Supernatant Samples Research Articles

Extracellular microRNAs in bronchoalveolar lavage samples from patients with lung diseases as predictors for lung cancer

ObjectiveThe detection of tumor-associated microRNA expression in bronchial lavage (BL) samples of lung cancer patients could improve the non-invasive tumor diagnostic. MethodsThe profile of extracellular microRNAs in bronchial lavage was evaluated using three pools for lung cancer group (malignant) and three pools for non-cancerous group (benign) of 10 patients each. To confirm the results for the selected microRNAs in a qRT-PCR the BL of 30 different lung cancer and non-cancerous patients was used. We examined total-RNA from cell-free supernatant of BL samples. For normalization we added exogenous cel-miR 39. ResultsUsing microRNA arrays we found a panel of eight microRNAs (hsa-miR 19b-1, 1285, 1289, 1303, 217, 29a-5p, 548-3p, 650) that were differentially expressed between the lung cancer and the non-cancerous group. Further investigation by qPCR revealed five microRNAs (U6 snRNA, hsa-miR 1285, 1303, 29a-5p, 650) that were significantly up-regulated in patients with lung cancer. ConclusionsIn bronchial lavage samples the five microRNAs identified in this study have a diagnostic potential for use as noninvasive biomarkers in lung cancer.

Extension of the celiac intestinal antibody (CIA) pattern through eight antibody assessments in fecal supernatants from patients with celiac disease

BackgroundDetection of anti-transglutaminase, anti-endomysium and anti-gliadin antibodies is commonly used to screen celiac disease patients. Besides that in serum, these antibodies are detectable in culture supernatants of oral, duodenal and colonic biopsy samples, saliva, gut lavage fluid samples, and fecal supernatants. Our aim was to extend the intestinal antibody pattern in fecal supernatants from patients with celiac disease. MethodsThe fecal supernatants obtained from 25 celiac disease patients and 12 healthy volunteers were used to determine IgA and IgG1 anti-endomysium by immunofluorescence analysis, IgA and IgG anti-transglutaminase, IgA and IgG anti-deamidated gliadin peptides, IgA/IgG anti-transglutaminase/deamidated gliadin peptides and IgA anti-actin by enzyme-linked immunosorbent assay. ResultsIgA anti-endomysium were found in 11 of 25 (44.0%) celiac disease patients and in none of healthy volunteers (p=0.0066). The levels of IgA anti-transglutaminase, IgA anti-deamidated gliadin peptides, IgA/IgG anti-transglutaminase/deamidated gliadin peptides and IgA anti-actin determined in celiac disease patients were significantly higher (p=0.0005, p=0.0018, p=0.0061 and p=0.0477, respectively) than those measured in healthy volunteers. The ROC curve analysis showed a diagnostic significance in IgA anti-transglutaminase (AUC=0.862, p<0.0001), IgA anti-deamidated gliadin peptides (AUC=0.822, p<0.0001) and IgA/IgG anti-transglutaminase/deamidated gliadin peptides (AUC=0.783, p=0.0003) fecal tests. ConclusionsOur data extend the intestinal antibody pattern detectable in fecal supernatants, thus increasing the knowledge in the humoral immunity of celiac disease. Further studies are needed to better evaluate the role of fecal antibody tests in identifying celiac disease patients.

Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules.

The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset—when the histone mark H2AT120 is dephosphorylated—INCENP and Aurora B switch from centromere to microtubule localization.

Improving Blood Plasma Glycoproteome Coverage by Coupling Ultracentrifugation Fractionation to Electrostatic Repulsion-Hydrophilic Interaction Chromatography Enrichment.

Blood plasma is considered to be an excellent source of disease biomarkers because it contains proteins, lipids, metabolites, cell, and cell-derived extracellular vesicles from different cellular origins including diseased tissues. Most secretory and membranous proteins that can be found in plasma are glycoproteins; therefore, the plasma glycoproteome is one of the major subproteomes that is highly enriched with disease biomarkers. As a result, the glycoproteome has attracted much attention in clinical proteomic research. The modification of proteins with glycans regulates a wide range of functions in biology, but profiling plasma glycoproteins on a global scale has been hampered by the presence of low stoichiometry of glycoproteins in a complex high abundance plasma proteome background and lack of effective analytical technique. This study aims to improve plasma glycoproteome coverage using pig plasma as a model sample with a two-step strategy. The first step involves fractionation of the plasma proteins using ultracentrifugation into supernatant and pellet that is believed to contain low abundant glycoproteins. In the second step, further enrichment of glycopeptides was achieved in both fractions by adopting electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled to tandem mass spectrometry (LC-MS/MS) analysis. The coverage of enriched glycoproteins in supernatant, pellet, and whole plasma sample as control was compared. Using this simple sample fractionation approach by ultracentrifugation and further ERLIC enrichment technique, sample complexity was reduced and glycoproteome coverage was significantly enhanced in supernatant and pellet fractions (by >50%) compared with whole plasma sample. This study showed that when ultracentrifugation is coupled to ERLIC glycopeptides enrichment and glycoproteome identification are significantly improved. This study demonstrates the combination of ultracentrifugation and ERLIC as a useful method for discovering plasma glycoprotein disease biomarkers.

Protein-poly(amino acid) precipitation stabilizes a therapeutic protein l-asparaginase against physicochemical stress

Long-term storage in aqueous solution has been demanded for the practical application of therapeutic proteins. Recently, a precipitation-redissolution method was proposed to prepare salt-dissociable protein-polyelectrolyte complex (PPC). To elucidate the utility of the complex for storage of proteins, we investigated the stress tolerance of PPC precipitates containing l-asparaginase (ASNase) and poly-l-lysine (polyK). PPC precipitate containing ASNase and polyK was prepared by precipitation-redissolution method. The sample was treated to three types of stress, i.e., heat, shaking, and oxidation. The protein concentration, enzyme activity, and CD spectrum of the supernatants of samples were measured after stressed. PPC precipitate consisting of ASNase and polyK showed tolerance against thermal and shaking stress compared to the native solution. In addition, PPC precipitate protected ASNase from inactivation by oxidation. PPC precipitate of ASNase/polyK complex successfully stabilized ASNase against physicochemical stresses. These results suggest that the PPC precipitate has great potential as a storage method in aqueous solution for unstable proteins.

Accelerator mass spectrometry analysis of 14C-oxaliplatin concentrations in biological samples and 14C contents in biological samples and antineoplastic agents

Accelerator mass spectrometry (AMS) is expected to play an important role in microdose trials. In this study, we measured the 14C concentration in 14C-oxaliplatin-spiked serum, urine and supernatant of fecal homogenate samples in our Yamagata University (YU) – AMS system. The calibration curves of 14C concentration in serum, urine and supernatant of fecal homogenate were linear (the correlation coefficients were ⩾0.9893), and the precision and accuracy was within the acceptance criteria.To examine a 14C content of water in three vacuum blood collection tubes and a syringe were measured. 14C was not detected from water in these devices. The mean 14C content in urine samples of 6 healthy Japanese volunteers was 0.144dpm/mL, and the intra-day fluctuation of 14C content in urine from a volunteer was little. The antineoplastic agents are administered to the patients in combination. Then, 14C contents of the antineoplastic agents were quantitated. 14C contents were different among 10 antineoplastic agents; 14C contents of paclitaxel injection and docetaxel hydrate injection were higher than those of the other injections.These results indicate that our quantitation method using YU-AMS system is suited for microdosing studies and that measurement of baseline and co-administered drugs might be necessary for the studies in low concentrations.

Degradation of 2,4,6-trinitrotoluene by P. aeruginosa and characterization of some metabolites.

Degradation of 2,4,6-trinitrotoluene (TNT), a nitroaromatic explosive found in the soil and ground water, was investigated using Pseudomonas aeruginosa in in vitro experiments . Biodegradable abilitiy of this bacteria was performed with 50 and 75 mg L −1 TNT concentrations in a defined liquid medium for 96 h time period. Treatment of TNT in supernatant samples taken at 0, 6, 12, 24, 48, 72 and 96 h from agitated vessels was followed by reverse-phase high-performance liquid chromatography (HPLC). In cultures supplemented with 50 and 75 mgL −1 TNT, after 96 h of incubation 46% and 59% reduction were detected respectively. Two metabolites as degradation intermediates with nitrite release into the medium, 2,4-dinitrotoluene (2,4-DNT) and 4-aminodinitrotoluene (4-ADNT), were elucidated by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). These findings clearly indicate that Pseudomonas aeruginosa can be used in bioremediation of TNT contaminated sites.

New multiplex interferon panel for simultaneous quantification of 13 human cytokines involved in anti-virus responses (TECH3P.934)

Abstract In response to pathogens, especially viruses, cells release interferons and other cytokines to fight the infections. Interferons are typically divided into three types: I (e.g., INF-α, IFN-β), II (e.g., IFN-γ), and III (e.g., IFN-λ1, IFN-λ2). All interferons are important for fighting viral infections and for regulating the immune system. In addition, interferons are critically involved in cancer and autoimmune diseases such as psoriasis, systemic lupus erythematosus, and multiple sclerosis. Expression profiling of interferons and other related cytokines is critical in achieving a deeper understanding of the immune responses and various disease processes. We have developed a multiplex Human Interferon Panel, using fluorescence-encoded beads that are suitable for use on various flow cytometers. This panel allows simultaneous quantification of 13 cytokines involved in anti-virus responses including interferons (α, β, γ, λ1 and λ2), interleukins (1β, 6, 8, 10, 12), TNF-α, IP-10 and GM-CSF. Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The panel has been validated by detecting expected changes in biological samples. Further advantages include high sensitivity, small sample volume, flexible assay configurations, and time- and cost-effectiveness. The Interferon Panel can be used for serum, plasma, cell culture supernatant and other sample types, offering a useful tool for biomedical research and drug discovery.

Loss of Infliximab Into Feces Is Associated With Lack of Response to Therapy in Patients With Severe Ulcerative Colitis

It is not clear why some patients with ulcerative colitis (UC) do not respond to treatment with anti-tumor necrosis factor (TNF) agents, such as infliximab. It could be that some patients have high level of inflammation, with large quantities of TNF to be neutralized by the drug. We investigated whether loss of anti-TNF agents through ulcerated intestinal mucosa reduces the efficacy of these drugs in patients with severe UC. We collected fecal samples from 30 consecutive patients with moderate to severely active UC during the first 2 weeks of infliximab therapy at the University of Amsterdam hospital. Infliximab concentrations were measured in serum and supernatants of fecal samples using an enzyme-linked immunosorbent assay (Sanquin Biologicals Laboratory, Amsterdam, The Netherlands). Clinical and endoscopic responses were assessed 2 and 8 weeks and 3 months after treatment began. Infliximab was detected in 129 of 195 fecal samples (66%); the highest concentrations were measured in the first days after the first infusion. Patients that were clinical nonresponders at week 2 had significantly higher fecal concentrations of infliximab after the first day of treatment than patients with clinical responses (median concentration, 5.01 μg/mL in nonresponders vs 0.54 μg/mL in responders; P= .0047). We did not observe a correlation between fecal and serum concentrations of infliximab. Infliximab is lost into stools of patients with UC. High fecal concentrations of infliximab in the first days after therapy begins are associated with primary nonresponse. Additional studies are needed to determine how therapeutic antibodies are lost through the intestinal mucosa and how this process affects treatment response. Clinical trial ID: NL41310.018.12.

Improved sensitivity of the urine CAA lateral-flow assay for diagnosing active Schistosoma infections by using larger sample volumes.

BackgroundAccurate determination of Schistosoma infection rates in low endemic regions to examine progress towards interruption of transmission and elimination requires highly sensitive diagnostic tools. An existing lateral flow (LF) based test demonstrating ongoing infections through detection of worm circulating anodic antigen (CAA), was improved for sensitivity through implementation of a protocol allowing increased sample input. Urine is the preferred sample as collection is non-invasive and sample volume is generally not a restriction.MethodsCentrifugal filtration devices provided a method to concentrate supernatant of urine samples extracted with trichloroacetic acid (TCA). For field trials a practical sample volume of 2 mL urine allowed detection of CAA down to 0.3 pg/mL. The method was evaluated on a set of urine samples (n = 113) from an S. mansoni endemic region (Kisumu, Kenya) and compared to stool microscopy (Kato Katz, KK). In this analysis true positivity was defined as a sample with either a positive KK or UCAA test.ResultsImplementation of the concentration method increased clinical sensitivity (Sn) from 44 to 98% when moving from the standard 10 μL (UCAA10 assay) to 2000 μL (UCAA2000 assay) urine sample input. Sn for KK varied between 23 and 35% for a duplicate KK (single stool, two slides) to 52% for a six-fold KK (three consecutive day stools, two slides). The UCAA2000 assay indicated 47 positive samples with CAA concentration above 0.3 pg/mL. The six-fold KK detected 25 egg positives; 1 sample with 2 eggs detected in the 6-fold KK was not identified with the UCAA2000 assay.ConclusionsLarger sample input increased Sn of the UCAA assay to a level indicating ‘true’ infection. Only a single 2 mL urine sample is needed, but analysing larger sample volumes could still increase test accuracy. The UCAA2000 test is an appropriate candidate for accurate identification of all infected individuals in low-endemic regions. Assay materials do not require refrigeration and collected urine samples may be stored and transported to central test laboratories without the need to be frozen.

Bone matrix vesicle-bound alkaline phosphatase for the assessment of peripheral blood admixture to human bone marrow aspirates

PurposePeripheral blood (PB) admixture should be minimized during numerical and functional, as well as cytokinetic analysis of bone marrow (BM) aspirates for research purposes. Therefore, purity assessment of the BM aspirate should be performed in advance. We investigated whether bone matrix vesicle (BMV)-bound bone alkaline phosphatase (ALP) could serve as a marker for the purity of BM aspirates. ResultsTotal ALP activity was significantly higher in BM serum (97 (176–124)U/L, median (range)) compared to PB serum (63 (52–73)U/L, p<0.001). Agarose gel electrophoresis showed a unique bone ALP fraction in BM, which was absent in PB. Native polyacrylamide gel electrophoresis revealed the high molecular weight of this fraction, corresponding with membrane-bound ALP from bone matrix vesicles (BMV), as evidenced by electron microscopy. A serial PB admixture experiment of bone cylinder supernatant samples, rich in BMV-bound ALP, confirmed the sensitivity of this proposed quality assessment method. Furthermore, a BMV ALP fraction of ≥15% is suggested as cut-off value for minimal BM quality. Moreover, the BM purity declines rapidly with larger aspirated BM volumes. ConclusionThe exclusive presence of BMV-bound ALP in BM could serve as a novel marker to assess purity of BM aspirates.

Method optimization for fecal sample collection and fecal DNA extraction.

This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses. Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I®, Norgen Biotek All-In-One®, MoBio PowerMag®) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures. The optimal MSM I protocol involves a 0.2 g stool sample and 1000 μL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20°C or -80°C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/μL. The protocol was reproducible in terms of DNA yield among different stool aliquots. We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65–100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates.

Dynamic changes of virus load in supernatant of primary CEK cell culture infected with different generations of avian infectious bronchitis virus strains Sczy3 as revealed by real-time reverse transcription-polymerase chain reaction

Infectious bronchitis virus (IBV) can multiply effectively in chick embryo kidney (CEK) cells after adapting to the chick embryo. To investigate the dynamic changes in IBV load in the supernatant of primary CEK cells, we developed an SYBR Green I-based real-time polymerase chain reaction assay to quantify nucleic copy numbers of the IBV-Sczy3 strain. The 20, 54, and 87th generations of CEK-adapted IBV-Sczy3 strains were used to infect CEK cells, and then nucleic copy numbers in the samples of supernatant collected at 12, 24, 36, 48, 60, and 72 h were detected. The results showed that the rapid growth period of the virus load of all the 3 generations was approximately 12-36 h post-infection; the peak of the virus load appeared at 36 h post-infection and then decreased gradually in the order of 20th > 54th > 87th for the 3 generations of CEK-adapted strains; the dynamic change curve of the IBV load in the supernatant of primary CEK cells showed a single peak. The results of this study provide a useful reference for CEK-adapted IBV field strains and the production of CEK-attenuated IBV vaccine.

Effects of scorched food leachates with or without activated charcoal pretreatment on AhR activation in cultured cells.

Aryl hydrocarbon receptor (AhR) is a transcription factor activated by xenobiotics, including dioxins and polycyclic aromatic hydrocarbons (PAHs). Although AhR is also activated by some dietary constituents, it has not been completely clarified in what circumstances AhR ligands are ingested in our daily life. Because PAHs are formed by the incomplete combustion of organic materials, we hypothesized that scorched foods might contain and leach out AhR ligands sufficient to stimulate AhR in vitro. To test this hypothesis, scorched foods (bread, cheese, etc.) were mixed vigorously with water, and the supernatants were retrieved as samples. The samples were added to HepG2 cells stably expressing an AhR-responsive reporter gene. Also, expression of CYP1A1, an endogenous AhR-responsive gene, was analyzed by RT-PCR in different cell lines treated with the samples. We further tested whether pretreatment of the samples with activated charcoal would alter their AhR-stimulating activity. All the supernatant samples tested induced AhR-dependent reporter gene activity and CYP1A1 mRNA expression. In some samples, these inductions were inhibited by pretreatment with activated charcoal. Our findings indicate that scorched food leachates stimulate AhR in cultured cells and that activated charcoal adsorbs the AhR-stimulating substances in some leachates. Thus, people who habitually eat scorched foods are exposed to AhR ligands on a regular basis. Further studies are needed to elucidate whether burnt foods actually exert biological effects on our health.

Artificial neural network associated to UV/Vis spectroscopy for monitoring bioreactions in biopharmaceutical processes

Currently, mammalian cells are the most utilized hosts for biopharmaceutical production. The culture media for these cell lines include commonly in their composition a pH indicator. Spectroscopic techniques are used for biopharmaceutical process monitoring, among them, UV-Vis spectroscopy has found scarce applications. This work aimed to define artificial neural networks architecture and fit its parameters to predict some nutrients and metabolites, as well as viable cell concentration based on UV-Vis spectral data of mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Off-line spectra of supernatant samples taken from batches performed at different dissolved oxygen concentrations in two bioreactor configurations and with two pH control strategies were used to define two artificial neural networks. According to absolute errors, glutamine (0.13 ± 0.14 mM), glutamate (0.02 ± 0.02 mM), glucose (1.11 ± 1.70 mM), lactate (0.84 ± 0.68 mM) and viable cell concentrations (1.89 10(5) ± 1.90 10(5) cell/mL) were suitably predicted. The prediction error averages for monitored variables were lower than those previously reported using different spectroscopic techniques in combination with partial least squares or artificial neural network. The present work allows for UV-VIS sensor development, and decreases cost related to nutrients and metabolite quantifications.

Decreased bacterial diversity characterizes the altered gut microbiota in patients with psoriatic arthritis, resembling dysbiosis in inflammatory bowel disease.

To characterize the diversity and taxonomic relative abundance of the gut microbiota in patients with never-treated, recent-onset psoriatic arthritis (PsA). High-throughput 16S ribosomal RNA pyrosequencing was utilized to compare the community composition of gut microbiota in patients with PsA (n = 16), patients with psoriasis of the skin (n = 15), and healthy, matched control subjects (n = 17). Samples were further assessed for the presence and levels of fecal and serum secretory IgA (sIgA), proinflammatory proteins, and fatty acids. The gut microbiota observed in patients with PsA and patients with skin psoriasis was less diverse when compared to that in healthy controls. This could be attributed to the reduced presence of several taxa. Samples from both patient groups showed a relative decrease in abundance of Coprococcus species, while samples from PsA patients were also characterized by a significant reduction in Akkermansia, Ruminococcus, and Pseudobutyrivibrio. Supernatants of fecal samples from PsA patients revealed an increase in sIgA levels and decrease in RANKL levels. Analysis of fatty acids revealed low fecal quantities of hexanoate and heptanoate in both patients with PsA and patients with psoriasis. Patients with PsA and patients with skin psoriasis had a lower relative abundance of multiple intestinal bacteria. Although some genera were concomitantly decreased in both conditions, PsA samples had a lower abundance of reportedly beneficial taxa. This gut microbiota profile in PsA was similar to that previously described in patients with inflammatory bowel disease and was associated with changes in specific inflammatory proteins unique to this group, and distinct from that in patients with skin psoriasis and healthy controls. Thus, the role of the gut microbiome in the continuum of psoriasis-PsA pathogenesis and the associated immune response merits further study.

Sustained Growth of Primary Myeloma Cells in Coculture with Whole Donor Bone Marrow Is Associated with Induced Secretion of the Microenvironmental Mediator of Cytokinesis, Hemicentin-1

Primary human myeloma (MM) cells do not survive in culture while current systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animals. The aim of the study was to establish a coculture system for studying long-term growth of primary MM and their interaction with whole BM microenvironment, namely normal bone marrow (NBM) system . Whole BM cells from healthy donors (n=20) were cultured in medium supplemented with serum (10% v/v) pooled from MM patients for 7 days followed by coculture with CD138-selected primary MM cells (4:1 NBM:MM ratio) or MM cell lines (10:1 NBM:MM ratio) for ≥7 days. This NBM system is composed of adherent and non-adherent compartments. Adherent cells were mainly macrophages and mesenchymal stem cells (MSCs) whereas non-adherent cells contained typical hematopoietic cells including CD19+, CD3+, CD11b+ and CD33+ cells. Growth of MM cells was determined by CD45/CD38 flow cytometry and by bioluminescence of luciferase-expressing MM cells. MM cells or subset of MM cells from all patients (n=60) survived and grew in this system regardless of molecular risk or subtype, and MM growth was comparable to coculture with the supportive osteoclasts or MSCs. Adherent and non-adherent compartments supported MM cells which required patient’s serum for optimal growth. In 14 of 20 experiments, number of MM plasma cells, quantified by flow cytometry or bioluminescence analysis was increased by 58±12% (p<0.0005) in the NBM system and cell proliferation was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of OPM2, H929 and ARP1 lines was also stimulated in the NBM system which protected these cells from dexamethasone (1-2.5µM) but not bortezomib (0.01-5nM), while the effect of lenalidomide varied (0.1-5µM). For identifying secreted proteins that may mediate MM growth in the NBM system, supernatant were collected from serum-free culture of NBM, MM cells and NBM/MM coculture (18 hrs, n=3). Proteomics analysis performed on supernatant samples identified 1843 proteins. The clinical markers B2M and LDHA were present at high levels and were significantly higher by 2-2.4 folds in NBM/MM coculture compared to cultured NBM (p<0.04). Further filtration revealed 89 proteins that were significantly changed upon NBM/MM coculture but minimally detected in the MM cells culture: 14 were significantly lower and 75 were higher in NBM/MM cocultures compared to cultured NBM. These factors include mediators of extracellular matrix, immunity, and inflammation. A microenvironmental secreted factor that was not detected in the supernatant from MM cells or NBM but was secreted in cocultures was hemicentin-1 (HMCN1), a unique extracellular matrix protein directly involved in cytokinesis (Xu and Vogel, Curr Biol 2011) but has yet not been implicated in MM. Hemicentin-1 gene expression was detected in cultured NBM and MSCs but not in primary MM cells, MM lines or CD11b+ NBM cells. Induction of hemicentin-1 expression in MSCs after coculture with MM cells was validated by immunohistochemistry. Hemicentin-1 expression is higher in random bone biopsies from newly diagnosed MM patients (n=406) compared to donor biopsies (n=25, p<0.008) and highest in MM focal lesion biopsies (n=49, q<0.0005 vs. paired random bone biopsies). Higher baseline HMCN1 expression in biopsies was associated with inferior overall survival in TT3b clinical trial (p<0.027). The NBM system demonstrates the ability of primary MM plasma cells to interact with and to survive in coculture with healthy allogeneic adult BM through secretion of factors involved in immune evasion and extracellular matrix modification. Ongoing work is underway to unravel the role of hemicentin-1 in MM growth. DisclosuresNo relevant conflicts of interest to declare.

Antibody against granulin-epithelin precursor sensitizes hepatocellular carcinoma to chemotherapeutic agents.

Granulin-epithelin precursor (GEP) overexpression has been shown in many cancers with functional role on growth, and recently on regulating chemoresistance and cancer stem cell (CSC) properties. Here, we investigate the combined effect of GEP antibody and chemotherapeutic agent. Combination therapy was compared with monotherapy using hepatocellular carcinoma (HCC) cells in vitro and orthotopic liver tumor models in vivo. CD133 and related hepatic CSC marker expressions were investigated by flow cytometry. Antiproliferative and apoptotic effects and signaling mechanisms were examined by immunohistochemistry, flow cytometry, and Western blot analysis. Secretory GEP levels in the serum and culture supernatant samples were measured by ELISA. We demonstrated that HCC cells that survived under chemotherapeutic agents showed upregulation of hepatic CSC markers CD133/GEP/ABCB5, and enhanced colony and spheroid formation abilities. Importantly, GEP antibody sensitized HCC cells to the apoptosis induced by chemotherapy for both HCC cell lines and the chemoresistant subpopulations, and counteracted the chemotherapy-induced GEP/ABCB5 expressions and Akt/Bcl-2 signaling. In human HCC orthotopic xenograft models, GEP antibody treatment alone was consistently capable of inhibiting the tumor growth. Notably, combination of GEP antibody with high dose of cisplatin resulted in the eradication of all established intrahepatic tumor in three weeks. This preclinical study demonstrated that GEP antibody sensitized HCC cells to apoptosis induced by chemotherapeutic agents. Combination treatment with GEP antibody and chemotherapeutic agent has the potential to be an effective therapeutic regimen for GEP-expressing cancers.

Urine sample preparation in 96-well filter plates to characterize inflammatory and infectious diseases of the urinary tract.

Urine has been an important body fluid source for diagnostic and prognostic biomarkers of diseases for a long time. Technological advances during the last two decades have enabled a fundamental shift from the discovery of candidate protein biomarkers using single-assay platforms to highly parallel liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomic analysis platforms. MS/MS-based approaches such as multiple reaction monitoring (MRM) are also being used increasingly for targeted protein biomarker validation. In large part due to the fact that the majority of protein in voided urine is soluble, such studies have focused on the analysis of urine supernatants, whereas the pellets were discarded after centrifugal sedimentation. Urine sediments are of particular value in the analysis of urinary tract infections (UTI). The LC-MS/MS methods now have sufficient resolving power and sensitivity to survey metaproteomes--the entirety of proteins derived from multiple organisms that interact with each other in mutualistic or antagonistic fashion. Challenges of proteomic analysis of urine include the high dynamic range of protein abundance, high levels of protein post-translational modifications, and high quantities of natural protease inhibitors. Recently, a robust and scalable workflow that can parallelize the processing of multiple urinary supernatant and sediment samples was developed and validated in our lab. This method utilizes 96-well format filter-aided sample preparation (96FASP) strategy and was shown to successfully identify large numbers of proteins from urine samples. Processing 10-50 µg total protein in single experiment, LC-MS/MS with a Q-Exactive mass spectrometer resulted in more than 1,100 distinct human protein identifications from urine supernatants, and around 400 microbial and 1,400 human protein identifications from urine sediments. The surveys are a rich data resource not only for biomarker discovery but also to interrogate mechanisms of pathogenesis in the urinary system.