Distal procto-colitis and n-3 polyunsaturated fatty acids: the mechanism(s) of natural cytotoxicity inhibition

BACKGROUND: Altered natural killer (NK) and lymphokine-activated killer (LAK) cell activities have been reported with ulcerative colitis (UC). Previously, we have shown that in patients with UC, the n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), specifically inhibit natural cytotoxicity with clinical improvement in disease activity. The aim of this study therefore was to evaluate the possible mechanism(s) involved in this inhibition, and in particular the alteration of production of interleukin 2 (IL2) and the arachidonic acid metabolite leukotriene B4 (LTB4), both known to modulate NK cell activity. MATERIALS AND METHODS: Each patient with procto-colitis received either fish oil extract (EPA 3.2 g, DHA 2.4 g; n = 9) or placebo (n = 9) daily for 6 months. Monthly assessment included disease activity using clinical and sigmoidoscopic scores. Peripheral blood mononuclear (PBMN) cells were isolated and NK cell cytotoxic activity in vitro was measured. Monthly serum samples were analysed for LTB4, IL2 and soluble IL2 receptors (sIL2R). RESULTS: The n-3 PUFAs group had significantly reduced NK cell activity, compared with the placebo group (P < 0.05, Mann-Whitney U-test). In the n-3 PUFA group, incubation of PBMN cells for 72 h with recombinant interleukin 2 (rIL2) reversed the NK inhibition. In patients with active proctocolitis, serum levels of LTB4 correlated positively with NK cell cytotoxicity (r = 0.873, P < 0.05, Kendall's correlation coefficient). After six months of n-3 PUFAs supplementation, serum levels of LTB4 were undetectable with concurrent significant reduction in NK cell cytotoxic activity. The latter was associated with significant reduction of serum IL2 and sIL2R levels (P < 0.05). CONCLUSION: This study has demonstrated both evidence of suppression of immune reactivity and concurrent reduction in disease activity in patients with proctocolitis receiving n-3 PUFAs supplementation. This may have important implications for therapy in patients with UC.     

Omega-3 fatty acids suppress the enhanced production of 5-Iipoxygenase products from polymorph neutrophil granulocytes in cystic fibrosis

Abstract. Pulmonary damage in cystic br osis (CF) is associated with chronic inflammation mediated in part by proinflammatory 5-lipoxygenase products (5-LOP, leukotrienes and 5-hydroxyeicosatetraenoic acid) from polymorph neutrophil granulocytes (PMN). The authors studied 5-LOP formation of PMN from CF patients and in vitro effects of added eicosapentae-noic acid (EPA) and fish oil. Circulating PMN were isolated from 10 CF patients without acute infections and 10 control persons of the same age (4–20 years). Total 5-LOP liberation from PMN of CF patients was significantly increased over controls after incubation with the calcium ionophore A23 (1 μmol L^-1) without arachidonic acid (AA) (380 ±24 vs. 294±28pmol mL^-1) and with 10μmolL^-1 AA (1303±104 vs. 1015 ± 104 pmolmL^-1), and there were nonsignificant trends to high values after incubation with 5μmolL^-1 platelet activating factor (PAF, 134% of controls) and 1 μmol L^-1 formyl-methionyl-leucyl-phenylalanine (FMLP, 125%). The addition of 100 mu;g mL^-1 fish oil to PMN of CF patients challenged with A23 completely suppressed synthesis of proinflammatory 5-LOP of the 4-series, while inactive 5-LOP metabolites of the 5-series were produced. Added EPA (10 μ molL^-1) also suppressed 4-series 5-LOP and significantly reduced leukotriene B₄ concentration by 48% from 39.9 ±3.2 to 20.6 ± 11.4 pmol L^-1 again with a concomittant increase of inactive 5-series metabolites. The authors conclude that the turnover of endogenous and exogenous AA is enhanced in CF, possibly due to stimulated phospho-lipase A₂ activity. The relatively small effect of the receptor dependent stimuli PAF and FMLP may be caused by a down-regulation of PMN receptors in CF. Supplementation of long-chain ω-3-fatty acids may be beneficial for reducing excessive inflammation in CF patients and should be further evaluated.     

Peroxidase activity within circulating neutrophils correlates with pulmonary phenotype in cystic fibrosis

Excess neutrophils are present in the airways of patients with cystic fibrosis (CF). Myeloperoxidase (MPO) activity of acid extracts of sputum is directly correlated with airflow obstruction in CF patients. We hypothesized that the sputum MPO was derived from the MPO of neutrophils that entered the airways from the circulation. Active MPO without protease activity injures airways. If MPO activity from circulating neutrophils that emigrate into the airways of these patients causes increased airway epithelial permeability and mucus-gland secretion, then (1) those patients with greater MPO activity per circulating neutrophil would be more likely to produce sputum and (2) the MPO activity per circulating neutrophil would positively correlate with airflow obstruction. We determined the MPO activity for both circulating and sputum neutrophils. Spirometry and respiratory cultures were obtained simultaneously with blood and sputum samples. CF patients with more MPO activity within their circulating neutrophils were more likely to produce sputum ( P =.001, chi 2 test), and the MPO activity per circulating neutrophil was positively correlated with airflow obstruction as measured on the basis of the ratio of 1-second forced expiratory volume to forced vital capacity ( P <. 03, Kruskal-Wallace test). These associations were independent of age, sex, the results of respiratory-tract culture, or protease activity in the circulating neutrophils. MPO activity in circulating neutrophils from CF patients homozygotic for the deletion of phenylalanine at position 508 in the CF transmembrane regulator protein is directly related to the severity of these patients' pulmonary disease. Our results are consistent with the hypothesis that circulating neutrophils deliver active MPO to the airway, producing airway injury and airflow obstruction in homozygotic delF508 CF patients.     

Molecular and Biological Characterization of the Murine Leukotriene B4 Receptor Expressed on Eosinophils

The movement of leukocytes into tissues is regulated by the local production of chemical mediators collectively referred to as chemoattractants. Although chemoattractants constitute a diverse array of molecules, including proteins, peptides, and lipids, they all appear to signal leukocytes through a related family of seven transmembrane–spanning G protein–coupled receptors. The eosinophil is a potent proinflammatory cell that is attracted into tissues during allergic inflammation, parasitic infection, and certain malignancies. Since the molecular mechanisms controlling eosinophil recruitment are incompletely understood, we performed a degenerate polymerase chain reaction on cDNA isolated from murine eosinophils to identify novel chemoattractant receptors. We report the isolation of a cDNA that encodes a 351–amino acid glycoprotein that is 78% identical to a human gene that has been reported to be a purinoceptor (P2Y₇) and a leukotriene B₄ (LTB₄) receptor (BLTR). Chinese hamster ovary (CHO) cells transfected with this cDNA specifically bound [³H]LTB₄ with a dissociation constant of 0.6 ± 0.1 nM. Furthermore, LTB₄ induced a dose-dependent intracellular calcium flux in transfected CHO cells. In contrast, [³⁵S]dATP did not specifically bind to these transfectants. This mRNA was expressed at high levels in interleukin 5–exposed eosinophils, elicited peritoneal macrophages and neutrophils, and to a lesser extent interferon γ stimulated macrophages. Low levels of expression were detected in the lung, lymph node, and spleen of unchallenged mice. Western blot analysis detected the mBLTR protein in murine eosinophils and alveolar macrophages as well as human eosinophils. In addition, elevated levels of mBLTR mRNA were found in the lungs of mice in a murine model of allergic pulmonary inflammation in a time course consistent with the influx of eosinophils. Our findings indicate that this murine receptor is an LTB₄ receptor that is highly expressed on activated leukocytes, including eosinophils, and may play an important role in mediating eosinophil recruitment into inflammatory foci.     

Selective monitoring of vitamin D2 and D3 supplementation with a highly specific 25-hydroxyvitamin D3 immunoassay with negligible cross-reactivity to 25-hydroxyvitamin D2

Background

The effects of vitamin D₂ and D₃ supplementation on circulating concentrations of 25(OH)D₃ require reliable analytical tools for specific determination of 25(OH)D₃ and 25(OH)D₂. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D₂.

Methods

25(OH)D₃ concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D₃ daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D₂ daily for 3 months.

Results

This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D₂, (0.7%), and demonstrated a high correlation (r² = 0.93) with 25(OH)D₃ determined by HPLC. 25(OH)D₃ increased by 14% in subjects receiving vitamin D₃ for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D₃ levels in those receiving vitamin D₂.

Conclusions

We report that 25(OH)D₃ ELISA was used for evaluation of 25(OH)D₃ concentrations in subjects receiving vitamin D₂ and D₃ supplementation. The increase of 25(OH)D₃ in circulation with vitamin D3 supplementation and lack of increase with vitamin D₂ supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D₃ status in humans.     

The effects of n-3 polyunsaturated fatty acid-rich total parenteral nutrition on neutrophil apoptosis in a rat endotoxemia

Although recruited neutrophils function as first-line defense to remove bacteria, delayed apoptosis is implicated in persistent inflammation leading to organ injury. Leukotrien B₄, n-6 polyunsaturated fatty acids (PUFAs) product, is one of the mediators that delay neutrophil apoptosis. The mechanism of the beneficial effects of supplementation of fish oil-based long-chain n-3 PUFAs in parenteral nutrition for critically ill patients has not been fully understood. One possible mechanism is the less inflammatory n-3 PUFAs products can compete with proinflammatory n-6 PUFAs products for access to the enzymes. The aim of this study was to determine whether n-3 PUFA rich parenteral nutrition may alter the composition of fatty acids in the neutrophil membrane and restore delay of neutrophil apoptosis during endotoxin-induced systemic inflammation in rats. The animals in group 1 were treated with 20% Hicaliq NC-N in Neoparen-2 for three days. The animals in group 2 (referred to as n-6 PUFA-rich parenteral nutrition) were given parenteral nutrition solutions containing 20% soybean oil in Neoparen-2 (n-6/n-3 = 10). The animals in group 3 (referred to as n-3 PUFA-rich parenteral nutrition) were administered parenteral nutrition consisting of 10% soybean oil and 10% fish oil emulsion (n-6/n-3 = 1.3). The n-3/n-6 ratio of the neutrophil membrane was significantly increased in group 3 and was associated with restored lipopolysaccharide-delayed-apoptosis of neutrophils in bone marrow cells and increased production of leukotriene B₅ from peritoneal neutrophils stimulated by lipopolysaccharide. Our preliminary results showed that n-3 PUFA-rich parenteral nutrition regulated neutrophil apoptosis and prevented synthesis of pro-inflammatory eicosanoids, explaining the protective effects seen in the clinical setting.     

A Thrombelastograph whole blood assay for clinical monitoring of NSAID-insensitive transcellular platelet activation by arachidonic acid

Optical platelet aggregation (OPA) with platelet-rich plasma (PRP) was compared with a Thrombelastograph (TEG) whole blood assay for monitoring arachidonic acid (AA)-induced platelet activation. Assays were performed on 47 interventional cardiology and 24 general surgery patients receiving aspirin therapy for cardiovascular disease, as well as 48 volunteers asked to take nonsteroidal anti-inflammatory drugs (NSAIDs) or 12 volunteers on chronic NSAID therapy unrelated to diagnosed cardiovascular disease. Whole blood TEG monitoring of NSAID inhibition detected NSAID-insensitive AA activation of platelets in a significantly higher number of cardiology (23%) and surgery (25%) patients and normal volunteers on chronic NSAID (25%) therapy relative to normal subjects not on chronic NSAID therapy (0%). Whole blood NSAID insensitivity was observed with cyclooxygenase-I inhibitors, such as aspirin and ibuprofen; was not affected by Celebrex, a cyclooxygenase-II inhibitor; but was completely inhibited by thromboxane-receptor antagonists. This was not due to platelet NSAID insensitivity, because complete inhibition of AA-activation responses in PRP was observed with either TEG or OPA assays. We confirmed that thromboxane B(2) formation in PRP from NSAID-insensitive subjects was completely inhibited by NSAIDs. However, significant amounts were formed in whole blood from NSAID-insensitive subjects, but not in whole blood from NSAID-sensitive subjects. Thromboxane formation after AA addition was not found in washed blood cells with 90% reduced platelet counts or in leukocyte-rich buffy coat fractions, but could be restored by addition of PRP. NSAID-insensitive activation was inhibited by nordihydroguaiaretic acid, with an IC(50) of 30 micromol, implicating 12- and/or 15-lipoxygenases in this transcellular pathway.     

PEGylation improves pharmacokinetic profile, liver uptake and efficacy of Interferon gamma in liver fibrosis

Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-proliferative, immunomodulatory, apoptotic and anti-fibrotic functions. However, its clinical use is limited to the treatment of few diseases due to the rapid clearance from the body. PEGylated IFN-alpha formulations are shown to be beneficial in viral hepatitis, but PEGylation of IFNγ to enhance its therapeutic effects in liver fibrosis is not yet explored. Liver fibrosis is characterized by the extensive accumulation of an abnormal extracellular matrix and is the major cause of liver-related morbidity and mortality worldwide. To date, there is no pharmacotherapy available for this disease. We modified IFNγ with different-sized linear PEG molecules (5, 10 and 20 kDa) and assessed the biological activity in vitro and in vivo. All PEGylated IFNγ constructs were biologically active and activated IFNγ signaling in vitro as determined with a nitric oxide release assay and a pGAS-Luc reporter plasmid assay, respectively. Similar to IFNγ, all PEGylated IFNγ induced a significant reduction of fibrotic parameters in mouse NIH3T3 fibroblasts as shown with immunohistochemical staining and quantitative PCR analyses. In vivo, the pharmacokinetic profile of radiolabeled ¹²⁵I-IFNγ-PEG conjugates revealed a decreased renal clearance and an increased plasma half-life with an increase of PEG size. Moreover, the liver accumulation of PEGylated IFNγ constructs was significantly higher than the unmodified IFNγ, which was also confirmed by increased MHC-II expression in the livers. Furthermore, in a CCl₄-induced acute liver injury model in mice, PEGylated constructs reduced the early fibrotic parameters more drastically than unmodified IFNγ. Of note, these effects were stronger with higher PEG-sized IFNγ constructs. These data nicely correlated with the pharmacokinetic data. In conclusion, PEGylation significantly improved the pharmacokinetics, liver uptake and anti-fibrotic effects of IFNγ. This study opens new opportunities to exploit the therapeutic applications of PEGylated IFNγ for the treatment of liver fibrosis and other diseases.     

Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation

Lycopene is a natural carotenoid antioxidant that is present in tomatoes and tomato products. The pharmacologic function of lycopene in platelets is not yet understood. Therefore, in this study we sought to systematically examine the effects of lycopene in the prevention of platelet aggregation and thrombus formation. We found that lycopene concentration-dependently (2-12 micromol/L) inhibited platelet aggregation in human platelets stimulated by agonists. Lycopene (6 and 12 micromol/L) inhibited phosphoinositide breakdown in platelets labeled with tritiated inositol, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane B2 formation stimulated by collagen. In addition, lycopene (6 and 12 micromol/L) significantly increased the formations of cyclic GMP and nitrate but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of 47,000 Da (P47), a marker of protein kinase C activation, was triggered by PDBu (60 nmol/L). This phosphorylation was markedly inhibited by lycopene (12 micromol/L) in phosphorus-32-labeled platelets. In an in vivo study, thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Lycopene (5, 10, and 20 mg/kg) significantly prolonged the latency period for the induction of platelet-plug formation in mesenteric venules. These results indicate that the antiplatelet activity of lycopene may involve the following pathways: (1) Lycopene may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane B2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization. (2) Lycopene also activated the formations of cyclic GMP/nitrate in human platelets, resulting in the inhibition of platelet aggregation. The results may imply that tomato-based foods are especially beneficial in the prevention of platelet aggregation and thrombosis.     

Mechanisms of platelet retention in the collagen-coated-bead column

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.     

Aspirin therapy for inhibition of platelet reactivity in the presence of erythrocytes in patients with vascular disease

Inhibition of erythrocyte (RBC) promotion of platelet reactivity could improve the antiplatelet effect of aspirin (ASA). We tested different ASA regimens for optimal inhibition of platelets and the effects of RBC in patients with a history of vascular diseases. Collagen-induced platelet activation (14C-5HT, TXA2 release) and platelet recruitment (proaggregatory activity of cell-free releasates from activated platelets) were measured in PRP, platelet-RBC (Hct 40%), and whole blood (WB) in 206 patients initially on 200-300-mg ASA/day. Their regimen was modified to biweekly 500 mg (loading dose, L) plus daily or twice-daily low-dose ASA (50 or 100 mg). TXA2 was inhibited with all regimens. Percentage of patients with suboptimal inhibition of platelet recruitment in WB was 200-300 ASA/day (41%), L-50/day (87%), L-100/day (58%), L-50/twice-daily (39%), and L-100/twice-daily (20%; P < 0.05 vs other regimens). 14C-5HT release was inhibited to the greatest extent with L-100/twice-daily in PRP + RBC or WB (P < 0.05 vs other regimens) due to greater inhibition of the RBC prothrombotic effect. Compared with other ASA regimens, L-100 twice-daily (equivalent to 221-mg ASA/day in the 14-day cycle), reduced by >50% the proportion of patients with suboptimal inhibition of platelet recruitment in WB and inhibited 14C-5HT release to the greatest extent.     

In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative

Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the α-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.     

Characterization of angiotensin II-receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125-labeled Ang II to monitor Ang II-receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant approximately 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II-receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P<.005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (approximately 75%) with lesser amounts of AT2 (approximately 25%). Up-regulation of Ang II-receptor number was associated with increased Ang II-receptor responsiveness, as evidenced by enhanced Ang II-stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.     

Past, present and future of A(2A) adenosine receptor antagonists in the therapy of Parkinson's disease

Several selective antagonists for adenosine A_2A receptors (A_2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson's disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D₂ and adenosine A_2A receptors in the basal ganglia. At present it is believed that A_2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson's patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A_2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized antiparkinsonian drug therapy, namely the existence of (hetero)dimers/oligomers of G protein-coupled receptors, a topic that is currently the focus of intense debate within the scientific community. Dopamine D₂ receptors (D₂Rs) expressed in the striatum are known to form heteromers with A_2A adenosine receptors. Thus, the development of heteromer-specific A_2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs.     

Oxycodone and morphine have distinctly different pharmacological profiles: radioligand binding and behavioural studies in two rat models of neuropathic pain

Previously, we reported that oxycodone is a putative kappa-opioid agonist based on studies where intracerebroventricular (i.c.v.) pre-treatment of rats with the kappa-selective opioid antagonist, nor-binaltorphimine (nor-BNI), abolished i.c.v. oxycodone but not morphine antinociception, whereas pretreatment with i.c.v. naloxonazine (mu-selective antagonist) produced the opposite effects. In the present study, we used behavioural experiments in rat models of mechanical and biochemical nerve injury together with radioligand binding to further examine the pharmacology of oxycodone. Following chronic constriction injury (CCI) of the sciatic nerve in rats, the antinociceptive effects of intrathecal (i.t.) oxycodone, but not i.t. morphine, were abolished by nor-BNI. Marked differences were found in the antinociceptive properties of oxycodone and morphine in streptozotocin (STZ)-diabetic rats. While the antinociceptive efficacy of morphine was abolished at 12 and 24 weeks post-STZ administration, the antinociceptive efficacy of s.c. oxycodone was maintained over 24 weeks, albeit with an approximately 3- to 4-fold decrease in potency. In rat brain membranes irreversibly depleted of mu- and delta-opioid binding sites, oxycodone displaced [(3)H]bremazocine (kappa(2)-selective in depleted membranes) binding with relatively high affinity whereas the selective mu- and delta-opioid ligands, CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)) and DPDPE ([D-Pen(2,5)]-enkephalin), respectively, did not. In depleted brain membranes, the kappa(2b)-ligand, leu-enkephalin, prevented oxycodone's displacement of high-affinity [(3)H]bremazocine binding, suggesting the notion that oxycodone is a kappa(2b)-opioid ligand. Collectively, the present findings provide further support for the notion that oxycodone and morphine produce antinociception through distinctly different opioid receptor populations. Oxycodone appears to act as a kappa(2b)-opioid agonist with a relatively low affinity for mu-opioid receptors.     

Glutamate abnormalities in obsessive compulsive disorder: neurobiology, pathophysiology, and treatment

Obsessive compulsive disorder is prevalent, disabling, incompletely understood, and often resistant to current therapies. Established treatments consist of specialized cognitive-behavioral psychotherapy and pharmacotherapy with medications targeting serotonergic and dopaminergic neurotransmission. However, remission is rare, and more than a quarter of OCD sufferers receive little or no benefit from these approaches, even when they are optimally delivered. New insights into the disorder, and new treatment strategies, are urgently needed. Recent evidence suggests that the ubiquitous excitatory neurotransmitter glutamate is dysregulated in OCD, and that this dysregulation may contribute to the pathophysiology of the disorder. Here we review the current state of this evidence, including neuroimaging studies, genetics, neurochemical investigations, and insights from animal models. Finally, we review recent findings from small clinical trials of glutamate-modulating medications in treatment-refractory OCD. The precise role of glutamate dysregulation in OCD remains unclear, and we lack blinded, well-controlled studies demonstrating therapeutic benefit from glutamate-modulating agents. Nevertheless, the evidence supporting some important perturbation of glutamate in the disorder is increasingly strong. This new perspective on the pathophysiology of OCD, which complements the older focus on monoaminergic neurotransmission, constitutes an important focus of current research and a promising area for the ongoing development of new therapeutics.     

The biology of Nociceptin/Orphanin FQ (N/OFQ) related to obesity,stress, anxiety,mood, and drug dependence

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995. The generation of specific agonists, antagonists and receptor deficient mice and rats has enabled progress in elucidating the biological functions of N/OFQ. Additionally, radio-imaging technologies have been advanced for investigation of this system in animals and humans. Together with traditional neurobehavioral techniques, these tools have been utilized to identify the biological significance of the N/OFQ system and its interacting partners. The present review focuses on the role of N/OFQ in the regulation of feeding, body weight homeostasis, stress, the stress-related psychiatric disorders of depression and anxiety, and in drug and alcohol dependence. Critical evaluation of the current scientific preclinical literature suggests that small molecule modulators of nociceptin opioid peptide receptors (NOP) might be useful in the treatment of diseases related to these biological functions. In particular, the literature data suggest that antagonism of NOP receptors will produce anti-obesity and antidepressant activities in humans. However, there are also contradictory data discussed. The current literature on the role of N/OFQ in anxiety and addiction, on the other hand points primarily to a role of agonist modulation being potentially therapeutic. Some drug-like molecules that function either as agonists or antagonists of NOP receptors have been optimized for human clinical study to test some of these hypotheses. The discovery of PET ligands for NOP receptors, combined with the pharmacological tools and burgeoning preclinical data set discussed here bodes well for a rapid advancement of clinical understanding and potential therapeutic benefit.     

Autonomic nerves and perivascular fat: Interactive mechanisms

The evidence describing the autonomic innervation of body fat is reviewed with a particular focus on the role of the sympathetic neurotransmitters. In compiling the evidence, a strong case emerges for the interaction between autonomic nerves and perivascular adipose tissue (PVAT). Adipocytes have been shown to express receptors for neurotransmitters released from nearby sympathetic varicosities such as adrenoceptors (ARs), purinoceptors and receptors for neuropeptide Y (NPY). Noradrenaline can modulate both lipolysis (via α₂- and β₃-ARs) and lipogenesis (via α₁- and β₃-ARs). ATP can inhibit lipolysis (via P₁ purinoceptors) or stimulate lipolysis (via P_2y purinoceptors). NPY, which can be produced by adipocytes and sympathetic nerves, inhibits lipolysis. Thus the sympathetic triad of transmitters can influence adipocyte free fatty acid (FFA) content. Substance P (SP) released from sensory nerves has also been shown to promote lipolysis. Therefore, we propose a mechanism whereby sympathetic neurotransmission can simultaneously activate smooth muscle cells in the tunica media to cause vasoconstriction and alter FFA content and release from adjacent adipocytes in PVAT. The released FFA can influence endothelial function. Adipocytes also release a range of vasoactive substances, both relaxing and contractile factors, including adiponectin and reactive oxygen species. The action of adipokines (such as adiponectin) and reactive oxygen species (ROS) on cells of the vascular adventitia and nerves has yet to be fully elucidated. We hypothesise a strong link between PVAT and autonomic fibres and suggest that this poorly understood relationship is extremely important for normal vascular function and warrants a detailed study.     

Activity of liver enzymes in multiple sclerosis patients with Hot-nature diet and co-supplemented hemp seed,evening primrose oils intervention

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