Effect of CB2 Stimulation on Gene Expression in Pediatric B-Acute Lymphoblastic Leukemia: New Possible Targets.

Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.

Effects of Iron Chelation in Osteosarcoma.

BACKGROUND: Osteosarcoma is an aggressive bone tumor. It represents the principal cause of cancer-associated death in children. Considering the recent findings on the role of iron in cancer, iron chelation has been investigated for its antineoplastic properties in many tumors. Deferasirox is the most used iron chelator compound and in previous studies showed an anticancer effect in hematologic and solid malignancies. Eltrombopag is a Thrombopoietin receptor used in thrombocytopenia that also binds and mobilize iron. It demonstrated an effect on iron overload conditions and also in contrasting cancer cell proliferation. OBJECTIVE: We analyzed the effects of deferasirox and eltrombopag in human osteosarcoma cells in an attempt to identify other therapeutic approaches for this tumor. METHODS: We cultured and treated with deferasirox and Eltrombopag, alone and in combination, two human osteosarcoma cell lines, MG63 and 143B. After 72h exposure, we performed RTqPCR, Western Blotting, Iron Assay and cytofluorimetric assays to evaluate the effect on viability, apoptosis, cell cycle progression and ROS production. RESULTS: The iron-chelating properties of the two compounds are also confirmed in osteosarcoma, but we did not observe any direct effect on tumor progression. DISCUSSION: We tested deferasirox and eltrombopag, alone and in combination, in human osteosarcoma cells for the first time and demonstrated that their iron-chelating activity does not influence biochemical pathways related to cancer progression and maintenance. CONCLUSION: Although further investigations on possible effects mediated by cells of the tumor microenvironment could be of great interest, in vitro iron chelation in osteosarcoma does not impair tumor progression.

The Role of Cannabinoid Receptor Type 2 in the Bone Loss Associated With Pediatric Celiac Disease.

OBJECTIVES: In this study, we investigated the role of the cannabinoid receptor type 2 (CB2) in the bone loss associated with celiac disease (CD) evaluating the effect of its pharmacological modulation on osteoclast activity. We previously demonstrated a significant association between the CB2 Q63R variant and CD, suggesting it as a possible disease biomarker. Moreover, CB2 stimulation is beneficial for reducing osteoclast activity in several bone pathologic conditions. METHODS: In vitro osteoclasts (OCs) were differentiated from peripheral blood mononuclear cells of healthy donors, CD children at diagnosis and after 1 year of gluten-free diet (GFD) and characterized by real-time PCR and western blot for the expression of CB2 and specific osteoclastic markers, TRAP and Cathepsin K. TRAP assay and Bone Resorption assay were performed to evaluate osteoclast activity before and after 48 h exposure to CB2 selective drugs (JWH-133 and AM630) and Vitamin D. RESULTS: We found in CD patients an osteoclast hyperactivation and low levels of CB2. CB2 stimulation with JWH-133 agonist is more effective than Vitamin D in reducing osteoclast activity whereas CB2 blockade with AM630 increases osteoclast activation. The anti-osteoporotic effect of JWH-133 decreases when used in co-treatment with vitamin D. GFD reduces osteoclast activity without restore CB2 expression. CONCLUSIONS: CB2 could be a molecular marker to predict the risk of bone alterations in CD and a pharmacological target to reduce bone mass loss in patients who need a direct intervention on bone metabolism, in addition to the GFD.

Can Denosumab be used in combination with Doxorubicin in Osteosarcoma?

Osteosarcoma is an aggressive bone tumor of the pediatric age. It is therefore important to improve conventional therapies (chemotherapy and surgery). Anticancer drugs often cause osteoporosis due to a misbalance of RANK/RANK-L/OPG pathway. Denosumab is a monoclonal antibody with high affinity and specificity to RANK-L, the ligand released by osteoblasts that enhances osteoclasts differentiation and bone resorption. It is used in osteoporosis and in other conditions characterized by bone mass loss. Doxorubicin is a chemotherapic drug used in several kinds of tumors, and also patients treated with it often develop osteoporosis. We investigated the effects of Denosumab alone and in combination with Doxorubicin, in two human osteosarcoma cell lines (MG63 and U-2 OS). We evaluated the effect of these treatments on apoptosis, cell cycle progression, invasion capacity and bone metabolism. We observed for the first time an anti-invasive effect of Denosumab in OS cells and confirmed its anti-osteoporotic activity also in Osteosarcoma. On the other hand, we demonstrate that Denosumab not only does not affect apoptosis and cell cycle progression, but when used in combination with Doxorubicin, it causes an unexpected reduction of its activity. These results indicate that the presence of Denosumab might inhibit the efficacy of the chemotherapic drug. In conclusion, while our results certainly support and confirm the efficacy of Denosumab in Osteoporosis, we discourage the use of Denosumab in addition to conventional chemotherapy in Osteosarcoma, even though, certainly further investigations are necessary to better clarify the clinical role of this monoclonal antibody in cancer.

Safety of Anticancer Agents Used in Children: A Focus on Their Off-Label Use Through Data From the Spontaneous Reporting System.

BACKGROUND: Among factors influencing the higher risk of developing unknown or rare adverse drug reactions (ADRs) among children and adolescents, there is the frequent off-label use of drugs that seems to be very common in pediatric oncological patients. Our study aim to collect and evaluate data on the safety profile of antineoplastic drugs and their off-label use in the pediatrics population using real life data. METHODS: We retrieved Individual Case Safety Reports (ICSRs) with an anticancer agent as suspected drug among those reported through the Campania spontaneous reporting system from 1 January 2013 to 30 September 2019. We classified ICSRs into four off-label categories: "age," "route of administration," "weight," and "therapeutic indication." We defined an ICSR as an off-label case if it met at least one of the aforementioned categories for at least one of the reported suspected antineoplastic drugs. RESULTS: A total of 18 ICSRs (7.6%) out of 236 were classified as off-label cases. The median age of patients was 13 years (interquartile range, IQR: 6-16), with 94.4% of cases occurring in male patients. In the classification of the off-label category, 16 ICSRs were categorized according to the "therapeutic indication" and two for the "age." No case was categorized for the off-label categories "route of administration" and "weight." The two off-label cases categorized as "age" were both related to the use of brentuximab vedotin for Hodgkin's lymphoma in patients aged 16 years. Twenty-nine ADRs (1.6 suspected adverse drug reactions per ICSR) were identified among off-label cases. Among ADRs, those reported more than one were diarrhea (N = 3), neutropenia (N = 3), nausea (N = 2), pyrexia (N = 2), and vomit (N = 2). CONCLUSIONS: Our findings showed a low number of ICSRs classified as off-label. The majority of off-label ICSRs were categorized for the "therapeutic indication." This low number of off-label ICSRs might be largely due to the underreporting phenomenon, which is a major limit in pharmacovigilance. Therefore, we believe that spreading pharmacovigilance knowledge and awareness might improve this aspect.

Cannabinoid Receptor Type 2: A Possible Target in SARS-CoV-2 (CoV-19) Infection?

In late December 2019, a novel coronavirus (SARS-CoV-2 or CoV-19) appeared in Wuhan, China, causing a global pandemic. SARS-CoV-2 causes mild to severe respiratory tract inflammation, often developing into lung fibrosis with thrombosis in pulmonary small vessels and causing even death. COronaVIrus Disease (COVID-19) patients manifest exacerbated inflammatory and immune responses, cytokine storm, prevalence of pro-inflammatory M1 macrophages and increased levels of resident and circulating immune cells. Men show higher susceptibility to SARS-CoV-2 infection than women, likely due to estrogens production. The protective role of estrogens, as well as an immune-suppressive activity that limits the excessive inflammation, can be mediated by cannabinoid receptor type 2 (CB2). The role of this receptor in modulating inflammation and immune response is well documented in fact in several settings. The stimulation of CB2 receptors is known to limit the release of pro-inflammatory cytokines, shift the macrophage phenotype towards the anti-inflammatory M2 type and enhance the immune-modulating properties of mesenchymal stromal cells. For these reasons, we hypothesize that CB2 receptor can be a therapeutic target in COVID-19 pandemic emergency.

Correction: Argenziano, M., et al. The Endocannabinoid System in Pediatric Inflammatory and Immune Diseases. Int. J. Mol. Sci. 2019, 20, 5875.

The authors wish to make the following corrections to this paper [...].

Mifamurtide and TAM-like macrophages: effect on proliferation, migration and differentiation of osteosarcoma cells.

Tumor-associated macrophages and their alternative activation states together with cytokines and growth factors trapped in tumor microenvironment contribute to the progression of OS. In contrast to other tumor types, M2 polarized macrophages, reduce metastasis and improve survival in osteosarcoma patients. Mifamurtide is an immunomodulatory drug given together with standard adjuvant chemotherapy in high-grade osteosarcoma to improve outcome. Macrophages obtained from peripheral blood mononucleated cells of healthy donors and MG63 cells were cultured alone and together, and treated with Mifamurtide. We analyzed the effects of Mifamurtide on macrophage polarization and on MG63 proliferation, migration and differentiation, evaluating the expression of M1/M2 and osteoblast markers and molecules involved in metastasis and proliferation pathways. Our data suggest that Mifamurtide, switching macrophage polarization towards a TAM-like intermediate M1/M2 phenotype, may modulate the delicate balance between pro-inflammatory and immunomodulatory macrophage functions. Moreover, Mifamurtide may inhibit the cellular proliferation and induce the tumor cell differentiation, probably through the down regulation of pSTAT3, pAKT and IL-17R.

The Endocannabinoid System in Pediatric Inflammatory and Immune Diseases.

Endocannabinoid system consists of cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors, their endogenous ligands, and the enzymes responsible for their synthesis and degradation. CB2, to a great extent, and CB1, to a lesser extent, are involved in regulating the immune response. They also regulate the inflammatory processes by inhibiting pro-inflammatory mediator release and immune cell proliferation. This review provides an overview on the role of the endocannabinoid system with a major focus on cannabinoid receptors in the pathogenesis and onset of inflammatory and autoimmune pediatric diseases, such as immune thrombocytopenia, juvenile idiopathic arthritis, inflammatory bowel disease, celiac disease, obesity, neuroinflammatory diseases, and type 1 diabetes mellitus. These disorders have a high social impact and represent a burden for the healthcare system, hence the importance of individuating more innovative and effective treatments. The endocannabinoid system could address this need, representing a possible new diagnostic marker and therapeutic target.

TNF-α effect on human delivery onset by CB1/TRPV1 crosstalk: new insights into endocannabinoid molecular signaling in preterm vs. term labor. Analysis of the EC/EV pathway and predictive biomarkers for early diagnosis of preterm delivery.

BACKGROUND: Endocannabinoids/endovanilloid (EC/EV) system and inflammation are recognized as key regulators of cell-signaling pathways in female reproduction. The knowledge of predictive biomarkers involved in preterm birth (PTB) represents an important goal to make an early diagnosis. The aim of the study was to investigate the role of EC/EV system and inflammation in human delivery, in placental samples from spontaneous deliveries. METHODS: We examined the expression of genes encoding for the components of EC/EV system (CB1, CB2, TRPV1, MAGL, FAAH, DAGL, NAPE-PLD) and for inflammatory cytokines (IL-6, TNF-α) with qRT-PCR techniques, in human placental samples from preterm delivery (at 30 and at 34 weeks) compared to term delivery (40 weeks, control group). RESULTS: We found a marked increase of CB1, anandamide, and inflammatory cytokines, mainly TNF-α, together with TRPV1 down-regulation in term delivery group, compared to preterm groups (P<0.05). CONCLUSIONS: Our findings highlighted the emergent pivotal role of the EC/EV system and inflammation in spontaneous term delivery and provided the framework for future studies that investigate the CB1/TRPV1 crosstalk in preterm birth. Particularly, we found a link between the stimulation of CB1 receptors and the antagonism of TRPV1 channels, that could be used in PTB prevention, through selected molecules.

The Endocannabinoid/Endovanilloid System in Bone: From Osteoporosis to Osteosarcoma.

Bone is a dynamic tissue, whose homeostasis is maintained by a fine balance between osteoclast (OC) and osteoblast (OB) activity. The endocannabinoid/endovanilloid (EC/EV) system's receptors are the cannabinoid receptor type 1 (CB1), the cannabinoid receptor type 2 (CB2), and the transient receptor potential cation channel subfamily V member 1 (TRPV1). Their stimulation modulates bone formation and bone resorption. Bone diseases are very common worldwide. Osteoporosis is the principal cause of bone loss and it can be caused by several factors such as postmenopausal estrogen decrease, glucocorticoid (GC) treatments, iron overload, and chemotherapies. Studies have demonstrated that CB1 and TRPV1 stimulation exerts osteoclastogenic effects, whereas CB2 stimulation has an anti-osteoclastogenic role. Moreover, the EC/EV system has been demonstrated to have a role in cancer, favoring apoptosis and inhibiting cell proliferation. In particular, in bone cancer, the modulation of the EC/EV system not only reduces cell growth and enhances apoptosis but it also reduces cell invasion and bone pain in mouse models. Therefore, EC/EV receptors may be a useful pharmacological target in the prevention and treatment of bone diseases. More studies to better investigate the biochemical mechanisms underlining the EC/EV system effects in bone are needed, but the synthesis of hybrid molecules, targeting these receptors and capable of oppositely regulating bone homeostasis, seems to be a promising and encouraging prospective in bone disease management.

CB2 Receptor Stimulation and Dexamethasone Restore the Anti-Inflammatory and Immune-Regulatory Properties of Mesenchymal Stromal Cells of Children with Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction, with a complex and unclear pathogenesis. The impaired immunosuppressive capacity of mesenchymal stromal cells in ITP patients (ITP-MSCs) might play a role in the development of the disease. Correcting the MSC defects could represent an alternative therapeutic approach for ITP. High-dose dexamethasone (HD-Dexa) is the mainstay of the ITP therapeutic regimen, although it has several side effects. We previously demonstrated a role for cannabinoid receptor 2 (CB2) as a mediator of anti-inflammatory and immunoregulatory properties of human MSCs. We analyzed the effects of CB2 stimulation, with the selective agonist JWH-133, and of Dexa alone and in combination on ITP-MSC survival and immunosuppressive capacity. We provided new insights into the pathogenesis of ITP, suggesting CB2 receptor involvement in the impairment of ITP-MSC function and confirming MSCs as responsive cellular targets of Dexa. Moreover, we demonstrated that CB2 stimulation and Dexa attenuate apoptosis, via Bcl2 signaling, and restore the immune-modulatory properties of MSCs derived from ITP patients. These data suggest the possibility of using Dexa in combination with JWH-133 in ITP, reducing its dose and side effects but maintaining its therapeutic benefits.

Iron chelating properties of Eltrombopag: Investigating its role in thalassemia-induced osteoporosis.

Chronic blood transfusions are responsible to cause iron overload, which leads to several complications to end organs and osteoporosis. Iron chelation is needed to remove iron excess and to contain bone-mass loss. Deferasirox is the most recent oral iron chelator that prevents transfusion related iron overload complications. Recently Eltrombopag (ELT) iron chelating properties are emerging. ELT is an agonist at Thrombopoietin receptor, used in treatment of thrombocytopenia. We tested ELT and Deferasirox in iron overloaded osteoclasts from thalassemic patients and donors measuring intracellular iron, TRAP expression and osteoclast activity. We confirmed ELT iron chelation capacity also in bone tissue and a synergic effect when used with Deferasirox. Moreover, having demonstrated its effects on osteoclast activity, we suggest for the first time that ELT could ameliorate bone tissue's health reducing bone mass loss.

Association between 14 bp insertion/deletion HLA-G functional polymorphism and insulin resistance in a cohort of Italian children with obesity.

BACKGROUND: The non-classical HLA-class I molecule-g (HLA-G) gene shows a deletion/insertion (del/ins) polymorphism of a 14-base-pair sequence (14 bp) in the exon 8 at the 3' untranslated region. The presence of the 14 bp insertion allele has been associated to lower soluble HLA-G protein production, a protein with anti-inflammatory activities. So far, no studies have investigated the relationship between HLA-G 14 bp del/ins polymorphism and metabolic features of obese children and adolescents. We aimed to assess if the HLA-G ins/del polymorphism, and in particular the HLA-G ins/ins genotype determining lower sHLA-G production, is associated to insulin resistance (evaluated by homeostasis model assessment [HOMA]) in a population of obese children. METHODS: We enrolled 574 obese children and adolescents. Anthropometric and laboratory data were collected. The white blood cell (WBC) count was evaluated as surrogate marker of inflammation. C-reactive protein (CRP) was available in 48 patients. HOMA was calculated. Patients were genotyped for the HLA-G del/ins polymorphism. RESULTS: Subjects carrying the HLA-G ins/ins genotype, presented with higher HOMA, WBC and CRP values, compared to del/ins and del/del genotypes (P ≤ 0.0009, ≤0.02 and ≤0.0001, respectively). Comparison of the regression line slopes, performed for HOMA and WBC on the basis of HLA-G genotypes, showed that subjects carrying the HLA-G ins/ins genotype presented with a stronger correlation between HOMA and WBC, compared to the other genotypes (Model r2 3.13%, P ≤ 0.006). CONCLUSIONS: We showed a strong association between HLA-G 14 bp ins/ins genotype and HOMA in obese children and adolescents. This association could be hypothetically modulated by subclinical inflammation.

Bortezomib and endocannabinoid/endovanilloid system: a synergism in osteosarcoma.

Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents. Bortezomib (BTZ) is an approved anticancer drug, classified as a selective reversible inhibitor of the ubiquitin-dependent proteasome system, that leads to cancer cell cycle arrest and apoptosis reducing the invasion ability of Osteosarcoma cells in vitro. It also regulates the RANK/RANKL/OPG system, involved in the pathogenesis of bone tumors and in cell migration. A side effect of BTZ is to induce painful sensory peripheral neuropathy which lead to cessation of therapy or dose reduction. Recently BTZ has been evaluated in combination with Cannabinoids targeting CB1 receptor, demonstrating a promising synergic effect. The Endocannabinoid/Endovanilloid (EC/EV) system includes two G protein-coupled receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel and their endogenous ligands and enzymes. CB1 and CB2 are expressed mainly in Central Nervous System and Immune Peripheral cells respectively. TRPV1 is also expressed in primary sensory neurons and is involved in pain modulation. EC/EV system induces apoptosis, reduces invasion and cell proliferation in Osteosarcoma cell lines and is involved in bone metabolism. We analyzed the effects of BTZ, alone and in combination with selective agonists at CB2 (JWH-133) and TRPV1 (RTX) receptors, in the Osteosarcoma cell line (HOS) on Apoptosis, Cell Cycle progression, migration and bone balance. We observed that the stimulation of CB2 and TRPV1 receptors increase the efficacy of BTZ in inducing apoptosis and reducing invasion, cell cycle progression and by modulating bone balance. These data suggest the possibility to use BTZ, in combination with EC/EV agonists, in Osteosarcoma therapy reducing its dose and its side effects.

Role of Cannabinoids in Obesity.

Obesity is an increasing health problem worldwide. Its related comorbidities imply a high cost for the National Health System and diminish a patient's life quality. Adipose tissue is composed of three types of cells. White adipocytes are involved in fat storage and secretion of hormones. Brown adipocytes are involved in thermogenesis and caloric expenditure. Beige adipocytes are transitional adipocytes that in response to various stimuli can turn from white to brown and could be protective against the obesity, enhancing energy expenditure. The conversion of white in beige adipose tissue is a potential new therapeutic target for obesity. Cannabinoid receptors (CB) regulate thermogenesis, food intake and inflammation. CB1 ablation or inhibition helps reducing body weight and food intake. Stimulation of CB2 limits inflammation and promotes anti-obesity effects by reducing food intake and weight gain. Its genetic ablation results in adiposity development. CB receptors are also responsible for transforming white adipose tissue towards beige or brown adipocytes, therefore their modulation can be considered potential anti-obesity target. CB1 principal localization in central nervous system represents an important limit. Stimulation of CB2, principally localized on peripheral cells instead, should facilitate the anti-obesity effects without exerting remarkable psychotropic activity.

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.

The DMT1 IVS4+44C>A polymorphism and the risk of iron deficiency anemia in children with celiac disease.

BACKGROUND: Iron deficiency anemia in celiac disease is related to impaired duodenal mucosal uptake, due to villous atrophy. Iron enters the enterocytes through an apical divalent metal transporter, DMT1. Different DMT1 transcripts have been identified, depending on the presence of an iron-responsive element that allows DMT1 up-regulation during iron starvation. An intronic DMT1 polymorphism, IVS4+44C>A, has been associated with metal toxicity, and the CC-carriers show high iron levels. AIMS: This study investigates the association between DMT1 IVS4+44C>A and anemia in a cohort of 387 Italian celiac children, and the functional role of the polymorphism. METHODS AND RESULTS: By association analysis, we found that DMT1 IVS4+44-AA genotype confers a four-fold risk of developing anemia, despite of atrophy degree. By analysis of mRNA from gastroesophageal biopsies, we found that total DMT1 is significantly upregulated in presence of mild, but not severe, atrophy, independently from IVS4+44C>A variant, and in normal but not in atrophic CC-biopsies. Moreover, we found that A-allele is associated to preferential expression of the DMT1 transcripts lacking the iron-responsive element, thus limiting the DMT1 overexpression that normally occurs to respond to iron starvation. DISCUSSION: Possibly, the IVS4+44-AA-related dysregulation of the iron-induced changes in DMT1 expression is not able to impair iron absorption in physiological condition. However, if exacerbated by the concomitant massive loss of functional absorbing tissue paralleling worsened stages of villus atrophy, it might be ineffective in counteracting iron deficiency, despite of DMT1 overexpression. CONCLUSION: We suggest, for the first time, that celiac disease may unmask the contribution of the DMT1 IVS4+44C>A polymorphism to the risk of anemia.

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.

Cannabinoid receptor 2-63 RR variant is independently associated with severe necroinflammation in HIV/HCV coinfected patients.

OBJECTIVE: This is the first study to analyze the impact of the rs35761398 variant of the CNR2 gene leading to the substitution of GLN (Q) of codon 63 of the cannabinoid receptor 2 (CB2) with ARG (R) on the clinical presentation of chronic hepatitis in HIV/HCV coinfected patients. METHODS: Enrolled in this study were 166 consecutive HIV/HCV coinfected patients, naïve for HCV treatment. A pathologist unaware of the patients' condition graded liver fibrosis, necroinflammation (Ishak) and steatosis. All patients were screened for the CB2 rs35761398 polymorphism. RESULTS: Of the 166 HIV/HCV coinfected patients, 72.9% were males, 42.5% were infected with HCV-genotype-3 and 60.2% had been intravenous drug users. The median age was 40.6 years and the immunological condition good (median CD4+ cells/mm3 = 507, IQR: 398.0-669.5). Thirty-five (21.1%) patients were naive for ART and 131(78.9%) were on ART. The CB2-RR variant was detected in 45.8% of patients, QR in 38.6% and QQ in 15.7%. Patients with CB2-RR showed a necroinflammation score (HAI) ≥9 more frequently than those with CB2-QQ or CB2-QR (32.9% vs. 11.5% and 14.1%, respectively, p≤0.001). In the multivariate analysis, the CB2-RR variant (p = 0.03) and liver fibrosis were both identified as independent predictors of the entity of liver necroinflammation (p = 0.0001). CONCLUSION: This study shows interesting interplay between the CB2-RR variant and liver necroinflammation in chronic hepatitis patients with HIV/HCV coinfection, an observation of clinical value that coincides with the interest in the use of the CB2 agonists and antagonists in clinical practice emerging from the literature.