Chronic smoke exposure is associated with autophagy in murine Peyer's patches

INTRODUCTION: Cigarette smoke causes oxidative stress, leading to smoke-induced autophagy in several organs. Autophagy is a homeostatic process regulating the turnover of proteins and cytoplasmatic organelles. However, recently it has also been associated with many autoimmune and inflammatory disorders, among which Crohn’s disease. The purpose of the present study was to investigate whether cigarette smoke exposure is associated with increased autophagy in Peyer’s patches and its epithelium. AIMS & METHODS: C57BL/6 mice were exposed to cigarette smoke or air. After 24 weeks, the animals were sacrificied and Peyer’s patches were collected. m RNA expression of autophagy-related genes was determined by RT-PCR. Transmission electron microscopy (TEM) was used to evaluate the presence of autophagic vesicles in the follicleassociated epithelium of Peyer’s patches. RESULTS: Expression of Beclin-1, a protein involved in the nucleation of autophagosomes, and of Atg5 and Atg7, which both play a role in the autophagosome vesicle elongation and completion, increased after chronic smoke exposure. Furthermore, electron microscopy of the follicle-associated epithelium demonstrated that the mean area of autophagic vesicles per epithelial cell increased considerably from 1.1 μm2 ± 0.4 μm2 in the air group to 2.4 μm2 ± 0.4 μm2 in the smoke group (p < 0.05). Epithelial cells had a significantly higher number of autophagic vesicles after smoke exposure (1.1 ± 0.1 after smoke exposure versus 0.5 ± 0.1 vesicles per cell after air exposure, p < 0.05), but the size of the vesicles did not differ between both groups. CONCLUSION: Here we provide the first evidence that chronic exposure to cigarette smoke is associated with autophagy in murine Peyer’s patches, and more in particular in the follicle-associated epithelium covering Peyer’s patches. Our findings can help to understand the role of smoking in the pathogenesis of inflammatory bowel disease, such as Crohn’s disease

Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593-0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73-2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83-7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice

Cigarette smoking alters intestinal barrier function and Peyer's Patch composition

Smokers have a two-fold increased risk to develop Crohn’s disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Interestingly, the Peyer’s patches in the terminal ileum are the sites where the first CD lesions develop. To investigate whether smoke exposure causes alterations in Peyer’s patches, we studied C57BL/6 mice after exposure to air or cigarette smoke for 24 weeks. First, barrier function of the follicle-associated epithelium overlying Peyer’s patches was evaluated. We demonstrate that chronic smoke exposure is associated with increased apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in the ileal Peyer’s patches. We observed significant increases in total dendritic cells (DC), CD4+ T-cells (including regulatory T-cells) and CD8+ T-cells after smoke exposure compared with air-exposed animals. The CD11b+ DC subset almost doubled. Interestingly, these changes were accompanied by an up-regulated mRNA expression of the chemokines CCL9 and CCL20, which are known to attract CD11b+ DC towards the subepithelial dome of Peyer’s patches. Our results demonstrate that cigarette smoke exposure induces apoptosis in follicle-associated epithelium and is associated with immune cell accumulation in Peyer’s patches, changes which can predispose to the development of CD

Cigarette smoke induces apoptosis in the follicle-associated epithelium of murine Peyer's patches

Background: Recently, cigarette smoking has been associated with the development of several auto-immune diseases, including rheumatoid arthritis and inflammatory bowel disease (IBD). The cellular and molecular mechanisms through which cigarette smoking predisposes to IBD are unknown. Cigarette smoke-induced apoptosis is described in several in vivo and in vitro experiments, and might play a role in the pathogenesis of several smoke-associated diseases. The aim of this study was to quantify apoptosis in normal murine Follicle-Associated Epithelium (FAE) and compare this to apoptosis in FAE of smoking mice. Methods: 8 C57BL/6 male mice were exposed to cigarette smoke for 24 weeks (chronic exposure); a control group of 8 mice was exposed to air during the same period. After 24 weeks the mice were sacrificed and Peyer’s patches of each mouse were dissected for histology. Immunohistochemistry for caspase-3 was performed on paraffin-embedded tissue sections of 11 Peyer’s patches of smoking animals and 11 Peyer’s patches of controls. To compare apoptotic activities between smokers and controls, the apoptotic index (percentage of apoptotic cells per 100 cells) in the FAE was calculated. An unpaired student T-test was applied. Results: A statistically significant increase in apoptosis of FAE cells was observed in smoking mice compared to air-exposed mice (P=0.002). In the FAE of smoking animals, the mean apoptotic index was 1.82 with a range of 1.11 to 3.00, whereas the mean apoptotic index in non-smoking animals was 0.92 (range 0.24 -2.06). Most apoptotic cells in both groups were seen at the apex of the FAE. Conclusion: We quantified rates of apoptosis in the FAE of murine Peyer’s patches. Furthermore we compared apoptosis in the FAE of smoking mice versus non-smoking siblings and observed an increased apoptotic index in the FAE of smoking animals. Our results demonstrate that cigarette smoke induces a significant increase of apoptosis in the FAE of murine Peyer’s patches and may point to a role for smoking in the pathogenesis of intestinal inflammation. Further investigation needs to clarify whether this increase in apoptosis influences normal function of the FAE

From ‘conflict minerals’ to peace? Reviewing mining reforms, gender, and state performance in eastern Democratic Republic of Congo

For the last two decades, the Congolese Artisanal and small-scale mining (ASM) sector has been undergoing reforms of its governing structures. A recurrent argument supporting the reforms - predominantly in the 3T ASM sector (i.e. tin, tantalum and tungsten) - is that artisanal mining feeds violence (including sexual violence), conflicts, underdevelopment, and poverty. The reforms have been extensively studied, and academic research in turn has triggered empirical policy and advocacy researches. Researchers have found different outcomes and interpretations of the effectiveness of the reforms. This study consists of a systematic review of the findings gathered from the literature on the changing role of the state in governing 3T mining. It argues that in the study areas (i.e. North-Kivu, South-Kivu and Tanganyika), the challenges faced by the state in governing artisanal mining tend to be similar, complex, and rooted in the mining history of eastern DRC. Recent armed conflicts have only added new layers to existing challenges. In addressing these challenges and given their complexity, the state has succeeded in some areas but has failed in putting in place a coherent `model' for governing the 3T ASM sector

Radiological sacroiliitis, a hallmark of spondylitis, is linked with CARD15 gene polymorphisms in patients with Crohn's disease

Background: Sacroiliitis is a common extraintestinal manifestation of Crohn's disease but its association with the HLA-B27 phenotype is less evident. Polymorphisms in the CARD15 gene have been linked to higher susceptibility for Crohn's disease. In particular, associations have been found with ileal and fibrostenosing disease, young age at onset of disease, and familial cases. Objectives: To investigate whether the presence of sacroiliitis in patients with Crohn's disease is linked to the carriage of CARD15 polymorphisms. Methods: 102 consecutive patients with Crohn's disease were clinically evaluated by a rheumatologist. Radiographs of the sacroiliac joints were taken and assessed blindly by two investigators. The RFLP-PCR technique was used to genotype all patients for three single nucleotide polymorphisms (SNP) in the CARD15 gene. Every SNP was verified by direct sequencing. The HLA-B27 phenotype was determined. Results: Radiological evidence of sacroiliitis with or without ankylosing spondylitis was found in 23 patients (23%), of whom only three were HLA-B27 positive. In contrast, 78% of patients with sacroiliitis carried a CARD15 variant v 48% of those without sacroiliitis (p = 0.01; odds ratio 3.8 (95% confidence interval, 1.3 to 11.5)). Multivariate analysis (logistic regression) showed that the association between sacroiliitis and CARD15 polymorphisms was independent of other CARD15 related phenotypes (ileal and fibrostenosing disease, young age at onset of disease, familial Crohn's disease) (p = 0.039). Conclusions: CARD15 variants were identified as genetic predictors of Crohn's disease related sacroiliitis. An association was demonstrated between these polymorphisms and an extraintestinal manifestation of Crohn's disease

High intensity interval training is associated with greater impact on physical fitness, insulin sensitivity and muscle mitochondrial content in males with overweight/obesity, as opposed to continuous endurance training: a randomized controlled trial

Objectives: To evaluate the effect of high intensity training (HIT) on physical fitness, basal respiratory exchange ratio (bRER), insulin sensitivity and muscle histology in overweight/obese men compared to continuous aerobic training (CAT). Material and methods: 16 male participants with overweight/obesity (age: 42-57 years, body mass index: 28-36 kg/m2) were randomized to HIT (n=8) or CAT (n=8) for 10 weeks, twice a week. HIT was composed of 10 minutes high intensity, 10 minutes continuous aerobic, 10 minutes high intensity exercises. CAT was composed of three times 10 minutes continuous exercising. Changes in anthropometry, physical and metabolic fitness were evaluated. Muscle histology (mitochondria and lipid content) was evaluated by transmission electron microscopy (TEM). Results: HIT showed a significant increase for peak VO2 (P=0.01), for insulin sensitivity (AUC glucose (P<0,001), AUC insulin (P<0,001), OGTT composite score (P=0.007)) and a significant decrease of bRER (P<0.001) compared to CAT. Muscle mitochondrial content was significantly increased after HIT at the subsarcolemmal (P=0.004 number and P=0.001 surface) as well as the intermyofibrillar site (P<0.001 number and P=0.001 surface). Conclusion: High intensity training elicits stronger beneficial effects on physical fitness, basal RER, insulin sensitivity, and muscle mitochondrial content, as compared to continuous aerobic training

Pathologic Assessment of Rectal Carcinoma after Neoadjuvant Radio(chemo)therapy: Prognostic Implications

Neoadjuvant radio(chemo)therapy is increasingly used in rectal cancer and induces a number of morphologic changes that affect prognostication after curative surgery, thereby creating new challenges for surgical pathologists, particularly in evaluating morphologic changes and tumour response to preoperative treatment. Surgical pathologists play an important role in determining the many facets of rectal carcinoma patient care after neoadjuvant treatment. These range from proper handling of macroscopic specimens to accurate microscopic evaluation of pathological features associated with patients&apos; prognosis. This review presents the well-established pathological prognostic indicators and discusses challenging features in order to provide both surgical pathologists and treating physicians with a checklist that is useful in a neoadjuvant setting