Background

Olive plant leaves (Olea europaea L.) have been used for centuries in folk medicine to treat diabetes, but there are very limited data examining the effects of olive polyphenols on glucose homeostasis in humans.

Objective

To assess the effects of supplementation with olive leaf polyphenols (51.1 mg oleuropein, 9.7 mg hydroxytyrosol per day) on insulin action and cardiovascular risk factors in middle-aged overweight men.

Design

Randomized, double-blinded, placebo-controlled, crossover trial in New Zealand. 46 participants (aged 46.4±5.5 years and BMI 28.0±2.0 kg/m2) were randomized to receive capsules with olive leaf extract (OLE) or placebo for 12 weeks, crossing over to other treatment after a 6-week washout. Primary outcome was insulin sensitivity (Matsuda method). Secondary outcomes included glucose and insulin profiles, cytokines, lipid profile, body composition, 24-hour ambulatory blood pressure, and carotid intima-media thickness.

Results

Treatment evaluations were based on the intention-to-treat principle. All participants took >96% of prescribed capsules. OLE supplementation was associated with a 15% improvement in insulin sensitivity (p = 0.024) compared to placebo. There was also a 28% improvement in pancreatic β-cell responsiveness (p = 0.013). OLE supplementation also led to increased fasting interleukin-6 (p = 0.014), IGFBP-1 (p = 0.024), and IGFBP-2 (p = 0.015) concentrations. There were however, no effects on interleukin-8, TNF-α, ultra-sensitive CRP, lipid profile, ambulatory blood pressure, body composition, carotid intima-media thickness, or liver function.

Conclusions

Supplementation with olive leaf polyphenols for 12 weeks significantly improved insulin sensitivity and pancreatic β-cell secretory capacity in overweight middle-aged men at risk of developing the metabolic syndrome.

Trial Registration

Australian New Zealand Clinical Trials Registry #336317.

The diagnosis of non-small cell lung carcinoma (NSCLC) at an early stage, as well as better prediction of outcome remains clinically challenging due to the lack of specific and robust non-invasive markers. The discovery of microRNAs (miRNAs), particularly those found in the bloodstream, has opened up new perspectives for tumor diagnosis and prognosis. The aim of our study was to determine whether expression profiles of specific miRNAs in plasma could accurately discriminate between NSCLC patients and controls, and whether they are able to predict the prognosis of resectable NSCLC patients. We therefore evaluated a series of seventeen NSCLC-related miRNAs by quantitative real-time (qRT)-PCR in plasma from 52 patients with I-IIIA stages NSCLC, 10 patients with chronic obstructive pulmonary disease (COPD) and 20-age, sex and smoking status-matched healthy individuals. We identified an eleven-plasma miRNA panel that could distinguish NSCLC patients from healthy subjects (AUC = 0.879). A six-plasma miRNA panel was able to discriminate between NSCLC patients and COPD patients (AUC = 0.944). Furthermore, we identified a three-miRNA plasma signature (high miR-155-5p, high miR-223-3p, and low miR-126-3p) that significantly associated with a higher risk for progression in adenocarcinoma patients. In addition, a three-miRNA plasma panel (high miR-20a-5p, low miR-152-3p, and low miR-199a-5p) significantly predicted survival of squamous cell carcinoma patients. In conclusion, we identified two plasma miRNA expression profiles that may be useful for predicting the outcome of patients with resectable NSCLC.

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.

Background

This usability test investigated the overall preference and usability of the novel NovoTwist® insulin pen needle versus conventional screw-thread needles, when used with Next Generation FlexPen®, in children and adolescents with diabetes.

Methods

This was an open-label, randomized, crossover usability test in children and adolescents with type 1 diabetes who administered insulin with an insulin pen. Test needles were NovoTwist and the participant’s current screw-thread needle (or NovoFine® needle). Following instruction, participants attached the needle to Next Generation FlexPen, made an injection into a foam cushion, and detached the needle. This procedure was conducted three times with both needles in a random order. Responses to 13 questions on user experience with each needle (including overall preference, ease of attachment/detachment of needle/cap, handling, learning, confidence in attachment, and convenience of use) were subsequently recorded on a six-point rating scale (1 = very difficult; 6 = very easy).

Results

Fifteen children aged ≥6 to ≤12 years and 15 adolescents aged ≥13 to ≤17 years participated in the test. A significantly higher proportion of children and adolescents (77%) indicated that they would prefer to use NovoTwist compared with screw-thread needles (p = .005). NovoTwist was preferred by most children and adolescents for overall ease of use (77%; p = .005), for ease of attachment (87%; p < .001) and detachment (83%; p < .001), and as the most appropriate needle to handle for daily injections (73%; p = .016). The mean rating for confidence in correct needle attachment was not significantly different between the two needle types. Seven out of eight parents of children who required assistance for their daily insulin injections stated that they would be “very likely” to allow their child to attach NovoTwist.

Conclusions

These factors may promote confidence in this needle, and thus in self-injecting, among younger patients and their parents.

It is noteworthy that bacterial or viral infections, and the resulting chronic inflammation, have been shown to predispose individuals to certain types of cancer. Remarkably, these microbes upregulated some transcription factors involved in the regulation of the epithelial to mesenchymal transition, referred herein as EMT. EMT is a cellular process that consists in the conversion of epithelial cell phenotype to a mesenchymal phenotype. Under physiological conditions EMT is clearly important for embryogenesis, organ development, wound repair and tissue remodeling. However, EMT may also be activated under pathologic conditions, more particularly in carcinogenesis and metastatic progression. In this review, we make a parallel between microbes- and growth factors-induced transcription factors. A unifying EMT model then emerges that may help in understanding the development of microbial pathogenesis and in defining new potential future therapeutic strategy in treating diseases linked to infections.

Introduction

The reproducibility of tractography is important to determine its sensitivity to pathological abnormalities. The reproducibility of tract morphology has not yet been systematically studied and the recently developed tractography contrast Tract Density Imaging (TDI) has not yet been assessed at the tract specific level.

Materials and Methods

Diffusion tensor imaging (DTI) and probabilistic constrained spherical deconvolution (CSD) tractography are performed twice in 9 healthy subjects. Tractography is based on common space seed and target regions and performed for several major white matter tracts. Tractograms are converted to tract segmentations and inter-session reproducibility of tract morphology is assessed using Dice similarity coefficient (DSC). The coefficient of variation (COV) and intraclass correlation coefficient (ICC) are calculated of the following tract metrics: fractional anisotropy (FA), apparent diffusion coefficient (ADC), volume, and TDI. Analyses are performed both for proximal (deep white matter) and extended (including subcortical white matter) tract segmentations.

Results

Proximal DSC values were 0.70–0.92. DSC values were 5–10% lower in extended compared to proximal segmentations. COV/ICC values of FA, ADC, volume and TDI were 1–4%/0.65–0.94, 2–4%/0.62–0.94, 3–22%/0.53–0.96 and 8–31%/0.48–0.70, respectively, with the lower COV and higher ICC values found in the proximal segmentations.

Conclusion

For all investigated metrics, reproducibility depended on the segmented tract. FA and ADC had relatively low COV and relatively high ICC, indicating clinical potential. Volume had higher COV but its moderate to high ICC values in most tracts still suggest subject-differentiating power. Tract TDI had high COV and relatively low ICC, which reflects unfavorable reproducibility.

Background

Magnetic resonance imaging (MRI) is an important tool for cardiac research, and it is frequently used for resting cardiac assessments. However, research into non-pharmacological stress cardiac evaluation is limited.

Methods

We aimed to design a portable and relatively inexpensive MRI cycle ergometer capable of continuously measuring pedalling workload while patients exercise to maintain target heart rates.

Results

We constructed and tested an MRI-compatible cycle ergometer for a 1.5 T MRI scanner. Resting and sub-maximal exercise images (at 110 beats per minute) were successfully obtained in 8 healthy adults.

Conclusions

The MRI-compatible cycle ergometer constructed by our research group enabled cardiac assessments at fixed heart rates, while continuously recording power output by directly measuring pedal force and crank rotation.

Background

We aimed to evaluate the incidence of type 1 diabetes mellitus in children <15 years of age (yr) in the Auckland region (New Zealand) over 20 years (1990–2009).

Methods

We performed a retrospective review of all patients <15 yr diagnosed with type 1 diabetes, from an unselected complete regional cohort.

Results

There were 884 new cases of type 1 diabetes, and age at diagnosis rose from 7.6 yr in 1990/1 to 8.9 yr in 2008/9 (r2 = 0.31, p = 0.009). There was a progressive increase in type 1 diabetes incidence among children <15 yr (p<0.0001), reaching 22.5 per 100,000 in 2009. However, the rise in incidence did not occur evenly among age groups, being 2.5-fold higher in older children (10–14 yr) than in the youngest group (0–4 yr). The incidence of new cases of type 1 diabetes was highest in New Zealand Europeans throughout the study period in all age groups (p<0.0001), but the rate of increase was similar in New Zealand Europeans and Non-Europeans. Type 1 diabetes incidence and average annual increase were similar in both sexes. There was no change in BMI SDS shortly after diagnosis, and no association between BMI SDS and age at diagnosis.

Conclusions

There has been a steady increase in type 1 diabetes incidence among children <15 yr in Auckland over 20 years. Contrary to other studies, age at diagnosis has increased and the greatest rise in incidence occurred in children 10–14 yr. There was little change in BMI SDS in this population, providing no support for the ‘accelerator hypothesis’.

Background

Type 1 diabetes mellitus (T1DM) may lead to severe long-term health consequences. In a longitudinal study, we aimed to identify factors present at diagnosis and 6 months later that were associated with glycosylated haemoglobin (HbA1c) levels at 24 months after T1DM diagnosis, so that diabetic children at risk of poor glycaemic control may be identified.

Methods

229 children <15 years of age diagnosed with T1DM in the Auckland region were studied. Data collected at diagnosis were: age, sex, weight, height, ethnicity, family living arrangement, socio-economic status (SES), T1DM antibody titre, venous pH and bicarbonate. At 6 and 24 months after diagnosis we collected data on weight, height, HbA1c level, and insulin dose.

Results

Factors at diagnosis that were associated with higher HbA1c levels at 6 months: female sex (p<0.05), lower SES (p<0.01), non-European ethnicity (p<0.01) and younger age (p<0.05). At 24 months, higher HbA1c was associated with lower SES (p<0.001), Pacific Island ethnicity (p<0.001), not living with both biological parents (p<0.05), and greater BMI SDS (p<0.05). A regression equation to predict HbA1c at 24 months was consequently developed.

Conclusions

Deterioration in glycaemic control shortly after diagnosis in diabetic children is particularly marked in Pacific Island children and in those not living with both biological parents. Clinicians need to be aware of factors associated with poor glycaemic control beyond the remission phase, so that more effective measures can be implemented shortly after diagnosis to prevent deterioration in diabetes control.

OBJECTIVE

We aimed to establish the ideal injection techniques using 5-mm needles to reliably inject insulin into the subcutaneous fat in both children and adults and to quantify the associated pain and leakage of the test medium.

RESEARCH DESIGN AND METHODS

A total of 259 subjects (122 children/adolescents and 137 adults) were injected with sterile air corresponding to 20 IU insulin (200 μl) with 32-G 5-mm needles at 90° or 45°, in the abdomen and thigh, and with or without a pinched skin fold. Injection depth was assessed via ultrasonography. Subjects rated pain on a visual analog scale. Test medium injections into the abdomen and thigh (0.2–0.6 ml) were also administered to assess injection leakage.

RESULTS

Among children, 5.5% of injections were intramuscular (IM) and 0.5% were intradermal, while in adults, the incidence was 1.3 and 0.6%, respectively. The frequency of IM injections was greater in boys and negligible among adult women. Subcutaneous fat thickness was the primary predictor of the likelihood of IM injections (P < 0.001). A third of all patients reported experiencing no pain during insulin injection, with children/adolescents experiencing considerably more discomfort than adults. Some leakage of medium was observed, but was unrelated to injection volume and was generally minimal.

CONCLUSIONS

5-mm needles are reliably inserted into subcutaneous fat in both adults and children. These needles were associated with reduced pain and minimal leakage. We recommend an angled injection with a pinched skin fold for children, while in adults, the technique should be left to patient preference.

Methods of classification using transcriptome analysis for case-by-case tumor diagnosis could be limited by tumor heterogeneity and masked information in the gene expression profiles, especially as the number of tumors is small. We propose a new strategy, EMts_2PCA, based on: 1) The identification of a gene expression signature with a great potential for discriminating subgroups of tumors (EMts stage), which includes: a) a learning step, based on an expectation-maximization (EM) algorithm, to select sets of candidate genes whose expressions discriminate two subgroups, b) a training step to select from the sets of candidate genes those with the highest potential to classify training tumors, c) the compilation of genes selected during the training step, and standardization of their levels of expression to finalize the signature. 2) The predictive classification of independent prospective tumors, according to the two subgroups of interest, by the definition of a validation space based on a two-step principal component analysis (2PCA). The present method was evaluated by classifying three series of tumors and its robustness, in terms of tumor clustering and prediction, was further compared with that of three classification methods (Gene expression bar code, Top-scoring pair(s) and a PCA-based method). Results showed that EMts_2PCA was very efficient in tumor classification and prediction, with scores always better that those obtained by the most common methods of tumor clustering. Specifically, EMts_2PCA permitted identification of highly discriminating molecular signatures to differentiate post-Chernobyl thyroid or post-radiotherapy breast tumors from their sporadic counterparts that were previously unsuccessfully classified or classified with errors.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca2+/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

Author Summary

The “death receptor” CD95 (also known as Fas) plays an essential role in ensuring immune tolerance of self antigens as well as in the elimination of the body's cells that have been infected or transformed. This receptor is engaged by the membrane-bound ligand CD95L, which can be released into blood circulation after cleavage by metalloproteases. Hitherto, most of the studies on the CD95 signal have been performed with chimeric CD95Ls that mimic the membrane-bound ligand and exhibit a level of aggregation beyond that described for the metalloprotease-cleaved ligand. Multi-aggregated CD95L elicits a caspase-driven apoptotic signal. In this study, we observe that levels of soluble and naturally processed CD95L in sera of patients suffering from lupus correlate with disease severity. Strikingly, although this soluble CD95L fails to trigger cell death unlike its chimeric version, it induces a “non-canonical” Ca2+/c-yes/PI3K-dependent signaling pathway that promotes the transmigration of T-lymphocytes across the endothelial barrier. These findings shed light on an entirely new role for the soluble CD95L that may contribute to local or systemic tissue damage by enhancing the infiltration of activated T-lymphocytes. Overall, these findings underline the importance of revisiting the role of this “apoptotic cytokine” in the context of chronic inflammatory disorders.

Objective

To prospectively assess the relation between carotid plaque characteristics and the development of new cerebral white matter lesions (WMLs) at MRI.

Methods

Fifty TIA/stroke patients with ipsilateral 30–69% carotid stenosis underwent MRI of the plaque at baseline. Total plaque volume and markers of vulnerability to thromboembolism (lipid-rich necrotic core [LRNC] volume, fibrous cap [FC] status, and presence of intraplaque hemorrhage [IPH]) were assessed. All patients also underwent brain MRI at baseline and after one year. Ipsilateral cerebral WMLs were quantified with a semiautomatic method.

Results

Mean WML volume significantly increased over a one-year period (6.52 vs. 6.97 mm3, P = 0.005). WML volume at baseline and WML progression did not significantly differ (P>0.05) between patients with 30–49% and patients with 50–69% stenosis. There was a significant correlation between total plaque volume and baseline ipsilateral WML volume (Spearman ρ = 0.393, P = 0.005). There was no significant correlation between total plaque volume and ipsilateral WML progression. There were no significant associations between LRNC volume and WML volume at baseline and WML progression. WML volume at baseline and WML progression did not significantly differ between patients with a thick and intact FC and patients with a thin and/or ruptured FC. WML volume at baseline and WML progression also did not significantly differ between patients with and without IPH.

Conclusion

The results of this study indicate that carotid plaque burden is significantly associated with WML severity, but that there is no causal relationship between carotid plaque vulnerability and the occurrence of WMLs.

Both external and internal exposure to ionizing radiation are strong risk factors for the development of thyroid tumors. Until now, the diagnosis of radiation-induced thyroid tumors has been deduced from a network of arguments taken together with the individual history of radiation exposure. Neither the histological features nor the genetic alterations observed in these tumors have been shown to be specific fingerprints of an exposure to radiation. The aim of our work is to define ionizing radiation-related molecular specificities in a series of secondary thyroid tumors developed in the radiation field of patients treated by radiotherapy. To identify molecular markers that could represent a radiation-induction signature, we compared 25K microarray transcriptome profiles of a learning set of 28 thyroid tumors, which comprised 14 follicular thyroid adenomas (FTA) and 14 papillary thyroid carcinomas (PTC), either sporadic or consecutive to external radiotherapy in childhood. We identified a signature composed of 322 genes which discriminates radiation-induced tumors (FTA and PTC) from their sporadic counterparts. The robustness of this signature was further confirmed by blind case-by-case classification of an independent set of 29 tumors (16 FTA and 13 PTC). After the histology code break by the clinicians, 26/29 tumors were well classified regarding tumor etiology, 1 was undetermined, and 2 were misclassified. Our results help shed light on radiation-induced thyroid carcinogenesis, since specific molecular pathways are deregulated in radiation-induced tumors.

Background

GH therapy requires daily injections over many years and compliance can be difficult to sustain. As growth hormone (GH) is expensive, non-compliance is likely to lead to suboptimal growth, at considerable cost. Thus, we aimed to assess the compliance rate of children and adolescents with GH treatment in New Zealand.

Methods

This was a national survey of GH compliance, in which all children receiving government-funded GH for a four-month interval were included. Compliance was defined as ≥85% adherence (no more than one missed dose a week on average) to prescribed treatment. Compliance was determined based on two parameters: either the number of GH vials requested (GHreq) by the family or the number of empty GH vials returned (GHret). Data are presented as mean ± SEM.

Findings

177 patients were receiving GH in the study period, aged 12.1±0.6 years. The rate of returned vials, but not number of vials requested, was positively associated with HVSDS (p<0.05), such that patients with good compliance had significantly greater linear growth over the study period (p<0.05). GHret was therefore used for subsequent analyses. 66% of patients were non-compliant, and this outcome was not affected by sex, age or clinical diagnosis. However, Maori ethnicity was associated with a lower rate of compliance.

Interpretation

An objective assessment of compliance such as returned vials is much more reliable than compliance based on parental or patient based information. Non-compliance with GH treatment is common, and associated with reduced linear growth. Non-compliance should be considered in all patients with apparently suboptimal response to GH treatment.

The role of chronic inflammation, acting as an independent factor, on the onset of gastrointestinal carcinogenesis is now well accepted. However, even if there is an increase in the number of elements directly involving polymorphonuclear leukocytes (PMNL), as a major actor in digestive carcinogenesis, the different cellular and molecular events occurring in this process are still not completely understood. The transepithelial migration of PMNL, which is the ultimate step of the afflux of PMNL into the digestive mucosa, is a complex phenomenon involving sequential interaction of molecules expressed both on PMNL and on digestive epithelial cells. Chronic inflammatory areas rich in PMNL [so-called (chronic active inflammation)] and iterative transepithelial migration of PMNL certainly evoke intracellular signals, which lead toward progressive transformation of epithelia. Among these different signals, the mutagenic effect of reactive oxygen species and nitrates, the activation of the nuclear factor-κB pathway, and the modulation of expression of certain microRNA are key actors. Following the initiation of carcinogenesis, PMNL are involved in the progression and invasion of digestive carcinomas, with which they interact. It is noteworthy that different subpopulations of PMNL, which can have some opposite effects on tumor growth, in association with different levels of transforming growth factor-β and with the number of CD8 positive T lymphocytes, could be present during the development of digestive carcinoma. Other factors that involve PMNL, such as massive elastase release, and the production of angiogenic factors, can participate in the progression of neoplastic cells through tissues. PMNL may play a major role in the onset of metastases, since they allow the tumor cells to cross the endothelial barrier and to migrate into the blood stream. Finally, PMNL play a role, alone or in association with other cell parameters, in the initiation, promotion, progression and dissemination of digestive carcinomas. This review focuses on the main currently accepted cellular and molecular mechanisms that involve PMNL as key actors in digestive carcinogenesis.

Background

Lung cancer in never smokers would rank as the seventh most common cause of cancer death worldwide.

Methods and Findings

We performed high-resolution array comparative genomic hybridization analysis of lung adenocarcinoma in sixty never smokers and identified fourteen new minimal common regions (MCR) of gain or loss, of which five contained a single gene (MOCS2, NSUN3, KHDRBS2, SNTG1 and ST18). One larger MCR of gain contained NSD1. One focal amplification and nine gains contained FUS. NSD1 and FUS are oncogenes hitherto not known to be associated with lung cancer. FISH showed that the amplicon containing FUS was joined to the next telomeric amplicon at 16p11.2. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. Other cancer genes present in aberrations included ARNT, BCL9, CDK4, CDKN2B, EGFR, ERBB2, MDM2, MDM4, MET, MYC and KRAS. Unsupervised hierarchical clustering with adjustment for false-discovery rate revealed clusters differing by the level and pattern of aberrations and displaying particular tumor characteristics. One cluster was strongly associated with gain of MYC. Another cluster was characterized by extensive losses containing tumor suppressor genes of which RB1 and WRN. Tumors in that cluster frequently harbored a central scar-like fibrosis. A third cluster was associated with gains on 7p and 7q, containing ETV1 and BRAF, and displayed the highest rate of EGFR mutations. SNP array analysis validated copy-number aberrations and revealed that RB1 and WRN were altered by recurrent copy-neutral loss of heterozygosity.

Conclusions

The present study has uncovered new aberrations containing cancer genes. The oncogene FUS is a candidate gene in the 16p region that is frequently gained in never smokers. Multiple genetic pathways defined by gains of MYC, deletions of RB1 and WRN or gains on 7p and 7q are involved in lung adenocarcinoma in never smokers.

Background and aims

Crohn's disease (CD) ileal lesions are colonised by pathogenic adherent-invasive Escherichia coli (AIEC) producing outer membrane vesicles (OMVs) that contribute to the bacterial invasion process. In addition, increased expression of endoplasmic reticulum (ER)-localised stress response proteins, due to ER stress, is observed in patients with CD. The expression of the ER-localised stress response protein Gp96 in patients with CD and its biological role with regards to the ability of AIEC to invade intestinal epithelial cells were analysed.

Methods and results

Immunohistochemistry on tissue arrays showed that, together with CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6) or the ER stress protein Grp78, Gp96 is also strongly expressed at the apical plasma membrane of the ileal epithelial cells of 50% of patients with CD. Invasion experiments in the presence of antibodies raised against Gp96, or after transfection of Intestine-407 cells with gp96 small interfering RNA (siRNA), indicated that Gp96 is essential to promote AIEC LF82 invasion, allowing, via the recognition of the outer membrane protein OmpA, OMVs to fuse with intestinal epithelial cells.

Conclusions

Gp96 is overexpressed on the apical surface of ileal epithelial cells in patients with CD and acts as a host cell receptor for OMVs, promoting AIEC invasion. From the results shown here, it is speculated that AIEC could take advantage of the abnormal expression of Gp96 in patients with CD to invade the ileal mucosa.

Background

The role(s) of mast cells (MC) in gastric mucosal inflammation caused by Helicobacterpylori is (are) still debated.

Aim

To determine whether there is an association between MC density and epithelial cell apoptosis in antral gastric mucosa infected by H pylori.

Patients and methods

Biopsy specimens from 122 H pylori‐positive subjects with chronic active gastritis, 84 patients with non‐steroidal anti‐inflammatory drug‐induced gastritis and 48 volunteers were included. H pylori genotypes were determined by PCR amplification of bacterial cultures. Immunohistochemical analysis was performed on tissue microarrays with anti‐CD117, anti‐chymase, anti‐tryptase, anti‐myeloperoxidase, anti‐Bcl‐2, anti‐Bcl‐x, anti‐Bax and anti‐caspase 3 antibodies.

Results

Of the 122 patients infected with H pylori, 76 (62.3%) harboured cagA positive strains. H pylori isolates belonged to the vacAs1/m1 genotype in 82 (67%) cases, to the vacAs2/m2 genotype in 23 (18.8%) cases and to the vacAs1/m2 genotype in 17 (13.9%) cases. 61 (50%) H pylori isolates were babA2+. In patients infected with H pylori, the density of MC, and in particular the number of MC‐associated epithelial cells, was correlated with a high number of apoptotic epithelial cells. Moreover, the density of MC was correlated with the number of neutrophils infiltrating the antral gastric mucosa, and was strongly increased in patients infected with cagA, vacAs1/m1 and babA2 positive strains.

Conclusions

Taken together, these data show that the density of MC can be considered as a histopathological criterion of gastritis activity in patients infected with H pylori.

OBJECTIVE

Type 2 diabetes is associated with left ventricular hypertrophy (LVH) and diastolic dysfunction, which may eventually lead to clinical heart failure. We sought to determine the cardiovascular effects of adolescent-onset type 2 diabetes.

RESEARCH DESIGN AND METHODS

We recruited diabetic girls (8 with type 2 and 11 with type 1 diabetes) from a hospital diabetes service and nondiabetic control subjects (9 lean and 11 overweight) from the schools of the diabetic subjects. Echocardiography and measurements were performed by a single observer, blinded to subject group allocation, and included M-mode left ventricular dimensions, two-dimensional left ventricular mass, Doppler diastolic flows, estimation of left ventricular filling pressure, and systolic longitudinal motion. Left ventricular mass was indexed to height and fat-free body mass. ANOVA was used to compare the groups.

RESULTS

The groups were similar in age and height, but significant differences in body composition were observed. Subjects with type 2 diabetes had larger left ventricular dimensions and left ventricular mass, which persisted when indexed to height. Diastolic filling was impaired in both diabetic groups, and systolic longitudinal function was lower in the type 2 diabetic group. Half of the group with type 2 diabetes met the published criteria for LVH and left ventricular dilatation; 25% had evidence of elevated left ventricular filling pressure in association with structural abnormalities.

CONCLUSIONS

This study has demonstrated preclinical abnormalities of cardiac structure and function in adolescent girls with type 2 diabetes, despite the short duration of diabetes and highlights the potential high cardiovascular risk occurring in adolescent type 2 diabetes.

Immunocompromised patients who develop invasive filamentous mycotic infections can be efficiently treated if rapid identification of the causative fungus is obtained. We report a case of fatal necrotic pneumonia caused by combined pulmonary invasive mucormycosis and aspergillosis in a 66 year-old renal transplant recipient. Aspergillus was first identified during the course of the disease by cytological examination and culture (A. fumigatus) of bronchoalveolar fluid. Hyphae of Mucorales (Rhizopus microsporus) were subsequently identified by culture of a tissue specimen taken from the left inferior pulmonary lobe, which was surgically resected two days before the patient died. Histological analysis of the lung parenchyma showed the association of two different filamentous mycoses for which the morphological features were evocative of aspergillosis and mucormycosis. However, the definitive identification of the associative infection was made by polymerase chain reaction (PCR) performed on deparaffinized tissue sections using specific primers for aspergillosis and mucormycosis. This case demonstrates that discrepancies between histological, cytological and mycological analyses can occur in cases of combined mycotic infection. In this regard, it shows that PCR on selected paraffin blocks is a very powerful method for making or confirming the association of different filamentous mycoses and that this method should be made available to pathology laboratories.

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

Background

Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology

VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal Findings

C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions

Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.

The proinflammatory effect of Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains have been recently demonstrated in vitro by showing that polymorphonuclear leukocyte (PMN) transepithelial migration is induced after bacterial colonization of apical intestinal monolayers. The effect of Afa/Dr DAEC-PMN interaction on PMN behavior has been not investigated. Because of the putative virulence mechanism of PMN apoptosis during infectious diseases and taking into account the high level of expression of the decay-accelerating factor (DAF, or CD55), the receptor of Afa/Dr DAEC on PMNs, we sought to determine whether infection of PMNs by Afa/Dr DAEC strains could promote cell apoptosis. We looked at the behavior of PMNs incubated with Afa/Dr DAEC strains once they had transmigrated across polarized monolayers of intestinal (T84) cells. Infection of PMNs by Afa/Dr DAEC strains induced PMN apoptosis characterized by morphological nuclear changes, DNA fragmentation, caspase activation, and a high level of annexin V expression. However, transmigrated and nontransmigrated PMNs incubated with Afa/Dr DAEC strains showed similar elevated global caspase activities. PMN apoptosis depended on their agglutination, induced by Afa/Dr DAEC, and was still observed after preincubation of PMNs with anti-CD55 and/or anti-CD66 antibodies. Low levels of phagocytosis of Afa/Dr DAEC strains were observed both in nontransmigrated and in transmigrated PMNs compared to that observed with the control E. coli DH5α strain. Taken together, these data strongly suggest that interaction of Afa/Dr DAEC with PMNs may increase the bacterial virulence both by inducing PMN apoptosis through an agglutination process and by diminishing their phagocytic capacity.