gamma by NK cells. It inhibited MIP-1beta in HTLV-1 positive B-cells but tripled IL-8, MIP-1alpha and MIP-1beta in B-cells and MIP-1beta in eosinophilic cells but doubled IL-8. Both cannabinoids strongly inhibited IL-10 production by HUT-78 T-cells. Results indicate that THC and nonpsychotropic CBD have complex lineage and derivative specific effects on cytokines consistent with previous animal studies. These effects while of potential benefits in some inflammatory/autoimmune diseases may worsen HIV infection, tumorigenesis and allergic inflammation in the lung.
Article
The ultimate reason for better characterizing the immune response to infectious agents is the hope that this knowledge may lead to the development of better preventative or therapeutic measures. As more information becomes available, it becomes possible to incorporate these findings into the design of better vaccines and treatments. Likewise, attempts to either enhance or suppress specific helper T-cell responses may be required to control immunopathologic reactions. Although cytokine intervention in the clinical setting remains theoretic at this time, future manipulation based on the TH1/TH2 paradigm is probable.
Article
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Article
Cannabidiol is the main nonpsychoactive component of marijuana. We examined the ability of in vivo and in vitro cannabidiol to interfere with the production of interleukin (IL)-12 and IL-10 by murine macrophages and to modulate macrophage chemotaxis. Cannabidiol added in vitro to peritoneal macrophages significantly increased IL-12 and decreased IL-10 production. The CB1 and CB2 receptor antagonists prevented this modulation. Macrophages from animals treated with cannabidiol at the dose of 30 mg kg(-1) either orally or i.p. produced higher levels of IL-12 and lower levels of IL-10 in comparison to controls, and the CB receptor antagonists did not prevent these effects. Cannabidiol dose-dependently decreased fMLP-induced chemotaxis of macrophages, and the CB2 receptor antagonist prevented this decrease.
Article
The increased vulnerability of foals to specific pathogens such as Rhodococcus equi is believed to reflect an innate immunodeficiency, the nature of which remains poorly understood. Previous studies have demonstrated that neonates of many species fail to mount potent Th1 responses. The current research investigates the ability of circulating and pulmonary lymphocytes of developing foals to produce interferon gamma (IFNgamma). Peripheral blood mononuclear cells (PBMC) were prepared from up to 10 horse foals at regular intervals throughout the first 6 months of life. Bronchoalveolar lavage (BAL) samples were collected at 1, 3 or 6 months of age from three groups of five foals. The PBMC and BAL cells were stimulated in vitro and IFNgamma production was measured by intracellular staining. In addition, RNA was extracted from freshly isolated and in vitro stimulated PBMC and BAL cells for quantitation of IFNgamma gene expression by real time PCR. Newborn foals exhibited a marked inability to express the IFNgamma gene and produce IFNgamma protein. This deficiency was observed in both circulating and pulmonary lymphocytes. However, IFNgamma gene expression and protein production increased steadily throughout the first 6 months of life, reaching adult levels within the first year of life. These findings suggest that foals are born with an inherent inability to mount a Th1-based cell mediated immune response which may contribute to their susceptibility to intracellular pathogens.
Article
We recently reported that cannabidiol (CBD) exhibited a generalized suppressive effect on T-cell functional activities in splenocytes directly exposed to CBD in vitro or isolated from CBD-administered mice. To investigate the potential mechanisms of CBD effects on T cells, we characterized the pro-apoptotic effect of CBD on primary lymphocytes. The apoptosis of splenocytes was markedly enhanced following CBD exposure in a time- and concentration-dependent manner, as evidenced by nuclear hypodiploidity and DNA strand breaks. Exposure of splenocytes to CBD elicited an early production of reactive oxygen species (ROS) with the peak response at 1 h post CBD treatment. In parallel with the ROS production, a gradual diminishment in the cellular glutathione (GSH) content was detected in CBD-treated splenocytes. Both CBD-mediated ROS production and GSH diminishment were remarkably attenuated by the presence of N-acetyl-L-cysteine (NAC), a thiol antioxidant. In addition, CBD treatment significantly stimulated the activation of caspase-8, which was abrogated in the presence of NAC or GSH. Pretreatment of splenocytes with a cell-permeable inhibitor for caspase-8 significantly attenuated, in a concentration-dependent manner, CBD-mediated apoptosis, but not ROS production. Collectively, the present study demonstrated that the apoptotic effect of CBD in primary lymphocytes is closely associated with oxidative stress-dependent activation of caspase-8.