Endocannabinoids and Reproductive Events in Health and Disease.

The lasting research on the endocannabinoid system (ECS) has now provided solid and convincing evidence that proves the detrimental effects of recreational drug abuse (a growing habit among teenagers) on fertility. Endocannabinoids (eCBs) affect reproductive events from gametogenesis to fertilization, from embryo implantation to the final outcome of pregnancy and, thus, they have been proposed as suitable biomarkers to predict the reproductive potential of male and female gametes in clinical practice. Novel tools for reproductive medicine are highly sought after, and here we report the latest findings on the impact of the ECS on fertility, demonstrating how basic research can be translated into new medical strategies.

Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors.

The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB(2)Rs) in the CNS. Here, we show that CB(2)Rs are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP(3)R-dependent opening of Ca(2+)-activated Cl(-) channels. To investigate the functional role of CB(2)R activation, we induced neuronal firing and observed a CB(2)R-mediated reduction in firing frequency. The description of this unique CB(2)R-mediated signaling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.

Endocannabinoids, related compounds and their metabolic routes.

Endocannabinoids are lipid mediators able to bind to and activate cannabinoid receptors, the primary molecular targets responsible for the pharmacological effects of the Δ9-tetrahydrocannabinol. These bioactive lipids belong mainly to two classes of compounds: N-acylethanolamines and acylesters, being N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, their main representatives. During the last twenty years, an ever growing number of fatty acid derivatives (endocannabinoids and endocannabinoid-like compounds) have been discovered and their activities biological is the subject of intense investigations. Here, the most recent advances, from a therapeutic point of view, on endocannabinoids, related compounds, and their metabolic routes will be reviewed.

Cellular and Molecular Effects of Microgravity on the Immune System: A Focus on Bioactive Lipids.

Microgravity is one of the main stressors that astronauts are exposed to during space missions. This condition has been linked to many disorders, including those that feature dysfunctional immune homeostasis and inflammatory damage. Over the past 30 years, a significant body of work has been gathered connecting weightlessness-either authentic or simulated-to an inefficient reaction to pathogens, dysfunctional production of cytokines and impaired survival of immune cells. These processes are also orchestrated by a plethora of bioactive lipids, produced by virtually all cells involved in immune events, which control the induction, magnitude, outcome, compartmentalization and trafficking of immunocytes during the response to injury. Despite their crucial importance in inflammation and its modulation, however, data concerning the role of bioactive lipids in microgravity-induced immune dysfunctions are surprisingly scarce, both in quantity and in variety, and the vast majority of it focuses on two lipid classes, namely eicosanoids and endocannabinoids. The present review aims to outline the accumulated knowledge addressing the effects elicited by microgravity-both simulated and authentic-on the metabolism and signaling of these two prominent lipid groups in the context of immune and inflammatory homeostasis.

The Effects of Ultrasonic Scaling and Air-Abrasive Powders on the Topography of Implant Surfaces: Scanning Electron Analysis and In Vitro Study.

OBJECTIVES: This in vitro study aimed to investigate the impact of bicarbonate air-abrasive powders and ultrasonic scaling with stainless steel tips on the micro- and nanotopography and roughness of three different implant-abutment junction titanium surfaces. MATERIALS AND METHODS: Three types of sterile and decontaminated titanium surfaces (RS, UTM, XA) were used for analysis. Nine disks per surface type were subjected to micro- and nanotopography analysis, scanning electron microscopy (SEM), roughness analysis, and fibroblast cultivation. Ultrasonic debridement and air polishing were performed on the surfaces. Human dermal fibroblasts were cultured on the surfaces for 5 days. STATISTICAL ANALYSIS: Data analysis adhered to ISO 25178 standards for surface texture assessment. SEM micrographs were used to reconstruct areas for extracting roughness parameters. Excel and Mex 6.0 software were utilized for quantitative and stereoscopic analysis. RESULTS: The study found varying effects on surface roughness posttreatment. RS Disco samples exhibited higher surface roughness compared with UTM and XA samples, both in average and nanoscale roughness. Decontamination led to increased surface roughness for all samples, particularly RS Disco. Fibroblast growth tests revealed enhanced cell network formation on decontaminated discs, possibly due to increased nanoscale roughness or the presence of bicarbonate salts. CONCLUSION: The study underscores the complex interplay between surface topography, microbial biofilm, and treatment efficacy in peri-implant disease management. While smoother surfaces may resist biofilm accumulation, increased nanoscale roughness postdecontamination can enhance fibroblast attachment and soft tissue integration. This dichotomy highlights the need for tailored treatment protocols that consider material-specific factors, emphasizing that successful implant therapy should balance microbial control with conducive surface characteristics for long-term osseointegration and soft tissue stability.

Identifying Clinical Phenotypes in People Who Are Hispanic/Latino With Chronic Low Back Pain: Use of Sensor-Based Measures of Posture and Movement, Pain, and Psychological Factors.

OBJECTIVE: The aim of this study was to identify clinical phenotypes using sensor-based measures of posture and movement, pain behavior, and psychological factors in Hispanic/Latino people with chronic low back pain (CLBP). METHODS: Baseline measures from an ongoing clinical trial were analyzed for 81 Hispanic/Latino people with CLBP. Low back posture and movement were measured using commercial sensors during in-person testing and 8 hours of ecological monitoring. Magnitude, frequency, and duration of lumbar movements, sitting and standing postures were measured. Movement-evoked pain was assessed during in-person movement testing. Psychological measures included the Pain Catastrophizing Scale and the Fear Avoidance Beliefs Questionnaire. Random forest analysis was conducted to generate 2 groups and identify important variables that distinguish groups. Group differences in demographics, pain, psychological, and posture and movement variables were examined using t-tests and chi-square analyses. RESULTS: Two subgroups of Hispanic/Latino people with CLBP were identified with minimal error (7.4% misclassification ["out-of-bag" error]). Ecological posture and movement measures best distinguished groups, although most movement-evoked pain and psychological measures did not. Group 1 had greater height and weight, lower movement frequency, more time in sitting, and less time in standing. Group 2 had a greater proportion of women than men, longer low back pain duration, higher movement frequency, more time in standing, and less time in sitting. CONCLUSION: Two distinct clinical phenotypes of Hispanic/Latino people with CLBP were identified. One group was distinguished by greater height and weight and more sedentary posture and movement behavior; the second group had more women, longer duration of low back pain, higher lumbar spine movement frequency, and longer duration of standing postures. IMPACT: Ecological measures of posture and movement are important for identifying 2 clinical phenotypes in Hispanic/Latino people with CLBP and may provide a basis for a more personalized plan of care. LAY SUMMARY: Wearable sensors were used to measure low back posture and movement in Hispanic/Latino people with chronic low back pain. These posture and movement measures helped to identify 2 different clinical subgroups that will give physical therapists more information to better personalize treatment for chronic low back pain in Hispanic/Latino patients.

Interleukin-1ß causes anxiety by interacting with the endocannabinoid system.

Interleukin-1ß (IL-1ß) is involved in mood alterations associated with inflammatory illnesses and with stress. The synaptic basis of IL-1ß-induced emotional disturbances is still unknown. To address the possible involvement of the endocannabinoid system in IL-1ß-induced anxiety, we performed behavioral and neurophysiological studies in mice exposed to stress or to intracerebroventricular injections of this inflammatory cytokine or of its antagonist. We found that a single intracerebroventricular injection of IL-1ß caused anxiety in mice, and abrogated the sensitivity of cannabinoid CB1 receptors (CB1Rs) controlling GABA synapses in the striatum. Identical behavioral and synaptic results were obtained following social defeat stress, and intracerebroventricular injection of IL-1 receptor antagonist reverted both effects. IL-1ß-mediated inhibition of CB1R function was secondary to altered cholesterol composition within membrane lipid rafts, and required intact function of the transient receptor potential vanilloid 1 (TRPV1) channel, another element of the endocannabinoid system. Membrane lipid raft disruption and inhibition of cholesterol synthesis, in fact, abrogated IL-1ß-CB1R coupling, and TRPV1-/- mice were indeed insensitive to the synaptic and behavioral effects of both IL-1ß and stress. On the other hand, cholesterol enrichment of striatal slices mimicked the synaptic effects of IL-1ß on CB1Rs only in control mice, while the same treatment was ineffective in slices prepared from TRPV1-/- mice. The present investigation identifies a previously unrecognized interaction between a major proinflammatory cytokine and the endocannabinoid system in the pathophysiology of anxiety.

5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment.

The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5-lipoxygenase (5-LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO-4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5-LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (∼3-fold compared to ground-based controls) and cleaved-poly (ADP-ribose) polymerase (PARP) protein expression (∼3-fold), as well as mRNA levels of apoptosis-related markers such as p53 (∼3-fold) and calpain (∼4-fold); these changes were paralleled by an early increase of 5-LOX activity (∼2-fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.

Subventricular zone neural progenitors protect striatal neurons from glutamatergic excitotoxicity.

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-induced epilepsy and ischaemic stroke. To test the role of subventricular zone adult neural stem and progenitor cells in protecting central nervous system tissue from glutamatergic excitotoxicity, neurophysiological recordings of spontaneous excitatory postsynaptic currents from single medium spiny striatal neurons were measured on acute brain slices. Indeed, lipopolysaccharide-stimulated, but not unstimulated, subventricular zone adult neural stem and progenitor cells reverted the increased frequency and duration of spontaneous excitatory postsynaptic currents by secreting the endocannabinod arachidonoyl ethanolamide, a molecule that regulates glutamatergic tone through type 1 cannabinoid receptor (CB(1)) binding. In vivo restoration of cannabinoid levels, either by administration of the type 1 cannabinoid receptor agonist HU210 or the inhibitor of the principal catabolic enzyme fatty acid amide hydrolase, URB597, completely reverted the increased morbidity and mortality of adult neural stem and progenitor cell-ablated mice suffering from epilepsy and ischaemic stroke. Our results provide the first evidence that adult neural stem and progenitor cells located within the subventricular zone exert an 'innate' homeostatic regulatory role by protecting striatal neurons from glutamate-mediated excitotoxicity.

Radiometric Assay of FAAH Activity.

Fatty acid amide hydrolase (FAAH) is an intracellular enzyme responsible for the hydrolysis of endogenous anandamide (AEA), a reaction that terminates the biological effects of this lipid mediator. The final products of AEA cleavage are arachidonic acid and ethanolamine. In the method described herein, FAAH activity is measured through the use of the radioactive substrate [14C-ethanolamine]-AEA and subsequent quantification of the reaction product [14C]-ethanolamine.

Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases.

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2. To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants, H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms.

The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release.

The endocannabinoid system and endocannabinoid receptor-driven modulation of glutamate release were studied in rat brain cortex astroglial gliosomes. These preparations contained the endocannabinoids N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, as well their major biosynthetic (N-acyl-phosphatidylethanolamines-hydrolyzing-phospholipase D and diacylglycerol-lipase) and catabolic (fatty acid amide-hydrolase and monoacylglycerol-lipase) enzymes. Gliosomes expressed type-1 (CB1R), type-2 (CB2R) cannabinoid, and type-1 vanilloid (TRPV1) receptors, as ascertained by Western blotting and confocal microscopy. Methanandamide, a stable analogue of anandamide acting as CB1R, CB2R, and TRPV1 agonist, stimulated or inhibited the depolarization-evoked gliosomal [(3)H]D: -aspartate release, at lower and higher concentrations, respectively. Experiments with ACEA (arachidonyl-2'-chloroethylamide), JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]-pyran) and capsaicin, selective agonists at CB1R, CB2R and TRPV1, respectively, demonstrated that potentiation of [(3)H]D: -aspartate release was due to CB1R while inhibition to CB2R and TRPV1 engagement. These findings were confirmed by using selective receptor antagonists. Furthermore, CB1R activation caused increase of intracellular IP3 and Ca(2+) concentration, suggesting an involvement of phospholipase C.

Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum.

Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.

Pulmonary function with expiratory resistive loading in healthy volunteers.

Expiratory flow limitation is a key characteristic in obstructive pulmonary diseases. To study abnormal lung mechanics isolated from heterogeneities of obstructive disease, we measured pulmonary function in healthy adults with expiratory loading. Thirty-seven volunteers (25±5 yr) completed spirometry and body plethysmography under control and threshold expiratory loading of 7, 11 cmH2O, and a subset at 20 cmH2O (n = 11). We analyzed the shape of the flow-volume relationship with rectangular area ratio (RAR; Ma et al., Respir Med 2010). Airway resistance was increased (p<0.0001) with 7 and 11 cmH2O loading vs control (9.20±1.02 and 11.76±1.68 vs. 2.53± 0.80 cmH2O/L/s). RAR was reduced (p = 0.0319) in loading vs control (0.45±0.07 and 0.47±0.09L vs. 0.48±0.08). FEV1 was reduced (p<0.0001) in loading vs control (3.24±0.81 and 3.23±0.80 vs. 4.04±1.05 L). FVC was reduced (p<0.0001) in loading vs control (4.11±1.01 and 4.14±1.03 vs. 5.03±1.34 L). Peak expiratory flow (PEF) was reduced (p<0.0001) in loading vs control (6.03±1.67 and 6.02±1.84 vs. 8.50±2.81 L/s). FEV1/FVC (p<0.0068) was not clinically significant and FRC (p = 0.4) was not different in loading vs control. Supra-physiologic loading at 20 cmH2O did not result in further limitation. Expiratory loading reduced FEV1, FVC, PEF, but there were no clinically meaningful differences in FEV1/FVC, FRC, or RAR. Imposed expiratory loading likely leads to high airway pressures that resist dynamic airway compression. Thus, a concave expiratory flow-volume relationship was consistently absent-a key limitation for model comparison with pulmonary function in COPD. Threshold loading may be a useful strategy to increase work of breathing or induce dynamic hyperinflation.

Dynamic changes of anandamide in the cerebrospinal fluid of Parkinson's disease patients.

A correct balance between endocannabinoid and dopamine-dependent systems is believed to underlie physiological motor control. We measured the levels of the endocannabinoid anandamide in the cerebrospinal fluid of Parkinson's disease (PD) patients. Subjects were divided into three groups: newly diagnosed de novo patients, subjects undergoing drug withdrawal, and patients under pharmacological therapy. These groups were compared to age-matched control subjects. Anandamide levels in untreated patients were more than doubled as compared to controls. However, chronic dopaminergic replacement restored control anandamide levels. Abnormal anandamide increase might reflect a compensatory mechanism occurring in course of PD, aimed at normalizing dopamine depletion.

Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy.

PURPOSE: The endocannabinoid system is involved in excitatory/inhibitory balance mechanisms within the central nervous system (CNS). Growing evidence shows that its perturbation leads to development of epileptic seizures in experimental models, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. Experimental data also demonstrate that the endocannabinoid anandamide (AEA) can antagonize epileptic discharges in hippocampal tissue. The objective of our study was to measure endocannabinoids levels in the cerebrospinal fluid (CSF) of drug-naive patients affected by temporal lobe epilepsy (TLE). METHODS: We measured the levels of both AEA and the other endocannabinoid, 2-arachidonoylglycerol (2-AG), in the CSF of drug-naive patients with TLE. RESULTS: A significant reduction of AEA was found in the CSF of patients with compared with healthy controls (epileptic patients = 2.55 +/- 1.78 pmol/ml; healthy controls = 11.65 +/- 7.53 pmol/ml; n = 9 for both groups, p < 0.01). 2-AG levels, however, were not affected (epileptic patients = 209.5 +/- 146.56; healthy controls = 159.6 +/- 110.2) (n = 6 for both groups, p = 0.48). DISCUSSION: Our findings seem to be consistent with experimental evidence demonstrating a significant prevention of epileptic seizures induced by endocannabinoids in models of epilepsy. Furthermore, they support the hypothesis that AEA may be involved in its pathogenesis, suggesting a hypothetical primary impairment of the endocannabinoid system in untreated TLE. The actual role of this in vivo dysregulation still remains unclear.

Bioactive Lipids in Health and Disease.

Although the primordial concept of lipids is associated with the role they play as key components of the cell membrane, growing research in the field of bioactive lipids and lipidomic technologies proves the prominent role of these molecules in other biological functions [...].

CSF Levels of the Endocannabinoid Anandamide are Reduced in Patients with Untreated Narcolepsy Type 1: A Pilot Study.

BACKGROUND: Endocannabinoids (ECs) modulate both excitatory and inhibitory components in the CNS. There is a growing body of evidence that shows ECs influence both hypothalamic orexinergic and histaminergic neurons involved in narcolepsy physiopathology. Therefore, ECs may influence sleep and sleep-wake cycle. OBJECTIVE: To evaluate EC levels in the CSF of untreated narcoleptic patients to test whether ECs are dysregulated in Narcolepsy Type 1 (NT1) and Type 2 (NT2). METHODS: We compared CSF Anandamide (AEA), 2-Arachidonoylglycerol (2-AG) and orexin in narcoleptic drug-naïve patients and in a sample of healthy subjects. RESULTS: We compared NT1 (n=6), NT2 (n=6), and healthy controls (n=6). We found significantly reduced AEA levels in NT1 patients compared to both NT2 and controls. No differences were found between AEA levels in NT2 versus controls and between 2-AG levels in all groups, although a trend toward a decrease in NT1 was evident. Finally, the CSF AEA level was related to CSF orexin levels in all subjects. CONCLUSION: We demonstrated that the EC system is dysregulated in NT1.

Lack of effect of cannabis-based treatment on clinical and laboratory measures in multiple sclerosis.

The endocannabinoid system (ECS) is involved in the pathophysiology of multiple sclerosis (MS), and relief from pain and spasticity has been reported in MS patients self-medicating with marijuana. A cannabis-based medication containing Delta(9)-tetrahydrocannabinol and cannabidiol (Sativex) has been approved in some countries for the treatment of MS-associated pain. The effects of this pharmaceutical preparation on other clinically relevant aspects of MS pathophysiology, however, are still unclear. In 20 MS patients, we measured the effects of Sativex on clinically measured spasticity and on neurophysiological and laboratory parameters that correlate with spasticity severity or with the modulation of the ECS. Sativex failed to affect spasticity and stretch reflex excitability. This compound also failed to affect the synthesis and the degradation of the endocannabinoid anandamide, as well as the expression of both CB1 and CB2 cannabinoid receptors in various subpopulations of peripheral lymphocytes.

N-Acyl Amino Acids: Metabolism, Molecular Targets, and Role in Biological Processes.

The lipid signal is becoming increasingly crowded as increasingly fatty acid amide derivatives are being identified and considered relevant therapeutic targets. The identification of N-arachidonoyl-ethanolamine as endogenous ligand of cannabinoid type-1 and type-2 receptors as well as the development of different -omics technologies have the merit to have led to the discovery of a huge number of naturally occurring N-acyl-amines. Among those mediators, N-acyl amino acids, chemically related to the endocannabinoids and belonging to the complex lipid signaling system now known as endocannabinoidome, have been rapidly growing for their therapeutic potential. Here, we review the current knowledge of the mechanisms for the biosynthesis and inactivation of the N-acyl amino acids, as well as the various molecular targets for some of the N-acyl amino acids described so far.