Skin dysbiosis and Cutibacterium acnes biofilm in inflammatory acne lesions of adolescents.

Acne vulgaris is a common inflammatory disorder affecting more than 80% of young adolescents. Cutibacterium acnes plays a role in the pathogenesis of acne lesions, although the mechanisms are poorly understood. The study aimed to explore the microbiome at different skin sites in adolescent acne and the role of biofilm production in promoting the growth and persistence of C. acnes isolates. Microbiota analysis showed a significantly lower alpha diversity in inflammatory lesions (LA) than in non-inflammatory (NI) lesions of acne patients and healthy subjects (HS). Differences at the species level were driven by the overabundance of C. acnes on LA than NI and HS. The phylotype IA1 was more represented in the skin of acne patients than in HS. Genes involved in lipids transport and metabolism, as well as potential virulence factors associated with host-tissue colonization, were detected in all IA1 strains independently from the site of isolation. Additionally, the IA1 isolates were more efficient in early adhesion and biomass production than other phylotypes showing a significant increase in antibiotic tolerance. Overall, our data indicate that the site-specific dysbiosis in LA and colonization by virulent and highly tolerant C. acnes phylotypes may contribute to acne development in a part of the population, despite the universal carriage of the microorganism. Moreover, new antimicrobial agents, specifically targeting biofilm-forming C. acnes, may represent potential treatments to modulate the skin microbiota in acne.

ent-Beyerane Diterpenes as a Key Platform for the Development of ArnT-Mediated Colistin Resistance Inhibitors.

Colistin is a last-resort antibiotic for the treatment of multidrug resistant Gram-negative bacterial infections. Recently, a natural ent-beyerene diterpene was identified as a promising inhibitor of the enzyme responsible for colistin resistance mediated by lipid A aminoarabinosylation in Gram-negative bacteria, namely, ArnT (undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase). Here, semisynthetic analogues of hit were designed, synthetized, and tested against colistin-resistant Pseudomonas aeruginosa strains including clinical isolates to exploit the versatility of the diterpene scaffold. Microbiological assays coupled with molecular modeling indicated that for a more efficient colistin adjuvant activity, likely resulting from inhibition of the ArnT activity by the selected compounds and therefore from their interaction with the catalytic site of ArnT, an ent-beyerane scaffold is required along with an oxalate-like group at C-18/C-19 or a sugar residue at C-19 to resemble L-Ara4N. The ent-beyerane skeleton is identified for the first time as a privileged scaffold for further cost-effective development of valuable colistin resistance inhibitors.

Sclerostin is expressed in the atherosclerotic plaques of patients who undergoing carotid endarterectomy.

BACKGROUND: Sclerostin (SC) is a monomeric glycoprotein expressed by osteocytes that affects bone formation. Recent studies have suggested a potential role for this protein in the pathophysiology of vascular diseases. The aim of the present study was to investigate SC expression in atherosclerotic plaques of patients affected by severe atherosclerotic disease who underwent carotid endarterectomy. We also evaluated possible differences in SC expression between patients with and without type 2 diabetes (T2D). METHODS: This was a cross-sectional study involving 46 patients aged 55 to 80 years (mean, 71.1 ± 6.7 years, 36 men, 15 patients with T2D) who underwent carotid endarterectomy. Immunohistochemical levels of SC were evaluated in the atherosclerotic plaques by double-staining immunochemistry, and serum SC levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: Sclerostin was present in the atherosclerotic plaques of all subjects investigated and increased significantly in the media compared with the intima (P < 0.0001) as well as in vascular smooth muscle cells (VSMCs) compared with the infiltrating macrophages (P < 0.0001). However, no significant difference in SC expression was observed between patients with and without T2D. No correlation was found between serum and immunohistochemical levels of SC; significantly increased SC serum levels were detected in males compared with females (P = 0.049). CONCLUSIONS: We have demonstrated, for the first time, the expression of SC in VSMCs of atherosclerotic plaques, suggesting a potential role for this protein in the development of atherosclerosis. Further studies are needed to understand if the role played by SC is detrimental or protective in the atherosclerotic disease process.

Evidence of diabetes-specific autoimmunity in obese subjects with normal glucose tolerance.

BACKGROUND: Recently, significant attention has been paid to the possible activation of an autoimmune response in the presence of obesity. The aim of this study was to evaluate and compare the frequencies of autoantibodies typical of autoimmune diabetes in obese patients with normal glucose tolerance (NGT), obese patients with type 2 diabetes (T2D) and controls. We also evaluated the presence of immunoreactivity to Hashimoto's thyroiditis and autoimmune gastritis. MATERIALS AND METHODS: Consecutive sera from obese patients, 444 with NGT, 322 with T2D, and 212 controls were analysed by radioimmunoassay or enzyme-linked immunosorbent assay for glutamic acid decarboxylase, protein tyrosine phosphatase islet antigen-2 (IA-2)IC and IA-2(256-760) , islet beta-cell zinc cation transporter (ZnT8), thyroid peroxidase, and anti-parietal cell autoantibodies. RESULTS: Altogether the presence of organ-specific autoantibodies was significantly more frequent in obese patients with NGT (128/444, 28.5%) and obese with T2D (79/322, 24.5%) than in controls (36/212, 17%; P = 0.002). Thyroid peroxidase immunoreactivity was prevalent in all groups of subjects investigated. The frequencies of diabetes-specific autoantibodies were slightly higher in obese patients with NGT (20/444, 4.5%) than in obese with T2D (12/322, 3.7%) and controls (4/212, 1.9%). The anti IA-2(256-760) was the most frequent islet autoantibody in obese subjects with NGT (14/20, 70%). CONCLUSIONS: We observed significant evidence of immunoreactivity specific to diabetes, thyroid, and gastric-parietal cells in obese patients with NGT. The relatively higher frequency of the diabetes-related IA-2(256-760) autoantibodies in obese patients with NGT may suggest that this autoantibody could be associated with obesity the presence of obesity itself.

Relation Between Wrist Circumference and Left Ventricular Structure in Overweight Children.

The aim of this study was to assess the relation of wrist circumference with changes in left ventricular (LV) structure in a population of overweight/obese children and adolescents. One hundred and six children and adolescents were consecutively enrolled. In all subjects body weight, height, wrist circumference, waist circumference, body mass index-standard deviation score, fasting glucose, insulin, lipid profile, and blood pressure were evaluated. All subjects underwent echocardiographic assessment, and the following parameters were evaluated: LV dimension at end diastole and LV dimension at end systole, LV posterior wall thickness at end diastole and LV posterior wall thickness at end systole, interventricular septal thickness at end diastole and interventricular septal thickness at end systole, LV mass, and epicardial adipose tissue (EAT). LV hypertrophy was defined as LVM Index ≥95th percentile. Wrist circumference correlated with all parameters of LV dimensions and LV mass (p <0.0001) and EAT (p = 0.04). The strongest correlations were reported between wrist circumference with LV dimension at end diastole and LV dimension at end systole (r = 0.73 and r = 0.68 respectively, p <0.0001, for both). Results of the multivariate regression analysis showed that wrist circumference was significantly associated with all parameters of LV dimensions, LV mass, and EAT (p ≤0.002). The logistic regression showed that wrist circumference was significantly associated with LV hypertrophy (odds ratio 1.39, p = 0.046). In conclusion, wrist circumference could be a useful measure of cardiovascular risk in obese children and adolescents, opening new perspectives in the prediction of cardiovascular diseases.

Wrist circumference is associated with increased systolic blood pressure in children with overweight/obesity.

Wrist circumference is a clinical marker for insulin-resistance in overweight/obese children and adolescents. Insulin resistance is considered a major determinant of increased vascular resistance and hypertension. The aim of the study was to investigate the association between wrist circumference and systolic (S) and diastolic (D) blood pressure (BP) in a population of overweight/obese children and adolescents. A population of 1133 overweight/obese children and adolescents (n = 1133) were consecutively enrolled. Multivariate regression analyses were used to investigate the influence of independent variables on the variance of BP. The prevalence of hypertension was 21.74% in males and 28.95% in females (p = 0.048). The results showed that SBP was significantly associated with wrist circumference in both genders (p < 0.0001 for both comparisons). We found no association between DBP and wrist circumference in either gender. Wrist circumference accounted for 17% of the total variance of SBP in males and 14% in females. Wrist circumference, a marker of insulin resistance, is associated with increased SBP in overweight/obese children and adolescents, suggesting a role of insulin resistance in the pathogenesis and development of hypertension.

ISA-2011B, a Phosphatidylinositol 4-Phosphate 5-Kinase α Inhibitor, Impairs CD28-Dependent Costimulatory and Pro-inflammatory Signals in Human T Lymphocytes.

Phosphatidylinositol 4,5-biphosphate (PIP2) is a membrane phospholipid that controls the activity of several proteins regulating cytoskeleton reorganization, cytokine gene expression, T cell survival, proliferation, and differentiation. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are the main enzymes involved in PIP2 biosynthesis by phosphorylating phosphatidylinositol 4-monophosphate (PI4P) at the D5 position of the inositol ring. In human T lymphocytes, we recently found that CD28 costimulatory molecule is pivotal for PIP2 turnover by recruiting and activating PIP5Kα. We also found that PIP5Kα is the main regulator of both CD28 costimulatory signals integrating those delivered by TCR as well as CD28 autonomous signals regulating the expression of pro-inflammatory genes. Given emerging studies linking alterations of PIP2 metabolism to immune-based diseases, PIP5Kα may represent a promising target to modulate immunity and inflammation. Herewith, we characterized a recently discovered inhibitor of PIP5Kα, ISA-2011B, for its inhibitory effects on T lymphocyte functions. We found that the inhibition of PIP5Kα lipid-kinase activity by ISA-2011B significantly impaired CD28 costimulatory signals necessary for TCR-mediated Ca2+ influx, NF-AT transcriptional activity, and IL-2 gene expression as well as CD28 autonomous signals regulating the activation of NF-κB and the transcription of pro-inflammatory cytokine and chemokine genes. Moreover, our data on the inhibitory effects of ISA-2011B on CD28-mediated upregulation of inflammatory cytokines related to Th17 cell phenotype in type 1 diabetes patients suggest ISA-2011B as a promising anti-inflammatory drug.

An electrochemical immunoassay for the screening of celiac disease in saliva samples.

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95% clinical sensitivity and 96% clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age.

Anti-transglutaminase immunoreactivity and histological lesions of the duodenum in coeliac patients.

Coeliac disease (CD) is characterized by several markers, including anti-transglutaminase auto-antibodies (tTGAb) directed against multiple epitopes of the gliadin protein. We aimed to investigate the correlation among CD duodenal lesions, tTGAb titres and the immunoreactivity against tTG constructs. A total of 345 CD patients (209 females, 136 males, overall median age: 7.3 years) were tested for full-length (fl) tTGAb with a fluid-phase radioimmunoassay. Out of the total, 231 patients were also tested for immunoreactivity against tTG fragments (F1: a.a. 227-687 and F2: a.a. 473-687). Patients were classified according to diffuse (D), patchy (P) or bulb (B) histological lesions. All sera were found fltTGAb positive. Patients with D, P and B lesions had a mean Ab index of 0.84±0.39, 0.57±0.39 and 0.45±0.24, respectively. Mean tTGAb titre varied between D and localized (P+B) patients (0.84±0.39 versus 0.52±0.34, P < 0.0001). Overall, 86.1% of patients were F1 auto-antibody (F1Ab) positive (D: 89%, P: 75%, B: 40%; D versus P+B: P = 0.004) and 49% of patients were F2 auto-antibody (F2Ab) positive (D: 53%, P: 19%, B: 10%; D versus P+B: P = 0.0006). Of the D patients 50.7% showed combined F1Ab-F2Ab (D versus P+B: P = 0.001), whereas 60% of B patients were negative for both F1Ab and F2Ab (B versus D: P < 0.0001). Coeliac-specific tTGAb immunoreactivity correlates with the grading and extension of histological duodenal lesions in CD patients at diagnosis. The immunoreactivity against single and combined tTG fragments is significantly higher in patients with D lesions. This is the first evidence of a distinct coeliac-specific immunoreactivity in patients with different duodenal involvement.

The celiac iceberg: characterization of the disease in primary schoolchildren.

OBJECTIVE: Celiac disease (CD) has a prevalence of 0.55% to 1% in Italy. Identifying CD in schoolchildren to characterize CD iceberg and evaluate the effect of diagnosis in screening-detected children. METHODS: A total of 7377 5- to 8-year-old children were invited to participate. A total of 5733 salivary samples were collected and tested for anti-transglutaminase antibodies (tTGAb), using a fluid-phase radioimmunoassay. Salivary tTGAb-positive children were analyzed for serum antibodies (anti-endomysium antibodies, radioimmunoassay, and enzyme-linked immunosorbent assay tTGAb). Positive children underwent endoscopy and then started gluten-free diet (GFD) and periodical follow-up. RESULTS: Forty-six subjects were found salivary tTGAb-positive and 16 border-line. Forty-five of 46 and 5 of 15 of them were also serum antibody-positive. Forty-two children showed duodenal villous atrophy and 1 had only type 1 lesions. Three children started GFD without performing endoscopy. CD prevalence (including 23 previously diagnosed children with CD) was 1.2%. Considering all 65 celiacs in our sample, a silent CD was found in 64%, typical in 28%, atypical in 7%, and potential in 1%. All patients showed strict adherence to GFD, weight and stature increase, and well-being improvement. Eighty-five percent and all but 2 screening-detected children with CD had Italian parents. CONCLUSIONS: Our sample size, representative of primary schoolchildren of our region, demonstrated that CD prevalence is growing in Italy, with a modified clinical spectrum and iceberg deepness.

IgA anti-transglutaminase autoantibodies at type 1 diabetes onset are less frequent in adult patients and are associated with a general celiac-specific lower immune response in comparison with nondiabetic celiac patients at diagnosis.

OBJECTIVE: To evaluate the celiac-associated humoral autoimmunity in child, adolescent, and adult patients at type 1 diabetes (DM1) onset and to determine whether DM1 celiac-specific humoral immunoreactivity occurs similarly to that in nondiabetic patients at celiac disease (CD) diagnosis. RESEARCH DESIGN AND METHODS: IgA anti-transglutaminase autoantibody (IgA-tTGAb) was detected in 654 new-onset DM1 sera. IgA-tTGAb(+) DM1 sera were subsequently analyzed for IgG-tTG, deamidated gliadin (DGP), and actin antibodies, and results were compared with those found in 83 screen-detected nondiabetic patients at CD diagnosis. RESULTS: A total of 12.8% DM1 sera were IgA-tTGAb(+), with a lower autoantibody frequency in adult patients aged >18 years (6.8 vs. 15.1%, aged ≤18 years; P = 0.005). IgA-tTGAb titers, IgG-tTGAb, and DGPAb frequency/titers and mean number of celiac-autoantibody positivities per patient were significantly lower in IgA-tTGAb(+) DM1 compared with nondiabetic CD patients. CONCLUSIONS: Age of diabetes onset is negatively associated with risk of CD. The celiac-specific humoral immunoreactivity at DM1 onset is significantly lower compared with that found in nondiabetic patients at CD diagnosis.

Endoscopic and histological gastric lesions in children with celiac disease: mucosal involvement is not only confined to the duodenum.

OBJECTIVES: Lymphocytic gastritis (LG) has been reported in patients with celiac disease (CD). The aim of the present study was to evaluate gastric mucosa involvement in celiac children and gastroenterological controls (GC). METHODS: In a retrospective study on 226 patients with CD (82  M; median age: 5.7 years) at diagnosis and 154 GC (66  M; median age: 7.4 years), the evaluation of gastric and duodenal mucosa was performed. CD was diagnosed according to the North America Society for Pediatric Gastroenterology, Hepatology, and Nutrition criteria. Gastric lesions were classified according to Updated Sydney System. Anti-gastric parietal cell antibodies (GPCA) were assayed by enzyme-linked immunosorbent assay. RESULTS: A total of 21.2% and 7% of patients with CD showed chronic superficial gastritis (CSG) and LG, respectively. Helicobacter pylori (Hp) infection was found in 6 (2.7%) children with CD (66.7% had CSG, 16.7% LG, and 16.7% interstitial gastritis). CSG was present in 21.4% of controls. No control subject showed LG. Hp infection was found in 24 (15.6%) children with GC (91.7% had CSG). Among patients with CSG, Hp infection was more frequent in controls than in celiac children (P < 0.0001). Ten of 90 patients with CD and 1 of 29 controls were positive for GPCA. CONCLUSIONS: Gastritis is a common finding in children with CD and adolescents. In celiac subjects, CSG is the most frequently detected. Our data suggest the hypothesis that LG may be related to a longer exposure to gluten. The presence of GPCA may suggest the presence of an underlying autoimmune process.

Gender-dependent association of type 2 diabetes with the vasoactive intestinal peptide receptor 1.

Type 2 diabetes is characterized by an inadequate pancreatic beta-cell response to the progressive insulin resistance. Its pathogenesis is complex and has been connected with a state of preclinical chronic inflammation. Vasoactive intestinal peptide (VIP) and its receptors play a relevant role in the homeostasis of insulin secretion as well as in the control of inflammation. In particular, VIP receptor 1 (VPAC1) has been found to be down-modulated during inflammation, and to be associated with several diseases. The objective of this study was to compare the distribution of SNPs mapping in the VIP receptor 1 gene in cases with type 2 diabetes and matched controls. Seven hundred cases with type 2 diabetes (423 males and 277 females) and 830 random controls (419 males and 411 females) were analyzed for the distribution of three common SNPs mapping in the VPAC1 gene. The results show a significantly different genotype distribution of the SNP rs9677 in the 3'-UTR of VPAC1 in female cases with type 2 diabetes compared to gender-matched controls (ptrend=6×10(-4)). The rs9677 CC genotype confers the highest risk (OR: 2.1) and correlates with worse clinical parameters such as higher level of total cholesterol, higher LDL/HDL ratio and a higher HbA1c concentration. The genetic association reported here indicates that VIP/VPAC1 signaling can be a relevant pathway in the pathogenesis of type 2 diabetes in females suggesting that at least some aspects of the genetic predisposition to this disease can be gender-specific.

First salivary screening of celiac disease by detection of anti-transglutaminase autoantibody radioimmunoassay in 5000 Italian primary schoolchildren.

OBJECTIVE: The high prevalence of celiac disease (CD) prompted us to evaluate a new, noninvasive disease screening strategy. The aim was to identify CD in 6- to 8-year-old children for a timely diagnosis, start gluten-free diet (GFD) in compliant subjects, achieve the growth target, and prevent CD complications. METHODS: Five thousand subjects were invited to participate in the study. Four thousand forty-eight saliva samples were tested for anti-tissue transglutaminase (tTG) immunoglobulin (Ig)A using a fluid-phase radioimmunoprecipitation method. Positive children were tested for serum radioimmunoassay tTG IgA, enzyme-linked immunosorbent assay tTG IgA, and anti-endomysium IgA. Children confirmed as positive by serum assays underwent endoscopy with duodenal biopsies and, at the diagnosis of CD, were suggested to start GFD. RESULTS: Consent was obtained from 4242 parents (84.8%) for the screening to be performed, and adequate saliva samples were collected from 4048 children (95.4%). Thirty-two children were found to be salivary tTG IgA positive and 9 with borderline autoantibody levels. Thirty-one of the 32 and 3 of the 9 subjects were also serum positive. Twenty-eight children showed villous atrophy when undergoing intestinal biopsy, whereas 1 had Marsh 1 lesions; 3 children were suggested to start GFD without performing endoscopy. CD prevalence in the population investigated (including 19 CD known cases) was 1.16%. The ratio between screening-detected patients and those diagnosed before the screening was 3:2. The ratio between symptomatic and asymptomatic patients was 1:1.6. CONCLUSIONS: We demonstrated that it is possible to perform a powerful, simple, well-accepted, and sensitive CD screening using saliva. Until now, the compliance with GFD in children with CD has been optimal.

GAD and IA-2 autoantibody detection in type 1 diabetic patient saliva.

Some attempts have been made in assaying glutamic-acid decarboxylase autoantibodies (GADA) in type 1 diabetic patient (T1DM) saliva. However, these salivary assays did not show sufficient sensitivity and specificity in comparison to serum assays. In this study we evaluated the ability of a fluid-phase (35)S-radioimmunoassay to detect GADA and tyrosine phosphatase 2 autoantibodies (IA-2A) in 70 T1DM, 24 T1DM first degree relatives (FDR) and 76 healthy subject saliva. Paired saliva and serum samples were collected from each subject and analyzed. GADA were detected in 45/70 (64.3%) sera and 43/70 (61.4%) T1DM saliva, respectively. IA-2A were detected in 33/70 (47.1%) sera and 30/70 (42.9%) T1DM saliva, respectively. All FDR serum/saliva samples were autoantibody negative. In conclusion, we here report that GADA and IA-2A are detectable with high sensitivity and specificity in human saliva, a specimen which can be easily collected by non-invasive procedures and may represent a reliable tool for the study of T1DM autoimmunity.