Inflammation and CB2 signaling drive novel changes in the ocular lipidome and regulate immune cell activity in the eye.

Uveitis is inflammation of the uvea which consists of the iris, ciliary body and the choroid of the eye. Uveitis can lead to impaired vision and is responsible for 10% of all cases of blindness globally. Using an endotoxin-induced uveitis (EIU) rodent model, our previous data implicated the endogenous cannabinoid system (ECS) in the amelioration of many of the components of the inflammatory response. Here, we test the hypothesis that the reduction in inflammatory mediators in the EIU model by the CB2 agonist, HU308, is associated with changes in ECS endogenous ligands as well as related lipids, prostaglandins (PGs), 2-acyl glycerols, and lipoamines. Analysis of leukocytes and neutrophils, CB2 mRNA, and 26 lipids in the eye of WT mice after EIU induction and HU308 treatment were compared to the same analyses in the CB2 knock-out (CB2 KO) mouse. Endothelial leukocyte adhesion and neutrophil migration were significantly increased in both WT and CB2 KO after EIU. HU308 significantly reduced the leukocyte adhesion and neutrophil recruitment in the WT animals. HU308 also significantly reduced leukocyte adhesion in the CB2 KO mouse, yet, had no effect on neutrophil infiltration suggesting an important off-target effect of HU308. Lipidomics profiles revealed significant increases in 6 non-ECS lipids after EIU in the WT and that HU308 effectively reduced these back to control levels; in addition, HU308 increased levels of 2-acyl glycerols and decreased all N-acyl glycines. CB2 KOs with saline-injection compared to WT had significantly elevated levels of 2-acyl glycerols, whereas levels of N-oleoyl ethanolamine (OEA), N-stearoyl ethanolamine (SEA), and PGE2 were reduced. CB2 KOs with EIU had 13 lipids that were significantly lower than WT with EIU including 4 N-acyl glycines. HU308 had no effect on lipid concentrations in the CB2 KOs with EIU, however, it did cause further reductions on 3 additional lipids compared to saline controls. HU308 appears to be acting at a non-CB2 target for the reduction of leukocyte infiltration in the EIU model; however, our data suggest that HU308 is working through CB2 to reduce neutrophil migration and for the regulation of multiple lipid signaling pathways including PGs, lipoamines, and 2-acyl glycerols. These data implicate ocular CB2 as a key component of lipid signaling in the eye and part of the regulatory processes of inflammation.

DIBI, a polymeric hydroxypyridinone iron chelator, reduces ocular inflammation in local and systemic endotoxin-induced uveitis.

BACKGROUND/OBJECTIVE: Non-infectious uveitis is an inflammatory disease of the eye commonly treated by corticosteroids, though important side effects may result. A main mediator of inflammation are oxygen free radicals generated in iron-dependent pathways. As such, we investigated the efficacy of a novel iron chelator, DIBI, as an anti-inflammatory agent in local and systemic models of endotoxin induced uveitis (EIU). METHODS: Firstly, the effects of DIBI in systemic EIU in Lewis rats were established. 2 hours post intravenous LPS or LPS/DIBI injections, leukocyte activation and functional capillary density (FCD) were examined using intravital microscopy (IVM) of the iridial microcirculation. Secondly, the toxicity of DIBI was evaluated in BALB/C mice for both acute and chronic dosages through gross ocular examination, intraocular pressure measurements and hematoxylin-eosin staining of ocular tissue. Lastly, three groups of BALB/C mice, control, LPS or DIBI + LPS, were studied to evaluate the effectiveness of DIBI in treating local EIU. Five hours post-local intravitreal (i.v) injection, leukocyte activation and capillary density were examined via IVM. RESULTS: Treatment of systemic EIU with DIBI resulted in a reduction of leukocyte activation and FCD improvement within the iridial microcirculation. Toxicity studies suggested that acute and chronic DIBI administration had no adverse effects in the eye. In the local EIU model, DIBI was shown to reduce leukocyte activation and restored the FCD/DCD ratio, providing evidence for its anti-inflammatory properties. CONCLUSIONS: Our study has provided evidence that DIBI has anti-inflammatory effects in experimental uveitis. Additionally, no local ocular toxicity was observed.

Cannabinoid 2 receptor activation reduces leukocyte adhesion and improves capillary perfusion in the iridial microvasculature during systemic inflammation.

BACKGROUND: Leukocyte adhesion to the endothelium and decreased microvascular blood flow causing microcirculatory dysfunction are hallmarks of systemic inflammation. We studied the impact of cannabinoid receptor activation on the iridial microcirculation, which is accessible non-invasively in vivo, in systemic inflammation induced by endotoxin challenge. METHODS: 40 Lewis rats were used in the experiments. Endotoxemia was induced by 2 mg/kg i.v. lipopolysaccharide (LPS). Cannabinoid receptors (CBRs) were stimulated by i.v. administration of WIN 55212-2 (WIN; 1 mg/kg). CB1R antagonist (AM281; 2.5 mg/kg i.v.) or CB2R antagonist (AM630; 2.5 mg/kg i.v.) treatment prior to WIN was applied to identify the anti-inflammatory effects underlying each CBR subtype. Leukocyte-endothelial interactions were examined in rat iridial microvas culature by intravital microscopy at baseline and 1 and 2 h post-LPS. Additionally, systemic (mean arterial pressure, heart rate) and local (laser Doppler flow) hemodynamic variables were measured prior to and during cannabinoid treatments. RESULTS: Endotoxemia resulted in severe inflammation as shown by significantly increased numbers of adherent leukocytes at 1 and 2 h observation time post-LPS challenge and decreased microcirculatory blood flow at 2 h within the iridial microcirculation. WIN treatment significantly reduced leukocyte adhesion in iridial microvessels with a diameter greater and less than 25 µm during endotoxemia (p <  0.05). Pre-treatment of animals by CB1R antagonist, AM281, did not affect WIN effects on LPS-induced leukocyte adhesion. When pre-treated with the CB2R antagonist, AM630, a reversal of the WIN-induced reduction in leukocyte adhesion was noticed in vessels with a diameter of less than 25 µm (p <  0.05). Cannabinoid treatment significantly increased the local iridial microcirculatory blood flow 2 hours after systemic LPS administration (p <  0.05). CONCLUSIONS: Systemic administration of the CBR agonist, WIN, decreased leukocyte-adhesion and improved iridial microvascular blood flow. This effect is most likely mediated by CB2R activation. Our findings indicate that the iris microvasculature can serve as a model to study the microcirculation during systemic inflammation and help to identify potential therapies to treat microcirculatory dysfunction in diseases such as sepsis.

Anti-inflammatory effects of cannabinoid CB(2) receptor activation in endotoxin-induced uveitis.

BACKGROUND AND PURPOSE: Cannabinoid CB2 receptors mediate immunomodulation. Here, we investigated the effects of CB2 receptor ligands on leukocyte-endothelial adhesion and inflammatory mediator release in experimental endotoxin-induced uveitis (EIU). EXPERIMENTAL APPROACH: EIU was induced by intraocular injection of lipopolysaccharide (LPS, 20 ng·µL(-1) ). Effects of the CB2 receptor agonist, HU308 (1.5% topical), the CB2 receptor antagonist, AM630 (2.5 mg·kg(-1) i.v.), or a combination of both compounds on leukocyte-endothelial interactions were measured hourly for 6 h in rat iridial vasculature using intravital microscopy. Anti-inflammatory actions of HU308 were compared with those of clinical treatments for uveitis - dexamethasone, prednisolone and nepafenac. Transcription factors (NF-κB, AP-1) and inflammatory mediators (cytokines, chemokines and adhesion molecules) were measured in iris and ciliary body tissue. KEY RESULTS: Leukocyte-endothelium adherence was increased in iridial microvasculature between 4-6 h after LPS. HU308 reduced this effect after LPS injection and decreased pro-inflammatory mediators: TNF-α, IL-1ß, IL-6, CCL5 and CXCL2. AM630 blocked the actions of HU-308, and increased leukocyte-endothelium adhesion. HU-308 decreased levels of the transcription factors NF-κB and AP-1, while AM630 increased levels of NF-κB. Topical treatments with dexamethasone, prednisolone or nepafenac, failed to alter leukocyte adhesion or mitigate LPS-induced increases in inflammatory mediators during the 6 h of EIU. CONCLUSION AND IMPLICATIONS: Activation of CB2 receptors was anti-inflammatory in a model of acute EIU and involved a reduction in NF-κB, AP-1 and inflammatory mediators. CB2 receptors may be promising drug targets for the development of novel ocular anti-inflammatory agents. LINKED ARTICLES: This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.

Histamine influence on apoptosis in trophoblast cell cultures.

INTRODUCTION: It has been demonstrated that histamine plays an important role in the pathogenesis of preeclampsia. Histamine regulates the process of differentiation of trophoblast cells; it also acts as a growth factor in malignant melanoma cells, and prevents monocytic apoptosis. Trophoblast research has shown that in preeclampsia placentas, trophoblast apoptosis is significantly increased. AIM OF THE STUDY: The aim of our study was to demonstrate the influence of histamine on the process of apoptosis in human trophoblast cell cultures. MATERIALS AND METHODS: Placentas were obtained after vaginal delivery. Tissue samples were excised from placentas and, with the use of modified Kliman's method, trophoblast cell cultures were established. The cultures were incubated with dexamethasone as an apoptosis inducer 48 hours prior to apoptosis detection assays. Along with dexamethasone, selected cell cultures were incubated with histamine (1 micromol/l) or histamine (1 micromol/l) and terfenadine (from 1 to 5 micromol/l), a H(1) receptor antagonist. For apoptotic activity detection, and quantitative analysis, we used an ELISA assay. M30-Apoptosense ELISA Kit is based on the M30 monoclonal antibody that binds only the caspase-cleaved cytokeratin 18 formed during apoptosis in trophoblast cells. RESULTS: Our investigation showed significantly (p < 0.05) increased apoptotic activity in cultures incubated with dexamethasone, histamine and terfenadine (% of reference value, +/-SEM): up to 113.1 +/- 4.33%. Cell cultures incubated with dexamethasone and histamine only showed significantly lower apoptotic activity 90.2 +/- 5.17%. We suggest that histamine may inhibit apoptotic activity in trophoblast cell cultures via H(1) receptor. Thus histamine may regulate the process of trophoblast differentiation (via integrin aV-b3 expression, as we previously suggested), and influence cell turnover in the placenta.

[Monitoring oxypurines levels in women with pregnancy induced hypertension and in women with physiological pregnancy]. / Monitorowanie stezenia oksypuryn u kobiet z nadcisnieniem indukowanym ciaza i w ciazy fizjologicznej.

In this paper the concentration of oxypurines in the plasma taken from women with PIH, regular pregnancies and control group was determined. In comparison to the other groups very high concentration of HX and low X in the plasma of women with PIH was found. Such a result allows for the conclusion proving the exsistance of hypoxia in the group of women with PIH. To confirm the claim the clinical evaluation of pregnant women, fetuses and newborns was used. The values of most parameters proved that fetuses and newborns coming from the pregnancies of women with complicated PIH are weak, in the clinical evaluation, than those coming from the regular pregnancies.

Prevalence of measles antibodies in patients with subacute sclerosing panencephalitis in Poland in the years 1971--1978.

Between 1971 and 1978, clinical centres in four provinces of Poland reported 107 cases of subacute sclerosing panencephalitis (SSPE). This series comprised 73.8% boys and 26.2% girls. Most children were aged 6--8 years, and 66% of them had had measles in the first two years of life. As compared with a control group the geometric means of the haemagglutination-inhibiting antibodies and neutralizing antibodies were between ten and twenty times higher in SSPE patients. Determinations of the levels of antibodies carried out several times during the disease showed a rise of the titres to values as high as 1 : 128,000 in the serum and 1 : 1,024 in the cerebrospinal fluid in some cases.