Immunosuppressive activity of non-psychoactive Cannabis sativa L. extract on the function of human T lymphocytes.

BACKGROUND: Cannabis sativa L. extracts (CSE) are used for treating inflammatory conditions, but little is known about their immunomodulatory effects. We investigated a novel CSE with high (14%) CBD and low (0.2%) THC concentration in comparison with pure CBD on primary human lymphocytes. METHODS: Proliferation, cell cycle distribution, apoptosis/necrosis and viability were analysed with standard methods. Genotoxicity was evaluated with the comet-assay. The effect on T lymphocyte activation was evaluated via CD25/CD69 marker expression, degranulation assays and the production of cytokines. The influence on the transcription factors was analysed using Jurkat reporter cell lines. Specific CB2 receptor antagonist SR144528 and TRPV1 receptor antagonist A78416B were used to study the involvement of CB2 or TRPV1 receptors. RESULTS: CSE inhibited the proliferation of activated T lymphocytes in a dose-dependent manner without inducing apoptosis, necrosis, or affecting cell viability and DNA integrity. The inhibitory effect was mediated via the suppression of T lymphocytes activation, particularly by the suppression of CD25 surface marker expression. Furthermore, CSE interferes with the functionality of the T lymphocytes, as indicated by inhibition of degranulation, IL-2, and IFN-γ production. AP-1-and-NFAT-reporter activation was reduced implicating an AP-1-and-NFAT-mediated mode of action. The effects were in part reversed by SR144528 and A78416B, showing that the effects were mainly mediated by CB2 and TRPV1 receptors. CONCLUSION: CSE and CBD have immunomodulatory effects and interfere with the activation and functionality of T lymphocytes. A comparison between CSE and CBD suggests that the immunosuppressive effect of CSE is mostly due to the effect of CBD.

Unusual and Unexpected Allergic Reactions Can Be Unraveled by Molecular Allergy Diagnostics.

The fifth class of immunoglobulin, immunoglobulin E (IgE) was discovered in 1967 and has had immense importance for the understanding, diagnosis, and treatment of allergic disease. More than 50 years have passed and efforts to characterize, standardize, and refine allergens with the aim to improve clinical diagnosis and allergen-specific immunotherapy are still ongoing. Another important breakthrough was made in 1999 with the introduction of component-resolved diagnostics (CRD), making it possible to quantify IgE antibodies against individual allergen proteins for diagnostic purposes at a molecular level. The progress and developments made in allergy diagnosis often originate from clinical observations and case studies. Observant physicians and health-care personnel have reported their findings in the medical literature, which in turn has inspired researchers to become involved in clinical research. Allergists continuously encounter new allergies and are often asked by their patients how to prevent new reactions. In the current article, we focus on recent clinical observations that can now be explained by CRD. The examples taken concern allergic reactions toward peanuts, tree nuts, lemon kernels, health drinks, meat, insects, dog dander, cannabis, and semen. We now have an improved understanding of why patients may react in a serious or unexpected way, as illustrated by these examples, yet many other clinical observations remain unexplained. The aim of this review is to highlight the importance of clinical observations among allergic patients, focusing on systemic, or unusual and unexpected allergic reactions, where component-testing has further refined the diagnosis of IgE-mediated allergy.

The Immunopathology of COVID-19 and the Cannabis Paradigm.

Coronavirus disease-19 caused by the novel RNA betacoronavirus SARS-CoV2 has first emerged in Wuhan, China in December 2019, and since then developed into a worldwide pandemic with >99 million people afflicted and >2.1 million fatal outcomes as of 24th January 2021. SARS-CoV2 targets the lower respiratory tract system leading to pneumonia with fever, cough, and dyspnea. Most patients develop only mild symptoms. However, a certain percentage develop severe symptoms with dyspnea, hypoxia, and lung involvement which can further progress to a critical stage where respiratory support due to respiratory failure is required. Most of the COVID-19 symptoms are related to hyperinflammation as seen in cytokine release syndrome and it is believed that fatalities are due to a COVID-19 related cytokine storm. Treatments with anti-inflammatory or anti-viral drugs are still in clinical trials or could not reduce mortality. This makes it necessary to develop novel anti-inflammatory therapies. Recently, the therapeutic potential of phytocannabinoids, the unique active compounds of the cannabis plant, has been discovered in the area of immunology. Phytocannabinoids are a group of terpenophenolic compounds which biological functions are conveyed by their interactions with the endocannabinoid system in humans. Here, we explore the anti-inflammatory function of cannabinoids in relation to inflammatory events that happen during severe COVID-19 disease, and how cannabinoids might help to prevent the progression from mild to severe disease.

Identification of IgE- binding pollen protein from Cannabis sativa in pollen-hypersensitive patients from north Pakistan.

Cannabis sativa (C.sativa) is well-known for its medicinal, industrial and recreational use. However, allergies in relation to Cannabis sativa (C.sativa) are rarely reported. C. sativa is one of the common weeds found in Pakistan and its pollen grains are common in spring and fall season. Although categorized as an aeroallergen, there are limited number of reports regarding allergenic potential in C. sativa. Therefore, the current study is aimed at exploring the IgE- binding potential among the C. sativa pollen in local pollen allergic patients. Initial screening of C. sativa sensitized individuals was carried out by dot blot from the sera of pollen allergic patients. Proteins from the pollen grains were extracted and resolved on 10% gel. Eight bands were visible on gel however only one protein fragment i.e. of 14KDa size was found to bind to IgE as analyzed through protein gel blot analysis. Strong IgE affinity of a 14 kDa protein fragment from C. sativa pollen extract suggests its allergenic potential. Further study is required to find the exact nature of this protein fragment.

[MEDICAL CANNABIS - A SOURCE FOR A NEW TREATMENT FOR AUTOIMMUNE DISEASE?].

Medical uses of Cannabis sativa have been known for over 6,000 years. Nowadays, cannabis is mostly known for its psychotropic effects and its ability to relieve pain, even though there is evidence of cannabis use for autoimmune diseases like rheumatoid arthritis centuries ago. The pharmacological therapy in autoimmune diseases is mainly based on immunosuppression of diffefent axes of the immune system while many of the drugs have major side effects. In this review we set out to examine the rule of Cannabis sativa as an immunomodulator and its potential as a new treatment option. In order to examine this subject we will focus on some major autoimmune diseases such as diabetes type I and rheumatoid arthritis.

Prevalence of sensitization to Cannabis sativa. Lipid-transfer and thaumatin-like proteins are relevant allergens.

BACKGROUND: Although allergy to Cannabis sativa was first reported over 40 years ago, the allergenicity has scarcely been studied. The objectives of this study were to investigate the frequency of sensitization to this plant, to analyze the clinical characteristics and allergenic profile of sensitized individuals and to identify the allergens involved. METHODS: Five hundred and forty-five individuals in Spain attending allergy clinics with respiratory or cutaneous symptoms underwent a skin-prick test (SPT) with C. sativa leaf extract. The extract was characterized by SDS-PAGE and 2-dimensional electrophoresis. Specific IgE to C. sativa was measured in positive SPT individuals. The clinical and allergenic profiles of sensitized individuals were investigated and the most-recognized allergens sequenced and characterized by liquid chromatography-mass spectrometry/mass spectrometry. RESULTS: Of this preselected population, 44 individuals had positive SPT to C. sativa (prevalence 8.1%). Prevalence was higher in individuals who were C. sativa smokers (14.6%). Two individuals reported mild symptoms with C. sativa. Twenty-one individuals from 32 available sera (65.6%) had positive specific IgE to C. sativa. Twelve sera recognized at least 6 different bands in a molecular-weight range of between 10 and 60 kDa. Six of them recognized a 10-kDa band, identified as a lipid transfer protein (LTP) and 8 recognized a 38-kDa band, identified as a thaumatin-like protein. CONCLUSIONS: There is a high prevalence of sensitization to C. sativa leaves. The clinical symptoms directly attributed to C. sativa were uncommon and mild. The sensitization profile observed suggests that C. sativa sensitization may be mediated by two mechanisms, i.e. cross-reactivity, mainly with LTP and thaumatin-like protein, and exposure-related 'de novo' sensitization.

Allergic hypersensitivity to cannabis in patients with allergy and illicit drug users.

BACKGROUND: Cannabis is the illicit drug most widely used by young people in high-income countries. Allergy symptoms have only occasionally been reported as one of the adverse health effects of cannabis use. OBJECTIVES: To study IgE-mediated response to cannabis in drug users, atopic patients, and healthy controls. METHODS: Asthmatic patients sensitised to pollen, and all patients sensitised to tobacco, tomato and latex, considered as cross-reacting allergens, were selected from a data base of 21,582 patients. Drug users attending a drug-rehabilitation clinic were also included. Controls were 200 non-atopic blood donors. Specific IgE determination, prick tests and specific challenge with cannabis extracts were performed in patients and controls. RESULTS: Overall, 340 patients, mean age 26.9±10.7 years, were included. Males (61.4%) were the most sensitised to cannabis (p<0.001). All cannabis-sensitised patients were alcohol users. Eighteen (72%) of the patients allergic to tomato were sensitised to cannabis, but a positive specific challenge to cannabis was highest in patients sensitised to tobacco (13/21, 61.9%), (p<0.001). Pollen allergy was not a risk factor for cannabis sensitisation. Prick tests and IgE for cannabis had a good sensitivity (92 and 88.1%, respectively) and specificity (87.1 and 96%) for cannabis sensitisation. CONCLUSIONS: Cannabis may be an important allergen in young people. Patients previously sensitised to tobacco or tomato are at risk. Cannabis prick tests and IgE were useful in detecting sensitisation.

Immunochemical approach using monoclonal antibody against Δ(9)-tetrahydrocannabinolic acid (THCA) to discern cannabis plants and to investigate new drug candidates.

A monoclonal antibody (MAb-4A4) against Δ9- tetrahydrocannabinolic acid (THCA) showing extensive cross-reactivity against various cannabinoids was prepared. Using this antibody, a competitive enzyme-linked immunoassay (ELISA) was developed to detect Δ9-THCA in the range of 1 to 100 mg/ml. Various cannabinoids including Δ9-THC (Δ9-tetrahydrocannabinolic acid), Δ8-THCA (Δ8-tetrahydrocannabinolic acid), Δ8-THC (Δ8-tetrahydrocannabinol), CBD (cannabidiol), and CBN (cannabinol) were recognized by MAb-4A4, and their cross-reactivities were 55-1600% compared with Δ9-THCA (100%). This novel characteristic of this MAb enabled detection of marijuana residues in biological samples by detection of residual cannabinoids. The ELISA using MAb-4A4 was found to be applicable even for withered samples which contained only trace amounts of Δ9-THCA and Δ9-THC. In addition, this method using MAb-4A4 could be useful in forensic analysis since the MAb-4A4 also shows cross-reactivities against cannabinoid metabolites in body fluids. As well as forensic applications using this MAb, an investigation of new drug candidates focusing on cannabinoid metabolites arising from biotransformation in plant tissue was performed using immunochemical screening. The resulting new drug candidates were cannabinoid glycosides biotransformed by Pinellia ternata whose bioactivity is as yet unidentified. Our results indicate the utility of the application of ELISA using MAb-4A4 for further experiments involving marijuana and cannabinoids not only in the forensic field but also in the context of drug discovery.

Sensitization and allergy to Cannabis sativa leaves in a population of tomato (Lycopersicon esculentum)-sensitized patients.

BACKGROUND: Cases of allergy to Cannabis sativa have occasionally been reported, but both the allergenic profile and eventual cross-reactivity pattern remain unknown. OBJECTIVE: To analyze the allergenic profile of a population of patients from Spain sensitized to C. sativa and to characterize the C. sativa leaf extract. METHODS: A total of 32 subjects were enrolled in the study: group A, 10 individuals sensitized to tomato, reporting reactions by contact or inhalation to Cannabis; group B, 14 individuals sensitized to tomato, without reactions to Cannabis; group C, 8 individuals not sensitized to tomato and without reactions to Cannabis. Sensitivity to Cannabis, tomato and peach peel, Platanus hybrida and Artemisia vulgaris pollen extracts was measured by skin tests and specific IgE. Individual immunoblots and inhibition experiments with a pool of sera were conducted. RESULTS: All tomato-sensitized subjects (and 1 negative) had positive skin tests to C. sativa leaves and hashish. Specific IgE to C. sativa and peach peel was more common than to tomato. Immunoblot experiments showed 2 prominent bands of 10 and 14 kDa and 2 weakly recognized bands of 30 and 45 kDa. Tomato, peach and A. vulgaris extracts inhibited most of the bands present in C. sativa. P. hybrida inhibited only the high-molecular-weight bands. CONCLUSION: Sensitization to C. sativa with or without symptoms is frequent among patients in Spain sensitized to tomato. C. sativa leaves are a potential allergenic source and their allergens may cross-react with other allergenic sources from plants (fruit peels and pollen).

Unheated Cannabis sativa extracts and its major compound THC-acid have potential immuno-modulating properties not mediated by CB1 and CB2 receptor coupled pathways.

There is a great interest in the pharmacological properties of cannabinoid like compounds that are not linked to the adverse effects of Delta(9)-tetrahydrocannabinol (THC), e.g. psychoactive properties. The present paper describes the potential immuno-modulating activity of unheated Cannabis sativa extracts and its main non-psychoactive constituent Delta(9)-tetrahydrocanabinoid acid (THCa). By heating Cannabis extracts, THCa was shown to be converted into THC. Unheated Cannabis extract and THCa were able to inhibit the tumor necrosis factor alpha (TNF-alpha) levels in culture supernatants from U937 macrophages and peripheral blood macrophages after stimulation with LPS in a dose-dependent manner. This inhibition persisted over a longer period of time, whereas after prolonged exposure time THC and heated Cannabis extract tend to induce the TNF-alpha level. Furthermore we demonstrated that THCa and THC show distinct effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibit the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. These results suggest that THCa and THC exert their immuno-modulating effects via different metabolic pathways.

Cannabinoid influence on cytokine profile in multiple sclerosis.

Cannabinoids have been suggested as possessing immunomodulatory properties, and cannabinoid receptors are present on leucocytes. Clinically, there is some evidence that cannabinoids may be therapeutically useful in treating multiple sclerosis, which is generally believed to be an autoimmune condition. This paper reports data derived from the Cannabinoids in MS (CAMS) study, which was the largest randomized controlled trial yet conducted to evaluate the therapeutic efficacy of cannabinoids. We found no evidence for cannabinoid influence on serum levels of interferon (IFN)-gamma, interleukin (IL)-10, IL-12 or C-reactive protein as measured using enzyme-linked immunosorbent assay (ELISA), in comparison to control values. Mitogenic stimulation experiments also failed to demonstrate any significant reduction in percentage of CD3+, IFN-gamma producing cells after exposure to cannabinoids in vivo, although numbers were small. Further work is needed to establish the functional significance of cannabinoid receptors on immune cells.

Cannabis (hemp) positive skin tests and respiratory symptoms.

BACKGROUND: We have noted several patients who had rhinitis and/or asthma symptoms when exposed to Cannabis plants in the summer months. Cannabis plants are common in the Midwest. OBJECTIVES: To examine whether Cannabis might be a clinically important allergen, we determined Cannabis pollination patterns in the Omaha area for 5 years, the prevalence of skin test positivity, and the association with respiratory symptoms. METHODS: Airborne Cannabis (and other weed) pollens were collected using a Rotorod air impactor, and pollen counts were done using a standardized protocol. RESULTS: Measurable Cannabis pollen count was not recorded until the last 2 weeks of July. Peak pollination typically occurred during mid- to late-August, and comprised up to 36% of the total pollen counts. Cannabis pollen was not observed after mid-September. To determine the prevalence of skin test positivity, we added Cannabis to the multi-test routine skin test battery. Seventy-eight of 127 patients tested (61%) were skin test positive. Thirty of the 78 patients were randomly selected to determine if they had allergic rhinitis and/or asthma symptoms during the Cannabis pollination period. By history, 22 (73%) claimed respiratory symptoms in the July through September period. All 22 of these subjects were also skin test positive to weeds pollinating during the same period as Cannabis (ragweed, pigweed, cocklebur, Russian thistle, marsh elder, or kochia). CONCLUSIONS: The strong association between skin test reactivity, respiratory symptoms, and pollination period suggests that Cannabis could be a clinically important aeroallergen for certain patients and should be further studied.

Identification of Cannabis pollens using an allergic patient's immunoglobulin E and purification and characterization of allergens in Cannabis pollens.

Cannabis pollen allergens were detected using the serum of an allergic patient. The allergens were then purified by sequential column chromatography (including DE52 cellulose and phenyl-Sepharose CL-4B) and preparative HPLC. The molecular weight of the allergens were determined as 10,050 and 13,706 by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We utilised Western blotting and development of an enzyme-linked immunosorbent assay for the detection of Cannabis pollen allergens.