Ultrastructure and 3D transmission electron tomography of collagen fibrils and proteoglycans of swollen human corneal stroma.

BACKGROUND: The transparency of the cornea is regulated by the unique organization of collagen fibrils (CFs) which is maintained by proteoglycans (PGs). The interlacing of CF lamellae in the anterior stroma provides the biomechanical properties of the cornea. OBJECTIVE: To investigate the alterations of CFs and PGs in the swollen cornea, with special reference to the anterior stroma by using electron microscopy and 3D ultrastructural tomography. METHOD: Nine healthy normal scleral corneal rings (age from 40 to 65 years) were hydrated individually in deionised water to induce swelling in the cornea. Three of them were hydrated for 2hr whereas the other three were hydrated for 48hr. The remaining three scleral normal corneal rings were used as a control.The corneas were processed for electron microscopy (EM) to study the CFs and PGs. Ultrathin sections were observed using transmission electron microscopy (JOEL 1400) and digital images of CFs, PGs and lamellae were captured using a bottom mounted Quemesa camera and iTEM Soft Imaging System. The software program 'Composer-x64, version 3.4.2.0' was used to construct individual 3D images from 120 digital images taken from -60 to + 60 degree angles. RESULTS: The 3D tomography showed the degeneration of microfibrils within the CFs of the swollen cornea. The CF diameter was significantly reduced and the interfibrillar spacing significantly increased in both the 2hr and 48hr hydrated corneas compared to the normal cornea. Within the hydrated corneas, the CF diameter was smaller and the interfibrillar spacing was increased in the middle and posterior stroma compared to the anterior stroma. The PG area in both the 2hr and the 48hr hydrated cornea was reduced in the anterior stroma, whereas it was increased in middle and posterior stroma compared to the normal cornea. The density of the PGs in both the 2hr and the 48hr samples, was reduced compared to the density of PGs in the normal cornea. CONCLUSION: The CFs, PGs and lamellae had degenerated, caused by swelling. 3D imaging demonstrated that the impairment of the microfibrils and PGs within the CF, is caused by the excessive hydration or swelling in the anterior as well as in the middle and posterior stroma. The lamellae of the anterior stroma which provides the biomechanical strength in the normal cornea, had degenerated in the swollen corneas due to the presence of the damaged CFs and PGs.

CB2 receptor activation causes an ERK1/2-dependent inflammatory response in human RPE cells.

A chronic low-level inflammation contributes to the pathogenesis of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in Western countries. The loss of central vision results from attenuated maintenance of photoreceptors due to the degeneration of retinal pigment epithelium (RPE) cells beneath the photoreceptor layer. It has been proposed that pathologic inflammation initiated in RPE cells could be regulated by the activation of type 2 cannabinoid receptors (CB2). Here, we have analysed the effect of CB2 activation on cellular survival and inflammation in human RPE cells. RPE cells were treated with the selective CB2 agonist JWH-133 in the presence or absence of the oxidative stressor 4-hydroxynonenal. Thereafter, cellular viability as well as the release of pro-inflammatory cytokines and potential underlying signalling pathways were analysed. Our results show that JWH-133 led to increased intracellular Ca2+ levels, suggesting that RPE cells are capable of responding to a CB2 agonist. JWH-133 could not prevent oxidative stress-induced cell death. Instead, 10 µM JWH-133 increased cell death and the release of proinflammatory cytokines in an ERK1/2-dependent manner. In contrast to previous findings, CB2 activation increased, rather than reduced inflammation in RPE cells.

Clearance of autophagy-associated dying retinal pigment epithelial cells - a possible source for inflammation in age-related macular degeneration.

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.

Insulin pump therapy with continuous glucose monitoring improves metabolic control in brittle type 1 diabetes.

AIM: to evaluate the combination of insulin pump therapy and continuous glucose monitoring in outcome on metabolic control in patients with brittle type 1 diabetes. MATERIALS AND METHODS: Insulin pump therapy was initiated in eleven brittle type 1 diabetics with poor metabolic control (mean Hba1c = 9.6%). Metabolic control was evaluated with CGMS and HbA1c in the following 6 months. RESULTS: Glycated haemoglobin showed a reduction in 1.4% in the 6 months following initialisation of pump therapy. Physical activity, various foods and insulin were tested with CGMS. There were no severe hypoglycaemia and occasional postprandial hyperglycaemia, where patients and their family learned the practical issues of carbohydrate counting. During the next 6 months on pump therapy, the patients successfully managed their diabetes. CONCLUSIONS: Insulin pump therapy can be initiated and used effectively in brittle type 1 diabetics to improve metabolic control and quality-of-life. When diabetes and pump management are appropriately individualized, this kind of therapy can help type 1 diabetics to achieve and to sustain metabolic control. Lifestyle flexibility, quality-of-life improvement, and independence can be maintained throughout young adulthood.

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes.

MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.

[Hypertension and diabetes mellitus]. / Hipertensija i dijabetes melitus.

AIM: To evaluate hypertension in patients with Diabetes Mellitus (DM) and its correlation with age, duration of DM, Body Mass Index (BMI) and HbA1C). MATERIALS AND METHODS: A retrospective study was made on 1211 patients with DM (male 554 and female 657), hospitalized at Clinic of Endocrinology between January 2001 and December 2002. Patients were divided in two groups: Control group (CG)-subdivided into 3 groups patients with DM type 1 (CG-1), DM type 2 on oral anti-hyper-glycemic agents (CG-2)and DM type 2 on insulin therapy (CG-3) and Examined Group (EG), the same groups for diabetes, including hypertension. RESULTS: We found hypertension in 12.6% patients with DM type 1, 30.5% in DM type 2 on oral anti-hyper-glycemic agents and 33.4% in DM type 2 on insulin therapy. CONCLUSION: Hypertension is mostly presented in DM type 2 patients (33,4%), instead of 12.6% in DM type 1. There is statistical significance (p<0.05) between duration of DM in patients with and without hypertension.