Selected cannabis cultivars modulate glial activation: in vitro and in vivo studies.

INTRODUCTION: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neuroinflammation, demyelination and axonal loss. Cannabis, an immunomodulating agent, is known for its ability to treat MS effectively. However, due to variations in the profile of secondary metabolites, especially cannabinoids, among cannabis cultivars, the effectiveness of cannabis treatment can vary, with significant variability in the effects on different biological parameters. For screening available cultivars, cellular in vitro as well as pre-clinical in vivo assays, are required to evaluate the effectiveness of the wide range of chemical variability that exists in cannabis cultivars. This study evaluated comparatively three chemically diverse cannabis cultivars, CN2, CN4 and CN6, containing different ratios of phytocannabinoids, for their neuroinflammatory activity in MS model. MATERIALS AND METHODS: In vitro experiments were performed with lipopolysaccharide (LPS)-activated BV-2 microglia and primary glial cells to evaluate the effect of different cannabis cultivars on nitric oxide (NO) and inflammatory cytokines, as well as inducible nitric oxide synthase (iNOS) protein expression. An in vivo experiment using the experimental autoimmune encephalomyelitis (EAE) MS model was conducted using Myelin oligodendrocyte glycoprotein (MOG) as the activating peptide. The cannabis extracts of the cultivars CN2, CN4, CN6 or vehicle, were intraperitoneally injected with clinical scores given based on observed symptoms over the course of study. At the end of the experiment, the mice were sacrificed, and splenocyte cytokine secretion was measured using ELISA. Lumbar sections from the spinal cord of treated MS mice were evaluated for microglia, astrocytes and CD4+ cells. RESULTS: Extracts of the CN2 cultivar contained tetrahydrocannabinolic acid (THCA) and tetrahydrocannabinol (THC) without cannabidiol (CBD), and a number of monoterpenes. CN4 contained cannabidiolic acid (CBDA) and tetrahydrocannabidiolic acid (THCA), with significant amounts of THC: CBD in a 1:1 ratio, as well as sesquiterpenes and some monoterpenes; and CN6 contained primarily CBDA and THCA, as well as THC and CBD in a 2:1 ratio, with some sesquiterpenes and no monoterpenes. All extracts were not cytotoxic in glial cells up to 50 µg/ml. Dose dependent inhibition of LPS-induced BV2 as well as primary microglial NO secretion confirmed the anti-inflammatory and anti-oxidative activity of the three cannabis cultivars. CN2 but not CN4 reduced both astrocytosis and microglial activation in lumbar sections of EAE mice. In contrast, CN4 but not CN2 significantly decreased the secretion of TNFα and Interferon γ (IFNγ) in primary splenocytes extracted from EAE mice. CONCLUSIONS: While both cannabis cultivars, CN2 and CN4, significantly reduced the severity of the clinical signs throughout the course of the study, they modulated different inflammatory mediators and pathways, probably due to differences in their phytocannabinoid composition. This demonstrates the differential potential of cannabis cultivars differing in chemotype to regulate neuroinflammation and their potential to treat MS.

An Emerging Strategy for Neuroinflammation Treatment: Combined Cannabidiol and Angiotensin Receptor Blockers Treatments Effectively Inhibit Glial Nitric Oxide Release.

Cannabidiol (CBD), the major non-psychoactive phytocannabinoid found in cannabis, has anti-neuroinflammatory properties. Despite the increasing use of CBD, little is known about its effect in combination with other substances. Combination therapy has been gaining attention recently, aiming to produce more efficient effects. Angiotensin II activates the angiotensin 1 receptor and regulates neuroinflammation and cognition. Angiotensin receptor 1 blockers (ARBs) were shown to be neuroprotective and prevent cognitive decline. The present study aimed to elucidate the combined role of CBD and ARBs in the modulation of lipopolysaccharide (LPS)-induced glial inflammation. While LPS significantly enhanced nitric oxide synthesis vs. the control, telmisartan and CBD, when administered alone, attenuated this effect by 60% and 36%, respectively. Exposure of LPS-stimulated cells to both compounds resulted in the 95% inhibition of glial nitric oxide release (additive effect). A synergistic inhibitory effect on nitric oxide release was observed when cells were co-treated with losartan (5 µM) and CBD (5 µM) (by 80%) compared to exposure to each compound alone (by 22% and 26%, respectively). Telmisartan and CBD given alone increased TNFα levels by 60% and 40%, respectively. CBD and telmisartan, when given together, attenuated the LPS-induced increase in TNFα levels without statistical significance. LPS-induced IL-17 release was attenuated by CBD with or without telmisartan (by 75%) or telmisartan alone (by 60%). LPS-induced Interferon-γ release was attenuated by 80% when telmisartan was administered in the absence or presence of CBD. Anti-inflammatory effects were recorded when CBD was combined with the known anti-inflammatory agent dimethyl fumarate (DMF)/monomethyl fumarate (MMF). A synergistic inhibitory effect of CBD and MMF on glial release of nitric oxide (by 77%) was observed compared to cells exposed to MMF (by 35%) or CBD (by 12%) alone. Overall, this study highlights the potential of new combinations of CBD (5 µM) with losartan (5 µM) or MMF (1 µM) to synergistically attenuate glial NO synthesis. Additive effects on NO production were observed when telmisartan (5 µM) and CBD (5 µM) were administered together to glial cells.

Therapeutic Potential of Phytocannabinoid Cannabigerol for Multiple Sclerosis: Modulation of Microglial Activation In Vitro and In Vivo.

Multiple sclerosis (MS) is a widespread chronic neuroinflammatory and neurodegenerative disease. Microglia play a crucial role in the pathogenesis of MS via the release of cytokines and reactive oxygen species, e.g., nitric oxide. Research involving the role of phytocannabinoids in neuroinflammation is currently receiving much attention. Cannabigerol is a main phytocannabinoid, which has attracted significant pharmacological interest due to its non-psychotropic nature. In this research, we studied the effects of cannabigerol on microglial inflammation in vitro, followed by an in vivo study. Cannabigerol attenuated the microglial production of nitric oxide in BV2 microglia and primary glial cells; concomitant treatment of the cells with cannabigerol and telmisartan (a neuroprotective angiotensin receptor blocker) decreased nitric oxide production additively. Inducible nitric oxide synthase (iNOS) expression was also reduced by cannabigerol. Moreover, tumor necrosis factor-α (TNF-α), a major cytokine involved in MS, was significantly reduced by cannabigerol in both cell cultures. Next, we studied the effects of cannabigerol in vivo using a mice model of MS, experimental autoimmune encephalomyelitis (EAE). The clinical scores of EAE mice were attenuated upon cannabigerol treatment; additionally, lumbar sections of EAE mice showed enhanced neuronal loss (relative to control mice), which was restored by cannabigerol treatment. Altogether, the set of experiments presented in this work indicates that cannabigerol possesses an appealing therapeutic potential for the treatment of MS.

Attenuation of neutrophil-mediated liver injury in mice by drug-free E-selectin binding polymer.

Inflammation with neutrophils infiltration is a prominent feature of alcohol-related liver disease (ARLD) and contributes to the severity of liver injury. Although an array of potential treatments has been studied, the standard treatment regimen is controversial and can induce severe side effects and infection-related complications. E-selectin, a cytokine inducible cell adhesion molecule, mediates the initial interaction of leucocytes with endothelial cells, and facilitates their further adhesion and extravasation into inflamed tissues. Given the important role of E-selectin in leukocytes trafficking, we hypothesized that a synthetic polymer presenting multiple copies of E-selectin binding peptide in a polyvalent manner (P-Esbp) may block the "roads" that facilitate neutrophil infiltration, inhibit the recruitment of neutrophils to the inflamed sites and reduce the extent of liver injury. We now demonstrate in vitro that P-Esbp reduced the recruitment of neutrophils (collected from blood of donors) on activated human umbilical vein endothelial cells (HUVEC) under flow conditions. Pre-treatment of mice with P-Esbp prior to alcohol binge attenuated alcohol-induced serum transaminase (ALT, AST) elevation, reduced pro-inflammatory cytokines (TNFα and IL-1ẞ) and chemokines (MIP-2/CXCL2 and MCP-1/CCL2) in National Institute on Alcohol Abuse and Alcoholism (NIAAA) model. Also, the up-regulation of neutrophil marker Ly6G and the number of MPO positive cells in the injured tissue was significantly reduced by the treatment, indicating diminished neutrophil infiltration. Moreover, as a result of P-Esbp treatment, E-selectin expression in the liver (mRNA and protein level) was downregulated, suggesting a potential to decrease ongoing local inflammatory response. Overall, our findings highlight the anti-inflammatory properties of the "drug-free" P-Esbp and its therapeutic potential to attenuate an excessive inflammation where infiltrating neutrophils can damage tissues and organs.

CD44-Targeted Polymer-Paclitaxel Conjugates to Control the Spread and Growth of Metastatic Tumors.

One of the greatest challenges in cancer therapy is to control metastatic spread, seeding, and growth of tumors in distant organs. Recently, we reported on the design of a novel "drug-free" therapeutic copolymer bearing the antimigratory A5G27 peptide, designated P-(A5G27)-FITC, that shows excellent specificity to cancer cells overexpressing CD44v3 and CD44v6 and inhibits cancer cell migration and invasion. We demonstrated that P-(A5G27)-FITC accumulated preferentially in subcutaneous (sc) implanted 4T1 tumors following parenteral administration. Moreover, we showed that pretreatment of mice with P-(A5G27)-FITC prior to 4T1 cell inoculation inhibited colonization of circulating 4T1 cells in the lungs. In this study, we designed a new polymer-peptide-drug conjugate to inhibit vigorously growing primary tumors and control invasive behavior of cancer cells. To this end, the antimitotic drug (paclitaxel, PTX) was conjugated to P-(A5G27)-FITC. The targeted polymer-drug conjugate (P-(A5G27)-PTX) was significantly more toxic toward CD44-overexpressing cancer cells than the nontargeted copolymer. In vivo, a single iv injection of P-(A5G27)-PTX prolonged the survival of C57BL/6 mice with established B16-F10 lung metastases. When injected intraperitoneally into BALB/c mice implanted sc with 4T1 tumors, P-(A5G27)-PTX significantly decreased the rate of primary tumor growth, increased the median survival of mice, and reduced the number of 4T1 metastases in the lungs when compared to nontargeted copolymer. Most interestingly, the CD44-targeted "drug-free" copolymer P-(A5G27) (without PTX) significantly inhibited the rate of tumor growth and further prolonged the median survival time of mice to the same extent as the PTX-containing formulations (P-(A5G27)-PTX or free PTX). Overall, this study highlights the therapeutic potential of the HPMA copolymer-A5G27 conjugates ("drug-free" and PTX-bearing copolymers) to control the metastatic spread of cancer.

Evaluation of biodegradable Zn-1%Mg and Zn-1%Mg-0.5%Ca alloys for biomedical applications.

Increasing interest in biodegradable metals (Mg, Fe, and Zn) as structural materials for orthopedic and cardiovascular applications mainly relates to their promising biocompatibility, mechanical properties and ability to self-remove. However, Mg alloys suffer from excessive corrosion rates associated with premature loss of mechanical integrity and gas embolism risks. Fe based alloys produce voluminous corrosion products that have a detrimental effect on neighboring cells and extracellular matrix. In contrast, Zn does not appear to exhibit a harmful mode of corrosion. Unfortunately, pure zinc possesses insufficient mechanical strength for biomedical structural applications. The present study aimed at examining the potential of two new zinc based alloys, Zn-1%Mg and Zn-1%Mg-0.5%Ca to serve as structural materials for biodegradable implants. This examination was carried out under in vitro conditions, including immersion testing, potentiodynamic polarization analysis, electrochemical impedance spectroscopy (EIS), and stress corrosion cracking (SCC) assessments in terms of slow strain rate testing (SSRT). In order to assess the cytotoxicity of the tested alloys, cell viability was evaluated indirectly using Saos-2 cells. The results demonstrate that both zinc alloys can be considered as potential candidates for biodegradable implants, with a relative advantage to the Zn-1%Mg alloy in terms of its corrosion resistance and SCC performance.

Higher Novel L-Cys Degradation Activity Results in Lower Organic-S and Biomass in Sarcocornia than the Related Saltwort, Salicornia.

Salicornia and Sarcocornia are almost identical halophytes whose edible succulent shoots hold promise for commercial production in saline water. Enhanced sulfur nutrition may be beneficial to crops naturally grown on high sulfate. However, little is known about sulfate nutrition in halophytes. Here we show that Salicornia europaea (ecotype RN) exhibits a significant increase in biomass and organic-S accumulation in response to supplemental sulfate, whereas Sarcocornia fruticosa (ecotype VM) does not, instead exhibiting increased sulfate accumulation. We investigated the role of two pathways on organic-S and biomass accumulation in Salicornia and Sarcoconia: the sulfate reductive pathway that generates Cys and l-Cys desulfhydrase that degrades Cys to H2S, NH3, and pyruvate. The major function of O-acetyl-Ser-(thiol) lyase (OAS-TL; EC 2.5.1.47) is the formation of l-Cys, but our study shows that the OAS-TL A and OAS-TL B of both halophytes are enzymes that also degrade l-Cys to H2S. This activity was significantly higher in Sarcocornia than in Salicornia, especially upon sulfate supplementation. The activity of the sulfate reductive pathway key enzyme, adenosine 5'-phosphosulfate reductase (APR, EC 1.8.99.2), was significantly higher in Salicornia than in Sarcocornia These results suggest that the low organic-S level in Sarcocornia is the result of high l-Cys degradation rate by OAS-TLs, whereas the greater organic-S and biomass accumulation in Salicornia is the result of higher APR activity and low l-Cys degradation rate, resulting in higher net Cys biosynthesis. These results present an initial road map for halophyte growers to attain better growth rates and nutritional value of Salicornia and Sarcocornia.

Cytotoxic characteristics of biodegradable EW10X04 Mg alloy after Nd coating and subsequent heat treatment.

Porous Mg scaffolds are considered as potential bone growth promoting materials. Unfortunately, the high rate of biocorrosion inherent to Mg alloys may cause a premature loss of mechanical strength, excessive evolution of hydrogen gas, and a rapidly shifting surface topography, all of which may hinder the ability of native cells to attach and grow on the implant surface. Here we investigated the cell cytotoxicity effects during corrosion of a novel magnesium alloy, EW10X04 (Mg-1.2%Nd-0.5%Y-0.5%Zr-0.4%Ca), following diffusion coating (DC) and heat treatment to reduce the corrosion rate. Cells were exposed either to corrosion products or to the corroding scaffold surface, in vitro. The microstructure characterization of the scaffold surface was carried out by scanning electron microscopy (SEM) equipped with a Noran energy dispersive spectrometer (EDS). Phase analyses were obtained by X-ray diffraction (XRD). We found that cell viability, growth, and adhesion were all improved when cultured on the EW10X04+DC surface or under corrosion product extracts due to lower corrosion rates relative to the EW10X04 control samples. It is therefore believed that the tested alloy after Nd coating and heat treatment may introduce a good balance between its biodegradation characteristics and cytotoxic effects towards cells.

The development of halophyte-based agriculture: past and present.

BACKGROUND: Freshwater comprises about a mere 2·5% of total global water, of which approximately two-thirds is locked into glaciers at the polar ice caps and on mountains. In conjunction with this, in many instances irrigation with freshwater causes an increase in soil salinity due to overirrigation of agricultural land, inefficient water use and poor drainage of unsuitable soils. The problem of salinity was recognized a long time ago and, due to the importance of irrigated agriculture, numerous efforts have been devoted towards improving crop species for better utilization of saline soils and water. Irrigating plants with saline water is a challenge for practitioners and researchers throughout the world. SCOPE: Recruiting wild halophytes with economic potential was suggested several decades ago as a way to reduce the damage caused by salinization of soil and water. A range of cultivation systems for the utilization of halophytes have been developed, for the production of biofuel, purification of saline effluent in constructed wetlands, landscaping, cultivation of gourmet vegetables, and more. This review critically analyses past and present halophyte-based production systems in the context of genetics, physiology, agrotechnical issues and product value. There are still difficulties that need to be overcome, such as direct germination in saline conditions or genotype selection. However, more and more research is being directed not only towards determining salt tolerance of halophytes, but also to the improvement of agricultural traits for long-term progress.

Effects of salinity on flowering, morphology, biomass accumulation and leaf metabolites in an edible halophyte.

The fresh water shortage in agriculture is an increasing problem worldwide, therefore the possibility of cultivating crops under saline conditions is of high importance. Crithmum maritimum, a halophytic plant naturally found on the rocky coastlines of the Atlantic Ocean and the Mediterranean Sea, has a long history of human consumption and was recently suggested as a cash crop for biosaline agriculture. In the present study, we compared the responses of different genotypes originating from France, Portugal and Israel to moderate saline irrigation (up to 100 mM NaCl). The genotypes varied greatly in the onset of flowering, their leaf appearance, growth habits and leaf metabolite content. Both Atlantic genotypes (from France and Portugal) flowered earlier than those from the Mediterranean, but the number of inflorescences decreased with salinity. Irrigation with 50 and 100 mM NaCl led to a reduction in biomass production in both the Israeli and the Portuguese genotypes, while the French genotype was found to produce maximum leaf yield at 50 mM NaCl. With increasing salinity, salt was accumulated by the plants, as indicated by increasing electrical conductivities of the leaf extracts. Concomitantly, antioxidant compounds (such as ascorbic acid), total polyphenols and ureides responded to salinity in a genotype-dependent manner; either they increased, decreased or were unaffected. Notably, the total fatty acid concentration increased with salinity in both Mediterranean genotypes, reaching 2.7 and 2.4 % total fatty acids (on a dry weight basis) at 100 mM NaCl. Moreover, the proportion assigned to omega-3 fatty acids in these genotypes was higher than in their Atlantic counterparts at the highest salinity tested. Our results highlight the variations existing among C. maritimum genotypes from different origins regarding salt-induced changes in plant growth, flowering behaviour and leaf metabolites with nutritional value. Thus, genotypic characteristics should be taken into account when evaluating a wild plant species for future crop cultivation.

Impairment in Sulfite Reductase Leads to Early Leaf Senescence in Tomato Plants.

Sulfite reductase (SiR) is an essential enzyme of the sulfate assimilation reductive pathway, which catalyzes the reduction of sulfite to sulfide. Here, we show that tomato (Solanum lycopersicum) plants with impaired SiR expression due to RNA interference (SIR Ri) developed early leaf senescence. The visual chlorophyll degradation in leaves of SIR Ri mutants was accompanied by a reduction of maximal quantum yield, as well as accumulation of hydrogen peroxide and malondialdehyde, a product of lipid peroxidation. Interestingly, messenger RNA transcripts and proteins involved in chlorophyll breakdown in the chloroplasts were found to be enhanced in the mutants, while transcripts and their plastidic proteins, functioning in photosystem II, were reduced in these mutants compared with wild-type leaves. As a consequence of SiR impairment, the levels of sulfite, sulfate, and thiosulfate were higher and glutathione levels were lower compared with the wild type. Unexpectedly, in a futile attempt to compensate for the low glutathione, the activity of adenosine-5'-phosphosulfate reductase was enhanced, leading to further sulfite accumulation in SIR Ri plants. Increased sulfite oxidation to sulfate and incorporation of sulfite into sulfoquinovosyl diacylglycerols were not sufficient to maintain low basal sulfite levels, resulting in accumulative leaf damage in mutant leaves. Our results indicate that, in addition to its biosynthetic role, SiR plays an important role in prevention of premature senescence. The higher sulfite is likely the main reason for the initiation of chlorophyll degradation, while the lower glutathione as well as the higher hydrogen peroxide and malondialdehyde additionally contribute to premature senescence in mutant leaves.

The effect of pH and calcium ions on the stability of amphiphilic and anionic ß-sheet peptide hydrogels.

Amphiphilic peptides can form bottom-up-designed self-assembled hydrogels composed of elongated fibril matrices that could find uses in various biologically-related systems, acting as platforms for drug delivery or scaffolds that mimic extracellular matrices in tissue regeneration systems. We have previously reported that the amphiphilic and anionic ß-sheet forming peptide, Pro-Asp-(Phe-Asp)5 -Pro, P(FD)-5, generates hydrogels that template calcium-phosphate mineral and as such, were able to enhance bone formation in vivo. Our earlier results prompted us to further exploit the effects of pH and calcium ion concentration on P(FD)-5 peptide in solution, in hydrogels and in mineral-loaded hydrogel compositions. Circular dichroism-based characterization of solutions of the peptide demonstrated transitions between the unfolded state to a ß-sheet structure as function of peptide concentration, pH and calcium ion concentration. FTIR measurements were employed to monitor differences between the structure of the peptide in solution and in hydrogels. Rheology and dissolution studies demonstrated the improved stability of hydrogels prepared by a two-step procedure, where the peptides are dissolved and self-assemble in the first step, while in the second step, calcium ions are allowed to adsorb onto the system. These results, highlighting the effects of a few central factors on the structure, assembly and stability of amphiphilic and anionic ß-sheet peptide systems, will contribute to the further development of designed self-assembled peptide systems from solutions to hydrogels and hydrogel-loaded matrices, such as mineral putty compositions.

The importance of iron supply during repetitive harvesting of Aster tripolium.

Aster tripolium L. is a salt marsh halophyte that has recently gained interest as a cash crop vegetable. Leaf yield and quality were investigated in plants grown with salinity in experiments with Perlite in pots and in plots on dune sand. Plants were repetitively harvested in a 14-day cycle. A. tripolium irrigated with 50mM NaCl exhibited the highest yield when grown in pots, whereas in the plot experiment no significant differences in biomass accumulation occurred up to 80mM NaCl in the irrigation water. Chemical leaf composition changed with salinity, exhibiting higher levels of electrical conductivity, total soluble solutes and the non-enzymatic antioxidant compounds ascorbic acid and polyphenols compared with control plants grown without NaCl supplementation. Using the repetitive harvest regime, leaf chlorosis occurred, a symptom shared by deficiencies in either nitrogen or iron. Comparative applications of five iron chelate formulations in plants grown with 50mM NaCl in pots revealed improved leaf colour and chlorophyll content for only two of the applied Fe-chelates. Concomitantly with leaf colour restoration, the activity of nitrate reductase, the first enzyme during nitrate assimilation, which requires heme-iron for its proper function, increased 3-fold as a result of the iron treatment in the plot experiment. Importantly, the enhancement of nitrate reductase activity was associated with a considerable decrease in the leaf nitrate concentration. Therefore, we concluded that iron deficiency, in addition to leaf chlorosis, reduces A. tripolium leaf quality as a vegetable by increasing the leaf nitrate content. Furthermore, nitrate reductase (NR) activity levels in A. tripolium leaves may act as an indicator of iron deficiency that manifests itself as reduced nitrate content owing to the higher NR activity upon proper iron nutrition. These results demonstrate the importance of salinity level and the application of an appropriate iron-chelating formulation to generate marketable yields of Aster tripolium leafy vegetable when grown commercially on dune sand.

A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants.

Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.