PEA-OXA restores cognitive impairments associated with vitamin D deficiency-dependent alterations of the gut microbiota.

There is a growing evidence suggesting the association of vitamin D deficiency (VDD) and cognitive impairment. In this study we evaluated the possible involvement of gut microbiota in the cognitive impairments mediated by VDD and investigated the effects of pharmacological treatment with the oxazoline derivative of the aliamide palmitoylethanolamide, 2-Pentadecyl-2-oxazoline (PEA-OXA). Mice were submitted to behavioural, biochemical and electrophysiological analysis to assess whether their vitamin D status affected cognitive performance together with gut microbiota composition. In VDD mice we found cognitive malfunctioning associated with reduced neuroplasticity, indicated by impaired long term potentiation, and neuroinflammation at the hippocampal level. Importantly, PEA-OXA counteracted the cognitive impairments and modified the biochemical and functional changes induced by VDD. Additionally, PEA-OXA treatment enhanced gut microbiota diversity, which tended to be decreased by VDD only in female mice, elevated the relative abundance of lactic and butyric acid-producing families, i.e. Aerococcaceae and Butyricicoccaceae, and reversed the VDD-induced decrease of butyrate-producing beneficial genera, such as Blautia in female mice, and Roseburia in male mice. These data provide novel insights for a better understanding of the cognitive decline induced by VDD and related gut dysbiosis and its potential therapeutic treatment.

Biphasic Hormetic-like Effect of Lebecetin, a C-type Lectin of Snake Venom, on Formalin-induced Inflammation in Mice.

BACKGROUND: Integrins, important extracellular matrix (ECM) receptor proteins, are affected by inflammation and can participate in the maintenance of many painful conditions. Although they are ubiquitous and changeable across all cell types, the roles of these cell adhesion molecules in pathological pain have not been fully explored. OBJECTIVE: We evaluated the effects of the subcutaneous injection of lebecetin, a C-type lectin isolated from Macrovipera lebetina snake venom, previously reported to inhibit α5ß1 and αv integrin activity, on different components of inflammation induced by the formalin administration in the hind paw of mice. METHODS: The formalin-induced nocifensive behavior, edema, and histopathological changes in the hind paw associated with cytokine, iNOS, and COX2 expression, nociceptive-specific neuron activity, and microglial activation analysis in the spinal cord were evaluated in mice receiving vehicle or lebecetin pretreatment. RESULTS: Lebecetin inhibited the nocifensive responses in the formalin test, related edema, and cell infiltration in the injected paw in a biphasic, hormetic-like, and dose-dependent way. According to that hormetic trend, a reduction in pro-inflammatory cytokines IL-6, IL-8, and TNF-alpha and upregulation of the anti-inflammatory cytokine IL-10 in the spinal cord were found with the lowest doses of lebecetin. Moreover, COX2 and iNOS expression in serum and spinal cord followed the same biphasic pattern of cytokines. Finally, nociceptive neurons sensitization and activated microglia were normalized in the dorsal horn of the spinal cord by lebecetin. CONCLUSION: These findings implicate specific roles of integrins in inflammation and tonic pain, as well as in the related central nervous system sequelae.

Effects of CB2 and TRPV1 receptors' stimulation in pediatric acute T-lymphoblastic leukemia.

T-Acute Lymphoblastic Leukemia (T-ALL) is less frequent than B-ALL, but it has poorer outcome. For this reason new therapeutic approaches are needed to treat this malignancy. The Endocannabinoid/Endovanilloid (EC/EV) system has been proposed as possible target to treat several malignancies, including lymphoblastic diseases. The EC/EV system is composed of two G-Protein Coupled Receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel, their endogenous and exogenous ligands and enzymes. CB1 is expressed mainly in central nervous system while CB2 predominantly on immune and peripheral cells, therefore we chose to selectively stimulate CB2 and TRPV1. We treated T-ALL lymphoblasts derived from 4 patients and Jurkat cells with a selective agonist at CB2 receptor: JWH-133 [100 nM] and an agonist at TRPV1 calcium channel: RTX [5 uM] at 6, 12 and 24 hours. We analyzed the effect on apoptosis and Cell Cycle Progression by a cytofluorimetric assays and evaluated the expression level of several target genes (Caspase 3, Bax, Bcl-2, AKT, ERK, PTEN, Notch-1, CDK2, p53) involved in cell survival and apoptosis, by Real-Time PCR and Western Blotting. We observed a pro-apoptotic, anti-proliferative effect of these compounds in both primary lymphoblasts obtained from patients with T-ALL and in Jurkat cell line. Our results show that both CB2 stimulation and TRPV1 activation, can increase the apoptosis in vitro, interfere with cell cycle progression and reduce cell proliferation, indicating that a new therapeutic approach to T-cell ALL might be possible by modulating CB2 and TRPV1 receptors.

Anti-proliferative, pro-apoptotic and anti-invasive effect of EC/EV system in human osteosarcoma.

Osteosarcoma is the most common and aggressive bone tumor in children. The Endocannabinoid/Endovanilloid system has been proposed as anticancer target in tumor of different origins. This system is composed of two receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel and their ligands and enzymes. CB1 is expressed mainly in central nervous system while CB2 predominantly on immune and peripheral cells. We investigated the effects of JWH-133 (CB2 agonist) and RTX (TRPV1 agonist) in six human Osteosarcoma cell lines: MG-63, U-2OS, MNNG/HOS, Saos-2, KHOS/NP, Hs888Lu, by Apoptosis and Migration-Assay. We also compared the effects of these compounds on Caspase-3, AKT, MMP-2 and Notch-1 regulation by Q-PCR and Western Blotting. We observed an anti-proliferative, pro-apoptotic, anti-invasive effect. Our results show that both CB2 stimulation and TRPV1 activation, in different Osteosarcoma cell lines, can act on the same pathways to obtain the same effect, indicating the Endocannabinoid/Endovanilloid system as a new therapeutic target in Osteosarcoma.

The Role of Mifamurtide in Chemotherapy-induced Osteoporosis of Children with Osteosarcoma.

BACKGROUND: Osteosarcoma is the most frequent malignant bone tumor in childhood and young adulthood. Long-term survivors of osteosarcoma patients show high prevalence of osteoporosis and fractures. The immunomodulatory mifamurtide, which modulates macrophages activity, improves disease outcome. OBJECTIVE: To evaluate the role of mifamurtide on macrophage component of bone, the osteoclasts, during chemotherapy in children with osteosarcoma. METHOD: Osteoclasts, obtained from peripheral blood cells of healthy donors were harvested in the presence or not of mifamurtide. Moreover, osteoclast cultures were obtained from osteosarcoma patients, at onset and during chemotherapy, alone or with mifamurtide. Pro-osteoporotic tartrateresistant acid phosphatase (TRAP), phosphokinase-ß-2 (PKCß2), vanilloid receptor type 1 (TRPV1), and anti-osteoporotic cannabinoid receptor type 2 (CB2) biomarkers were analyzed by bio-molecular (qPCR), biochemical (Western Blotting), and morphological (TRAP assay) approaches. RESULTS: Osteoclasts from osteosarcoma patients show significant increase of TRAP and decrease of CB2 with respect to osteoclasts from healthy donors. This osteoclast hyperactivity is more evident in osteoclasts from osteosarcoma patients during chemotherapy. Mifamurtide reduces pro-osteoporotic TRAP, PKCß2, TRPV1 levels and increases CB2 in osteoclasts from healthy donors. Moreover, chemotherapy-induced effects on bone resorption markers are fully reverted in osteoclasts derived from osteosarcoma patients in chemotherapy plus mifamurtide. CONCLUSION: Our data suggest a new therapeutic role for mifamurtide as possible anti-resorption agent in chemotherapy-induced osteoporosis in children with osteosarcoma.

PKCßII-mediated cross-talk of TRPV1/CB2 modulates the glucocorticoid-induced osteoclast overactivity.

In this study, we investigated the role of the endovanilloid/endocannabinoid system in the glucocorticoid-induced osteoclast overactivity. Receptorial and enzymatic component of the endovanilloid/endocannabinoid system are expressed in bone cells, and dysregulated when bone mass is reduced. Moreover, blockade or desensitization of vanilloid receptor 1 (TRPV1) and/or stimulation of cannabinoid receptor 2 (CB2) are beneficial for reducing number and activity of the bone cells modulating resorption, the osteoclasts. We have treated in vitro healthy woman derived osteoclasts with methylprednisolone in presence or not of CB2 or TRPV1 agonists/antagonists, analysing the effect on osteoclast function and morphology through a multidisciplinary approach. Moreover, a treatment with a protein kinase C inhibitor to evaluate osteoclast activity and endovanilloid/endocannabinoid component expression levels was performed in osteoclasts derived from healthy subjects in presence of not of methylprednisolone. Our results show, for the first time, that the endovanilloid/endocannabinoid system is dysregulated by the treatment with methylprednisolone, that the osteoclast activity is increased and that pharmacological compounds stimulating CB2 or inhibiting TRPV1 might reduce, possible inhibiting protein kinase C beta II, the methylprednisolone-induced osteoclast over-activation, suggesting their therapeutic use for protecting from the glucocorticoid-induced bone mass loss.

Cannabinoid Receptor 2 as Antiobesity Target: Inflammation, Fat Storage, and Browning Modulation.

CONTEXT: Obesity is associated with a low-grade inflammatory state and adipocyte (ADP) hyperplasia/hypertrophy. Obesity inhibits the "browning" of white adipose tissue. Cannabinoid receptor 2 (CB2) agonists reduce food intake and induce antiobesity effect in mice. A common missense CB2 variant, Q63R, causes CB2-reduced function. OBJECTIVE: To evaluate the influence of CB2 receptor on the modulation of childhood obesity and of ADP activity and morphology. DESIGN: CB2-Q63R variant was analyzed in obese Italian children. The effects of an inflammatory stimulus and those of drugs selectively acting on CB2 were investigated on in vitro ADPs obtained from mesenchymal stem cells of adult healthy donors or from sc adipose biopsies of adult nonobese and obese subjects. SETTING: Department of Women, Child and General and Specialist Surgery of the Second University of Naples. PATIENTS OR OTHER PARTICIPANTS: A total of 501 obese Italian children (age 11 ± 2.75). Twelve healthy bone marrow donors (age 36.5 ± 15); and 17 subjects, 7 lean (age 42 ± 10) and 10 obese (age 37.8 ± 12) underwent sc adipose tissue biopsies. MAIN OUTCOME MEASURES: Effects of CB2 stimulation on adipokine, perilipin, and uncoupling protein-1 expression. RESULTS: The less-functional CB2-R63 variant was significantly associated with a high z-score body mass index. CB2 blockade with AM630 reverse agonist increased inflammatory adipokine release and fat storage and reduced browning. CB2 stimulation with JWH-133 agonist reversed all of the obesity-related effects. CONCLUSION: CB2 receptor is a novel pharmacological target that should be considered for obesity.

CB(2) and TRPV(1) receptors oppositely modulate in vitro human osteoblast activity.

In the current study, we have investigated the effect of CB2 and TRPV1 receptor ligands on in vitro osteoblasts from bone marrow of human healthy donors. A pivotal role for the endocannabinoid/endovanilloid system in bone metabolism has been highlighted. We have demonstrated a functional cross-talk between CB2 and TRPV1 in human osteoclasts, suggesting these receptors as new pharmacological target for the treatment of bone resorption disease as osteoporosis. Moreover, we have shown the presence of these receptors on human mesenchimal stem cells, hMSCs. Osteoblasts are mononucleated cells originated from hMSCs by the essential transcription factor runt-related transcription factor 2 and involved in bone formation via the synthesis and release of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. For the first time, we show that CB2 and TRPV1 receptors are both expressed on human osteoblasts together with enzymes synthesizing and degrading endocannabinoids/endovanilloids, and oppositely modulate human osteoblast activity in culture in a way that the CB2 receptor stimulation improves the osteogenesis whereas TRPV1 receptor stimulation inhibits it.

The cannabinoid receptor type 2 as mediator of mesenchymal stromal cell immunosuppressive properties.

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians. Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes. In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells. We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.

The 17-ß-oestradiol inhibits osteoclast activity by increasing the cannabinoid CB2 receptor expression.

Bone is a highly metabolically active tissue and its formation and resorption is at the base of bone remodelling. The critical importance of a balanced bone remodelling is demonstrated by human diseases, i.e. osteoporosis, in which a net increase in bone resorption is responsible of skeleton weakening and fracture risk. Oestrogens display anti-resorptive properties on bone metabolism. Indeed, the so-called post-menopausal osteoporosis occurs after interruption of gonad function and benefits from hormonal replacement treatment. Recently, an important role for the endocannabinoid system in the regulation of skeletal remodelling in human has also been shown. In particular, we showed that CB2 stimulation is able to reduce the number of human OCs in vitro. Here, we provide unprecedented evidence that 17-ß-oestradiol administration inhibits activity and formation of human OCs in vitro, demonstrating that oestrogens are able to induce an increase of CB2 expression probably through the recruitment of a putative oestrogens responsive element in the CB2 encoding for gene.