We found 2 studies conducted in healthy volunteers’ samples, conducted by the same research team, Pacifi et al. [23, 24], in 2003  and 2006 . Both were longitudinal observational studies. On the 2003 article , 61 volunteers were included and 3 distinct groups were analyzed: polydrug users, cannabis users, and a control group with no drug use. The aim was to compare the cell-mediated immune response and cytokine release in cannabis users in relation to the control group. The major finding was that cannabis users had lower function on immune response, with a considerable decrease in inflammatory cytokine serum levels. The authors state that the small sample size might have been a limitation of that study. On the 2006 article , 94 volunteers were included and divided into the same 3 groups of the 2003 article . The article analyzed the cell-mediated immune function and the occurrence of mild infectious diseases. It reported 3 important findings: (i) polydrug users had a big decrease in immune response and a considerable increase in anti-inflammatory transforming growth factor β1; therefore, (ii) polydrug users had an increase in mild common infections; finally, (iii) cannabis users had an intermediate decrease in immune response in relation to the control group. This article also had the small sample size limitation, and it did not consider the possible effect of lifestyle on immune function. Detailed information is presented in Table 2.
Finally, the third study was performed by Sexton et al. . It was a longitudinal study aiming to describe the immunological effects of chronic cannabis use among MS patients, measuring the levels of 10 cytokines. It found that cannabis users showed inhibited monocyte migrations, decrease in serum IL-7, decrease in levels of lymphocyte T-helper type 1 (TH1) and lymphocyte T-helper type 2 (TH2) cytokines, and increase in anandamide (AEA) in comparison to controls. Small sample size was also a limitation.
Five out of the 6 papers in our review were longitudinal studies. In 4 of the papers, cannabis or its extracts were found to be negatively associated with immunity status, indicating that the higher the cannabis consumption, the lower the immunity cytokine levels went. The remaining 2 articles showed no association between cannabis use and serum immunity cytokine levels. The most commonly reported exposure covariates were age, sex, cannabis use, alcohol use, and tobacco use.
Sexton et al.  performed a cross-sectional study, evaluating 10 cannabis users. The aim of this study was to evaluate the migratory potential of isolated monocytes from cannabis users. It found that cannabinoids inhibited the migration of monocytes in both groups (naïve and nonnaïve to cannabis), and the monocytes from subjects nonnaïve to cannabis expressed more CB1 messenger ribonucleic acid (mRNA). Although the authors report no limitations, we can infer that unknowing about the acute and long-term effects of phytocannabinoid (pCB) on human circulating monocytes limits the comprehension of the study findings.
We found 3 articles using volunteers with generalized medical conditions treated with medical cannabis extracts. All of them included patients with MS. Detailed information is presented in Table 2.
Koppel et al.  published a systematic review analyzing publications between 1948 and 2003 about the use of CBD on the treatment of multiple sclerosis (MS) and chronic pain, which registered the efficacy of oral extracts from cannabis that used combinations of THC/CBD or CBD only. Furthermore, a study conducted by Klein  found an anti-inflammatory potential for the cannabinoids, which would be helpful in treatment of the inflammatory diseases, such as rheumatoid arthritis, lupus erythematosus, and MS.
We identified 65 articles in our preliminary search. Of these references, 21 were excluded on the first analysis because of duplicates and nonrelevant articles. All 44 remaining articles were further reviewed, and 40 of these were excluded because they did not fulfill the inclusion criteria. The remaining 4 articles met our inclusion criteria and were fully reviewed, and data were extracted from them. Also, we identified 34 relevant publications after title review of all article’s references in which only 2 publications met our inclusion criteria and were fully reviewed, and data were extracted from them.
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For reactive oxygen intermediate (ROI) assay, RAW 264.7 cells were removed from the culture dish by scraping, and were washed and resuspended at 10 6 cells/mL in Hank’s balanced salt solution without phenol red. Cells (5×10 5 ) were added to a luminometer tube together with various concentrations of the essential oils (5, 10, 20, or 40 μg/mL). After 5 min, 10 μL luminol (Sigma) and 30 μL zymosan (Sigma) were added to each tube and the chemiluminescence was measured immediately in a luminometer (Biolumate LB 95; Berhold, Wilbad, Germany). A second set of samples was incubated for 24 h with the essential oils before adding luminol and zymosan. All experiments were done in duplicates.
Zymosan-induced generation of ROIs by RAW 264.7 macrophages was inhibited by essential Cannabis oils from each of the three chemotypes Tisza (T1), Felina (T2), and Ferimon (T3). RAW 264.7 macrophages (5×10 5 /500 μL HBSS) were either untreated (Control) or incubated with 20 or 40 μL essential oils for 5 min or 24 h before ROI induction by zymosan. The ROI was measured by luminol chemiluminescence. The percentage inhibition of ROI production is presented. *p<0.05. ROI, reactive oxygen intermediate; HBSS, Hank’s Balanced Salt Solution.
Nitric oxide (NO • ) determination and MTT evaluation of viability
TNFα in the sera of mice treated with zymosan and essential oils. Twenty-four hours after injecting zymosan and/or an intraperitoneal dose of CBD (5 mg/kg) or essential oils (10, 25, or 50 mg/kg) dissolved in vehicle containing ethanol:Cremophore:saline at a ratio of 1:1:18, the TNFα concentration in the serum was determined by ELISA. N=3 for each treatment group. **p<0.01. TNFα, tumor necrosis factor alpha.
1 The Lautenberg Center for General and Tumor Immunology, The Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel.
Similar to ROI inhibition, the T1 and T2 essential oils significantly suppressed LPS-induced NO • production by RAW macrophages when applied at a concentration of 40 μg/mL ( Fig. 3 ). Lower concentrations of T1 and T2 had almost no effect. The T3 essential oil had barely any effect at the concentrations used.