An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

A systematic review assessing soft tissue augmentation techniques

AIM: The aim of the present review was to systematically assess the dental literature in terms of soft tissue grafting techniques. The focused question was: is one method superior over others for augmentation and stability of the augmented soft tissue in terms of increasing the width of keratinized tissue (part 1) and gain in soft tissue volume (part 2). METHODS: A Medline search was performed for human studies focusing on augmentation of keratinized tissue and/or soft tissue volume, and complemented by additional hand searching. Relevant studies were identified and statistical results were reported for meta-analyses including the test minus control weighted mean differences with 95% confidence intervals, the I-squared statistic for tests of heterogeneity, and the number of significant studies. RESULTS: Twenty-five (part 1) and three (part 2) studies met the inclusion criteria; 14 studies (part 1) were eligible for comparison using meta-analyses. An apically positioned flap/vestibuloplasty (APF/V) procedure resulted in a statistically significantly greater gain in keratinized tissue than untreated controls. APF/V plus autogenous tissue revealed statistically significantly more attached gingiva compared with untreated controls and a borderline statistical significance compared with APF/V plus allogenic tissue. Statistically significantly more shrinkage was observed for the APF/V plus allogenic graft compared with the APF/V plus autogenous tissue. Patient-centered outcomes did not reveal any of the treatment methods to be superior regarding postoperative complications. The three studies reporting on soft tissue volume augmentation could not be compared due to lack of homogeneity. The use of subepithelial connective tissue grafts (SCTGs) resulted in statistically significantly more soft tissue volume gain compared with free gingival grafts (FGGs). CONCLUSIONS: APF/V is a successful treatment concept to increase the width of keratinized tissue or attached gingiva around teeth. The addition of autogenous tissue statistically significantly increases the width of attached gingiva. For soft tissue volume augmentation, only limited data are available favoring SCTGs over FGG.

Tissue coverage and neointimal hyperplasia in overlap versus nonoverlap segments of drug-eluting stents 9 to 13 months after implantation: in vivo assessment with optical coherence tomography

BACKGROUND Histologic experimental studies have reported incomplete neointimal healing in overlapping with respect to nonoverlapping segments in drug-eluting stents (DESs), but these observations have not been confirmed in human coronary arteries hitherto. On the contrary, angiographic and optical coherence tomography studies suggest that DES overlap elicits rather an exaggerated than an incomplete neointimal reaction. METHODS Optical coherence tomography studies from 2 randomized trials including sirolimus-eluting, biolimus-eluting, everolimus-eluting, and zotarolimus-eluting stents were analyzed at 9- to 13-month follow-up. Coverage in overlapping segments was compared versus the corresponding nonoverlapping segments of the same stents, using statistical pooled analysis. RESULTS Forty-two overlaps were found in 31 patients: 11 in sirolimus-eluting stents, 3 in biolimus-eluting stents, 17 in everolimus-eluting stents, and 11 in zotarolimus-eluting stents. The risk ratio of incomplete coverage was 2.35 (95% CI 1.86-2.98) in overlapping versus nonoverlapping segments. Thickness of coverage in overlaps was only 85% (95% CI 81%-90%) of the thickness in nonoverlaps. Significant heterogeneity of the effect was observed, especially pronounced in the comparison of thickness of coverage (I(2) = 90.31). CONCLUSIONS The effect of overlapping DES on neointimal inhibition is markedly heterogeneous: on average, DES overlap is associated with more incomplete and thinner coverage, but in some cases, the overlap elicits an exaggerated neointimal reaction, thicker than in the corresponding nonoverlapping segments. These results might help to understand why overlapping DES is associated with worse clinical outcomes, both in terms of thrombotic phenomena and in terms of restenosis and revascularization.

Heterogeneity analysis of Metastasis Associated in Colon Cancer 1 (MACC1) for survival prognosis of colorectal cancer patients: a retrospective cohort study.

BACKGROUND Metastasis of colorectal cancer (CRC) is directly linked to patient survival. We previously identified the novel gene Metastasis Associated in Colon Cancer 1 (MACC1) in CRC and demonstrated its importance as metastasis inducer and prognostic biomarker. Here, we investigate the geographic expression pattern of MACC1 in colorectal adenocarcinoma and tumor buds in correlation with clinicopathological and molecular features for improvement of survival prognosis. METHODS We performed geographic MACC1 expression analysis in tumor center, invasive front and tumor buds on whole tissue sections of 187 well-characterized CRCs by immunohistochemistry. MACC1 expression in each geographic zone was analyzed with Mismatch repair (MMR)-status, BRAF/KRAS-mutations and CpG-island methylation. RESULTS MACC1 was significantly overexpressed in tumor tissue as compared to normal mucosa (p < 0.001). Within colorectal adenocarcinomas, a significant increase of MACC1 from tumor center to front (p = 0.0012) was detected. MACC1 was highly overexpressed in 55% tumor budding cells. Independent of geographic location, MACC1 predicted advanced pT and pN-stages, high grade tumor budding, venous and lymphatic invasion (p < 0.05). High MACC1 expression at the invasive front was decisive for prediction of metastasis (p = 0.0223) and poor survival (p = 0.0217). The geographic pattern of MACC1 did not correlate with MMR-status, BRAF/KRAS-mutations or CpG-island methylation. CONCLUSION MACC1 is differentially expressed in CRC. At the invasive front, MACC1 expression predicts best aggressive clinicopathological features, tumor budding, metastasis formation and poor survival outcome.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.