Advanced glycation end-products affect transcription factors regulating insulin gene expression.

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic beta-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

Glucagon-like peptide-1 counteracts the detrimental effects of Advanced Glycation End-Products in the pancreatic beta cell line HIT-T 15.

Advanced Glycation End-Products (AGEs), a group of compounds resulting from the non-enzymatic reaction of reducing sugars with the free amino group of proteins, are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T 15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases proinsulin biosynthesis, stimulates insulin secretion, and improves pancreatic beta-cell viability. The aim of this work was to investigate the effects of GLP-1 on the function and viability of HIT-T 15 cells cultured with AGEs. HIT-T 15 cells were cultured for 5days in presence of AGEs alone, or supplemented with 10nmol/l GLP-1. Cell viability, insulin secretion, redox balance, and expression of the AGEs receptor (RAGE) were then determined. The results showed that GLP-1 protected beta cell against AGEs-induced cell death preventing both apoptosis and necrosis. Moreover, addition of GLP-1 to the AGEs culture medium restored the redox balance, improved the responsiveness to glucose, and attenuated AGEs-induced RAGE expression. These findings provide evidence that GLP-1 protects beta cells from the dangerous effects of AGEs.

Caveolin-1 is essential for glimepiride-induced insulin secretion in the pancreatic betaTC-6 cell line.

The K(ATP) channels play a pivotal role in the complex mechanism of insulin secretion. K(ATP) channels represent the target of sulphonylureas, a class of drugs widely used in type 2 diabetes to stimulate insulin secretion. We previously showed that caveolin-1 depletion impairs action of the sulphonylurea glimepiride in human endothelial cells. The aim of this work was to investigate the possible role of caveolin-1 in glimepiride-induced insulin secretion. Caveolin-1 was depleted using siRNA method in the pancreatic betaTC-6 cell line. Then stimulation of insulin secretion was performed with different secretagogues (glucose, KCl, and glimepiride). Here, we show that betaTC-6 caveolin-1 depleted cells maintained high rate of insulin secretion after KCl, but not after glucose and glimepiride stimulation. Moreover, we find a direct interaction between caveolin-1 and Kir6.2, one of the K(ATP) channel subunit. These results demonstrate that Cav-1 plays a critical role for glucose and sulfonylurea-stimulated insulin secretion.

Effect of conalbumin on phytomitogen stimulation and E-rosette formation of human peripheral lymphocytes in normal subjects.

An immunological in vitro study was carried out on conalbumin, an iron binding protein structurally similar to lactoferrin, which is a well-known bacterial inhibitor in human milk. Conalbumin itself has been proved to have bacteriostatic activity against a broad spectrum of bacteria responsible for gastrointestinal infections. The activity of conalbumin on the in vitro response to PHA, PWM and Con A and on the E-rosette formation ability of peripheral lymphocytes of 10 normal subjects was studied. The results showed that conalbumin did not affect the lymphocytes' E-rosette formation ability and did not induce blastic transformation of lymphocytes. However, conalbumin was able to produce a significant increase in the in vitro response of lymphocytes to PHA and PWM, suggesting an action on both T and B lymphocytes.

General features of the Adriatic Sea: the key role of carbon cycling.

Major inputs into the northern Adriatic Sea and critical problems affecting this basin are revised strengthening the need for a thorough knowledge of organic carbon cycling. Results gathered in the context of the PRISMA 2 project by our research group give information on the distribution and composition of organic matter in terms of major biochemical components, molecular weight sizes and biodegradability characteristics. Seasonal accumulation of dissolved organic matter in summer along with an increased role of carbohydrate and colloidal matter (being on average 15-20 and 60-65% of DOC, respectively) and restricted bacterial activity due to P deficiency are all priority subjects to be further investigated.

Pioglitazone attenuates the detrimental effects of advanced glycation end-products in the pancreatic beta cell line HIT-T15.

Pioglitazone is an anti-diabetic agent that preserves pancreatic beta cell mass and improves their function. Advanced Glycation End-Products (AGEs) are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The aim of this work was to investigate the effects of Pioglitazone on the function and viability of HIT-T15 cells cultured with AGEs. HIT-T15 cells were cultured for 5 days in the presence of AGEs alone, or supplemented with 1 µmol/l Pioglitazone. Cell viability, insulin secretion and insulin content, redox balance, expression of the AGE receptor (RAGE), and NF-kB activation were then determined. The results showed that Pioglitazone protected beta cells against AGEs-induced apoptosis and necrosis. Moreover, Pioglitazone restored the redox balance and improved the responsiveness to low glucose concentration. Adding Pioglitazone to the AGEs culture attenuated NF-kB phosphorylation, and prevented AGEs to down-regulate IkBα expression. These findings suggest that Pioglitazone protects beta cells from the dangerous effects of AGEs.

Total upper and lower eyelid replacement following thermal burn using an ALT flap--a case report.

Upper and lower eyelid unilateral full thickness reconstruction in a patient with no available adjacent tissues because of burns or trauma sequelae is a surgical challenge. A case of severe thermal burn with unilateral complete defect of both upper and lower eyelids is reported, together with the surgical technique of reconstruction. The patient was a 65-year-old man who sustained deep burns of the head and neck with upper airway burns after falling into a fireplace. After tracheostomy and acute resuscitation, he underwent escharectomy and coverage of his head and neck burns with split thickness skin grafts and with full thickness skin grafts to the eyelids. There was incomplete take of the skin grafts to the upper and lower left eyelids. In these areas, infection and loss of the tarsum and subsequent eyelid retraction led to exposure keratitis and blurred vision. After healing and respiratory rehabilitation, he was referred to our microsurgical unit for upper and lower eyelid reconstruction. A free forearm flap was first considered, but the Allen test was negative. Therefore, a free anterolateral thigh (ALT) flap was chosen to provide skin eyelid coverage. The flap was harvested including fascia and centred on one perforator. The levator muscle stump and conjunctiva from both upper and lower cul-de-sacs were dissected and advanced. Flap vessels were anastomosed to the superficial temporal artery and vein. The conjunctiva and the fascia replaced the new inner upper and lower lamella. To our knowledge, this is the first report of the use of a perforator flap, the ALT flap, in full thickness reconstruction of both upper and lower eyelids and may be a reliable option in such selected and challenging situations.

Free anterolateral thigh flap versus free forearm flap: Functional results in oral reconstruction.

Nowadays, microsurgery performed for oral reconstruction of cancer patients, has become the standard treatment in restoring oral functions. The free radial forearm flap (FRFF) is still apparently the first reconstructive choice in oral cavity cancers. Recently the anterolateral thigh flap (ALTF) seemed to challenge the superiority of FRFF. The lack of functional data on which to base this recent supposition is the reason for this new research. Twenty reconstructed patients were enrolled for this study. Speech, swallowing, and donor site complications were studied to assess differences between the two techniques. Results show that difference in function between ALTF and FRFF groups is statistically insignificant. Donor site risks and complications seem to be the only variables among groups. These variables may be used as indicators when making a surgical choice.

Rat serum improves rat pseudoislet formation and insulin gene expression.

Rat islet cells in culture are able to form tridimensional aggregates with an architecture and functional activity similar to native islets: pseudoislets. Pseudoislets represent an alternative source for islet transplantation, because their transplant results in a long term allograft acceptance without immunosuppression of the host. Use of pseudoislets has been limited by their reduced yield and by poor reaggregation mass. Since culture conditions have been reported to affect reaggregation, the aim of this study was to evaluate the effects of different concentrations of two sera (Fetal Bovine Serum [FBS] and Rat Serum [RS]) on reaggregation and insulin gene expression in pseudoislets. Islets were isolated from male Lewis rat by means of histopaque gradient centrifugation. The day after islets were disrupted into single cells and cultured in RPMI 1640 5.6 mM glucose with 2%, 5% and 10% solutions of both FBS and RS. Cells spontaneously reaggregated to form pseudoislets. After seven days of culture, pseudoislets were counted and analysed for insulin secretion and insulin gene expression using RT-PCR. Rat serum increased the number of aggregates and their diameters. Insulin gene expression of pseudoislets cultured with RS showed a ten fold increase in comparison to those cultured with FBS. These data show that the culture medium supplemented with RS improves total reaggregate volume and increases insulin gene expression. With the perspective of pseudoislets' use in transplantation RS is better indicated than FBS for the production of rat pseudoislets.

[Effect of conalbumin on stimulation by phytomitogens and on rosette E formation of human peripheral lymphocytes of normal subject]. / Effectto della conalbumina sulla stimolazione da fitomitogeni e sulla formazione delle rosette-E di linfociti periferici umani di soggetti normali.

We carried on an immunological in vitro study on conalbumin, an iron binding protein structurally similar to lactoferrin, a well know bacterial inhibitor of human milk. Conalbumin itself has been proved to have bacteriostatic activity against a broad spectrum of bacteria responsible for gastrointestinal infections. The activity of conalbumin on the in vitro response to PHA, PWM and Con A and on the E-rosette formation ability and does not induce blastic transformation of lymphocytes. However, conalbumin has been able to significantly increase the in vitro response of lymphocytes to PHA and PWM, suggesting an action on both T and B lymphocytes.

Cultured tibial rat osteoblasts: in vitro production and topography of osteonectin, biglycan and decorin.

Rat osteoblasts in culture undergo differentiative changes culminating in the formation of mineralized foci. We here report on the pattern of temporal expression and compartmentalization of osteonectin and of the two small proteoglycans, byglican and decorin. They were constitutively synthesized during in vitro differentiation of rat osteoblasts. The 3 proteins were detected in the conditioned medium and associated with the cell-matrix compartment. Within this compartment they showed prevalent cytoplasmic location and differential distribution on unmineralized noduli was detected for osteonectin and byglican, while decorin was detected throughout the nodules. Along with known functions in the matrix, a possible role in the cytoplasm may have to be sought for these bone cells components.

Osteoblastic cells from rat long bone. II: Adhesion to substrata and integrin expression in primary and propagated cultures.

Propagation in vitro of rat tibial osteoblasts (ROB) is accompanied by increased expression of the early osteogenic marker alkaline phosphatase (AP) and maturation of the osteogenic phenotype. In order to establish the pattern of the integrin expressed in ROB during progression to the mature osteoblastic phenotype, we have used biosynthetic, immunoblotting and immunohistochemical assays. We immunoprecipitated from osteoblasts, expanded for 1.5- and 7.5-doubling, alpha 5 beta 1, alpha v beta 3, alpha 3 beta 1, alpha 6 beta 1 and alpha 1 beta 1 integrin heterodimers; furthermore beta 5, alpha 2 and alpha 4 chains were detected by immunoblots and indirect immunofluorescence. alpha v, alpha 1, alpha 6 subunits in most cells, and beta 3 and beta 1 subunits in a minority, were found to be associated with adhesion plaques in osteoblasts of 1.5-, 4.5- and 7.5-doubling grown in the presence of FCS, while all other subunits stained diffusely all the cells. Adhesion to fibronectin (FN), laminin (LN), collagen type I (COL I) and III(COL III) by ROB at different doubling (1.5-11) was dependent on substratum concentration, and after 2.5 h at 55 nM 60% of the cells adhered to all substrata. Arg-Gly-Asp-Ser (RGDS) containing peptides inhibited adhesion of cells differentially, according to substratum; no dependence on extent of progation in vitro was observed. In conclusion, ROB cultured in vitro for 1.5- to 11-doubling had an unchanged pattern of expression of integrin subunits, heterodimer association and cellular distribution. Adhesion specificity and affinity were also unchanged. These results suggest that the phenotypic maturation, detected as an increase in AP expression, is not accompanied by major changes in the potential for cell-matrix interactions, and does not correspond to changes in the type of integrin subunits expressed by osteoblasts.

Two novel mutations (10410 T-->G; 10296 del C) at carboxy-terminus of the dystrophin gene associated with mental retardation. Mutations in brief no. 149. Online.

In this study we have carried out a mutational screening of exons 62-79 of the dystrophin gene by SSCP in 38 Italian patients with DMD/BMD and found two novel mutations at exon 70, in 2 mentally retarded DMD patients.

[Clinical and immunological study on 4 pediatric patients with chronic mucocandidiasis associated with hypoparathyroidism]. / Studio clinico ed immunologico di quattro pazienti in età pediatrica con mucocandidosi cronica associata ad ipoparatiroidismo.

Four paediatrics patients affected by Chronic Mucocutaneous Candidiasis associated with hypoparathyroidism have been studied. In vivo and in vitro responses to Candida albicans antigens and to other common antigens (Dermatophytin T, Varidase), the percentage of T and B cells and the response to the selective polyclonal phytomitogens (PHA, ConA, PWM) have been investigated in order to quantitate and functionally characterize the B and T lymphocyte classes. The HLA A an B typing has been also performed. Our results show a remarkable and most likely primitive impairment of the T cells, unresponsive either to the C. albicans and the two other skin test antigens or to the selective polyclonal mitogens in vitro. The infectious disease (Mucocandidiasis) is most likely secondary to the T-cell failure. The association between the Mucocandidiasis, hypoparathyroidism and the T-cell deficiency is probably due to a common primitive disembryogenetic defect.