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G Basta

Publications and source records attributed to G Basta.

At least 19 recordsLinked to original sources
PubMed2006

Circulating levels of Nepsilon-(carboxymethyl)lysine are increased in systemic sclerosis.

OBJECTIVE: Advanced glycation endproducts (AGEs), including Nepsilon-(carboxymethyl)lysine-protein adducts (CML) are involved in micro/macrovascular changes and are co-localized with adhesion molecules in inflamed tissues. Serum levels of CML were investigated in systemic sclerosis (SSc) characterized by microvascular modifications and correlated with indices of micro/macrovascular damage. METHODS: In 66 SSc patients (limited SSc, n = 55; diffuse SSc, n = 11) and 20 controls, CML serum levels were measured by enzyme-linked immunosorbent assay. Nailfold capillaroscopy, intima-media thickness (IMT) and the ankle-brachial index (ABI) were also recorded, to characterize micro/macrovascular involvement. RESULTS: CML levels were significantly higher in SSc (79.2 +/- 39 mg/ml vs 49.6 +/- 26.1 mg/ml, mean +/- s.d.; P<0.01), without significant differences between SSc subsets. CML levels were significantly higher in all capillaroscopic patterns: the 'early' pattern showed higher levels than 'active' and 'late' patterns. IMT was significantly higher in SSc (P<0.01) than in controls, whilst ABI was no different from controls. CONCLUSIONS: These data indicate that although both CML formation and macrovascular involvement are increased in SSc, there is no correlation between these two parameters. However, the characteristic early nailfold capillaroscopy changes of SSc are associated with proportionally greater CML formation, suggesting that AGEs are involved in SSc microangiopathy.

Adult↗
PubMed2006

Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with type 1 diabetes mellitus.

To comply with regulatory restrictions, with regard to graft of human islets immunoprotected within artificial microcapsules, into patients with type 1 diabetes mellitus (T1DM) with no recipient immunosuppression, we have prepared standard protocols on: (1) sodium alginate purification (clinical grade) for microcapsule fabrication; (2) preparation of biocompatible and permselective microcapsules containing human islets; and (3) minimally invasive techniques for grafting of the encapsulated human islets into the recipients' peritoneal cavity. As to no. 1, starting from pharmaceutical grade, raw sodium alginate powder, we prepared a pyrogen- and endotoxin-free 1.6% alginate solution by means of dialysis, multiple filtrations, and dilution/osmolality adjustments. As to no. 2, we have selected human islet preparations associated with >80% purity/viability, which underwent careful functional quality control testing prior to encapsulation; namely, most capsules contained one islet. As for no. 3, we have devised a simple intraperitoneal injection method under abdominal echography guidance with only local anesthesia to deposit the encapsulated islets in saline within the peritoneal leaflets. These technical protocols were officially approved by the Italian Ministry of Health which has released permission to conduct a phase I, closed human trial in 10 patients using encapsulated human islet grafts into nonimmunosuppressed patients with T1DM.

Capsules↗
PubMed2006

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Animals↗
PubMed2004

Neonatal pig pancreatic duct-derived insulin-producing cells: preliminary in vitro studies.

Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantation trials. Because functional islet beta-cells could derive from precursors situated in the ductal system, and neonatal animals are better suitable than adults for recovering such elements, we have examined whether isolated neonatal pancreatic ducts (NPD) could form insulin-producing cells. NPD, retrieved from the pancreas by collagenase digestion, were cultured for 2 weeks. A compact tissue monolayer detached by trypsin was re-incubated to form upon culture. The primary tissue monolayer was plated, yielding secondary monolayers that were supplemented in culture with the following factors: insulin transferrin selenium, niacinamide, keratinocyte growth factor, and high glucose, which promoted formation of islet cell-like clusters during 30 days of culture. Upon reaching 50 to 100 microm in diameter, the cell clusters were subjected to morphologic examination (assessment of viability by staining with ethidium bromide+fluorescein diacetate [EB+FD]; staining for insulin with diphenylthiocarbazone [DTZ]); DNA assay; insulin radioimmunoassay both in the basal state and after in vitro static incubation with high glucose; immunolabeling with anti-insulin fluorescent antibodies. Of the cell clusters, 80% were composed of viable cells that faintly showed DTZ staining. Basal insulin was 16.7 microU/mL, but no insulin response was elicited by stimulation with high glucose. Acid-ethanol extraction showed high insulin levels in the clusters. Finally, immunofluorescence for insulin was positive, indicating the presence of beta-cell-like committed elements. In conclusion, NPD may differentiate into insulin-producing cells, which are at a very early stage when the glucose-sensing apparatus is still immature.

Animals↗
PubMed2004

Transdifferentiation molecular pathways of neonatal pig pancreatic duct cells into endocrine cell phenotypes.

Restrictions in availability of cadaveric human donor pancreata have intensified the search for alternate sources of pancreatic endocrine tissue. We have undertaken to assess whether nonendocrine pancreatic tissue, with special regard to ducts, including epithelial cells, and retrieved from neonatal pig pancreata that are used for islet isolation, may under special in vitro culture conditions generate endocrine cell phenotypes. Special care was taken to identify the time-related appearance of molecular and biochemical markers associated with beta-cell specificity, in terms of glucose-sensing apparatus and insulin secretion. For this purpose, established ductal origin monolayer cell cultures were incubated with a battery of mono- or polyvalent growth factors. Morphological, immunocytochemical, molecular, and functional assays indicated that under special culture conditions ductal origin cells acquired an endocrine identity, based upon expression of key gene transcripts that govern the stimulus-coupled insulin secretory activity. Among factors eliciting transdifferentiation of ductal epithelial into endocrine cells, Sertoli cell (SC)-conditioned medium seemed to be the most powerful inducer of this process. In fact, the resulting cultures not only expressed beta-cell-oriented metabolic markers but also were associated with insulin and C-peptide output at equimolar ratios. This finding indicates that SC coincubation, more than other conditions, caused originally ductal cell cultures to gradually differentiate and mature into beta-cell-like elements. In vivo studies with this early cell differentiation product will test whether our approach may be suitable for correction of hyperglycemia in diabetic animal models.

Animals↗
PubMed2004

Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus.

Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.

Alginates↗
PubMed2003

Sertoli cell-induced adult rat islet beta-cell mitogenesis: causative pathways.

We have previously observed that in vitro co-incubation of rat pre-pubertal Sertoli cells (SC), or their dialyzed/concentrated secretory products with homologous islets, resulted in significant stimulation of the islet beta-cell mitotic index. Aim of the present work was to assess both the specificity and nature of the mechanisms underlying this phenomenon. For this purpose, first we tested astrocytes (AA), separated and purified from the rat brain cortex, where they are known to release a number of growth factors and neurotrophic cytokines, for co-incubation with the islets. However, under the same experimental conditions used for SC, AA did not induce any changes in the beta-cell life cycle, thereby confirming specificity of SC, with respect to induction of beta-cell mitogenicity. For the second purpose, we examined the products of PD-1, a gene located in the cytoplasm of SC, where it promotes spermatogenesis. By blocking the protein encoded by PD-1, under appropriate culture conditions, we observed that the SC-induced increase in beta-cell mitotic activity lost its statistical significance, which suggested a role of PD-1 with respect to SC-related mitogenic properties on beta-cells. These findings corroborate the idea that SC, by either direct contact, or by means of their secretory products, clearly affect the islet beta-cell mitotic rate. Preliminarily, PD-1 gene, located in the cytoplasm of SC, might be one of the factors involved with the induction of beta-cell mitotic activity. In conclusion, SC-induced beta-cell mitotic activity is specific, seemingly mediated by humoral factors whose acting mechanisms have started being unfolded.

Animals↗
PubMed2001

Improved function of rat islets upon co-microencapsulation with Sertoli's cells in alginate/poly-L-ornithine.

The purpose of this study was to assess whether Sertoli's cells would improve functional performance of homologous pancreatic islets within microcapsules. Purified rat Sertoli's cells were co-enveloped with islets in microcapsules that had been fabricated with alginic acid and poly-L-ornithine. Confocal laser microscopy was used to determine any mitogenic effects of Sertoli's cells on islets beta-cells. Insulin secretion from islets, with or without Sertoli's cells, was examined, and grafts of Sertoli's cells with islets in microcapsules into diabetic mice were carried out. Co-incubation of Sertoli's cells with islets resulted in a significant increase in the islet beta-cell mitotic rate, which was coupled with significantly higher insulin release under glucose stimulation, as compared to controls. Grafts of co-microencapsulated Sertoli's cells with islets resulted in prolongation of the achieved normoglycemia in the animals receiving Sertoli's cells with islets as compared to controls that received islets only. Sertoli's cells do promote mitogenic activities upon in vitro co-incubation with islets, whose in vitro functional and in vivo post-transplant consequences were evident. Sertoli's cells could, therefore, be co-microencapsulated with islets for transplantation in diabetic recipients.

Alginates↗
PubMed2001

Soluble E-selectin in essential hypertension: a correlate of vascular structural changes.

BACKGROUND: Increased expression of the endothelial leukocyte adhesion molecule E-selectin is implicated in vascular disease and may accompany the development of hypertension. We evaluated plasma soluble (s) E-selectin to assess its relationship with endothelium-dependent and endothelium-independent vasodilation in patients with hypertension. METHODS: Thirty-one previously untreated and uncomplicated essential hypertensive patients were compared with 16 normotensive controls for changes in forearm blood flow (by strain-gauge plethysmography) in response to brachial artery infusion of the endothelium-dependent vasodilator acetylcholine, and of the endothelium-independent vasodilator sodium nitroprusside. As an index of structural changes, minimal forearm vascular resistances were calculated as the ratio between maximal vasodilation after 13 min of ischemia and mean blood pressure. RESULTS: Responses to acetylcholine were significantly lower and minimal forearm vascular resistances higher in hypertensives versus controls, whereas responses to nitroprusside were comparable. Baseline sE-selectin concentrations were (mean +/- SEM) 37.4 +/- 1.8 ng/mL in hypertensives and 27.8 +/- 0.7 ng/mL in normotensives (P < .001). In essential hypertensive patients, a significant (P < .01) correlation with the response to nitroprusside (r = -0.47) was found, but not with the response to acetylcholine or minimal forearm vascular resistances. sE-selectin was also positively correlated with age and LDL cholesterol. At multivariate analysis, sE-selectin remained significantly correlated with nitroprusside responses and LDL cholesterol. CONCLUSIONS: In patients with essential hypertension, plasma levels of sE-selectin are higher than in normotensive controls and mostly related to structural vascular changes.

Acetylcholine↗
PubMed2001

Differential ability of human endothelial cells to internalize and express exogenous DNA.

Vascular endothelium gene expression regulates blood-vessel wall interactions, vascular permeability, smooth muscle cell growth and tone. The possibility to introduce exogenous DNA or RNA sequences in endothelial cells represents a novel therapeutic approach of vascular disease. The aim of the work was to investigate the ability of endothelial cells to internalize and express exogenous DNA sequences. Human umbilical vein endothelial cells (HUVEC) were transfected with either a 780 bp fluorescein-labeled DNA (FITC-DNA) or pEGFP-C1 plasmid encoding for a green fluorescent protein (GFP), using the cationic liposome DOTAP as transfection reagent. The transfected cell population was passed through a FACScan apparatus and percentage of fluorescent cells was determined using a FACScan analysis programme. The SW620 tumor-derived cell line was used as control. The percentage of FITC-DNA positive cells was 66.0% for HUVEC and 45.0% for SW620 cells. On the contrary, the percentage of GFP-positive cells was 13.8% and 43% for HUVEC and SW620, respectively. By increasing the amount of DNA as well as the protocol of administration the percentage of GFP-positive HUVEC was enhanced suggesting a rapid degradation of DNA in the HUVEC cytoplasm.

Cells, Cultured↗
PubMed2001

Endothelial cell adhesion on bioerodable polymers.

This paper presents the results of a preliminary screening of a new class of bioerodable polymers, partial esters of alternating copolymers of maleic anhydride and mono-methoxyoligoethyleneglycol vinyl ethers (PAM) for use in engineered vascular tissue. Different initial concentrations of PAM and human serum albumin (HSA) were spin-coated onto glass substrates and the surface properties of the resulting films and their relationship to endothelial cell adhesion was examined. An optimum PAM/HSA blend for use as the cell contact surface of a bioerodable scaffold was identified.

Journal Article↗
PubMed2001

Cellular support systems for alginate microcapsules containing islets, as composite bioartificial pancreas.

To improve the functional performance of microencapsulated islets, we examined the effects of putative cellular support systems, consisting of rat purified Sertoli cells (SC) and astrocytes (AA), on coenveloped allogeneic islets. Coincubation of islets with SC but not AA, resulted in significant stimulation of beta cell mitogenesis, coupled with a significant increase in in vitro glucose-stimulated insulin release. Preliminarily, the xenotransplantation of coencapsulated rat islets and homologous SC significantly prolonged remission of hyperglycemia in diabetic mice.

Alginates↗
PubMed2000

Estrogens and glucocorticoids inhibit endothelial vascular cell adhesion molecule-1 expression by different transcriptional mechanisms.

The antiatherogenic effect of estrogen is mediated, in part, by inhibitory effects on endothelial vascular cell adhesion molecule-1 (VCAM-1) expression. To determine the mechanism by which estrogen regulates VCAM-1 expression, we compared the effect of 17beta-estradiol (E(2)) and of the glucocorticoid dexamethasone (Dex) on lipopolysaccharide (LPS)-induced VCAM-1 expression in human endothelial cells. E(2) decreased LPS-induced VCAM-1 mRNA and protein expression to a greater extent than Dex. Dex, but not E(2), stabilized VCAM-1 mRNA. This correlated with inhibition of monocytoid U937 cell adhesion to endothelial cells. Transfection of endothelial cells with a functional VCAM-1 promoter construct showed that E(2) inhibited LPS-induced VCAM-1 gene transcription more potently than did Dex. However, using a truncated construct containing only the nuclear factor-kappaB (NF-kappaB)-responsive elements but lacking the consensus sequences for activator protein-1 (AP-1) and GATA, E(2) and Dex had similar inhibitory effects. Consistently, gel-shift assays showed that E(2) and Dex comparably inhibit LPS-induced activation of NF-kappaB, whereas E(2) inhibited LPS-induced activation of AP-1 and GATA to a greater extent than Dex. E(2) inhibition of NF-kappaB after LPS treatment was associated with decreased inhibitor kappaB (IkappaB) kinase activity and with a stabilization of the NF-kappaB inhibitor IkappaBalpha. These results indicate that E(2) decreases VCAM-1 gene expression through the inhibition of NF-kappaB, AP-1, and GATA and suggest novel mechanisms for the antiatherogenic effect of estrogen on the vascular wall.

Animals↗
PubMed2000

Inhibition of endothelial cell activation by nitric oxide donors.

Because nitric oxide (NO) inhibits the expression of endothelial leukocyte adhesion molecules, NO-generating compounds have major therapeutic potential for use outside their classical indications. We report on the in vitro potential antiatherogenicity of two novel cysteine-containing NO donors, SP/W 3672, a fast spontaneous NO releaser, and its prodrug SP/W 5186, which liberates NO after bioactivation. The ability of these two compounds to inhibit monocyte adhesion and surface expression of endothelial adhesion molecules was evaluated and compared with that of other NO donors. SP/W 5186 and SP/W 3672 inhibited the adhesion of U937 monocytes to cultured human endothelial cells more potently than S-nitrosoglutathione (GSNO) or spermine NONOate, whereas nitroglycerin and isosorbide dinitrate were ineffective at comparable concentrations. A similar rank order of potency was found for the inhibition of expression of the adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin as well as for major histocompatibility complex class II antigen expression. Estimated IC(50) values for vascular cell adhesion molecule-1 were >400 microM for SP/W 4744 (control for SP/W 3672 lacking the cysteine moiety), 200 microM for GSNO and spermine NONOate, 80 microM for SP/W 3672, and 50 microM for SP/W 5186. Moreover, SP/W 5186 inhibited VCAM-1 mRNA levels more potently than GSNO. This effect was likely to be transcriptional because mRNA degradation was not affected. In conclusion, SP/W 3672 and SP/W 5186 are novel potent inhibitors of endothelial activation, and this effect appears to relate to their ability to liberate NO for prolonged periods of time, either spontaneously or after conversion to active hydrolytic products.

Cell Adhesion↗
PubMed2000

Sertoli cell-induced reversal of adult rat pancreatic islet beta-cells into fetal-like status: potential implications for islet transplantation in type I diabetes mellitus.

BACKGROUND: Clinical success of pancreatic islet allograft (TX) for the therapy of diabetes mellitus is hampered by several pitfalls, primarily including the restricted availability of donor tissue and the immune- and/or non-immune-related TX's early loss, with the latter not necessarily being prevented by the host's general immunosuppression. Finally, adult islet beta-cells normally exhibit minimal proliferation capacity, which would not permit restoration of an eventually declining TX mass. METHODS: To address the limited beta-cell growth capacity, we have examined whether in vitro co-culturing adult rat islets (I) with prepubertal homologous Sertoli cells (SC) would stimulate I beta-cell expansion. SC-derived effects on the islets were studied in vitro, both morphologically (confocal laser microscopy) and functionally (glucose-stimulated insulin release). We have also preliminarily examined the in vivo impact of microencapsulated SC + I co-cultures on TX in diabetic mice. RESULTS: In vitro, we observed that SCs promoted significant beta-cell replication, as I beta-cell mitotic activity increased from 1% to greater than 8%, which coincided with the adult elements reversing into fetal-like status. This finding was coupled with significantly greater insulin release either in basal or in response to glucose, as compared with controls. CONCLUSIONS: Addition of SC to islets promotes reversal of the adult beta-cell elements into fetal-like conditions, thereby providing a new, potentially powerful tool that could significantly enhance the functional performance of islet TX in diabetic recipients.

Animals↗
PubMed2000

Effects of anti-oxidizing vitamins on in vitro cultured porcine neonatal pancreatic islet cells.

Oxidative stress may cause severe cellular damage to both allo- and xeno-transplanted islets, additional to islet graft-directed immunity, in diabetic patients. We thus aimed to examine the effects of antioxidants on in vitro culture-maintained, neonatal porcine cell clusters (NPCCs). NPCCs were treated with antioxidants (vitamins D3 and E) by a certain time of their maturation and differentiation process. Insulin recovery showed that both vitamins D3 and E, unlike untreated controls, resulted in preservation of the islet function for significantly long periods of time. Such effects were also confirmed during NPCCs in vitro static incubation with high glucose. Furthermore, morphologic examination of NPCCs demonstrated that at 16 days of cell culture beta-cell clusters were significantly larger and more intact when exposed to the vitamins as compared to controls. According to these preliminary results, because the employed vitamins, known to retain anti-oxidizing properties, seemed to clearly improve NPCCs morphology and function, they may represent a potentially useful tool for islet culture maintenance in the pre-transplant time period.

Animals↗
PubMed1999

Transplantation of pancreatic islets contained in minimal volume microcapsules in diabetic high mammalians.

To minimize technical problems relating to excessive size (600-800 mu in diameter) of standard alginate microcapsules (CSM) for pancreatic islet graft immunoisolation, we have developed two novel minimal volume, chemically identical, capsule prototypes (MVC): 1) coherent microcapsules (CM), and 2) medium-size microcapsules (300-400 mu, MSM). CM, which envelop each individual islet within a thin alginate hydrogel cast, are prepared by emulsification, whereas MSM are made by atomizing the islet-alginate suspension through a special microdroplet generator. Upon graft into diabetic rodents, CM have shown to immunoprotect both allo- and xenogeneic nondiscordant islets, and restored normoglycemia. In higher mammals, at subtherapeutic doses, CM fully immunoprotected islet allografts (pig-->pig), but only temporarily xenografts (dog-->pig). We then used MSM to immunoisolate canine islet allografts in the peritoneal cavity of dogs with spontaneous insulin-dependent diabetes. Of three grafted dogs, two showed full remission of hyperglycemia with insulin withdrawal. MSM could represent an intermediate solution between CSM and CM for peritoneal immunoisolated islet transplants.

Animals↗