The miR-27a-calreticulin axis affects drug-induced immunogenic cell death in human colorectal cancer cells.

Immunogenic cell death (ICD) evoked by chemotherapeutic agents implies emission of selected damage-associated molecular patterns (DAMP) such as cell surface exposure of calreticulin, secretion of ATP and HMGB1. We sought to verify whether miR-27a is implicated in ICD, having demonstrated that it directly targets calreticulin. To this goal, we exposed colorectal cancer cell lines, genetically modified to express high or low miR-27a levels, to two bona fide ICD inducers (mitoxantrone and oxaliplatin). Low miR-27a-expressing cells displayed more ecto-calreticulin on the cell surface and increased ATP and HMGB1 secretion than high miR-27a-expressing ones in time-course experiments upon drug exposure. A calreticulin target protector counteracted the miR-27a effects while specific siRNAs mimicked them, confirming the results reported. In addition, miR-27a negatively influenced the PERK-mediated route and the late PI3K-dependent secretory step of the unfolded protein response to endoplasmic reticulum stress, suggesting that miR-27a modulates the entire ICD program. Interestingly, upon chemotherapeutic exposure, low miR-27a levels associated with an earlier and stronger induction of apoptosis and with morphological and molecular features of autophagy. Remarkably, in ex vivo setting, under the same chemotherapeutic induction, the conditioned media from high miR-27a-expressing cells impeded dendritic cell maturation while increased the secretion of specific cytokines (interleukin (IL)-4, IL-6, IL-8) and negatively influenced CD4(+) T-cell interferon γ production and proliferation, all markers of a tumor immunoevasion strategy. In conclusion, we provide the first evidence that miR-27a impairs the cell response to drug-induced ICD through the regulatory axis with calreticulin.

Proteomic screening identifies calreticulin as a miR-27a direct target repressing MHC class I cell surface exposure in colorectal cancer.

Impairment of the immune response and aberrant expression of microRNAs are emerging hallmarks of tumour initiation/progression, in addition to driver gene mutations and epigenetic modifications. We performed a preliminary survey of independent adenoma and colorectal cancer (CRC) miRnoma data sets and, among the most dysregulated miRNAs, we selected miR-27a and disclosed that it is already upregulated in adenoma and further increases during the evolution to adenocarcinoma. To identify novel genes and pathways regulated by this miRNA, we employed a differential 2DE-DIGE proteome analysis. We showed that miR-27a modulates a group of proteins involved in MHC class I cell surface exposure and, mechanistically, demonstrated that calreticulin is a miR-27a direct target responsible for most downstream effects in epistasis experiments. In vitro miR-27a affected cell proliferation and angiogenesis; mouse xenografts of human CRC cell lines expressing different miR-27a levels confirmed the protein variations and recapitulated the cell growth and apoptosis effects. In vivo miR-27a inversely correlated with MHC class I molecules and calreticulin expression, CD8(+) T cells infiltration and cytotoxic activity (LAMP-1 exposure and perforin release). Tumours with high miR-27a, low calreticulin and CD8(+) T cells' infiltration were associated with distant metastasis and poor prognosis. Our data demonstrate that miR-27a acts as an oncomiRNA, represses MHC class I expression through calreticulin downregulation and affects tumour progression. These results may pave the way for better diagnosis, patient stratification and novel therapeutic approaches.

Metformin improves hemodynamic and rheological responses to L-arginine in NIDDM patients.

OBJECTIVE: The endothelium plays a pivotal role in the regulation of vascular tone by releasing nitric oxide (NO). Increased availability of L-arginine, the natural precursor of NO, induces vasodilatation and inhibits platelet activity. We studied the effect of metformin on hemodynamic and rheological responses to L-arginine in patients with NIDDM. RESEARCH DESIGN AND METHODS: Ten newly diagnosed NIDDM patients with mild fasting hyperglycemia (7.5 +/- 0.3 mmol/l) and without evidence of both micro- and macrovascular complications were investigated. They received an intravenous infusion of L-arginine (1 g/min for 30 min) with evaluation of plasma glucose and insulin, systolic (sBP) and diastolic (dBP) blood pressure, heart rate and plasma catecholamines, platelet aggregation, and blood viscosity and filterability. The L-arginine test was repeated after an 8-week treatment with metformin (850 mg b.i.d.). RESULTS: Metformin treatment significantly reduced basal fasting plasma glucose, HbA1c, and platelet aggregation to ADP (P < 0.05); the other parameters did not change. During pretreatment test, L-arginine infusion decreased sBP (from 137 +/- 4.1 to 129 +/- 4.5 mmHg, P < 0.01) and dBP (from 79 +/- 1.9 to 75 +/- 1.2 mmHg, P < 0.01) without affecting heart rate or plasma catecholamines. Both platelet aggregation and blood viscosity showed significant decrements after L-arginine, while blood filterability did not change. After metformin treatment, the decrease in blood pressure after L-arginine infusion was significantly enhanced, with a maximal decrease of sBP of 12 +/- 3.4 mmHg (8 +/- 2.5 mmHg pretreatment, P < 0.05) and dBP of 9.5 +/- 2.4 mmHg (4.5 +/- 1.9 mmHg pretreatment, P < 0.01). Heart rate, plasma norepinephrine levels, and blood filterability also rose significantly (P < 0.05-0.01). The decrease in both platelet aggregation and blood viscosity after L-arginine was significantly amplified after metformin. CONCLUSIONS: We conclude that L-arginine infusion in newly diagnosed NIDDM patients without vascular complications produces relevant hemodynamic and theological changes, which are amplified by an 8-week treatment with metformin. Whether these vascular effects of metformin will improve the poor cardiovascular outlook of the diabetic patient is still unknown.

The role of pre-expanded free flaps in revision of burn scarring.

The authors present two patients affected by scars resulting from burning of over 60 per cent of the total body area, in which the pre-expansion of a free flap has been used to increase the tissue surface useful for transfer from the only area of residual healthy skin (left forearm, left parascapular region). In both cases it was possible to transfer abundant healthy tissue into the desired areas, obtaining a rapid release of the region, which made possible an early physical rehabilitation of the patient starting after the second postoperative week. One of the main problems encountered, when facing surgical rehabilitation for the seriously burned patient, is the poor availability of skin donor areas suitable for reconstructive flaps. The pre-expansion of free flaps provides an advantage in that it allows the few integral residual areas to be used, improving vascularization and therefore increasing the available surface. Furthermore, as pre-expansion reduces tension on the margins, it allows for the easier closing of the donor area, with a minor risk of complications and a better scar outcome.

Tissue expansion in the treatment of pressure ulcers.

The authors report their experience using skin expanders in 11 patients with severe bed sores. The expanders, with different volumes, from 250 to 1000 cc, were generally overfilled using the cutaneous tonometer. In fact, with the information revealed by this apparatus on the skin in expansion, the authors were able to reduce the filling intervals without risking ulceration. In their experience, the results obtained were satisfactory: All patients treated achieved surgical recovery. The authors see a wide future for skin-expander use in pressure-ulcer treatment. They have a working hypothesis about using expanders to progressively advance sensitive skin in areas subject to ulceration. This hypothesis is based on the possibility of reexpanding the same flap several times, as has been seen in the treatment of other types of pathology.

Multiple CT imaging in pressure sores.

In patients affected by pressure sores, bones can be reached easily by the infection. This suggested a new way for a more precise diagnostic evaluation, looking at the most recent technological knowledge, which can offer a better evaluation of single lesions for a better planning of surgical operations. The possibility of performing multiple imaging under a CT guide, as well as a simple technique for contrast permanence within the sore, led the authors to demonstrate the validity of modern CT scanning as a main diagnostic method.

The use of a modified tonometer in burn scar therapy.

We report on the use of a modified Schiotz tonometer to evaluate the effects of therapies on burn scars. It is now possible to quantify the course of cicatrization with tonometry. Because tonometry affords a precise evaluation of burn scar diagnosis and prognosis, it is possible to avoid arbitrary clinical evaluation of the burn scarring process.

[Importance of cleansing in the topical treatment of skin lesions. A parallel study carried out in 20 patients and 30 rats]. / Importanza della detersione nel trattamento topico delle lesioni cutanee. Studio parallelo condotto su 20 pazienti e su 30 ratti.

There are frequent reports of the chronic nature of skin ulcers of varying etiology (burns, bedsores, wound diastasis, etc.); these heal with considerable difficulty probably due to problems related to the excessive use of disinfectants which, in spite of having a good bacterial action, interfere with re-epithelialisation processes. The aim of this study was to assess the value of simple wound cleansing instead of disinfection in those cases where there are no manifest signs of bacterial contamination. A widely sold solution, Katoderm (Devergè, Turin), was used in this study. The study was performed in two stages; the first in laboratory animals and the second in patients with small persistent lesions. The results of the study carried out in rats showed a significant prolongation of healing time in the group treated with polyvinylpyrrolidone-iodine compared to those in which lesions were only cleansed with Katoderm. The results of the clinical study also highlighted the faster speed of healing in patients treated with cleansing alone.

Reconstruction of ischial pressure ulcers by skin expansion.

Ten patients, eight of whom were paraplegic, have had ischial pressure ulcers treated by skin expansion. Skin expanders 680 ml in size were inserted subcutaneously on the posterior aspect of the thigh. They were filled during a short period without complications, the filling being monitored with a cutaneous tonometer. At a second operation the ulcers were repaired easily with the expanded flaps. After a few weeks the patients were able to undergo a programme of rehabilitation that included sitting with the expanded flaps. No ulcers recurred during a median follow-up of 12 months (range 8-34).

Ambulant vacuum-assisted closure of skin-graft dressing in the lower limbs using a portable mini-VAC device.

A skin graft may fail to adhere to the recipient site because of fluid collecting between the graft and the area being treated. We have devised a simple procedure, consisting of a vacuum-sealed dressing, to fix skin grafts on the lower limbs. A fully portable, battery-operated aspirator continuously draws secretions through a vacuum-sealed dressing, preventing accumulation of fluid underneath the graft. Patients are not confined to bed, thus reducing nursing time. The procedure was applied successfully in seven out of nine patients treated for ulcers of the lower limbs.

L-arginine for testing endothelium-dependent vascular functions in health and disease.

The objective of this study was to assess the role of L-arginine, the natural precursor of nitric oxide, for testing endothelial function in physiological and pathophysiological conditions. In an initial study of 20 healthy subjects, mean blood pressure decreases in response to increasing doses of L-arginine (1, 2, 3, and 5 g) were 1.1 +/- 1.3, 2.6 +/- 1.5, 7.6 +/- 1.3, and 7.7 +/- 2 mmHg, respectively, P < 0.01. The enantiomer D-arginine (3 g) did not produce any change in mean blood pressure and platelet aggregation (n = 10), whereas the infusion of the L-arginine analog NG-monomethyl-L-arginine (6 mg/min) reduced by 70% the vascular effects of L-arginine. In the whole population of 52 healthy subjects, there was an inverse correlation between age and blood pressure or platelet aggregation changes after L-arginine. Compared with matched controls (n = 20), the changes in mean blood pressure and platelet aggregation after L-arginine were significantly lower in non-insulin-dependent diabetic (n = 20) and hypercholesterolemic (n = 16), but not in hypertensive (n = 20), subjects. Changes in blood viscosity were significantly lower only in hypercholesterolemic subjects. Our findings suggest that an intravenous bolus of 3 g L-arginine may be a simple and useful tool to assess the endothelial control of blood pressure and platelet activity in health and disease.

Effect of insulin on blood rheology in non-diabetic subjects and in patients with Type 2 diabetes mellitus.

We evaluated the effect of insulin on platelet function, blood viscosity, and filterability in healthy subjects and in patients with Type 2 (non-insulin-dependent) diabetes mellitus. Fifteen diabetic patients were free from cardiovascular complications (group A), while the other 15 patients had both clinical and measured evidence of coronary or peripheral vascular disease (group B); 15 non-diabetic subjects served as controls. On blood samples taken without stasis, maximal platelet aggregation to 1.25 micromol l(-1) ADP, blood and plasma viscosity, and blood filterability were measured in basal conditions, and after incubation of blood, plasma or platelet-rich plasma with insulin at two physiological concentrations (120 and 480 pmol l(-1)). Compared with healthy subjects, the diabetic patients of group B had higher values of blood (p < 0.01) and plasma (p < 0.05) viscosity, and platelet aggregation response to ADP (p < 0.01), as well as lower values of blood filterability (p < 0.01). The diabetic patients of group A had values intermediate between normal subjects and the patients of group B. In non-diabetic subjects, insulin significantly decreased platelet aggregation and blood viscosity at low shear rates (22.5 s(-1)) (p < 0.01 for both), and had no significant effects on other parameters. In the diabetic patients of group A, insulin decreased blood viscosity at high (225 s(-1)) rates of shear (p < 0.01) and increased blood filterability (p < 0.01). The effects of insulin were not dose-related. In the diabetic patients of group B, none of the parameters evaluated was significantly influenced by insulin. Type 2 diabetic patients present many abnormalities of the rheologic properties of blood. The beneficial effects of insulin on platelet aggregation and blood viscosity are not evident in Type 2 diabetic patients, especially those with vascular complications and this may be relevant to the development of those complications.

Metabolic and cardiovascular effects of carvedilol and atenolol in non-insulin-dependent diabetes mellitus and hypertension. A randomized, controlled trial.

BACKGROUND: Diabetic patients are considered less suitable than nondiabetic patients for beta-blocker therapy because of the risk for worsened glucose and lipid metabolism and more severe hypoglycemic attacks. OBJECTIVE: To compare the metabolic and cardiovascular effects of carvedilol with those of atenolol in diabetic patients with hypertension. DESIGN: Randomized, double-blind, 24-week trial. SETTING: University hospital clinic. PATIENTS: 45 patients with non-insulin-dependent diabetes mellitus and hypertension. INTERVENTION: After a 4- to 6-week run-in period during which placebo was given in a single-blind manner, patients were randomly assigned to carvedilol or atenolol. MEASUREMENTS: An oral glucose tolerance test; assessment of insulin sensitivity and hormonal responses to insulin hypoglycemia; and assessment of lipid levels, blood pressure, left ventricular mass, and lipid peroxidation. RESULTS: Changes in systolic and diastolic blood pressure and left ventricular mass index were similar with carvedilol and atenolol (P > 0.2). Fasting plasma glucose and insulin levels decreased with carvedilol and increased with atenolol. Responses to carvedilol were greater than those to atenolol, as follows: increase in total glucose disposal, 9.54 mumol/kg of body weight per minute (95% CI, 7 to 11.9 mumol/kg per minute); decrease in plasma glucose response to oral glucose, 61 mmol/L x 180 minutes (CI, -101 to -21 mmol/L x 180 minutes); decrease in insulin response to oral glucose, 6.2 nmol/L x 180 minutes (CI, -9.8 to -2.6 nmol/L x 180 minutes); decrease in triglyceride level, 0.56 mmol/L (CI, -0.75 to -0.37 mmol/L; P < 0.001); increase in high-density lipoprotein cholesterol level, 0.13 mmol/L (CI, 0.09 to 0.17 mmol/L; P < 0.001); and decrease in lipid peroxidation, 0.25 mumol/L (CI, -0.34 to -0.16 mumol/L). CONCLUSIONS: By improving glucose and lipid metabolism and reducing lipid peroxidation, carvedilol may offer advantages in patients with diabetes and hypertension.