Depletion Technique Research Articles

Regulatory T Cells As Potential Targets for HIV Cure Research.

T regulatory cells (Tregs) are a key component of the immune system, which maintain a delicate balance between overactive responses and immunosuppression. As such, Treg deficiencies are linked to autoimmune disorders and alter the immune control of pathogens. In HIV infection, Tregs play major roles, both beneficial and detrimental. They regulate the immune system such that inflammation and spread of virus through activated T cells is suppressed. However, suppression of immune activation also limits viral clearance and promotes reservoir formation. Tregs can be directly targeted by HIV, thereby harboring a fraction of the viral reservoir. The vital role of Tregs in the pathogenesis and control of HIV makes them a subject of interest for manipulation in the search of an HIV cure. Here, we discuss the origin and generation, homeostasis, and functions of Tregs, particularly their roles and effects in HIV infection. We also present various Treg manipulation strategies, including Treg depletion techniques and interventions that alter Treg function, which may be used in different cure strategies, to simultaneously boost HIV-specific immune responses and induce reactivation of the latent virus.

Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy.

Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.

Folate-receptor-positive circulating tumor cells as an efficacious biomarker for the diagnosis of small pulmonary nodules.

The objective of this study is to investigate the clinical significance of folate-receptor-positive circulating tumor cells (FR+CTC) for the diagnosis of lung cancer, especially in early-stage patients. A total of 72 lung cancer patients, including 31 with stage I diseases and two with stage 0 diseases, were enrolled in this study. Twenty-four patients with benign lung diseases and two healthy volunteers served as the control group. Three milliliters of peripheral blood were collected from each participant for FR+CTC analysis on enrollment. FR+CTC enumeration was performed using immunomagnetic leukocyte depletion and ligand-targeted polymerase chain reaction techniques. The study results revealed that using a cutoff value of 8.7 CTC Units/3 mL, the sensitivity, and specificity of FR+CTC for diagnosis of lung cancer were 81.94% and 73.08%, respectively. Such high sensitivity (74.19%) and specificity (73.08%) persisted even if only stage I lung cancer patients were retained in the analysis. In receiver operating characteristic analysis, the performance of FR+CTC (area under the curve = 0.8153) was superior to other clinical biomarkers such as carcinoembryonic antigen, neuron-specific enolase, and cytokeratin 19 fragments. In a subgroup analysis, patients with nodule size of >3 cm showed an improved sensitivity (88.46%); although, the specificity appeared to decrease (40%). All five patients with benign diseases in this subgroup had inflammatory diseases, indicating that large inflammatory nodules may also release FR -expressing cells into the circulatory system. FR+CTC is a reliable biomarker for the early diagnosis of small-sized lung cancer. Further study with larger sample size is required to assess the diagnostic efficiency of FR+CTC in patients with large nodule sizes.

Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry.

In proteomics, dodecyl sulfate (DS-) as sodium salt is commonly used in protein solubilization prior to tryptic digestion, but the presence of the DS- hampers the electrospray ionization mass spectrometric (ESI-MS) analysis. The development of DS- depletion techniques is therefore important especially when dealing with small samples where there could be poor sensitivity due to sample loss or dilution during sample preparation. Here, we present a simple and fast electrokinetic removal method of DS- from small volumes of peptide and digested protein samples prior to ESI-MS. The selective removal was accomplished using an acidic extraction solution (ES) containing acetonitrile (ACN) inside a fused-silica capillary that was dipped into the sample. The use of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic movement of DS- monomers and micelles into the capillary. The high amount of ACN present at the tip of the capillary served to collapse the micelles migrating into the capillary, thereby releasing the peptides that were bound to these micelles, facilitating peptide retention in the sample and efficient DS- removal. Increased % MS signal intensity (SI) restoration of the peptide was observed, while DS- removal was unaffected when the amount of ACN in the ES was increased. This is because of the micelle to solvent stacking mechanism (effective electrophoretic mobility reversal) working at high concentration of ACN for the improved recovery of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin peptides were 75-83% while % MS SI reduction of DS- was up to 99% under optimal conditions, that is, 40% ACN in the ES. Higher % peptide recoveries from digested protein samples were obtained using the proposed method compared to the conventional cold acetone precipitation method.

Unraveling complex nanoscale lipid dynamics in simple model biomembranes: Insights from fluorescence correlation spectroscopy in super-resolution stimulated emission depletion mode.

Dynamic heterogeneity (DH) at nanoscale due to lipid-lipid and/or lipid-protein interactions in cell membranes plays a crucial role in determining a broad range of important cell functions. In cell membranes, the dimensions of these nanodomains have been postulated to be in the order of 10's of nm and transient in nature. While the structural features of membranes have been studied in detail, little is known about their dynamical characteristics due to paucity of techniques which can probe nanoscale phenomena with simultaneous high temporal resolution. A combination of super-resolution stimulated emission depletion (STED) and fluorescence correlation spectroscopy (FCS) technique can overcome this limitation and provide information about the nanoscale dynamic heterogeneity in cell membranes. Using STED-FCS and FCS diffusion law, we provide an understanding of how nanoscale dynamically organizing lipid platforms can emerge in minimal system of model biomembranes. To illustrate the utility of the technique we have chosen cholesterol containing supported lipid bilayers and demonstrated the role of cholesterol concentration and/or added pore-forming protein, Listeriolysin O (LLO) in determining onset of lipid DH. In addition we have also looked at multi-component lipid bilayers with and without cholesterol to infer about the role of phospholipid and cholesterol composition on lipid dynamics. These results on simple biomimetic systems provide insights into fundamental pathways for the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events and depict the significant contribution that STED-FCS can make in resolving several outstanding issues in membrane biology.

<b>Aerobic bacteria in oral cavity of Lancehead snakes (<i>Bothrops atrox</i>) with stomatitis

Stomatitis is a common disease found on snake farms, and Gram-negative bacilli are the main etiological agents that play an important role as secondary sources of viral or parasitic infections. The purpose of this work was to identify the aerobic bacteria in the oral cavity of Bothrops atrox with stomatitis. Samples for microbiological examination were collected from 12 snakes bred on a commercial snake farm for venom extraction. Samples of the secretion in the oral cavity of each serpent presenting stomatitis were collected from fang sheath, using a cotton swab with sterile alginate. The samples were incubated and cultured on Petri dishes containing blood agar and XLD agar using the agar depletion technique. Bacterial growth occurred in all analyzed samples collected from the oral cavity of Bothrops atrox with stomatitis, and some of the samples contained more than one microorganism. The following Gram-negative bacteria were isolated: Escherichia coli (26.31%), Citrobacter spp. (21.05%), Proteus spp. (15.78%) and Salmonella spp. (10.52%). The only Gram-positive bacterium that was isolated was Staphylococcus spp., which was present in 26.31% of the analyzed samples.

Adsorption/Desorption Transition of Recombinant Human Neurotrophin 4: Physicochemical Characterization.

Bulk physicochemical properties of neurotrophin 4 (NT-4) in electrolyte solutions and its adsorption/desorption on/from mica surfaces have been studied using dynamic light scattering (DLS), microelectrophoresis, a solution depletion technique (enzyme-linked immunosorbent assay, ELISA), and AFM imaging. Our study presents a determination of the diffusion coefficient, hydrodynamic diameters, electrophoretic mobility, and isoelectric point of the NT-4 under various ionic strength and pH conditions. The size of the NT-4 homodimer for an ionic strength of 0.015 M was substantially independent of pH and equal to 5.1 nm. It has been found that the number of electrokinetic charges per NT-4 molecule was equal to zero for all studied ionic strengths at pH 8.1, which was identified as the isoelectric point (iep). The protein adsorption/desorption on/from mica surfaces was examined as a function of ionic strength and pH. The kinetics of neurotrophin adsorption/desorption were evaluated at pH 3.5, 7.4, and 11 by direct AFM imaging and the ELISA technique. A monotonic increase in the maximum coverage of adsorbed NT-4 molecules with ionic strength (up to 5.5 mg/m2) was observed at pH 3.5. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA). Our measurements revealed a significant role of ionic strength, pH, and electrolyte composition in the lateral electrostatic interactions among differently charged NT-4 molecules. The transition between adsorption/desorption processes is found for the region of high pH and low surface concentration of adsorbed neurotrophin molecules at constant ionic strength. Additionally, results presented in this work show that the adsorption behavior of neurotrophin molecules may be governed by intrasolvent electrostatic interactions yielding an aggregation process. Understanding polyvalent neurotrophin interactions may have an impact on the reversibility/irreversibility of adsorption, and hence they might be useful for obtaining well-ordered protein layers, targeting the future development of drug delivery systems for treating neurodegenerative diseases.

Abstract 32: Ly6C Lo Monocyte/Macrophages Are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis

Venous thrombosis (VT) results in vein wall injury by promoting inflammation and fibrosis leading to venous reflux, swelling, pain, and potentially, recurrent thrombosis. While prior work has shown that infiltrating leukocytes are important for VT resolution, as of yet, the precise roles of different leukocyte subsets are not well understood. Monocyte/macrophages (Mo/MΦs) are essential for the repair and resolution of tissue injury in other models, and come in inflammatory (Ly6C Hi ) or pro-resolution (Ly6C Lo ) subtypes. We hypothesized that infiltrating Mo/MΦs would be critical to VT resolution. In order to study this in vivo , we utilized a conditional macrophage depletion technique, using CD11b-DTR mice, to examine the effects of Mo/MΦs in a murine model of stasis VT by inferior vena cava ligation. Administration of 10ng/g diphtheria toxoid (DTx) every 48 hours by intra-peritoneal injection in CD11b-DTR mice resulted in an 89% and 55% decrease in circulating monocytes at 24hrs and 48hrs, respectively. When compared to saline controls, DTx injection had no effects on thrombogenic response or IVC thrombus cell populations in C57BL/6 control mice. At 8 days’ post-ligation, DTx treated CD11b-DTR mice had preferentially decreased vein wall-thrombus Ly6C Lo Mo/MΦs as compared with controls. DTx treated mice had significantly larger thrombi (1.7-fold) and less TGF-β, FSP-1, and plasminogen by western immunoblotting (all P-values ≤ 0.01). Consistent with a reduction in Ly6C Lo Mo/MΦs was a significant decrease in cellular TGF-β by intra-cellular flow cytometry. These findings suggest that Ly6C Lo Mo/MΦs are essential for normal VT resolution and may promote thrombus resolution via a plasminogen-mediated mechanism.

Macrophage depletion decreases tumorigenesis and alters gut microbiota in the AOM/DSS mouse model of colon cancer

Abstract Macrophages are recognized as major players in the connection between inflammation and cancer; they represent up to 50% of the tumor mass and produce a wide array of inflammatory mediators with pro-tumoral functions. Emerging evidence links gut microbiota to the development of colorectal cancer and host-derived immune and inflammatory responses are important driving forces that can shape microbial community composition. We used a macrophage depletion technique to examine the role of macrophages on colon tumorigenesis and gut microbiota. To induce colorectal cancer, male C57BL/6 mice (n=32) received a single injection of AOM followed by three cycles of DSS supplemented water at concentrations of 2%, 1%, 1% at weeks 1, 4, and 7, respectively. Prior to the final DSS cycle (week 7) and twice weekly until sacrifice, mice (n=16/group) received 200ul i.p. of clodronate (CL2MDP) or PBS encapsulated liposomes to deplete macrophages. Mice were sacrificed at 12 weeks following the initial AOM injection and colon tissue was analyzed for tumor burden, macrophage markers, and inflammatory mediators. Stool samples were collected and DNA was isolated and subsequently sequenced for 16S rRNA. Macrophage depletion decreased tumor number by ~35% and specifically large (≥1mm) tumors by ~35% (p<0.05). This was consistent with a decrease in gene expression of EMR1 (overall macrophage marker) as well as expression of markers associated with M2 macrophages (IL-13, IL-10, TGFβ & CCL17) (p<0.05) but not M1 markers (NOS2, IL-12a & IL-23). Macrophage depletion also increased relative abundance of the Firmicutes phylum (p<0.05) in stool. We demonstrate that macrophages are important mediators of tumor growth and changes in gut microbiota in colorectal cancer.

Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques

The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL−1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL−1. Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers.

Antibacterial Activity and Toxicological Analysis In silico of 7- Hydroxycitronellal

The pharmacological selectivity imposed by the micro-organisms enabled the emergence of new perspectives in the research field of natural products, in which the monoterpenic phytoconstituents, especially the 7-hydroxycitronellal (7- OH) present antimicrobial activity. Therefore, this study assessed the in silico antimicrobial and toxicological activity of the monoterpene 7-OH. The product was solubilized in tween 80 and dimethylsulfoxide (DMSO). Later it was diluted in sterile distilled water up to the concentration of 2048µg/mL. The minimum inhibitory concentration (MIC) of the product was determined by microdilution in doubly concentrated brain heart infusion (BHI) medium. The minimum bactericidal concentration (MBC) was determined by the depletion technique in nutrient agar (NA) with aliquots of 10µL of the MIC, MIC × 2 and MIC × 4. The MIC and the MBC of the 7-OH was 64µg/mL for S. aureus (ATCC 6538). However, for S. epidermides (LPM 35), S. aureus (LPM 45) and S. aureus (LPM 55), were 512 and 1024µg/mL respectively. In the in silico toxicological analysis the absence of mutagenic, tumorigenic effects and damage to the reproductive system were observed. Therefore, the 7-OH is bactericidal for the strains used in this study and presents a theoretical low oral toxicity.

Study of the antifungal potential of (r)-(+)-citronellal and its association with therapeutic agents used in the treatment of vulvovaginal candidiasis

Vulvovaginal candidiasis (VVC) is a common fungal infection that affects healthy women of all ages. At least 75% of women will develop one or more infections once during their lifetime, with 6 to 9% of those individuals developing recurrent infections. In view of this context, this study sought to evaluate the antifungal potential of the isolated ( R )-(+)-citronellal [( R )-(+)-CT] and associated to therapeutic agents of clinical importance. The enantiomer was solubilized in tween 80 and dimethylsulfoxide (DMSO). Posteriorly diluted in sterile distilled water up to the concentration of 2048µg/mL. The minimum inhibitory concentration (MIC) of the product was determined by microdilution in RPMI-1640 obtaining dilutions of 1024-4µg/mL. The minimum fungicidal concentration (MFC) was determined by the Sabouraud dextrose agar (SDA) depletion technique from aliquots of 1µL of the MIC, MIC × 2 and MIC × 4. The MIC and the MFC values of ( R )-(+)-CT for 90% of the C. albicans strains were 16 and 32µg/mL respectively. In the susceptibility test, C. albicans presented a high resistance to fluconazole and to itraconazole, 12 (92.30%) of the strains. However, for ketoconazole and miconazole the resistance was of 4 (30.76%) and 3 (23.07%) of the strains respectively. In the combination testing of the ( R )-(+)-CT with ketoconazole and miconazole, the resistance was completely reverted. For fluconazole and itraconazole, the resistance was reverted in 9 (75%) and 7 (58.33%) of the strains respectively. The ( R )-(+)-CT presented fungicide activity with MFC of MIC × 2. When in combination with ketoconazole, fluconazole, itraconazole and miconazole increased the inhibition zones of these antifungal drugs, reducing the resistance against C. albicans .

Managing the Introduction of Super-Resolution Microscopy into a Core Facility.

Super resolution techniques place the resolution of fluorescence microscopy closer to the size of the underlying cell structure or molecular machine being studied. Structured illumination techniques will give users a set of tools that are close to their past experience and relatively simple and quick to learn. The present dyes can be used. Resolution approaching 100nm XY can be achieved. In contrast, stochastic methods such as PALM/STORM typically require the choice of new dyes and a much greater learning curve to master the technology and calculations. However, a further fivefold resolution improvement is possible. Stimulated depletion techniques such as STED offer a third set of approaches that will again require the use of new dyes. All these approaches require substantial investment in new equipment and in user training. There is no free lunch in the search for better resolution.

The mechanisms and research progress of laser fabrication technologies beyond diffraction limit

Laser is recognized as one of the top technological achievements of 20th century and plays an important role in many fields, such as medicine, industry, entertainment and so on. Laser processing technology is one of the earliest and most developed applications of laser. With the rapid development of nanoscience and nanotechnology and micro/nano electronic devices, the micro/nanofabrication technologies become increasingly demanding in manufacturing industries. In order to realize low-cost, large-area and especially high-precision micro-nanofabrication, it has great scientific significance and application value to study and develop the laser fabrication technologies that can break the diffraction limit. In this article, the super resolution laser fabrication technologies are classified into two groups, far-filed laser direct writing technologies and near-field laser fabrication technologies. Firstly, the mechanisms and progress of several far-field laser direct writing technologies beyond the diffraction limit are summarized, which are attributed to the lasermatter nonlinear interaction. The super-diffraction laser ablation was achieved for the temperature-dependent reaction of materials with the Gaussian distribution laser, and the super-diffraction laser-induced oxidation in Metal-Transparent Metallic Oxide grayscale photomasks was realized by the laser-induced Cabrera-Mott oxidation process. Besides, the multi-photon polymerization techniques including degenerate/non-degenerate two-photon polymerization are introduced and the resolution beyond the diffraction limit was achieved based on the third-order nonlinear optical process. Moreover, the latest stimulated emission depletion technique used in the laser super-resolution fabrication is also introduced. Secondly, the mechanisms and recent advances of novel super diffraction near-field laser fabrication technologies based on the evanescent waves or surface plasmon polaritons are recommended. Scanning near-field lithography used a near-field scanning optical microscope coupled with a laser to create nanoscale structures with a resolution beyond 100 nm. Besides, near-field optical lithography beyond the diffraction limit could also be achieved through super resolution near-field structures, such as a bow-tie nanostructure. The interference by the surface plasmon polariton waves could lead to the fabrication of super diffraction interference fringe structures with a period smaller than 100 nm. Moreover, a femtosecond laser beam could also excite and interfere with surface plasmon polaritons to form laser-induced periodic surface structures. Furthermore, the super-resolution superlens and hyperlens imaging lithography are introduced. Evanescent waves could be amplified by using the superlens of metal film to improve the optical lithography resolution beyond the diffraction resolution. The unique anisotropic dispersion of hyperlens could provide the high wave vector component without the resonance relationship, which could also realize the super resolution imaging. Finally, prospective research and development tend of super diffraction laser fabrication technologies are presented. It is necessary to expand the range of materials which can be fabricated by laser beyond the diffraction limit, especially 2D materials.

The Duality of Kupffer Cell Responses in Liver Metabolic States.

Kupffer cells (KCs) are vital innate immune system cells in the liver that participate in various metabolic states. Although several studies have directly evaluated the role of KCs in different metabolic situations in the liver, the definite characteristics of KCs remain unknown. KC depletion techniques are used to determine the functions of KCs under metabolic challenges; however, there is debate about their precise role even after successful ablation. While some KC ablation studies showed improvement in insulin resistance, fatty liver and metabolic parameters, other reports, under the similar conditions, have not. Some studies have rationalized the KCs dual actions in liver metabolic states by arguing their M1 and M2 biases. Activated M1 KCs secrete pro-inflammatory cytokines, growth factors and other mediators to create inflammatory stress which initiates inflammatory signalling in hepatocytes to disrupt the metabolic scenario. In contrast, M2 KCs generate anti-inflammatory cytokines and mediators which improve glucose and lipid metabolic states within the liver. Unfortunately, the M1/M2 bias does not provide reliable explanation as KCs depletion shows strikingly different results. Despite many investigational studies on this topic, a comprehensive review of these studies is lacking. This review attempts to address the issues related with the dichotomy of KCs effects in lipid metabolism. This review not only warns the future studies to carefully analyze the results that are drawn from KCs effects on lipid metabolism but also suggest to evaluate animal models and KCs depletion techniques as an equally important cofactor.

Comparison of Early Post-Transplant Outcomes in Ex Vivo αβ+ T Cell-Depleted Haploidentical HSCT with or without Pharmacologic Gvhd Prophylaxis

Background and purpose: One of the key obstacles to successful haploidenitcal hematopoietic cell transplantation (HHCT) is a development of fatal GVHD. Although much progress in immunosuppressant (IS) has effectively prevented the development of acute GVHD, they have many serious toxicity and drug interactions requiring serial monitoring of drug levels. Recent advances in ex vivo depletion technique enabled to effectively reduce T cells or their subset, αβ+ T cells, leading to residual αβ+ T cells in grafts well below 5×104/kg of recipient weight. We eliminated post-transplant pharmacologic GVHD prophylaxis along with targeting αβ+ T cell dose ≤ 5×104/kg since November 2015. In this study, we compared early post-transplant outcomes between with (IS+) or without (IS-) post-transplant immunosuppressants after ex vivo αβ+ T cell-depleted HHCT.Methods: Between May 2012 and July 2016, 69 pediatric patients received HHCT using TCRαβ-depleted grafts from haploidentical family donors at Asan Medical Center Children’s Hospital. Fifty patients received tacrolimus and mycophenolate mofetil to prevent acute GVHD, while 19 did not receive any immunosuppressant after transplant. All donors received G-CSF for 4 consecutive days and peripheral blood stem cells were collected on days -1 and 0. The αβ+ T cells were depleted by negative selection using the CliniMACS® system (Miltenyi-BioTec, Bergisch-Gladbach, Germany) according to manufacturer’s instruction. In the earlier trial of IS+, the final doses of αβ+ T cells were adjusted to 1-5×105 cells/kg by add-back from the raw bag. Since November 2015, the cell dose was targeted at ≤ 5×104 αβ+T cells/kg with no post-transplant immunosuppressants (IS-).Results: The median infused CD34+ cells, αβ+ T cells, γδ+ T cells and CD3-CD56+ NK cells per kg of recipient weight were 8.9×106, 33.8×104, 20.0×106, 45.9×106 in IS+ group and 6.1×106, 4.6×104, 17.5×106, 24.6×106 in IS- group, respectively. All 69 patients achieved neutrophil engraftment at a median of 10 days (range, 9-17). Three patients out of 50 in IS+ group experience graft rejection (GR), while no GR occurred in IS- group. The cumulative incidences of acute GVHD grade II-IV were similar (31% vs 33%). Severe acute GVHD ≥ grade III developed in 7 in IS+ group, while none in IS- group developed ≥ grade III. As of July 2016, the median follow-ups were 24 months (range 9.5-50.8) for IS+ group and 5 months (0.5-9.1) for IS- group. Two out of 50 patients in IS+ group died of TRM leading to 2.2% at 6 months and 4.9% at 1 year after HHCT, while no patients in IS- group died of TRM during the follow-up period. The mean time from transplant to discharge were longer in IS+ group compared to IS- group (32 days versus 21 days, P=0.049). While the mean time of hospital stay within 100 days post-HHCT for patients who survived more than 100 days was not different between two groups (47 days versus 34 days, P>0.05).Conclusions: The major findings of our study were less severe acute GVHD and shorter hospital stay from HHCT to discharge in IS- group, even with less T cell dose, compared to IS+ group. Therefore, this HHCT using ex vivo αβ-depleted graft containing αβ+ T cells ≤ 5×104/kg is an effective treatment strategy to prevent acute GVHD without post-transplant IS. In addition, the early clinical outcomes were comparable between with and without post-transplant IS. DisclosuresNo relevant conflicts of interest to declare.

Gas Transportation and Enhanced Coalbed Methane Recovery Processes in Deep Coal Seams: A Review

Coalbed methane (CBM) is a potential green energy supply for addressing the worldwide energy crisis. However, the recovery of economically viable amounts of methane requires the application of production-enhancement techniques. The greater effectiveness of enhanced coalbed methane (ECBM) recovery compared to traditional pressure depletion and hydraulic stimulation techniques has been identified in terms of higher CBM recovery with minimal pollution risk and the ability to contribute to CO2 sequestration. Gas transport behavior in a coal seam is the governing factor for ECBM recovery, which includes sorption/desorption and diffusion in the matrix and advective flux in cleats. The interactions among sorption, diffusion, and flow indicate the complexity and abstruseness of gas transport in coal. Therefore, the purpose of this paper is to provide comprehensive knowledge of the gas transport process in deep coal seams, particularly in relation to the ECBM process. According to the review, the dual-porosity sys...

How Does Competition between Anionic Pollutants Affect Adsorption onto Mg–Al Layered Double Hydroxide? Three Competition Schemes

Competitive adsorption of three anionic orange-type dyes (i.e., methyl orange, orange II, orange G) and three inorganic anions (HPO42–, SO42–, CrO42–) onto Mg–Al layered double hydroxide (LDH) from bisolute aqueous solutions has been studied in view of potential application of the sorption-based technology in dye removal from textile wastewater effluents. The individual solute affinities for the LDH sample were inferred from the calorimetry measurements of the cumulative enthalpy of displacement in single-solute systems, thus leading to the following affinity order: CrO42– ≈ SO42– < OG2– ≈ HPO42– < OII– ≪ MO–. The individual and composite adsorption isotherms from bisolute aqueous solutions were determined by a solution depletion technique and supplemented by XRD studies on the well-defined adsorption systems corresponding to selected points on isotherms. Based on the shape of the individual adsorption isotherms for a dye and inorganic anion, it was possible to distinguish three different competition sche...

Macrophage depletion decreases inflammation and tumorigenesis in the AOM/DSS mouse model of colon cancer

Abstract Macrophages have recently emerged as major players in the connection between inflammation and cancer; they represent up to 50% of the tumor mass and produce a wide array of inflammatory mediators with pro-tumoral functions. We used a macrophage depletion technique to examine the role of macrophages in colon cancer. To induce colon cancer, male C57BL/6 mice (n=20) received a single injection of AOM followed by three cycles of DSS supplemented water at concentrations of 2%, 1%, 1% at weeks 1, 4, and 7, respectively. Prior to the final DSS administration (week 7) and twice weekly thereafter for a duration of 5 weeks, mice (n=10/group) were administered 200μl of clodronate (CL2MDP) or PBS encapsulated liposomes via an intra-peritoneal route to deplete macrophages. Body weight, body composition, and symptom severity score were evaluated throughout the macrophage depletion period. Mice were sacrificed at 12 weeks following the initiation of colon cancer and colon tissue was analyzed for tumor burden, macrophage markers, and inflammatory mediators. There were no differences in body weight, body composition, or symptom severity score between the groups. Macrophage depletion decreased tumor number by ~25% and specifically medium tumor number by 50% (p&amp;lt;0.05). This was consistent with a decrease in expression of EMR1 (overall macrophage marker) as well as expression of markers associated with M2 macrophages (IL-13, IL-10, TGFβ &amp; CCL17) (p&amp;lt;0.05) but not M1 markers (NOS2, IL-12a &amp; IL-23). In addition, expression of the inflammatory mediators IL-6 and TNF-α were decreased following macrophage depletion. We demonstrate that macrophages are an important mediator of tumor growth and inflammation in colon cancer.

Solubility enhancement of dioxins and PCBs by surfactant monomers and micelles quantified with polymer depletion techniques

Partitioning of super-hydrophobic organic contaminants (SHOCs) to dissolved or colloidal materials such as surfactants can alter their behaviour by enhancing apparent aqueous solubility. Relevant partition constants are, however, challenging to quantify with reasonable accuracy. Partition constants to colloidal surfactants can be measured by introducing a polymer (PDMS) as third phase with known PDMS-water partition constant in combination with the mass balance approach. We quantified partition constants of PCBs and PCDDs (log KOW 5.8–8.3) between water and sodium dodecyl sulphate monomers (KMO) and micelles (KMI). A refined, recently introduced swelling-based polymer loading technique allowed highly precise (4.5–10% RSD) and fast (<24 h) loading of SHOCs into PDMS, and due to the miniaturisation of batch systems equilibrium was reached in <5 days for KMI and <3 weeks for KMO. SHOC losses to experimental surfaces were substantial (8–26%) in monomer solutions, but had a low impact on KMO (0.10–0.16 log units). Log KMO for PCDDs (4.0–5.2) were approximately 2.6 log units lower than respective log KMI, which ranged from 5.2 to 7.0 for PCDDs and 6.6–7.5 for PCBs. The linear relationship between log KMI and log KOW was consistent with more polar and moderately hydrophobic compounds. Apparent solubility increased with increasing hydrophobicity and was highest in micelle solutions. However, this solubility enhancement was also considerable in monomer solutions, up to 200 times for OCDD. Given the pervasive presence of surfactant monomers in typical field scenarios, these data suggest that low surfactant concentrations may be effective long-term facilitators for subsurface transport of SHOCs.