Droplet digital PCR of serum miR-499, miR-21 and miR-208a for the detection of functionally relevant coronary artery disease.

BACKGROUND: microRNAs (miRNAs) have shown promise as potential new biomarkers for myocardial injury and myocardial ischemia. New digital polymerase chain reaction (PCR) techniques allow for highly precise and reliable absolute direct quantification. METHODS: In this pilot study we used droplet digital PCR (ddPCR) to assess if miRNAs might be released into circulation in patients with functionally relevant coronary artery disease (CAD). Blood samples for measurement of high-sensitivity cardiac troponin I (hs-cTnI) and miRNAs were obtained before, immediately after peak stress, and 2 h after stress testing in a blinded manner in consecutive patients referred for rest/stress myocardial perfusion single-photon emission tomography/computer tomography (MPI-SPECT/CT). ddPCR was used to directly quantify the serum concentrations of miR-21, miR-208a, and miR-499 as potential markers of myocardial injury/ischemia. Functionally relevant CAD was determined by expert interpretation of MPI-SPECT/CT, coronary angiography and fractional flow reserve, if performed. RESULTS: Overall, 200 patients were included and functionally relevant CAD was detected in 85 of them (42%). Neither miR-21, miR-208a, nor miR-499 concentrations differed at rest, stress, or 2-h after stress when comparing patients with versus without functionally relevant CAD, while hs-cTnI concentrations were significantly higher in patients with functionally relevant CAD (P < 0.001). Exercise-induced changes in miRNA or hs-cTnI concentrations did not have diagnostic utility and were similar in patients with versus without functionally relevant CAD. CONCLUSION: miR-208a, miR-21 and miR-499 concentrations at rest, after exercise and exercise-induced changes do not provide additional clinical value regarding the detection of functionally relevant CAD.

Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction.

BACKGROUND: micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. AIMS: We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). METHODS: A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR. We used ddPCR and qRT-PCR to quantify the serum levels of miR-21, miR-208a and miR-499 between STEMI patients (n=24) and stable coronary artery disease (CAD) patients (n=20). In STEMI, I/R injury was assessed via measurement of ST-segment resolution. RESULTS: In the dilution series, ddPCR demonstrated superior coefficient of variation (12.1%vs.32.9%) and limit of detection (0.9325 vs.2.425copies/µl). In the patient cohort, ddPCR demonstrated greater differences in miR-21 levels (2190.5 vs. 484.7copies/µl; p=0.0004 for ddPCR and 136.4 vs. 122.8copies/µl; p=0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1copies/µl, p=0.0013 for ddPCR and 0 vs. 0copies/µl, p=0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5copies/µl, p<0.0001 for ddPCR and 0 vs. 19.41copies/µl, p<0.0001 for qRT-PCR). ddPCR also more accurately defined STEMI for all miRNAs (area under the curve (AUC) of 0.8021/0.7740/0.9063 for miR-21/208a/499 with ddPCR vs. AUC of 0.6083/0.6917/0.8417 with qRT-PCR). However, there was no association between miR-21/208a/499 levels and ischemia-reperfusion injury. CONCLUSION: ddPCR demonstrates superiority in both technical performance and diagnostic potential compared to qRT-PCR. Ultimately, this supports its use as a diagnostic method for quantifying micro-RNAs, particularly in large multi-center trials.

Levels of activated platelet-derived microvesicles in patients with soft tissue sarcoma correlate with an increased risk of venous thromboembolism.

BACKGROUND: Microvesicles are small vesicles expressing specific antigens from their cells of origin. Elevated levels of microvesicles have been shown to be associated with coagulation disorders as well as with different types of malignancies. This study aims to evaluate a possible correlation of different microvesicle subpopulations with a positive history of venous thromboembolism (VTE) in patients with soft tissue sarcoma. METHODS: Annexin V - positive microvesicles, leukocyte (CD45-positive), platelet (CD61-positive), activated platelet (CD62P-, CD63-positive), endothelium-derived (CD62E-positive) and tissue-factor (CD142-positive) microvesicles were identified in the peripheral blood of patients with soft tissue sarcoma (n = 39) and healthy controls (n = 17) using fluorescence-activated cell sorting (FACS). RESULTS: Both the total amount of Annexin V-positive microvesicles and levels of endothelium-derived (CD62E-positive) microvesicles were shown to decrease significantly after tumor resection (n = 18, p = 0.0395 and p = 0.0109, respectively). Furthermore, the total amount of Annexin V - positive microvesicles as well as leukocyte (CD45-positive) and endothelium-derived (CD62E-positive) microvesicles were significantly higher in patients with grade 3 (G3) soft tissue sarcoma (n = 9) compared to healthy controls (n = 17) (p = 0.0304, p = 0.0254 and p = 0.0357, respectively). Moreover, patients with G3 soft tissue sarcoma (n = 9) presented higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles compared to patients with grade 2 (G2) soft tissue sarcoma (n = 8) (p = 0.0483 and p = 0.0045). Patients with grade 1 (G1) soft tissue sarcoma (n = 3) presented with significantly lower levels of platelet (CD61-positive) microvesicles than patients with G3 soft tissue sarcoma (n = 9) (p = 0.0150). In patients with a positive history of VTE (n = 11), significantly higher levels of activated platelet (CD62P- and CD63-positive) microvesicles (p = 0.0078 and p = 0.0450, respectively) were found compared to patients without a history of VTE (n = 28). CONCLUSION: We found significantly higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles to be circulating in the peripheral blood of patients with G3 soft tissue sarcoma compared to patients with G2 soft tissue sarcoma. Furthermore, we showed that high counts of activated platelet-derived microvesicles correlate with the occurrence of VTE. Thus, the detection of these microvesicles might be an interesting new tool for early diagnosis of soft tissue sarcoma patients with increased risk for VTE, possibly facilitating VTE prevention by earlier use of thromboprophylaxis.

Partial break in tolerance of NKG2A-/LIR-1- single KIR+ NK cells early in the course of HLA-matched, KIR-mismatched hematopoietic cell transplantation.

Natural killer (NK) cell subpopulations from 8 HLA-matched but killer cell immunoglobulin-like receptor (KIR)/HLA-ligand-mismatched patient-donor pairs were analyzed in the course of allogeneic hematopoietic stem cell transplantation (HCT). The patients' post-transplantation NKG2A-/LIR-1- NK cells, which expressed only inhibitory KIRs for which the patient had no HLA class I ligands, showed higher cytotoxic capacity than the NKG2A-/LIR-1- NK cells lacking any inhibitory KIRs that remained tolerant throughout the course of HCT. The NKG2A+ NK cell subpopulations displayed the highest levels of cytotoxic activation, which appeared to be significantly enhanced in comparison with that in allogeneic graft's donors. LIR-1- NK cells were much more frequent after HCT than LIR-1+ NK cells and LIR-1 expression on NKG2A+ or NKG2A- NK cells was associated with significantly lower cytotoxic activities. Thus NKG2A-/LIR-1- NK cells expressing only HLA-mismatched KIRs show a partial break in tolerance in the first year following HCT. The failure to exclude LIR-1+ cells within the NKG2A- NK cell subset in previous studies could explain the earlier conflicting results. Thus systemic immune activation in patients following HCT augments the GvL effect through both increasing overall NK cell activities and partially breaking tolerance of unlicensed NK cells.

Synovial Sarcoma Microvesicles Harbor the SYT-SSX Fusion Gene Transcript: Comparison of Different Methods of Detection and Implications in Biomarker Research.

Background. Synovial sarcoma is an aggressive soft-tissue malignancy. This study examines the presence of the SYT-SSX fusion transcript in synovial sarcoma microvesicles as well as its potential role as a biomarker for synovial sarcoma. Patients and Methods. Microvesicle release of synovial sarcoma cells was examined by transmission electron microscopy. RNA-content was analyzed by qPCR, nested PCR, nested qPCR, and droplet digital PCR to compare their sensitivity for detection of the SYT-SSX fusion gene transcript. Whole blood RNA, RNA of mononuclear cells, and microvesicle RNA of synovial sarcoma patients were analyzed for the presence of the fusion gene transcripts. Results. Electron microscopic analysis revealed synovial sarcoma cells releasing membrane-enclosed microvesicles. In vitro, the SYT-SSX fusion gene transcript was detected in both synovial sarcoma cells and microvesicles. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA. In contrast, the fusion gene transcript was not detected in peripheral blood cells and microvesicles of synovial sarcoma patients. Conclusion. Synovial sarcoma cells release microvesicles harboring the SYT-SSX fusion transcript. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA; however, more sensitive assays are needed to detect cancer-specific microvesicles in the peripheral blood of cancer patients.

The role of APC/C(Cdh1) in replication stress and origin of genomic instability.

It has been proposed that the APC/C(Cdh1) functions as a tumor suppressor by maintaining genomic stability. However, the exact nature of genomic instability following loss of Cdh1 is unclear. Using biochemistry and live cell imaging of single cells we found that Cdh1 knockdown (kd) leads to strong nuclear stabilization of the substrates cyclin A and B and deregulated kinetics of DNA replication. Restoration of the Cdh1-dependent G2 DNA damage checkpoint did not result in G2 arrest but blocked cells in prometaphase, suggesting that these cells enter mitosis despite incomplete replication. This results in DNA double-strand breaks, anaphase bridges, cytokinesis defects and tetraploidization. Tetraploid cells are the source of supernumerary centrosomes following Cdh1-kd, leading to multipolar mitosis or centrosome clustering, in turn resulting in merotelic attachment and lagging chromosomes. Whereas some of these events cause apoptosis during mitosis, surviving cells may accumulate chromosomal aberrations.

Novel Broad-Spectrum Antimicrobial Photoinactivation of In Situ Oral Biofilms by Visible Light plus Water-Filtered Infrared A.

Antimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtained in situ; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 µg ml(-1) toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm(-2) for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log10 CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.

In vivo study of the initial bacterial adhesion on different implant materials.

OBJECTIVE: Biofilm formation on implant materials plays a major role in the aetiology of periimplantitis. The aim of this study was to examine in vivo the initial bacterial adhesion on six different implant materials. METHODS: The implant materials Ti-m, TiUnite®, ZiUnite®, ATZ-m, ATZ-s, TZP-A-m were tested using bovine enamel slabs as controls. All materials, fixed on splint systems, were examined after 30 min and 120 min of oral exposure. DAPI staining was used for quantitative analysis of the initially adherent microorganisms. Initial adherent microorganisms were visualised by fluorescence In situ-hybridisation (FISH) and quantified by confocal laser scanning microscopy (CLSM). The targets of the oligonucleotide probes were Eubacteria, Veillonella spp., Fusobacterium nucleatum, Actinomyces naeslundii and Streptococcus spp. RESULTS: DAPI analysis showed that increasing the time of oral exposure resulted in an increasing amount of initial adherent bacteria. The highest level of colonisation was on ZiUnite®, with the lowest occurring on the bovine enamel, followed by Ti-m. This early colonisation correlated significantly with the surface roughnesses of the materials. FISH and CLSM showed no significant differences relating to total bacterial composition. However, Streptococcus spp. was shown to be the main colonisers on each of the investigated materials. CONCLUSION: it could be shown that within an oral exposure time of 30 min and 120 min, despite the salivary acquired pellicle initial biofilm formation is mainly influenced directly or indirect by the material surface topography. Highly polished surfaces should minimise the risk of biofilm formation, plaque accumulation and possibly periimplantitis.

Microscope-based imaging platform for large-scale analysis of oral biofilms.

A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grown in situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.

Activation of nuclear inositide signalling pathways during erythropoietin therapy in low-risk MDS patients.

Inositide signaling pathways can have a role in the Myelodysplastic Syndromes (MDS) progression to acute myeloid leukemia. Erythropoietin (EPO) is currently used in low-risk MDS, where it successfully corrects anemia in 50-70% of patients. However, some MDS patients are refractory to this treatment and little is known about the exact molecular mechanisms underlying the effect of EPO in these subjects. Here, we investigated the role of inositide pathways in low-risk MDS treated with EPO, mainly focusing on the Akt/PI-PLC (Phosphoinositide-Phospholipase C) gamma1 axis, which is activated by the EPO receptor, and PI-PLCbeta1/Cyclin D3 signaling, as Cyclin D3 is associated with hematopoietic proliferation and differentiation. Interestingly, EPO responder patients showed a specific activation of both the Akt/PI-PLCgamma1 pathway and beta-Globin gene expression, while nonresponders displayed an increase in PI-PLCbeta1 signaling. Moreover, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and beta-Globin expression. Overall, these findings suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of Akt/PI-PLCgamma1 pathway in EPO signaling, therefore contributing to the comprehension of the effect of EPO in low-risk MDS and possibly paving the way to the identification of MDS patients at higher risk of refractoriness to EPO treatment.

Epigenetic regulation of nuclear PI-PLCbeta1 signaling pathway in low-risk MDS patients during azacitidine treatment.

Phosphoinositide-phospholipase C (PI-PLC) beta1 can be considered a specific target for demethylating therapy in high-risk myelodysplastic syndrome (MDS) patients, as azacitidine treatment has been associated with a PI-PLCbeta1-specific promoter demethylation, and induction of PI-PLCbeta1 gene and protein expression. However, little is known about the molecular effect of azacitidine in low-risk MDS or the functional mechanisms linked with azacitidine effect on PI-PLCbeta1 promoter. In the present study, we further investigated the role of epigenetic regulation of PI-PLCbeta1, mainly focusing on the structure of the PI-PLCbeta1 promoter. We first examined the effect of azacitidine on PI-PLCbeta1 promoter methylation and gene expression in low-risk MDS. Moreover, we studied the expression of key molecules associated with the nuclear inositide signaling pathways, such as cyclin D3. By applying a chromatin immunoprecipitation method, we also studied the correlation between the demethylating effect of azacitidine and the degree of recruitment to PI-PLCbeta1 promoter of some transcription factors implicated in hematopoietic stem cell proliferation and differentiation, as well as of the methyl-CpG-binding domain proteins, which specifically interact with methylated DNA. Taken together, our results hint at a specific involvement of PI-PLCbeta1 in epigenetic mechanisms, and are particularly consistent with the hypothesis of a role for PI-PLCbeta1 in azacitidine-induced myeloid differentiation.

Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress.

A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

Synergistic induction of PI-PLCß1 signaling by azacitidine and valproic acid in high-risk myelodysplastic syndromes.

The association between azacitidine (AZA) and valproic acid (VPA) has shown high response rates in high-risk myelodysplastic syndromes (MDS) cases with unfavorable prognosis. However, little is known about the molecular mechanisms underlying this therapy, and molecular markers useful to monitor the disease and the effect of the treatment are needed. Phosphoinositide-phospholipase C (PI-PLC) ß1 is involved in both genetic and epigenetic mechanisms of MDS progression to acute myeloid leukemia. Indeed, AZA as a single agent was able to induce PI-PLCß1 expression, therefore providing a promising new tool in the evaluation of response to demethylating therapies. In this study, we assessed the efficacy of the combination of AZA and VPA on inducing PI-PLCß1 expression in high-risk MDS patients. Furthermore, we observed an increase in Cyclin D3 expression, a downstream target of PI-PLCß1 signaling, therefore suggesting a potential combined activity of AZA and VPA in high-risk MDS in activating PI-PLCß1 signaling, thus affecting cell proliferation and differentiation. Taken together, our findings might open up new lines of investigations aiming at evaluating the role of the activation of PI-PLCß1 signaling in the epigenetic therapy, which may also lead to the identification of innovative targets for the epigenetic therapy of high-risk MDS.

Transgenic expression of human cathepsin B promotes progression and metastasis of polyoma-middle-T-induced breast cancer in mice.

Elevated expression of the cysteine protease cathepsin B (CTSB) has been correlated with a poor prognosis for cancer patients. In order to model high CTSB expression in mammary cancer, transgenic mice expressing human CTSB were crossed with transgenic polyoma virus middle T oncogene breast cancer mice (mouse mammary tumor virus-PymT), resulting in a 20-fold increase in cathepsin B activity in the tumors of double-transgenic animals. CTSB expression did not affect tumor onset, but CTSB transgenic mice showed accelerated tumor growth with significant increase in weight for end-stage tumors, as well as an overall worsening in their histopathological grades. Notably, the lung metastases in the CTSB transgenic animals were found to be both significantly larger and to occur at a significantly higher frequency. Ex vivo analysis of primary PymT tumor cells revealed no significant effects from elevated CTSB levels on tumor cell characteristics, that is, the formation of tumor cell colonies and the sprouting of invasive strands from PymT cell spheroids. However, tumors from CTSB-overexpressing mice showed increased numbers of tumor-associated B cells and mast cells. In addition, more CD31+ endothelial cells were detected in these tumors, correlating with higher levels of vascular endothelial growth factor (VEGF) being present in the tumor and serum. We conclude that elevated proteolytic CTSB activity facilitates progression and metastasis of PymT-induced mammary carcinomas, and is associated with increased immune cell infiltration, enhanced VEGF levels and the promotion of tumor angiogenesis.

Biofilm formation and composition on different implant materials in vivo.

Biofilm formation was evaluated on the following titanium and zirconia implants in vivo: machined titanium (Ti-m), modified titanium (TiUnite), modified zirconia (ZiUnite), machined alumina-toughened zirconia (ATZ-m), sandblasted alumina-toughened zirconia (ATZ-s), and machined zirconia (TZP-A-m). Bovine enamel slabs were used as controls. Surface morphologies were examined by atomic force (AFM) and scanning electron microscopy (SEM). The surface wettability was also determined. Twelve healthy volunteers wore a splint system with the tested materials. After 3 and 5 days the materials were examined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). The levels of Streptococcus spp., Veillonella spp., Fusobacteriaum nucleatum, and Actinomyces naeslundii were quantitatively determined. The biofilm thickness was found to be between 19.78 and 36.73 µm after 3 days and between 26.11 and 32.43 µm after 5 days. With the exception of Ti-m the biofilm thickness after 3 days was correlated with surface roughness. In addition to Streptococcus spp. as the main component of the biofilm (11.23-25.30%), F. nucleatum, A. naeslundii, and Veillonella spp. were also detected. No significant differences in biofilm composition on the implant surfaces could be observed. In total, the influence of roughness and material on biofilm formation was compensated by biofilm maturation.

Detection of disseminated tumour cells in mediastinoscopic lymph node biopsies and endobronchial ultrasonography-guided transbronchial needle aspiration in patients with suspected lung cancer.

PURPOSE: Ultrasound-guided transbronchial needle aspiration of mediastinal lymph nodes (EBUS-TBNA) is apparently more accurate for cancer diagnosis than standard transbronchial needle aspiration (TBNA), but it is less sensitive than mediastinoscopy. The detection of disseminated tumour cells in transbronchial needle aspiration and mediastinoscopic biopsies could improve staging and might be helpful concerning indications for neoadjuvant regimen. The goal of this study was to develop a quantitative method for the detection of disseminated tumour cells (DTCs) in lymph node samples from patients with suspected lung cancer. PATIENTS AND METHODS: We compared in a prospective trail EBUS-TBNA (n=58 patients, 86 samples) and mediastinoscopy (n=22 patients, 37 samples) in two largely independent cohorts of lung cancer patients. Eleven patients, 14 samples were analysed using both methods. Patients without evidence of malignant disease were available as controls for EBUS-TBNA (n=20 patients, 28 samples) and mediastinoscopy (n=6 patients, 8 samples). Real-time quantitative mRNA analysis was performed for the cytokeratin 19 (CK19) and MAGE-A genes (MAGE-A 1-6, MAGE-A12) as markers, using a LightCycler 480 instrument. RESULTS: CK19 mRNA expression in EBUS-TBNA samples was detected in 84/86 (98%) and in 28/28 control samples (100%). After mediastinoscopy 16/37 (43%) samples of lung cancer patients were CK19 mRNA positive while controls showed no CK19 mRNA expression (0/8). MAGE-A expression was detectable in 42/86 (49%) EBUS-TBNA samples and in 13/37 (35%) mediastinoscopy samples. MAGE-A expression was detected in EBUS-TBNA controls in 3/28 (11%) and 1/8 (12%) mediastinoscopy controls. High MAGE-A expression correlated with increased tumour stage. CONCLUSION: Since CK19 expression was detected in all EBUS-TBNA samples from the control patients, but not in mediastinoscopy samples, we conclude that CK19 is not suitable as a marker for disseminated tumour cells in samples attained by EBUS-TBNA. One possible explanation is a contamination with epithelial cells from the bronchial tubes. MAGE-A genes are promising markers for disseminated tumour cells in lymph nodes in patients with suspected lung cancer which merit further investigation.

The novel Akt inhibitor, perifosine, induces caspase-dependent apoptosis and downregulates P-glycoprotein expression in multidrug-resistant human T-acute leukemia cells by a JNK-dependent mechanism.

A significant impediment to the success of cancer chemotherapy is the occurrence of multidrug resistance, which, in many cases, is attributable to overexpression of membrane transport proteins, such as the 170-kDa P-glycoprotein (P-gp). Also, upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway is known to play an important role in drug resistance, and has been implicated in the aggressiveness of a number of different cancers, including T-acute lymphoblastic leukemia (T-ALL). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine (a synthetic alkylphospholipid), on human T-ALL CEM cells (CEM-R), characterized by both overexpression of P-gp and constitutive upregulation of the PI3K/Akt network. Perifosine treatment induced death by apoptosis in CEM-R cells. Apoptosis was characterized by caspase activation, Bid cleavage and cytochrome c release from mitochondria. The proapoptotic effect of perifosine was in part dependent on the Fas/FasL interactions and c-Jun NH(2)-terminal kinase (JNK) activation, as well as on the integrity of lipid rafts. Perifosine downregulated the expression of P-gp mRNA and protein and this effect required JNK activity. Our findings indicate that perifosine is a promising therapeutic agent for treatment of T-ALL cases characterized by both upregulation of the PI3K/Akt survival pathway and overexpression of P-gp.