Plasma Of Healthy Donors Research Articles

19 Effect of nasopharyngeal carcinoma-derived exosomes on human regulatory T cells

Introduction Exosomes are nanovesicles found in large quantities in biological fluids and tumors of patients with nasopharyngeal carcinoma (NPC). These tumor exosomes play an important role in tumor progression due to their immunosuppressive properties. In addition, it has been reported that the frequency and suppressor functions of CD4 + CD25highFOXP3 + CD127low regulatory T cells (Treg) are also higher in NPC patients than healthy donors. Interactions between NPC-derived exosomes and Treg remain unknown. Here we investigated their ability to induce, expand, activate and recruit human Treg. Material and methods Treg recruitment by exosomes derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients’ plasma (Patient-Exo), and CCL20 was tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model ( N = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). Results CCL20 allowed the intra-tumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34-fold increase, P P P = 0.0048) the expansion of human Treg, inducing the generation of Tim3Low Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75. These results were consistent with a stronger suppression of responder cells’ proliferation ( P Conclusion Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.

Detection of high mobility group A2 specific mRNA in the plasma of patients affected by epithelial ovarian cancer.

Ovarian cancer is the most lethal gynecological malignancy and the high mortality rate is associated with advanced-stage disease at the time of the diagnosis. In order to find new tools to make diagnosis of Epithelial Ovarian Cancer (EOC) at early stages we have analyzed the presence of specific HMGA2 mRNA in the plasma of patients affected by this neoplasm. HMGA2 overexpression represents a feature of several malignances including ovarian carcinomas. Notably, we detected HMGA2 specific mRNA in the plasma of 40 out 47 patients with EOC, but not in the plasma of healthy donors. All cases found positive for HMGA2 mRNA in the plasma showed HMGA2 protein expression in EOC tissues. Therefore, on the basis of these results, the analysis of circulating HMGA2 specific mRNA might be considered a very promising tool for the early diagnosis of EOC.

Facilitated biosensing via direct electron transfer of myoglobin integrated into diblock copolymer/multi-walled carbon nanotube nanocomposites.

Nanocomposite materials were prepared by sequential drop-casting of multi-walled carbon nanotube (MWCNT) suspensions and amphiphilic polybutadiene-block-poly(2-(N,N-dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer micellar solution on screen-printed electrodes (SPEs). These nanocomposite materials were found to be very favorable for integration of myoglobin (Mb) and facilitate direct electron transfer from the electrodes to heme proteins. In that respect, PB290-b-PDMAEMA240 was demonstrated to be a well-suited binding agent. In aqueous solutions, it forms core-corona micelles (shown by cryogenic transmission electron microscopy, cryo-TEM, and nanoparticle tracking analysis, NTA), which at pH 7 in phosphate buffer exhibit good adhesion to carbon materials (shown by atomic force microscopy, AFM, scanning electron microscopy, SEM, and scanning transmission electron microscopy, STEM) and build up uniform thin films on a hydrophobic graphite-based substrate. As demonstrated by a quartz crystal microbalance with dissipation monitoring, QCM-D, attractive interactions between Mb and PB290-b-PDMAEMA240 take place when both components are subsequently deposited onto a solid substrate. Spectroscopic studies confirmed that the absorption maximum of Mb remains unaltered, suggesting that at least some protein globules retain their tertiary structure. Cyclic voltammetry, CV, and square wave voltammetry, SWV, show a remarkable (ca. 180-fold) increase of the reductive current of Mb after its incorporation into the SPE/MWCNT/PB290-b-PDMAEMA240 matrix. The herein developed analytical approach was used for the detection of cardiac myoglobin as a very early marker of acute myocardial infarction (AMI) both in plasma of healthy donors and patients with AMI.

Activation of U937 cells in vitro is modified by fresh frozen plasma of healthy donors. Possible role of age and inflammatory profile

Activation of U937 cells in vitro is modified by fresh frozen plasma of healthy donors. Possible role of age and inflammatory profile

Influence of resveratrol and dihydroquercetin inclusion into phospholipid nanopatricles on their bioavailability and specific activity

The effects of natural polyphenols, resveratrol (RES) and dihydroquercetin (DHQ), included in phospholipid nanoparticles, have been compared with free substances of RES and DHQ in in vitro and in vivo experiments. Preincubation of healthy donor plasma low density lipoproteins (LDL) with RES or DHQ included in phospholipid nanoparticles caused a more pronounced decrease in Cu2+ induced lipid oxidation compared with the free substances, and reduced the formation of lipid peroxides products. Bioavailabilities of RES and DHQ in phospholipid formulations after oral administration in rats were increased by 1.5-2 times. In an acute hypoxia model in mice prophylactic two-week administration of RES or DHQ phospholipid formulations resulted in 25% increase in survival and 1.5-fold increase in catalase activity in brain homogenates compared to free substances. Using the model of endothelial dysfunction in rats induced by L-NAME it was shown, that RES markedly attenuated the inhibition effect of L-NAME on NO synthesis. RES in phospholipid nanoparticles had the same action at a dose 10 times lower compared to free RES. Load test with resistance (clamping of the ascending aorta for 30 sec) showed that phospholipid formulation of RES possessed more pronounced protective effect due to the stimulation of endothelial NO-synthase.

Chronic Lymphocytic Leukemia-Derived Extracellular Vesicles Contain a Distinctive Proteome, As Well As Specific Micro RNAs and Y RNAs

The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Of note, CLL cells themselves induce changes in surrounding cells, and extracellular vesicles (EVs) released by CLL cells represent a newly discovered mechanism of cell-cell communication. EVs are membrane enclosed nanoparticles 30 to 1000 nm in size and are able to reprogram recipient cells by transferring proteins and RNA molecules from their cell of origin. Thus, we aimed to analyze CLL cell-derived EVs present in blood plasma of CLL patients as well as those released by the CLL cell line MEC-1, in order to understand their role within the microenvironment.EVs were isolated from blood plasma of CLL patients and healthy donors, as well as from MEC-1 cell culture supernatant by serial centrifugation and density-based separation. Characterization of EVs by electron microscopy and immunoblot analysis revealed vesicles 20 to 300 nm in size, which are positive for various EV marker proteins such as Rab5a and Hsp70. Proteome analysis via mass spectrometry indicated differences in the composition of plasma derived EVs and peripheral blood mononuclear cells (PBMCs) from the respective patient, as well as between plasma derived EVs from healthy donors compared to CLL patients. Regarding the later, CLL EVs were specifically enriched for proteins involved in antigen presentation, endocytosis signaling as well as integrin mediated signaling and leukocyte extravasation. RNA analysis by BioAnalyzer profiling indicated an enrichment of small RNAs in EVs compared to cells. Subsequent small RNA sequencing revealed a unique microRNA signature of MEC-1 EVs with the 5 most abundant miRNAs encompassing about 60% of all detected miRNAs. Among them, the CLL-relevant miR-21 and miR-155, as well as miR-146a were selectively enriched in EVs. Moreover, Y RNAs, another class of small non-coding, regulatory RNAs, were highly enriched in MEC-1 EVs and their presence was also observed in plasma EVs of CLL patients. We uncovered a rapid uptake of CLL cell-derived EVs by human monocytes and macrophages. Whether the identified proteins and RNA transcripts shown to be enriched in CLL EVs induce phenotypic changes in targeted cells is being investigated. Further, quantification of plasma EVs in a large cohort of CLL patients is currently conducted and differences in the amount of EVs in correlation to disease outcome are analyzed.Harboring a distinct RNA and protein profile, EVs are potent vehicles for shuttling RNA and proteins to recipient cells and might be involved in the establishment of a supportive microenvironment in CLL. Functional analyses regarding possible effects of CLL EVs on target cells will broaden the knowledge of CLL pathogenesis and might help to identify new therapeutic options for CLL. DisclosuresNo relevant conflicts of interest to declare.

Intravenous immunoglobulin for Alzheimer's disease.

The foundation for using intravenous immunoglobulin (IVIg) to treat Alzheimer's disease (AD) can be traced back to the discovery, in the early 1990s, of naturally occurring anti-amyloid antibodies in human blood. A decade later, a number of independent investigators reported reduced levels of naturally occurring anti-amyloid antibodies in the spinal fluid and blood of AD patients relative to age-matched controls 1–3. Dodel subsequently reported that IVIg contained elevated levels of antibodies against amyloid-β (Aβ) monomers and proposed its use as a potential treatment for AD 4.

Interleukin-1 beta induces the expression and production of stem cell factor by epithelial cells: crucial involvement of the PI-3K/mTOR pathway and HIF-1 transcription complex.

Potential crosslinks between inflammation and leukaemia have been discussed for some time, but experimental evidence to support this dogma is scarce. In particular, it is important to understand the mechanisms responsible for potential upregulation of proto-oncogenic growth factor expressions by inflammatory mediators. Here, we investigated the ability of the highly inflammatory cytokine interleukin-1 beta (IL-1β) to induce the production of stem cell factor (SCF), which is a major hematopoietic growth factor that controls the progression of acute myeloid leukaemia upon malignant transformation of haematopoietic myeloid cells. We found that human IL-1β induced the expression/secretion of SCF in MCF-7 human epithelial breast cancer cells and that this process depended on the hypoxia-inducible factor 1 (HIF-1) transcription complex. We also demonstrated a crucial role of the phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in IL-1β-induced HIF-1α accumulation in MCF-7 cells. Importantly, mTOR was also found to play a role in IL-1β-induced SCF production. Furthermore, a tendency for a positive correlation of IL-1β and SCF levels in the plasma of healthy human donors was observed. Altogether, our results demonstrate that IL-1β, which normally bridges innate and adaptive immunity, induces the production of the major haematopoietic/proleukaemic growth factor SCF through the PI-3K/mTOR pathway and the HIF-1 transcription complex. These findings strongly support a cross-talk between inflammation and acute myeloid leukaemia.

Satellite DNA transcripts in blood plasma as potential markers of tumor growth

Recent studies of human and animal tumor tissues revealed a high transcriptional activity of pericentromeric satellite DNA repeats; i.e., they were found to produce up to half of all transcripts in tumor cells, which is many times more than in normal cells. It was also found that the two subtypes of satellite DNA (HSATII and GSATII) are transcribed reciprocally, i.e., HSATII transcripts strongly dominate in tumors, while GSATII transcripts are more abundant in the corresponding normal tissues. Since different RNAs are present in blood plasma, and some of them serve as efficient tumor markers, this study was undertaken to evaluate for the first time satellite HSATII and GSATII RNA levels in the plasma of healthy donors and cancer patients. The RT-PCR protocol designed for this purpose enabled detection of both HSATII and GSATII transcripts. It was shown that HSATII transcripts were more abundant than GSATII RNAs in the plasma of healthy donors, and this relationship was inverse in the plasma of cancer patients; these ratios were exactly opposite to those that exist within normal and tumor cells. Some notions concerning the probable origins of circulating satellite RNAs and their role as potential tumor markers are discussed.

Polyclonal Antibody Therapies for Clostridium difficile Infection

Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

Abstract 1870: Development of a standard operating procedure for exosome isolation and analysis using clinical samples: Application to cancer biomarker discovery

Abstract Exosomes are small vesicles (30-150 nm) found in abundance in human body fluids which function as carriers of different species of RNA and protein between diverse locations in the body. The spectrum of current scientific interest in exosomes is wide and ranges from studying their functions and pathways to utilizing them in diagnostics and therapeutics development. As such, there is a growing need for quick and easy methods for both isolation of exosomes and analysis of their cargo. We present herein a workflow for exosome isolation and analysis which entails: (i) fast and efficient isolation of exosomes from serum, plasma, and urine of both healthy donors and patients with prostate cancer, using Total Exosome Isolation reagents; (ii) characterization of their size distribution and count with Nanosight LM10 instrument; (iii) extraction of exosome “cargo” with Total Exosome RNA and Protein Isolation kit; (iv) characterization of exosomal RNA content using the Ion Torrent PGM sequencing and qRT-PCR. The protocol described herein lays the groundwork for the development of a standardized operating procedure (SOP) for isolation of exosomes and downstream analysis of their constituents, using clinical samples. We demonstrate that cancer-specific RNA signatures residing within the exosomes can be delineated from different patient cohorts. This is the first step towards developing a method whereby performance characteristics can be measured and used to optimize a validated assay useful for routine testing of clinical samples. Citation Format: Robert A. Setterquist, Alex J. Rai, Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Susan Magdaleno, Alexander V. Vlassov. Development of a standard operating procedure for exosome isolation and analysis using clinical samples: Application to cancer biomarker discovery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1870. doi:10.1158/1538-7445.AM2014-1870

Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: Apoptotic bodies, microvesicles, and exosomes

BackgroundExtracellular vesicles (EVs) are cell-derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell-derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments.MethodsEV subpopulations were isolated from three PCa cell lines (LNCaP, PC-3, and RC92a/hTERT) and the plasma of PCa patients and healthy donors, and characterized by transmission electron microscopy, nanoparticle tracking analysis and total protein content. gDNA fragments of different genes were detected by real time quantitative PCR and confirmed by DNA sequencing.ResultsWe report that the concentration of EVs was higher in the cancer patients than in the healthy controls. EV subpopulations differed from each other in terms of total protein and DNA content. Analysis of gDNA fragments of MLH1, PTEN, and TP53 genes from the PCa cell-derived EV subpopulations showed that different EVs carried different gDNA content, which could even harbor specific mutations. Altogether, these results suggest that both nucleic acids and proteins are selectively and cell-dependently packed into the EV subtypes.ConclusionsEVs derived from PCa cell lines and human plasma samples contain double-stranded gDNA fragments which could be used to detect specific mutations, making EVs potential biomarkers for cancer diagnostics and prognostics.

Identification of molecular species of cholesteryl ester hydroperoxides in very low-density and intermediate-density lipoproteins

Oxidation of lipoproteins is thought to play a crucial role in atherogenesis. Role for triglyceride-rich lipoproteins in atherogenesis is unclear. Thus, we aimed to investigate whether cholesteryl ester hydroperoxides (CEOOH) are present in very low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) by using highly sensitive liquid chromatography/mass spectrometry. Total lipids were extracted from the plasma of healthy donors (n = 6) and their fractions of VLDL and IDL. Additional three plasma samples were analysed freshly for CEOOH. Detection and identification of CEOOH was conducted by liquid chromatography/LTQ ion trap mass spectrometry/Orbitrap high mass accuracy mass spectrometry. Authentic standards of CEOOH were used for unequivocal identification on the basis of their mass spectra. We identified six molecular CEOOH species overall, namely, Ch18:1-OOH, Ch18:2-OOH, Ch18:3-OOH, Ch20:4-OOH, Ch20:5-OOH and Ch22:6-OOH. Of them, Ch18:2-OOH, Ch20:5-OOH, Ch20:4-OOH and Ch22:6-OOH were detected in all IDL samples, while only Ch20:4-OOH was detected in all VLDL samples. All of CEOOH species except for Ch18:3-OOH were detected in plasma, with constant detection of Ch20:5-OOH, and Ch22:6-OOH in all plasma samples. The presence of CEOOH species in VLDL and IDL was confirmed with the analytical sensitivity of 0.1 pmol, showing the constant appearance of more CEOOH species in IDL than VLDL. This finding might add biochemical evidences of atherogenicity of these lipoproteins. Clinical utility of measuring CEOOH level in these lipoproteins need to be investigated for the risk assessment of the cardiovascular disease.

Knot security and tensile strength of suture materials

To evaluate knot security and tensile failure load of suture tied in simple interrupted, beginning continuous, and ending continuous patterns for 11 suture materials commonly used in small animal surgery. Mechanical study. For each of 11 suture material types, and 5 knot sizes (2, 3, 4, 5, and 6 throws) 2 surgeons each tied 6 knots (n = 12 for each knot size in 11 suture materials). Three types of patterns were evaluated: a simple interrupted square knot, a square knot beginning a simple continuous pattern, and the knot ending a simple continuous pattern. All knots were incubated in healthy canine donor plasma at 40°C for a minimum of 24 hours. Sutures were evaluated for knot security (knots untied, suture failed by breaking, suture slipped from the clamps, or suture untied before testing) and maximum load carried before knot slippage or knot failure (termed tensile failure load). Significant differences were found in knot security and tensile failure load among suture types. There was no significant difference between the simple interrupted knots and the knots at the beginning of a simple continuous pattern; however, both were significantly less likely to fail than the knots tied at the end of a simple continuous pattern. The number of throws per knot had a significant effect for knot security and tensile failure load. Surgeon experience had a significant effect on failure mode and tensile failure load. Suture type, number of throws per knot (knot size), suture pattern, and surgeon experience play an important role in knot security and should be considered when performing surgery.

A stable isotope-labeled internal standard is essential for correcting for the interindividual variability in the recovery of lapatinib from cancer patient plasma in quantitative LC–MS/MS analysis

The development and validation of a LC-MS/MS method is often performed using pooled human plasma, which may fail to account for variations in interindividual matrices. Since calibrator standards and quality control samples are routinely prepared in pooled human plasma, variations in the extraction recovery and/or matrix effect between pooled plasma and individual patient plasma can cause erroneous measurements. Using both pooled human plasma as well as individual healthy donor and cancer patient plasma samples, we evaluated the analytical performance of two classes of internal standards (i.e., non-isotope-labeled and isotope-labeled) in the quantitative LC-MS/MS analysis of lapatinib. After exhaustive extraction with organic solvent, the recovery of lapatinib, a highly plasma protein-bound drug, varied up to 2.4-fold (range, 29-70%) in 6 different donors of plasma and varied up to 3.5-fold (range, 16-56%) in the pretreatment plasma samples from 6 cancer patients. No apparent matrix effects were observed for lapatinib in both pooled and individual donor or patient plasma samples. The calibration curve range was 5-5000ng/ml of lapatinib in plasma. Both the non-isotope-labeled (zileuton) and isotope-labeled (lapatinib-d3) internal standard methods showed acceptable specificity, accuracy (within 100±10%), and precision (<11%) in the determination of lapatinib in pooled human plasma. Nevertheless, only the isotope-labeled internal standard could correct for the interindividual variability in the recovery of lapatinib from patient plasma samples. As inter- and intra-patient matrix variability is commonly presented in the clinical setting, this study provides an example underscoring the importance of using a stable isotope-labeled internal standard in quantitative LC-MS/MS analysis for therapeutic drug monitoring or pharmacokinetic evaluation.

Abstract A79: The adenosine pathway in ovarian carcinoma: Tumor cells and tumor-derived exosomes express CD39 and CD73 ectonucleotidases, produce adenosine and mediate immune suppression

Abstract Background: Ectonucleotidases CD39/CD73 have been reported to play an important role in functional supression of various immune cells via adenosine that is generated locally in the tumor microenvironment. In patients with ovarian cancer (OvCa) exosomes released by tumor cells (TEX) are abundant in body fluids, including the plasma or ascites and may be involved in tumor progression. Based on the observations suggesting that TEX carry proteins that are expressed on tumor cells from which TEX originate, we hypothesized that CD39+ and CD73+ TEX could deliver these enzymes to distant immune cells. Adenosine produced fom ATP in the presence of TEX could suppress functions of these cells or elevate suppressor activity of regulatory T cells (Treg) , The aim of the study was to: (1) investigate the expression and clinical significance of CD39, CD73, adenosine deaminase (ADA) and CD26 in OvCa tissues and in TEX isolated from OvCa body fluids; (2) determine whether OvCa TEX metabolize exogeneous ATP to adenosine. (3) characterize the molecular profile of TEX; and (4) test whether OvCa TEX can suppress activities of NK cells and up-regulate suppressive activity of Treg. Methods: The expression of CD39, CD73, ADA and CD26 in OvCa tissues was determined by immunohistochemistry (IHC). The relationship between expression of these enzymes and clinicopathological characteristics was analyzed. TEX were isolated from supernatant of two OvCa cell lines (A2780, SKOV-3) and from the patients' plasma by exclusion chromatpgraphy followed by ultracentrifugation, as previously described.. Western blots were used for molecular characterization of TEX. NK cells and Treg were separated from the PMBC of normal donors and co-cultured with TEX. The phenotype of NK cells and Treg was evaluated by flow cytometry. ATP hydrolysis was measured using a luciferase detection assay. Results: By IHC in tissue sections, 70% of tumor cells were CD39+, 77% were CD73+ and 100% were CD26+ADA+.Expression levels of the ectonucleotidases varied from strong to moderate, and patients with a more advanced disease stage had tumors showing strongest CD73 expression (p&amp;lt;0,05). Exosomes isolated from plasma of OvCa patients were enriched in TEX which carried LAMP-1, CD63, TGF-β1, MAGE3/6, CD39, CD73, ADA and Ep-CAM. In contrast, exosomes isolated from the plasma of healthy donors carried LAMP-1 and CD63. TEX obtained from OvCa patients hydrolyzed more exogeneous ATP than did TEX from OvCa cell line supernatants (p&amp;lt;0.05) and produced more adenosine (p&amp;lt;0,05). After co-incubation with TEX, normal NK cells downregulated expression of NKG2D, NKp44 and NKp46 (p&amp;lt;0,05) and Treg up-regulated expression of Perforin, FasL, CCR7 (p&amp;lt;0,05). Co-incubation of Treg with TEX resulted by increased suppression of responder cells (p&amp;lt;0,01). Conclusion: Similar to OvCa tumor cells in tissue exosomes isolated from the plasma of OvCa patients were found to carry enzymatically-active ectonucleotidases and to produce extracellular adenosine. These exosomes were capable of down-regulating NK cell functions and up-regulating Treg activity in vitro. The same TEX-mesiated mechanisms could contribute to tumor-induced immune suppression characteristic of OvCa and resulting in tumor immune escape and OvCa progression. Note: This abstract was not presented at the conference. Citation Format: Marta E. Szajnik-Szczepanski, Magdalena Derbis, Michal Lach, Paulina Patalas, Marcin Michalak, Hanna Drzewiecka, Marta Glura, Ewa Nowak-Markwitz, Marek Spaczynski, Theresa L. Whiteside. The adenosine pathway in ovarian carcinoma: Tumor cells and tumor-derived exosomes express CD39 and CD73 ectonucleotidases, produce adenosine and mediate immune suppression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A79.

Endogenous morphine‐6‐glucuronide (M6G) is present in the plasma of patients: Validation of a specific anti‐M6G antibody for clinical and basic research

Endogenous morphine and its derivatives (morphine-6-glucuronide [M6G]; morphine-3-glucuronide [M3G]) are formed by mammalian cells from dopamine. Changes in the concentrations of endogenous morphine have been demonstrated in several pathologies (sepsis, Parkinson's disease, etc.), and they might be relevant as pathological markers. While endogenous morphine levels are detectable using enzyme-linked immunosorbant assay (ELISA), mass spectrometry (MS) analysis was, so far, the only approach to detect and quantify M6G. This study describes the preparation of a specific anti-M6G rabbit polyclonal antibody and its validation. The specificity of this antibody was assessed against 30 morphine-related compounds. Then, a M6G-specific ELISA-assay was tested to quantify M6G in the plasma of healthy donors, morphine-treated, and critically ill patients. The antibody raised against M6G displays a strong affinity for M6G, codeine-6-glucuronide, and morphine-3-6-glucuronide, whereas only weak cross-reactivities were observed for the other compounds. Both M6G-ELISA and LC-MS/MS approaches revealed the absence of M6G in the plasma of healthy donors (controls, n = 8). In all positive donors treated with morphine-patch (n = 5), M6G was detected using both M6G-ELISA and LC-MS/MS analysis. Finally, in a study on critically ill patients with circulating endogenous morphine (n = 26), LC-MS/MS analysis revealed that 73% of the positive-patients (19 of 26), corresponding to high M6G-levels in M6G-ELISA, contained M6G. In conclusion, we show that endogenous M6G can be found at higher levels than morphine in the blood of morphine-naive patients. With respect to the interest of measuring endogenous M6G in pathologies, we provide evidences that our ELISA procedure represents a powerful tool as it can easily and specifically detect endogenous M6G levels.

Clinical evaluation of microRNA expression profiling in non small cell lung cancer

Deregulation of miRNAs expression levels has been detected in many human tumor types, and recent studies have demonstrated the critical roles of miRNAs in cancer pathogenesis. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathological conditions. The high relative stability of miRNAs in biofluids such as plasma and serum, and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising non-invasive new tumor biomarkers. In this study, we used liquid bead array technology (Luminex) to profile the expression of 320 mature miRNAs in a pilot testing group of 19 matched fresh frozen cancerous and non-cancerous tissues from NSCLC patients. We further validated our results by RT-qPCR for differentially expressed miRNAs in an independent group of 40 matched fresh frozen tissues, 37 plasma samples from NSCLC patients and 28 healthy donors.We found that eight miRNAs (miR-21, miR-30d, miR-451, miR-10a, miR-30e-5p and miR-126*, miR-126, miR-145) were differentially expressed by three different statistical analysis approaches. Two of them (miR-10a and miR-30e-5p) are reported here for the first time. Bead-array results were further verified in an independent group of 40 matched fresh frozen tissues by RT-qPCR. According to RT-qPCR miR-21 was significantly up-regulated (P=0.010), miR-126* (P=0.002), miR-30d (P=0.012), miR-30e-5p (P<0.001) and miR-451 (P<0.001) were down-regulated, while miR-10a was not differentiated (P=0.732) in NSCLC tissues. However, in NSCLC plasma samples, only three of these miRNAs (miR-21, miR-10a, and miR-30e-5p) displayed differential expression when compared to plasma of healthy donors. High expression of miR-21 was associated with DFI and OS both in NSCLC tissues (P=0.022 and P=0.037) and plasma (P=0.045 and P=0.065), respectively. Moreover, we report for the first time that low expression of miR-10a in NSCLC plasma samples was associated with worse DFI (P=0.050) and high expression of miR-30e-5p was found to be associated with shorter OS (P=0.048). In conclusion, circulating miR-21, miR-10a and miR-30e-5p in plasma should be further evaluated as potential non-invasive biomarkers in NSCLC.

Prothrombin complex concentrate: Urgent anticoagulation reversal

Prothrombin complex concentrate: Urgent anticoagulation reversal

Plasma proteome changes associated with refractory anemia and refractory anemia with ringed sideroblasts in patients with myelodysplastic syndrome

BackgroundRefractory anemia and refractory anemia with ringed sideroblasts are two myelodysplastic syndrome (MDS) subgroups linked with anemia. MDS is a group of heterogeneous oncohematological bone marrow disorders characterized by ineffective hematopoiesis, blood cytopenias, and progression of the disease toward acute myeloid leukemia. The aim of this study was to search for plasma proteome changes in MDS patients with refractory anemia and refractory anemia with ringed sideroblasts.ResultsA total of 26 patient and healthy donor plasma samples were depleted of fourteen high-abundant plasma proteins, separated with 2D electrophoresis, and statistically processed with Progenesis SameSpots software. 55 significantly differing spots were observed and corresponded to 39 different proteins identified by nanoLC-MS/MS. Changes in the fragments of the inter-alpha-trypsin inhibitor heavy chain H4 protein were observed. Using mass spectrometry-based relative label-free quantification of tryptic peptides, there were differences in alpha-2-HS-glycoprotein peptides, while no differences were observed between the control and patient sample groups for retinol-binding protein 4 peptides.ConclusionsThis study describes plasma proteome changes associated with MDS patients with refractory anemia and refractory anemia with ringed sideroblasts. Changes observed in the inter-alpha-trypsin inhibitor heavy chain H4 fragments were in agreement with our previous studies of other MDS subgroups: refractory cytopenia with multilineage dysplasia and refractory anemia with excess blasts subtype 1. Mass spectrometry-based relative quantification of retinol-binding protein 4 peptides has shown that there are differences in the modification of this protein between refractory anemia with excess blasts subtype 1 patients and MDS patients with refractory anemia and refractory anemia with ringed sideroblasts. Alpha-2-HS-glycoprotein seems to be a new potential MDS biomarker candidate.