Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

Lower miR-21/ROS/HNE levels associate with lower glycemia after habit-intervention: DIAPASON study 1-year later.

BACKGROUND: The prevalence of prediabetes is increasing in the global population and its metabolic derangements may expose to a higher risk to develop type 2 diabetes (T2D) and its cardiovascular burden. Lifestyle modifications might have considerable benefits on ameliorating metabolic status. Alternative biomarkers, such as circulating miR-21, has been recently discovered associated with dysglycemia. Here we evaluated, in a longitudinal cohort of dysglycemic population the relation between the circulating miR-21/ROS/HNE levels and the habit-intervention (HI) after 1 year of follow-up. METHODS: 1506 subjects from DIAPASON study were screened based on the Findrisc score. Of them, 531 subjects with Findrisc ≥ 9 were selected for dysglycemia (ADA criteria) and tested for circulating miR-21, ROS and HNE levels, as damaging-axis. 207 subjects with dysglycemia were re-evaluated after 1-year of habit intervention (HI). Repeated measures tests were used to evaluate changes from baseline to 1-year of follow-up. The associations between glycemic parameters and miR-21/ROS/HNE were implemented by linear regression and logistic regression models. RESULTS: After HI, we observed a significant reduction of miR-21/ROS/HNE axis in dysglycemic subjects, concomitantly with ameliorating of metabolic parameters, including insulin resistance, BMI, microalbuminuria, reactive hyperemia index and skin fluorescence. Significant positive interaction was observed between miR-21 axis with glycaemic parameters after HI. Lower miR-21 levels after HI, strongly associated with a reduction of glycemic damaging-axis, in particular, within-subjects with values of 2hPG < 200 mg/dL. CONCLUSIONS: Our findings demonstrated that HI influenced the epigenetic changes related to miR-21 axis, and sustain the concept of reversibility from dysglycemia. These data support the usefulness of novel biological approaches for monitoring glycemia as well as provide a screening tool for preventive programmes.

Skin substitutes based on allogenic fibroblasts or keratinocytes for chronic wounds not responding to conventional therapy: a retrospective observational study.

Chronic wounds are an expression of underlying complex pathologies and have a high incidence. Skin substitutes may represent an alternative approach to treat chronic ulcers. The aim of this retrospective observational study was to evaluate the wound reduction using skin substitutes based on allogenic fibroblasts or keratinocytes in 30 patients not responding to conventional therapy. Wound bed was prepared, then keratinocytes on Laserskin(®) to treat superficial wounds or fibroblasts on Hyalograft 3D(R) to treat deep leg ulcers were applied, and finally wounds were treated with a secondary dressing composed of nanocrystalline silver. Once a week constructs were removed and new bioengineered products were applied, as well as nanocrystalline silver medication. In none of the cases under examination did any complications arise relating to the treatment. We also achieved a reduction in wound dimension and exudates, and an increase in wound bed score. Postoperative assessment shows a degree of healing that is statistically higher in the group treated with keratinocytes as compared with the fibroblast group. This retrospective study improves our understanding and defines the clinical indications for the various uses of the two types of skin substitutes.

SARS-CoV-2 Immunization Orchestrates the Amplification of IFNγ-Producing T Cell and NK Cell Persistence.

A successful vaccination would represent the most efficient means to control the pandemic of Coronavirus Disease-19 (COVID-19) that led to millions of deaths worldwide. Novel mRNA-based vaccines confer protective immunity against SARS-CoV-2, but whether immunity is immediately effective and how long it will remain in recipients are uncertain. We sought to assess the effectiveness of a two-dose regimen since the boosts are often delayed concerning the recommended intervals. Methods: A longitudinal cohort of healthcare workers (HCW, N = 46; 30.4% men; 69.6% women; mean age 36.05 ± 2.2 years) with no SARS-CoV-2 infection as documented by negative polymerase chain reaction was immunophenotyped in PBMC once a week for 4 weeks from the prime immunization (Pfizer mRNA BNT162b2) and had received 2 doses, to study the kinetic response. Results: We identified three risk groups to develop SARS-CoV-2 infection IgG+-based (late responders, R-; early responders, R+; pauci responders, PR). In all receipts, amplification of B cells and NK cells, including IL4-producing B cells and IL4-producing CD8+ T cells, is early stimulated by the vaccine. After the boost, we observed a growing increase of NK cells but a resistance of T cells, IFNγ-producing CD4+T cells, and IFNγ-producing NK cells. Also, hematologic parameters decline until the boost. The positive association of IFNγ-producing NK with IFNγ-producing CD4+T cells by the multiple mixed-effect model, adjusted for confounders (p = 0.036) as well as the correlation matrix (r = 0.6, p < 0.01), suggests a relationship between these two subsets of lymphocytes. Conclusions: These findings introduce several concerns about policy delay in vaccination: based on immunological protection, B cells and the persistent increase of NK cells during 2 doses of the mRNA-based vaccine could provide further immune protection against the virus, while CD8+ T cells increased slightly only in the R+ and PR groups.

Towards ideal magnetofluorescent nanoparticles for bimodal detection of breast-cancer cells.

An increasing number of novel molecular markers based on nanomaterials for tumor diagnostics have been developed in recent years. Many efforts have focused on the achievement of site-targeted bioconjugated nanoparticles. In contrast, the mechanisms of toxicity, endocytosis, and degradation pathways are still poorly understood, despite their primary importance for clinical translation. In this study, three different model nanoscale magnetofluorescent particle systems (MFNs) are designed and fabricated. These nanoparticles are evaluated in terms of size, morphology, zeta potential, fluorescence efficiency, capability of enhancing T(2) relaxivity of water protons, and stability. Accordingly, two are developed and the mechanism of internalization, the intracellular fate, and the toxicity in MCF-7 adenocarcinoma cells are studied. Besides the well-documented size effect, the anionic charge seems to be a crucial factor for particle internalization, as MFN penetration through the cell membrane could be modulated by surface charge. Ultrastructural analysis of transmission electron micrographs combined with evidence from confocal microscopy reveals that MFNs are internalized by clathrin-mediated endocytosis and macropinocytosis. Moreover, MFNs are found in EEA1-positive endosomes and in lysosomes, indicating that they follow a physiological pathway of endocytosis. Magnetorelaxometric analysis demonstrates that MFNs enable the detection of 5 x 10(5) cells mL(-1) after treatment with particle dosages as low as 30 microg mL(-1). Hence, MFNs appear to be a valuable and safe bimodal contrast agent that can be developed for the noninvasive diagnosis of breast cancer.

Femtomolar detection of autoantibodies by magnetic relaxation nanosensors.

The development of nanosystems applied to rapid and sensitive measurement of biomarkers in fluid samples is a current major goal in diagnostic biomedicine. In this article, we report the accurate and reliable detection of anti-HSA (human serum albumin) antibodies by protein-functionalized magnetic nanospherical probes due to the reversible alteration of their microaggregation state induced by protein antibody-specific interaction, sensed as changes in the T(2) relaxation time of surrounding water molecules. Once the optimal parameters were adjusted, the method proved to be very sensitive, providing concentration- and time-dependent responses. Furthermore, we demonstrate that the developed immunoassay is able to quantitatively determine the biomarker concentration from T(2) linear correlation, thereby supplying a rapid, yet accurate, assay with sensitivity in the femtomolar range. The high susceptibility and stability of these magnetic nanoparticles, as well as their accessible synthetic preparation, make these nanosensors a promising new tool for versatile and effective medical diagnostics.

Ibuprofen-arginine generates nitric oxide and has enhanced anti-inflammatory effects.

Ibuprofen, a chiral non-steroidal anti-inflammatory drug chemically related to fenoprofen and naproxen, has moderate but definite anti-inflammatory, analgesic and antipyretic properties, with considerably less gastrointestinal adverse effect than other drugs in the same family. Currently available in the market are preparations in which bioavailability of ibuprofen is increased by salification with various salts. We have investigated the pharmacological properties of one such salt, ibuprofen-arginine, of biological interest because l-arginine acts as substrate of the nitric oxide (NO) synthesising enzymes. Using epithelial HeLa cells expressing the endothelial NO synthase we show that ibuprofen-arginine releases NO and that this NO protects against the cytotoxic apoptogenic effects of staurosporine. We also found that ibuprofen-arginine is endowed with enhanced anti-inflammatory effects with respect to ibuprofen, as shown by reduced hind paw oedema, neutrophil infiltration and chondrocyte apoptosis in collagen-induced mouse arthritis, a model of chronic inflammation. NO has pleiotropic beneficial effects that may contribute to limit inflammation and anti-inflammatory compounds able to release NO display higher efficacy than the parent drugs in defined clinical settings. Our results open the possibility that NO generation contributes to the enhanced anti-inflammatory effects of ibuprofen-arginine vs. ibuprofen, suggesting co-administration of anti-inflammatory drugs and arginine as an additional way to exploit the beneficial effects of NO.

An uncommon cause of corrosive esophageal injury.

We present an unusual case of corrosive esophageal injury following liquid glue ingestion. The endoscopic findings were tissue sloughing and blackened appearance of the esophagogastric junction, due to caustic esophageal injuries following ingestion of glue containing toluene.

Over-the-catheter precut to gain access to the biliary duct during ERCP rendezvous.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) rendezvous during laparoscopic cholecystectomy is an efficient and safe method to treat cholecystocholedocholithiasis. Advancing a guidewire through the cystic duct into the duodenum and withdrawing it in the accessory channel of duodenoscope may be, however, laborious. Moreover, rendezvous performed in the typical manner needs the use of several costly accessories. We herein describe a simpler and cheaper method to gain access to the biliary duct at rendezvous. METHODS: Twenty-four consecutive patients undergoing ERCP rendezvous during laparoscopic cholecystectomy were considered. A catheter was introduced in the cystic duct and advanced into the duodenum. Access to the bile duct was than achieved by means of a precut sphincterotomy performed over the catheter emerging from the papilla. RESULTS: Cannulation was successful in all but two patients, in whom ERCP was performed in the conventional manner. The only complication was a case of mild post-sphincterotomy bleeding. In comparison with the typical rendezvous technique our procedure allowed savings of about 250, since its performance only requires a catheter and a knife sphincterotome. CONCLUSIONS: Over-the-catheter precut during ERCP rendezvous is a feasible and safe method which avoids the need for the manipulation of several accessories and guidewires, and thus results in money and time savings.

[Successful management of severe unremitting chylothorax after thoraci trauma]. / Trattamento efficace di chilotorace grave e persistente dopo trauma toracico.

Post-traumatic chylothorax needs surgical approach when conservative treatment is not successful to reduce chyle leakage. Thoracic duct ligation requires thoracoscopic or thoracotomic access. The authors report on a surgical thoracotomic approach to a severe and unremitting thoracic duct lesion after IX and X ribs and vertebral fractures.

Gastric outlet obstruction due to inflammatory fibroid polyp.

INTRODUCTION: Inflammatory fibroid polyp (IFP) is a rare mesenchymal tumor of the gastrointestinal tract. The lesion is characterized by spindle-shaped stromal cells and an inflammatory infiltrate rich in eosinophils. CASE REPORT: The AA. present a case of the IFP of the stomach. A 85-year-old woman was admitted to the hospital for early vomiting and epigastric pain. The patient underwent a gastroduodenoscopy that revealed a polypoid lesion mimicking cancer of the antrum and subtotal obstruction of the gastric transit. CONCLUSIONS: The Endoscopic Ultrasound Sonography suggested the presence of a lesion of the submucosal layer such as lymphoma or other type of malignancy like gastrointestinal stromal tumor (G.I.S.T).

[Inflammatory colitis: classification and surgical treatment]. / Le coliti infiammatorie. Classificazione e trattamento chirurgico.

The development of clinical and histopathological criteria for the diagnosis of Crohn's disease (CD) and ulcerative colitis (UC), pushed the scientists to identify a new category: the indeterminate colitis (CI). This term is used when definitive diagnosis of UC or CD has not been made by colonoscopy, colonic biopsy or colectomy. The distinction between these forms has major implications including the choice of medical treatment, timing of surgery, prognosis and disease course. The role of surgery in inflammatory bowel disease differs between the three main forms: in CD is primarily to treat complications of the disease process; in UC surgery is curative for intestinal manifestations and nearly eliminates the risk of future malignancy; in IC is actually discussed: the current guidelines identify in surgery the best treatment for fulminate disease, intractability of disease symptoms or failure of medical therapy. Although there is a few number of studies in the literature, selective criteria for the diagnosis and successful treatment must be revisited. The term CI should be used as a pending tray diagnosis, representing diagnostic inadequacy and not as specific nosological entity. Evidence emerging from the studies of serological markers (ASCA and P-ANCA) suggests that a subgroup of patients initially diagnosed as IC maybe identified as a separate group, and so they need a specific treatment for their disease.

Outcome of non-variceal acute upper gastrointestinal bleeding in relation to the time of endoscopy and the experience of the endoscopist: a two-year survey.

AIM: To prospectively assess the impact of time of endoscopy and endoscopist's experience on the outcome of non-variceal acute upper gastrointestinal (GI) bleeding patients in a large teaching hospital. METHODS: All patients admitted for non-variceal acute upper GI bleeding for over a 2-year period were potentially eligible for this study. They were managed by a team of seven endoscopists on 24-h call whose experience was categorized into two levels (high and low) according to the number of endoscopic hemostatic procedures undertaken before the study. Endoscopic treatment was standardized according to Forrest classification of lesions as well as the subsequent medical therapy. Time of endoscopy was subdivided into two time periods: routine (8 a.m.-5 p.m.) and on-call (5 p.m.-8 a.m.). For each category of experience and time periods rebleeding rate, transfusion requirement, need for surgery, length of hospital stay and mortality we compared. Multivariate analysis was used to discriminate the impact of different variables on the outcomes that were considered. RESULTS: Study population consisted of 272 patients (mean age 67.3 years) with endoscopic stigmata of hemorrhage. The patients were equally distributed among the endoscopists, whereas only 19% of procedures were done out of working hours. Rockall score and Forrest classification at admission did not differ between time periods and degree of experience. Univariate analysis showed that higher endoscopist's experience was associated with significant reduction in rebleeding rate (14% vs 37%), transfusion requirements (1.8+/-0.6 vs 3.0+/-1.7 units) as well as surgery (4% vs 10%), but not associated with the length of hospital stay nor mortality. By contrast, outcomes did not significantly differ between the two time periods of endoscopy. On multivariate analysis, endoscopist's experience was independently associated with rebleeding rate and transfusion requirements. Odds ratios for low experienced endoscopist were 4.47 for rebleeding and 6.90 for need of transfusion after the endoscopy. CONCLUSION: Endoscopist's experience is an important independent prognostic factor for non-variceal acute upper GI bleeding. Urgent endoscopy should be undertaken preferentially by a skilled endoscopist as less expert staff tends to underestimate some risk lesions with a negative influence on hemostasis.

Plasma cholecystokinin levels after vertical banded gastroplasty: effects of an acidified meal.

BACKGROUND: Although cholecystokinin (CCK) is involved in the short-term regulation of satiety, it has not been investigated in obese patients subjected to bariatric restrictive operations. METHODS: 8 morbidly obese patients (BMI 49.1 +/- 6.9), 7F and 1M, were investigated before and after vertical banded gastroplasty (VBG). 6 healthy lean volunteers served as the control group. CCK was determined (RIA) after an overnight fast and after the administration of an acidified (pH 3) liquid meal. Blood samples were taken 45 min before the meal, 5 min after it and then every 30 min for 3 hours. RESULTS: There were no differences between groups in basal CCK levels. However, the peak of CCK after the meal was significantly higher (P <0.01) in obese patients after VBG (24.9 +/- 18 pmol/l) than before VBG (9.8 +/- 6.7 pmol/l) and when compared with the control group (8.0 +/- 6.3 pmol/l). The time needed to reach the peak was longer in healthy volunteers (105 +/- 24.9 min) than in obese patients before VBG (45 +/- 40 min) and after VBG (7.5+/- 12 min) (P<0.01). CONCLUSIONS: VBG increases the peak of CCK secretion and shortens the time to reach it. These changes could contribute to the satiety effects of gastric restrictive operations.

Plasma leptin levels after vertical banded gastroplasty for morbid obesity: effects of an acidified meal.

BACKGROUND: Leptin is considered one of the anorectic messengers to the central nervous system in lean subjects. Although it is secreted by the gastric mucosa, there are contradictory evidences of its involvement in mediating the acute satiety effect of the meal in obese patients. The effects of restrictive operations on meal-stimulated leptin secretion are unknown. METHODS: The effects of a standard acidified (pH 3) meal on leptin release were investigated in obese patients, before and after vertical banded gastroplasty (VBG). 8 morbidly obese patients (BMI 49.1+/-6.5) had serum leptin determination after an overnight fast. Samples were taken basally and every 30 minutes after the meal for 3 hours. The test was repeated after 20% BMI reduction. 5 lean volunteers (BMI 22.5+/-1.7) served as the control group. RESULTS: In obese patients, basal serum leptin fell from 62+/-20.4 to 23.8+/-15.7 ng/ml after the operation (P <0.01) but still with significant differences vs the control group (5.6+/-3 ng/ml). The meal was associated with a significant decrease of serum leptin (ANOVA test, P <0.01), and significant differences between obese patients after surgery and lean subjects were found. CONCLUSION: Serum leptin was reduced by the meal in obese patients and VBG did not attain satiety through serum leptin changes.

Acute parathyroid hormone increase by oral peptones administration after roux-en-Y gastric bypass surgery in obese subjects: role of phosphate in the rapid control of parathyroid hormone release.

BACKGROUND: It is generally considered that changes in serum phosphate levels do not alter parathyroid hormone (PTH) secretion in the absence of concomitant changes in ionized serum calcium level in humans. An acute rise in PTH was shown after phosphate administration by intraduodenal gavage in rats. We aimed to study gastrin, phosphate, PTH, ionized calcium (iCa), and blood pH responses to oral peptones in morbidly obese patients before and after roux-en-Y gastric bypass (RYGB) surgery. METHODS: These parameters were evaluated in response to an oral peptone load in 24 (18 male and 6 female) obese subjects before and 6 months after RYGB surgery. In 12 gastric bypass patients, we also evaluated PTH and phosphate after peptones plus aluminum hydroxide administration to suppress phosphate absorption. RESULTS: Before RYGB, peptones increased gastrin (P < .001), and decreased iCa (P < .01) without changes in PTH or pH. Both phosphate and PTH markedly increased after RYGB with the peptones oral load (P < .01), without changes in pH, iCa, or gastrin. There was a significant, direct relationship between the increase of phosphate and the increase of PTH in the patients treated with aluminum hydroxide (r(2) = 0.78; P < .0001). CONCLUSION: Rapid delivery of peptones in the jejunum in bypassed obese patients results in a significant rise in phosphate and PTH, in the absence of changes of other PTH regulators, possibly mediated by a signaling from the gastrointestinal tract. RYGB patients provide an opportunity to study the control of PTH secretion, with potential relevant clinical implications.

Closure of the cystic duct by ultrasonic energy: an electron-microscopic and biomechanical study in man.

BACKGROUND: Ultrasonic activated devices are currently used both for sealing of blood vessels and dissection of parenchymal organs. Recently, ultrasonic energy has been used to seal the cystic duct during successful clip-less cholecystectomy, but no study examined the mechanisms of tissue welding by ultrasonic energy or the biomechanical properties of the seal. This was the aim of our investigations. PATIENTS AND METHODS: Eight patients (7 women and 1 man, mean age 42+/-7 y) subjected to laparoscopic cholecystectomy had a cystic duct sealed and sectioned by Ultracision; after removal of the specimens, the distal end was processed for scanning electron microscopy and transmission electron microscopy, whereas the proximal end to the gallbladder was used for determination of the bursting pressure. RESULTS: The bursting pressure of the cystic duct sealed by ultrasonic energy was 168+/-47 mm Hg, well above the maximal pressure of the common bile duct. At scanning electron microscopy, the end of the cystic duct was closed by an amorphous, bundled, dense substance. The section proximal to the end showed destruction of the epithelial layer with dense amorphous bridges connecting the opposite sites of the wall. When the lumen was observed, it was filled with bile microaggregates. At transmission electron microscopy, 2 different findings were evident: (1) coagulative necrosis in the sectioned area and (2) a combination of coagulative necrosis and collagen denaturation in the more distal sections. The connective tissue was characterized by attenuation of collagen birefringence and swelling of fibers and bundles due to shrinkage of collagen. CONCLUSIONS: Ultrasonic energy can be applied to suitable tissues to obtain sealing of the walls with good biomechanical effects.

Isolated ampullary adenoma causing biliary obstruction.

This is an interesting case of an isolated ampullary adenoma causing biliary obstruction that required surgical excision. We describe a patient who presented with a six month history of recurrent attacks of typical biliary pain radiating from the right upper quadrant of the abdomen to the back, nausea and vomiting, which we attributed to a large pedunculated tubulovillous adenoma. Abdominal ultrasound and endoscopic ultrasonography provided useful information in the diagnostic assessment of ampullary adenoma. Sporadic duodenal adenomas are an increasingly recognized condition in those with familial adenomatous polyposis syndromes as well as sporadic cases.

Surgical approach in a rare case of coloboma-choristoma.

Embryogenic eyelid defects can be isolated or associated with malformative diseases, such as Tessier craniofacial clefts. We describe the exceptional coexistence of upper eyelid coloboma and lower eyelid dermolipoma in a 45-day-old infant with a Tessier no. 0-1 cleft. The surgical intervention carried out on this patient, which used a flap of subconjunctival choristoma and grafting of reshaped cutaneous and tarsal portions of a preauricular anlage, is presented as a technique for correcting congenital palpebral coloboma. Adoption of this technique allowed virtually complete repair of the defect and gave an acceptable functional and cosmetic result.