M.C. Lopes

l-Arginine transport in retinas from streptozotocin diabetic rats: correlation with the level of IL-1β and NO synthase activity

Several evidences suggest that the pro-inflammatory cytokines IL-1 beta and the radical NO are implicated as effectors molecules in the pancreatic beta-cells dysfunction; an event preceding the pathogenesis of diabetes. IL-1 beta induces the expression of the inducible isoform of NO synthase (iNOS), which use L-arginine as substrate to overproduce NO. However, it is not known whether these events may participate in the development of diabetic retinopathy, which is the main cause of blindness. In this work, we found an increased level of IL-1 beta in retinas from streptozotocin-induced (STZ) diabetic rats. We also observed that the activity of the NO synthase (NOS) and the L-arginine uptake are enhanced in retinas from STZ-induced diabetic rats as compared to retinas from control rats. We found that the uptake of L-arginine in retinas from control and diabetic rats occurs through a transporter resembling the Y + system, i.e. it is saturable, not affected over the pH range 6.5 to 7.4, and is independent of the extracellular Na+. Nevertheless, the L-arginine transport in retinas from diabetic rats occurs through a carrier with lower affinity (K(m) = 25 microM) and higher capacity (Vmax = 295 +/- 22.4 pmol L-arginine/mg protein) than in retinas from control rats (K(m) = 5 microM and Vmax = 158 +/- 12.8 pmol L-arginine/mg protein) which is correlated with the increased NOS activity and consequent depletion of the intracellular pool of L-arginine.

Quantification of Retinal Microvascular Density in Optical Coherence Tomographic Angiography Images in Diabetic Retinopathy

Importance Quantitative measurements based on optical coherence tomographic angiography (OCTA) may have value in managing diabetic retinopathy (DR), but there is limited information on the ability of OCTA to distinguish eyes with DR. Objective To evaluate the ability of measurements of retinal microvasculature using OCTA to distinguish healthy eyes from eyes with DR. Design, Setting, and Participants In this prospective cross-sectional study, OCTA was used to examine the eyes of participants with type 2 diabetes with or without DR and the eyes of participants without diabetes from September 17, 2015, to April 6, 2016. Density maps based on superficial retinal layer (SRL) and deeper retinal layer (DRL) images were generated after a method to remove decorrelation tails was applied to the DRL images. Exposures Both eyes of each participant were examined by means of a 3-mm OCTA scan and 7-field fundus photography using the Diabetic Retinopathy Severity Scale. Main Outcomes and Measures Two measures were examined: perfusion density, based on the area of vessels, and vessel density, based on a map with vessels of 1-pixel width. The size of the foveal avascular zone was also calculated automatically, and so was the area under the receiver operating characteristic curve. Results A total of 50 eyes from 26 participants with diabetes (10 women and 16 men; mean [SD] age, 64.9 [7.5] years) and 50 healthy eyes from 25 participants without diabetes (14 women and 11 men; mean [SD] age, 64.0 [7.1] years) were imaged. All participants were white. Vessel density measured in the SRL had the highest area under the receiver operating characteristic curve (0.893 [95% CI, 0.827-0.959]), compared with perfusion density in the SRL (0.794 [95% CI, 0.707-0.881]), foveal avascular zone area (0.472 [95% CI, 0.356-0.588]), and vessel density in the DRL (0.703 [95% CI, 0.601-0.805]). Vessel density in the SRL negatively correlated with best-corrected visual acuity (r = –0.28; P = .05) and severity of DR (r = –0.46; P = .001). Density metrics correlated with age. No correlation was detected between vascular density or foveal avascular zone metrics and hemoglobin A1C or duration of diabetes. Conclusions and Relevance Vessel density measured by OCTA provides a quantitative metric of capillary closure that correlates with severity of DR and may allow staging, diagnosis, and monitoring that do not require subjective evaluation of fundus images.

Antifungal, antioxidant and anti-inflammatory activities of Oenanthe crocata L. essential oil

The present study reports the chemical composition, antifungal, antioxidant and anti-inflammatory properties as well as the cytotoxicity of Oenanthe crocata essential oil and one of its main compounds. The essential oil was obtained from the aerial parts of the plant by hydrodistillation and analysed by GC and GC/MS. The oil was predominantly composed of monoterpene hydrocarbons (85.8%), being the main compounds trans-β-ocimene (31.3%), sabinene (29.0%) and cis-β-ocimene (12.3%). For the antifungal activity, the minimal inhibitory and minimal lethal concentrations (MICs and MLCs) were determined. The oil was particularly active against dermatophytes and Cryptococcus neoformans, with MIC values ranging from 0.08 to 0.16 μL/mL. Regarding the anti-inflammatory activity, both the oil and sabinene demonstrated strong anti-inflammatory activity through nitric oxide (NO) production inhibition in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-triggered macrophages. Furthermore, the essential oil showed a potent NO scavenging effect and inhibited inducible NO synthase expression. Interestingly, and although we detected a cytotoxic effect in macrophages and keratinocytes for the highest concentrations tested of the oil and sabinene, we also disclosed bioactive and safe concentrations to be further explored for therapeutic proposes. Taking together, these results support the use of the oil and sabinene for the management of dermatophytosis and/or inflammatory-related diseases.

Dexamethasone prevents interleukin-1beta-induced nuclear factor-kappaB activation by upregulating IkappaB-alpha synthesis, in lymphoblastic cells.

AIMS: Glucocorticoids (GCs) exert some of their anti-inflammatory actions by preventing the activation of the transcription factor nuclear factor (NF)-kappaB. The GC-dependent inhibition of NF-kappaB may occur at different levels, but the mechanisms involved are still incompletely understood. In this work, we investigated whether the synthetic GC, dexamethasone (Dex), modulates the activity of NF-kappaB in the lymphoblastic CCRF-CEM cell line. We also evaluated the ability of Dex to prevent the activation of NF-kappaB in response to the potent proinflammatory cytokine, interleukin (IL)-1beta. RESULTS: Exposure of the cells to Dex (1 microM) induced the rapid degradation of IkappaB-alpha, leading to the transient translocation of the NF-kappaB family members p65 and p50 from the cytoplasm to the nucleus, as evaluated by western blot. Electrophoretic mobility shift assays revealed that, in the nucleus, these NF-kappaB proteins formed protein-DNA complexes, indicating a transient activation of NF-kappaB. Additionally, Dex also induced de novo synthesis of IkappaB-alpha, following its degradation. Finally, when the cells were exposed to Dex (1 microM) prior to stimulation with IL-1beta (20 ng/ml), Dex was efficient in preventing IL-1beta-induced NF-kappaB activation. The GC antagonist, RU 486 (10 microM), did not prevent any of the effects of Dex reported here. CONCLUSION: Our results indicate that, in CCRF-CEM cells, Dex prevents NF-kappaB activation, induced by IL-1beta, by a mechanism that involves the upregulation of IkappaB-alpha synthesis, and that depends on the early and transient activation of NF-kappaB.

Dexamethasone-induced and estradiol-induced CREB activation and annexin 1 expression in CCRF-CEM lymphoblastic cells: evidence for the involvement of cAMP and p38 MAPK

AIMS Annexin 1 (ANXA1), a member of the annexin family of calcium-binding and phospholipid-binding proteins, is a key mediator of the anti-inflammatory actions of steroid hormones. We have previously demonstrated that, in the human lymphoblastic CCRF-CEM cell line, both the synthetic glucocorticoid hormone, dexamethasone (Dex), and the estrogen hormone, 17beta-estradiol (E2beta), induce the synthesis of ANXA1, by a mechanism independent of the activation of their nuclear receptors. Recently, it was reported that the gene coding for ANXA1 contains acAMP-responsive element (CRE). In this work, we investigated whether Dex and E2beta were able to induce the activation of CRE binding proteins (CREB) in the CCRF-CEM cells. Moreover, we studied the intracellular signalling pathways involved in CREB activation and ANXA1 synthesis in response to Dex and E2beta; namely, the role of cAMP and the p38 mitogen activated protein kinase (MAPK). RESULTS The results show that Dex and E2beta were as effective as the cAMP analogue, dBcAMP, in inducing CREB activation. On the contrary, dBcAMP induced ANXA1 synthesis as effectively as these steroid hormones. Furthermore, the cAMP antagonist, Rp-8-Br-cAMPS, and the specific p38 MAPK inhibitor,SB203580, effectively prevented both Dex-induced, E2beta-induced and dBcAMP-induced CREB activation and ANXA1 synthesis. CONCLUSIONS Taken together, our results suggest that,in CCRF-CEM cells, Dex-induced and E2beta-inducedANXA1 expression requires the activation of the transcription factor CREB, which in turn seems to be mediated by cAMP and the p38 MAPK. These findings also suggest that, besides the nuclear steroid hormone receptors, other transcription factors, namely CREB, may play important roles in mediating the anti-inflammatory actions of glucocorticoids and oestrogen hormones.

Contact sensitizers downregulate the expression of the chemokine receptors CCR6 and CXCR4 in a skin dendritic cell line

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response, namely in allergic contact dermatitis (ACD). In this work, we investigated by flow cytometry the effect of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulfate (NiSO4), on the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of skin DC. Our results show that all the skin sensitizers studied decreased the membrane expression of the chemokine receptors CCR6 and CXCR4. In contrast, 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB without contact sensitizing properties, was without effect on the surface expression of these receptors. Lipopolysaccharide (LPS), which induces the maturation of DC, also reduced surface CCR6 and CXCR4 expression.

Effect of Skin Sensitizers on Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Skin Dendritic Cells: Role of Different Immunosuppressive Drugs

Nitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.

A5.1 Culture OF human chondrocytes in high glucose induces inflammatory markers and impairs autophagy

Background and Objectives Accumulating evidence indicates that Diabetes Mellitus is an independent risk factor for severe osteoarthritis (OA). Understanding the mechanisms involved is essential for designing preventive strategies and targeted therapies that can halt OA progression. Our previous studiesshowed that culture of human chondrocytes under excess glucose favours catabolic responses and oxidative stress. This study aims at elucidating whether exposure of chondrocytes to high glucose favours inflammatory responses and impairs autophagy, a crucial mechanism for the elimination of damaged molecules and organelles. Materials and Methods Human chondrocytes isolated from knee cartilage obtained from multi-organ donors at the Orthopaedics Department of the University and Hospital Center of Coimbra and the human chondrocytic cell line, C28/I2, were cultured in medium containing regular (10 mM) or excess (30 mM) glucose for various periods. The expression of pro-inflammatory and pro-catabolic markers (iNOS, IL-1β and TNF-α) was evaluated by qRT-PCR. iNOS protein levels and the nuclear translocation of p65, a marker of NF-κB activation, were evaluated by western blot. NO production was assessed by the Griess reaction. Autophagy was assessed by determining the protein levels of LC3-I and II in the presence and absence of the lysosome inhibitor, chloroquine. To rule out possible osmotic effects, parallel experiments were performed in the presence of the cell-impermeable polyol, mannitol. Results Culture of human chondrocytes in high glucose (30 mM) for 24h significantly increased iNOS mRNA and protein levels, as well as NO production relative to cells maintained in regular glucose. IL-1β and TNF-α mRNA levels were also significantly increased, while nuclear levels of NF-κB p65 increased in a time-dependent manner, peaking after 1h treatment. On the other hand, LC3-II levels were significantly reduced by culture of C28/I2 cells in high glucose for 24h, either in the presence or absence of chloroquine, indicating that its synthesis was decreased relative to cells maintained in regular glucose medium. Conclusions Hyperglycemia-like glucose concentrations are sufficient to induce the inflammatory process and to impair autophagy in human chondrocytes which can contribute to the development and progression of OA. Work supported by grants PEst-C/SAU/LA0001/2011 and PTDC/EME-TME/113039/2009 from FEDER through COMPETE and FCT.

Multimodal Evaluation of the Fellow Eye of Patients with Retinal Angiomatous Proliferation

Introduction: We conducted a multimodal, cross-sectional evaluation. Methods: Eyes were divided into 4 study groups: controls, early/intermediate age-related macular degeneration (AMD), fellow eyes of retinal angiomatous proliferation (RAP), and RAP eyes. Patients were evaluated with spectral-domain optical coherence tomography (OCT), enhanced depth imaging-OCT, and OCT angiography (OCTA). OCTA images were processed to generate maps of the vessel density and perfusion density of the superficial and deep retinal layers (SRL and DRL) and the choriocapillaris level (CL). The thickness of the outer nuclear layer and choroid was manually assessed. Results: We included 135 eyes of 100 patients (51 controls, 30 AMD, 42 RAP, and 12 fellow eyes). The fellow eyes showed a significantly lower vascular perfusion of the SRL, DRL, and CL (p < 0.02) than the early/intermediate AMD and control eyes did. Similarly, RAP eyes presented a lower vascular perfusion of the DRL and CL (p < 0.05). Besides, structural analyses of the fellow eyes and RAP eyes revealed a significantly higher prevalence of macular pigmentary changes, atrophy of the retinal pigment epithelium, hyperreflective “clumps” above flat drusen, amongst others, than early/intermediate AMD and control eyes (p < 0.05). Conclusion: We present the first report on the OCTA analysis of the fellow eye of patients with RAP. The reduced perfusion density and vessel density observed contributes, in association with clearly defined structural changes, to a wider characterization of RAP as a distinctive phenotype.

195 ROLE OF HIGH EXTRACELLULAR GLUCOSE CONCENTRATIONS IN MODULATING ANABOLIC AND CATABOLIC GENE EXPRESSION IN NORMAL AND OSTEOARTHRITIC HUMAN CHONDROCYTES

inserted in sham-operated rats. The immobilized rats and sham-operated rats made up the immobilized-remobilized (Im-Re) group and control group, respectively. Five-μm sections at the medial midcondylar region in sagittal plane were obtained and stained with H-E and Safranin-O (S-O). Six areas (NC area, T area, C area in the femur and tibia) were set and modified Mankin’s score, thickness of the articular cartilage, and number of chondrocytes were evaluated at each area. Mechanical properties of the articular cartilage were assessed by the scanning acoustic microscope (SAM). Results: [C Area] Chondrocytes decreased and disappeared after 1W Im-Re group (Figs. 1, 2). A marked reduction of S-O staining was observed (Figs. 4, 5). Mankin’s score was significantly higher after 1W Im-Re group (Fig. 7). Number of chondrocytes was significantly smaller after 2W Im-Re group (Fig. 9). The articular cartilage in the Im-Re group was almost blue (low sound speed) compared to the control (Figs. 10, 11). [T Area] Hypertrophy and cloning of chondrocytes were observed after 2W Im-Re group (Fig. 3). Thickness of the cartilage was significantly higher at 4W Im-Re group (Figs 8). [NC Area] Reduction of S-O staining in the non-calcified cartilage was almost restored but it was not restored around the tidemark (Fig. 6).