Culture Medium Modulates Proinflammatory Conditions of Human Pancreatic Islets Before Transplantation

A portion of transplanted islets is lost during engraftment as a result of stressful events, involving hypoxia and production of proinflammatory molecules by islets. Two of these molecules (monocyte chemoattractant protein-1, CCL2/MCP-1 and tissue factor, TF) are directly correlated with reduced graft function. We evaluated which factors reduce islet proinflammatory conditions. In particular the effects of different culture media supplemented with proteins or antioxidant agents on CCL2/MCP-1 and TF human islet release were evaluated. We observed that human islets after culture in final wash culture medium (FW) significantly decreased CCL2/MCP-1 release and TF production compared with CMRL and M199. These effects were independent from the type of protein added to the media (human serum, human albumin, fetal calf serum). Glutathione in FW further decreased CCL2/MCP-1 in a dose-dependent manner. Culture conditions can modulate the proinflammatory state of islets, and could be used in clinical islet transplantation to reduce inflammation during engraftment.     

Secondary failure of oral hypoglycaemic agents in lean patients with type 2 diabetes mellitus: insulin sensitivity, insulin response to different stimuli, and the role of cyclic-AMP.

The aim of this study was to evaluate the insulin (IRI) response to different stimuli and insulin sensitivity in Type 2 diabetic patients responsive to oral hypoglycaemic agents (OHA) and in Type 2 diabetic patients with secondary failure of OHA (SF), all patients being of normal body weight (relative body weight less than 120%), and the possible role of cyclic AMP in the reduced IRI release. SF patients, without islet cell antibodies (ICA), with hyperglycaemia lasting more than 3 months, underwent tests with i.v. tolbutamide (n = 21), i.v. glucose (n = 14), i.v. glucagon (n = 19), i.v. arginine infusion (n = 18); the arginine infusion was repeated in 12 patients during administration of aminophylline, an inhibitor of phosphodiesterase. The same tests were performed in groups of 8 to 15 OHA patients and in groups of 6 to 17 healthy subjects. During all the tests, blood glucose levels were higher in SF patients, than in OHA patients and in healthy subjects. Both SF patients and OHA patients had no IRI response to glucose; SF patients, in contrast to OHA patients, had a reduced IRI response to tolbutamide and to glucagon. The IRI response to arginine was not different in OHA, in SF patients and in healthy controls, but was significantly enhanced by aminophylline only in healthy controls. Insulin infusions (1.66 mU/Kg/min for 90 min) were performed in OHA patients and in SF patients at blood glucose levels of 150 and of 250 mg/dl: during the last 60 min, the amount of glucose metabolized (M), and the insulin sensitivity (M/I) index were greater in OHA than in SF patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pancreas and kidney transplantation: the San Raffaele Hospital (Milan, Italy) experience

Results of 33 simultaneous pancreas and kidney transplantations performed at the San Raffaele Hospital, Milan, Italy are presented. In 26 cases segmental neoprene duct-injected grafts were transplanted and in seven cases, duodenopancreatic bladder-drained grafts. Five-year patient, kidney and pancreas survival were respectively, 89%,72% and 58%. Five-year survival in patients with technically successful pancreas transplants was 73%. Thrombosis occured in 20% of cases. Mortality was 6% and overall morbidity 76%. Surgical complications were present in 51% of cases.     

Eosinophil cationic protein in tears of normal subjects and patients affected by vernal keratoconjunctivitis

The objectives of this study were to determine: 1) levels of tear eosinophil cationic protein (ECP) in patients with vernal keratoconjunctivitis (VKC); 2) the effect of pharmacologic therapy on ECP release; and 3) the correlation of this mediator with the severity of the disease. Tears were collected from 10 controls and 20 VKC patients before and after therapy for cytologic analysis and ECP measurement by radioimmunoassay. Ocular signs and symptoms were evaluated before tear collection. Mean ECP levels in controls were 7.5 ± 0.4 μg/l, and in VKC patients, 988.3 ± 128 μg/l before therapy ( P <0.001) and 566.3 ± 121 μg/l after therapy ( P <0.005). In dexamethasone (Dex) 0.1%, or cyclosporin A (CsA) 2%, patients (five per group), tear ECP decreased significantly after 7–14 days of treatment. Disodium cromoglycate (DSCG) 4% (five patients) for 14 days did not significantly affect ECP levels. ECP levels were significantly correlated with allergic signs ( P <0.001), symptoms ( P <0.001), and the number of eosinophils in tears (P<0.005). The results of this study suggest that tear ECP levels accurately reflect the clinical status of VKC patients. The measurement of ECP may prove useful not only in the diagnosis and monitoring of allergic disease, but also as an objective parameter for the evaluation of new antiallergic therapies.     

Thy 1.2 antigen inducing capacity of a calf thymus acid hydrolysate and its fractions

Splenic null cells of normal mice have been shown to undergo phenotypic induction of Thy 1.2 antigen by various thymic extracts. This experimental model has been employed in characterizing the Thy 1.2 inducing ability of an acid lysate from calf thymus and its various fractions. Incubation of splenocytes from C3H/He mice with unfractionated Thymomodulin induces a dose-response percent increase in the Thy 1.2 antigen bearing cells (9.28 +/- 2.6%, p less than 0.05). Comparable increase appears in fractions 5B (9.46 +/- 0.55%, p less than 0.001) and 5C (8.09 +/- 3%, p less than 0.005), obtained by ultrafiltration and containing peptides of m.w. 600-10,000 d and less than 600 d, respectively. The activity is confined to the acid fraction (6.61 +/- 0.54%, p less than 0.001) isolated by isoelectrofocusing. Control lysate from pig duodenal mucosa was devoid of Thy 1.2 inducing activity. The present findings indicate that Thymomodulin possesses Thy 1.2 inducing capacity on murine null cells and that such activity is maintained and enhanced after various fractionation procedures.     

Soluble Antiapoptotic Molecules and Immune Activation in Chronic Heart Failure and Unstable Angina Pectoris

Programmed myocyte cell death and activation of the immune system have been shown to occur in patients with congestive heart failure. Besides, unstable angina episodes are likely to be associated with immune activation. Our aim was to evaluate the role of changes in circulating levels of soluble Fas (sFas), suggestive of an enhanced inhibitory response to ongoing apoptosis, and soluble IL2 receptor (sIL2-R), indicative of T-lymphocyte activation, in chronic heart failure and unstable angina pectoris. Thirty patients affected by chronic heart failure (20 idiopathic and 10 ischemic cardiomyopathy) and 13 patients with unstable angina were evaluated. Twenty healthy individuals matched for age and gender were used as controls. A complete biochemical determination of indexes of myocardial damage including cardiac troponin I (cTnI) and creatine kinase (MB/CK) was performed. The results demonstrated that mean levels of sFas and sIL2-R were significantly increased in patients affected by chronic heart failure and unstable angina and were not associated with changes in renal function or with serum levels of cTnI. Highest values of sFas were found in NYHA class IV patients (IV NYHA class = 7.39 ± 0.52 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and more elevated in idiopathic than in ischemic cardiomyopathy (3.64 ± 0.40 vs. 1.82 ± 0.37 ng/ml; P < 0.01). Moreover, in chronic heart failure patients sFas and ejection fraction were negatively correlated (P = 0.01), whereas sFas and sIL2-R were positively correlated (P < 0.01). In unstable angina patients too, sFas and sIL2-R appeared to be correlated (P = 0.03); whereas sFas (angina group = 3.18 ± 0.39 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and sIL2-R (angina group = 0.46 ± 0.11 vs. controls = 0.00 UI/ml; P < 0.01) were higher in angina group than in controls. In most of the cases, the increase of sFas was associated with comparable changes in sIL2-R serum levels, indicating that the activation of Fas system is strictly associated with autoimmune–inflammatory reactions. This phenomenon, both in chronic heart failure and in unstable angina, occurs in the absence of biochemical evidences of myocardial damage and seems to parallel the activation of T cell. Soluble Fas could have a role in sustaining inflammatory response and in prolonging the detrimental effects correlated with it in chronic heart failure and angina pectoris.     

Persistence of anomalies in the growth hormone-releasing hormone-stimulated growth hormone response in diabetic-uremic patients after combined kidney-pancreas transplantation

Increased circulating growth hormone (GH) levels and aberrant response to different stimuli characterize both type 1 diabetes mellitus and chronic uremia and are associated with severe retinal, kidney and heart complications. Combined kidney and pancreas transplantation is a therapy that restores the endogenous, closed-loop, insulin secretion in diabetes and cure uremia. To evaluate if combined transplantation can restore a normal secretion and response of GH to growth hormone releasing hormone (GH-RH), we studied four groups of subjects: (1) seven type 1 diabetic patients with end-stage renal failure who had received pancreas and kidney transplantation (KPTx); (2) six diabetic uremic subjects, candidates for combined transplantation (IDDUP); (3) nine patients with chronic uveitis on immunosuppressive therapy comparable to pancreas recipients, six of whom treated only with prednisone (UVEST), while three (4) were treated with both prednisone and cyclosporin (UVESTCY). All subjects underwent a GH-RH test (50 microg intravenously, i.v., at 13:00 h). Serum insulin levels were significantly higher in IDDUP compared to UVEST (P=0.05) both at baseline and post GH-RH stimulus, while were similar to KPTx (P=0.2) and UVESTCY (P=0.7). In contrast, plasma free fatty acids were similar in all groups. In IDDUP baseline plasma glycerol was higher than in KPTx (P=0.04) and UVEST (P=0.02) and similar to UVESTCY (P=0.36); glycerol concentration did not change after GH-RH (P=0.08). Before and after GH-RH, serum GH levels tended to be higher in IDDUP (P=0.5) and KPTx (P=0.2) compared to UVEST and UVESTCY. Our results indicate that: 1) kidney-pancreas transplantation does not normalize the GH response to GH-RH; 2) GH abnormalities are not due either to the chronic immunosuppressive therapy or to the insulin effect on GH release; 3) GH abnormalities are probably secondary to functional and/or organic complications of the hypothalamus and/or pituitary as a sequela of diabetes mellitus.     

Preliminary evaluation of particle systems visualization on the skin surface by scanning electron microscopy and transparency profilometry

Background: There is a rising debate concerning the possible side effects arising from the use of particles at nanosize since the production of nanomaterials is increasing worldwide. Nanoparticles are able to enter the body through the skin, lungs or intestinal tract, depositing in several organs, and the risk associated with exposure to them, the routes of entry and the molecular mechanisms of any cytotoxicity need to be well understood. The aim of this work was to evaluate the suitability of skin replica as a method to study the colloidal systems visualization and distribution on skin surface. Methods: Solid lipid nanoparticles (SLN) were used as carrier systems. Skin replicas on healthy volunteers, before and after SLN application, were prepared and visualized using profilometry and scanning electron microscopy (SEM). Results: The results obtained in our study show that skin replica represents a suitable method to study the colloidal systems and their interaction with the skin surface. Conclusion: Profilometry enabled us to observe the systems distribution on a cutaneous texture. In addition, SEM, thanks to its high magnifications and field depth, allowed us to evaluate particles' distribution on the skin texture and the interaction between particles of different compositions and replica silicone.