Standard Centrifugation Research Articles

Comparative evaluation of growth factors from platelet concentrates: An in vitro study.

Aim:The aim of the study was to compare and evaluate the various growth factors released for a period of 23 days from platelet rich fibrin (PRF) and platelet rich fibrin matrix (PRFM).Materials and Methods:A total of 15 systemically healthy controls were recruited and 10 ml of blood sample was withdrawn from the individual. Following the standard centrifugation protocol, PRF and PRFM were prepared. The platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and insulin growth factor (IGF) were evaluated for 23 days.Results:The PDGF released from PRFM was statistically significant from PRF till the 15th day of release. The VEGF too had an increased release up till the 15th day from PRFM group as compared to PRF, but no statistically significant difference could be obtained. EGF from the 15th day to 23rd day had a greater release from PRFM group as compared to PRF group. FGF from 7th day to 23rd day had statistically significant difference in the PRFM group as compared to PRF group. TGF and IGF had statistically significant difference in PRFM group as compared to PRF group from 11th day to 23rd day and 1st to 17th day, respectively.Conclusion:The initial and robust release of GFs was seen in PRFM group at earlier days, whereas a steady and constant release of six GFs could be appreciated from PRF group upto 23rd day. Therefore, for a rapid and early healing and regeneration, both the platelet concentrates can be utilized in periodontal therapy.

Comparison of sperm selection techniques in donkeys: motile subpopulations from a practical point of view.

The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).

Tag7-Mts1 Complex Induces Lymphocytes Migration via CCR5 and CXCR3 Receptors

The discovery of new chemokines that induce the migration of lymphocytes to the infection site is important for the targeted search for therapeutic agents in immunotherapy. We recently showed that Tag7 (PGLYRP1), an innate immunity protein, forms a stable complex with the Ca2+ -binding protein Mts1 (S100A4), which is able to induce lymphocyte movement, although the individual Tag7 and Mts1 do not have this activity. The purpose of this study is to identify receptors that induce the migration of lymphocytes along the concentration gradient of the Tag7-Mts1 complex, and the components of this complex capable of interacting with these receptors. The study investigated the migration of human PBMC under the action of the Tag7-Mts1complex. PBMC of healthy donors were isolated using a standard Ficoll-Hypaque gradient centrifugation procedure. It has been established that the movement of PBMC along the concentration gradient of the Tag7-Mts1 complex is induced by the classical chemotactic receptors CCR5 and CXCR3. It has been shown that only Mts1 is able to bind to the extracellular domain of CCR5, however, this binding is not enough to induce cell movement. A comparative analysis of the primary and 3D structures of the three proteins revealed the homology of the amino acid sequence fragments of the Tag7-Mts1 protein complex with different sites of the CCR5 receptor ligand - MIP1α protein. In conclusion, it should be noted that the Tag7-Mts1 complex can be considered as a new ligand of the classical chemotactic receptors CCR5 and CXCR3.

Using Machine Learning to Classify the “Goodness” of Hmsc-Derived and AML-Derived EV's

IntroductionExtracellular vesicles (EVs) form a unique class of messengers for intercellular communication. Depending on their cell of origin, EVs have the ability to induce a phenotypic change in the recipient cell. For example, EVs from explant prostate cancer induce a neoplastic phenotype in normal prostate cell lines. Conversely, EVs from human mesenchymal stem cells (hMSC) reverse the malignant phenotype in prostate and colorectal cancer and mitigate radiation damage to the marrow. Characterization of EVs as “good” or “bad” has the potential to be a very important diagnostic tool in regard to direct therapy and biomarker identification. Currently, there is no way of characterizing the “goodness” of an EV sample. We leveraged advances in the area of machine learning to develop a novel therapeutic tool that can classify the goodness of an EV particle distribution in a serum sample.MethodsEVs were harvested from three sources: hMSC primary progenitor cells, Kasumi Acute Myeloid Leukemia (AML) cells lines, and patient samples. Using a standard centrifugation isolation, EVs were isolate, resuspended in 1% DMSO, and frozen. All samples were analyzed using the NanoSight N500. We collected biophysical properties of the EVs such as diameter and diffusion coefficient. The results were summarized in a distribution based on either size or diffusion coefficient.Summary statistics from each distributions were calculated. Summary statistics included mean, mode, and the diameter at which 10%, 50%, and 90% of size or diffusion coefficient is comprised of smaller particles (D10, D50, and D90, respectively). These served as inputs into a softmax Multilayer Perceptron. This neural network classifier was trained on only the hMSC-derived EVs and Kasumi AML-derived EVs, which served to represent a healthy patient and leukemic patient respectively.Results/ConclusionThe mean accuracy after 10 fold cross validation was 90.16% ± 9.26%. For each validation run, a Receiver Operating Characteristic (ROC) curve was drawn and the area under the curve (AUC) was calculated. The mean AUC (after 10 fold cross validation) was 95.97% ± 5.38%.We programmed the algorithm, when given a patient sample, to calculate and return similarity to the Kasumi AML-derived EVs. The algorithm was given three patient sample representing three leukemic disease processes: AML, Chronic Myelomonocytic Leukemia (CMML), and Multiple myeloma (MM). The result was a calculate % similarity of 100%, 100%, and 66% for AML, CMML, and MM respectively. These results are promising and have prompted us to begin collecting and test the algorithms on normal, active leukemic, and recovered leukemic patients. We endeavor to evolve our predictive algorithms to include disease and patient specific information, allowing us to adapt our learning models towards clinically relevant endpoints. DisclosuresReagan:Alexion: Honoraria; Pfizer: Research Funding; Takeda Oncology: Research Funding. Olszewski:Spectrum Pharmaceuticals: Consultancy, Research Funding; TG Therapeutics: Research Funding; Genentech: Research Funding.

Microfluidic Sorting Selects Sperm for Clinical Use With Reduced DNA Damage Compared to Density Gradient Centrifugation in Split Semen Samples

Capsule Microfluidic sorting of unprocessed semen allows for the selection of clinically usable sperm with lower DNA fragmentation than standard processing. Objective To determine if microfluidic sorting improves the selection of sperm with lower DNA fragmentation over standard density-gradient centrifugation. Design Blinded, controlled laboratory study. Setting Academic medical center. Specimens Consecutively collected routine semen analysis samples (n=70). Intervention(s) For each sample, the unprocessed semen was tested for DNA fragmentation and split for processing by density-gradient centrifugation and sorting by a microfluidic chip. DNA fragmentation was assessed in unprocessed and processed samples by Sperm Chromatin Dispersion (SCD) test. Outcome Measure DNA Fragmentation Index (DFI) was calculated as number of cells with fragmented DNA divided by the number of cells counted per slide. Results The median DFI in unprocessed samples was 21% (IQR 14-30). In paired analyses of all samples, those processed by the microfluidic chip demonstrated significantly decreased DFI compared to those processed by density-gradient centrifugation and unprocessed samples. The median DFI for chip specimens was 0% (IQR 0–2.4) while those processed by density gradient centrifugation had a median DFI of 6% (IQR 2-11). Unprocessed samples in the highest DFI quartile (DFI range 31–40%) had median DFI of 15% (IQR 11–19%) after density-gradient centrifugation and DFI 0% (IQR 0–1.9%) after processing with the microfluidic chip (p = 0.02). Conclusion Microfluidic sorting of unprocessed semen allows for the selection of clinically usable, highly motile sperm with nearly undetectable levels of DNA fragmentation. Future studies will be expending the sample size, and validate if the use of chip can be translated into difference in pregnancy rates. Conflict of Interest Disclosures Utkan Demirci, Ph.D. is the Co-founder and Scientific Advisor for DxNow Inc., LevitasBio Inc., and Koek Biotech. Mitchell Rosen, M.D. is a member of the Clinical Advisory Board for DxNow Inc. Funding None.

Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins.

Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144-1155, 2018.

Niacin prevents mitochondrial oxidative stress caused by sub-chronic exposure to methylmercury

Humans and animals can be exposed to different chemical forms of mercury (Hg) in the environment. For example, methylmercury (MeHg)-contaminated fish is part of the basic diet of the riparian population in the Brazilian Amazon Basin, which leads to high total blood and plasma Hg levels in people living therein. Hg induces toxic effects mainly through oxidative stress. Different compounds have been used to prevent the damage caused by MeHg-induced reactive oxygen species (ROS). This study aims to investigate the in vivo effects of sub-chronic exposure to low MeHg levels on the mitochondrial oxidative status and to evaluate the niacin protective effect against MeHg-induced oxidative stress. For this purpose, Male Wistar rats were divided into four groups: control group, treated with drinking water on a daily basis; group exposed to MeHg at a dose of 100 µg/kg/day; group that received niacin at a dose of 50 mg/kg/day in drinking water, with drinking water being administered by gavage; group that received niacin at a dose of 50 mg/kg/day in drinking water as well as MeHg at a dose of 100 µg/kg/day. After 12 weeks, the rats, which weighed 500–550 g, were sacrificed, and their liver mitochondria were isolated by standard differential centrifugation. Sub-chronic exposure to MeHg (100 µg/kg/day for 12 weeks) led to mitochondrial swelling (p < 0.05) and induced ROS overproduction as determined by increased DFCH oxidation (p < 0.05), increased gluthatione oxidation (p < 0.05), and reduced protein thiol content (p < 0.05). In contrast, niacin supplementation inhibited oxidative stress, which counteracted and minimized the toxic MeHg effects on mitochondria.

Abstract 1969: Cancer exosomes as a new mediator to spread drug resistance to EGFR tyrosine kinase inhibitors

Abstract Aim: The study aims to identify new therapeutic targets from cancer exosomes, which may spread drug resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in the treatment of non-small-cell lung cancer (NSCLC). Background: EGFR TKIs have exhibited extraordinary clinical efficacy in advanced lung cancer. However, their usefulness is compromised by drug resistance mediated by various mechanisms, including the emergence of a secondary EGFR T790M mutation. Exosomes are small membrane vesicles derived from the cellular phenomenon of membrane blebbing under both normal and pathological conditions. They mediate intercellular cross-talk by transferring receptors, antigens and cytokines from donor to recipient cells. It has been reported that EGFR TKI resistance mutation exists before drug exposure. Selective pressure from EGFR TKI therapy may thus cause the expansion of the latent resistant subclones, resulting in the emergence of a resistant phenotype in the entire tumor population. We hypothesize that exosomes may mediate the transfer of the secondary EGFR mutant or other mediators from the resistant subclones to neighboring sensitive cells. Method: The possible exosome-mediated transfer of secondary EGFR mutant (or other protein mediators) from EGFR T790M-bearing NSCLC (H1975) cells to a sensitive NSCLC cell line (HCC827) was evaluated. Exosomes were isolated from H1975 cells-conditioned medium by the standard differential centrifugation method. The secretion of protein mediators into exosomes by resistant H1975 cells was studied by protein array. The EGFR kinase activity of the exosomes was evaluated by biochemical assay. The uptake of fluorescence-labeled exosome from the resistant cells by the sensitive cells was visualized by fluorescence microscopy. The activation of the EGFR signaling pathway in the recipient sensitive cells after incubation with exosomes derived from the resistant cells was examined by Western blot analysis. Result: Exosomes were successfully isolated from H1975-conditioned medium and confirmed by the expression of exosomal markers CD63 and HSP70. Compared with exosomes derived from sensitive HCC827 cells, exosomes secreted from H1975 cells were enriched with apoptosis-regulating proteins including Src. H1975-derived exosomes increased the viability of HCC827 cells in the presence of gefitinib and inhibited the apoptosis induced by gefitinib. Western blot analysis showed that the exosomes can block the reduction of BCL-2 and full-length PARP, while abolishing the increase of BAX and cleaved PARP induced by gefitinib in HCC827 cells. Conclusion: H9175-derived exosomes may be involved in the spreading of drug resistance to EGFR TKIs by regulating cell apoptosis. New treatment strategies may be designed to target and inhibit this exosome-mediated transfer of mediators implicated in the acquired resistance to EGFR TKIs. Citation Format: Kenneth KW To, Christy WS Tong, Mia MX Wu. Cancer exosomes as a new mediator to spread drug resistance to EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1969.

Chlamydia trachomatis inhibits the production of pro-inflammatory cytokines in human PBMCs through induction of IL-10.

Previous research demonstrated that IL-10 was up-regulated in Chlamydia trachomatis-infected cells and that exogenous IL-10 was able to inhibit the secretion of pro-inflammatory cytokines by infected cells. However, the mechanisms are not well understood. The aim of this study was to investigate the mechanisms for up-regulation of IL-10 and inhibition of pro-inflammatory cytokine secretion in C. trachomatis-stimulated peripheral blood mononuclear cells (PBMCs). Human PBMCs were isolated from the blood of healthy human donors by standard Ficoll-Hypaque density gradient centrifugation. Cells were exposed to C. trachomatis in the presence or absence of MEK inhibitor U0126, the p38 inhibitor SB203580, the STAT3 inhibitor Ruxolitinib or anti-human IL-10 antibody. Cytokines were measured from culture supernatants using ELISA kits. Cells were harvested for real-time quantitative PCR to determine IL-10 mRNA levels and for Western blot assay to detect the expression of ERK1/2, p-ERK1/2, p38, p-p38, STAT3 and p-STAT3. Both mRNA and protein levels of IL-10 were up-regulated in stimulated cells, and the production of IL-10 was reduced when cells were treated with U0126 or SB203580. The expression of cytokines IL-6, IL-8 and TNF-α was enhanced in stimulated cells treated with anti-human IL-10 antibody. Moreover, neutralization of IL-10 resulted in a significant decrease of phosphorylated STAT3 in stimulated cells. Ruxolitinib caused a significant increase in the production of IL-6, IL-8 and TNF-α in stimulated cells. IL-10 is up-regulated in an ERK- and p38-dependent fashion in stimulated human PBMCs. IL-10 inhibits the production of pro-inflammatory cytokines by activating the JAK/STAT signalling pathway.

Enrichment of Extracellular Vesicle Subpopulations Via Affinity Chromatography.

Extracellular vesicles (EVs) are secreted nanoscale particles that transfer biomolecular cargo between cells in multicellular organisms. EVs play a variety of roles in intercellular communication and are being explored as potential vehicles for delivery of therapeutic biomolecules. However, EVs are highly heterogeneous in composition and biogenesis route, and this poses substantial challenges for understanding the role of EVs in biology and for harnessing these mechanisms for therapeutic applications, for which purifying therapeutic EVs from mixed EV populations may be necessary. Currently, technologies for isolating EV subsets are limited by overlapping physical properties among EV subsets. To meet this need, here we report an affinity chromatography-based method for enriching a specific EV subset from a heterogeneous EV starting population. By displaying an affinity tagged protein (tag-protein) on the EV surface, tagged EVs may be specifically isolated using simple affinity chromatography. Moreover, recovered EVs are enriched in the tag-protein relative to the starting population of EVs and relative to EVs purified from cell culture supernatant by standard differential centrifugation. Furthermore, chromatographically enriched EVs confer enhanced delivery of a cargo protein to recipient cells (via enhancing the amount of cargo protein per EV) relative to EVs isolated by centrifugation. Altogether, affinity chromatographic enrichment of EV subsets is a viable and facile strategy for investigating EV biology and for harnessing EVs for therapeutic applications.

Comparative Evaluation of Antimicrobial Efficacy of Platelet-rich Plasma, Platelet-rich Fibrin, and Injectable Platelet-rich Fibrin on the Standard Strains of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

Context:Platelet concentrates are commonly used to promote periodontal soft- and hard-tissue regeneration. Recently, their antimicrobial efficacy is also explored. Various platelet concentrates have evolved which differ in the centrifugation protocols. Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) have been found to have an important role in periodontal pathology.Aims:In this study, PRP, PRF, and I-PRF are compared for their antibacterial effect against Pg and Aa.Materials and Methods:Blood samples were obtained from ten systemically and periodontally healthy individuals. Platelet concentrates were prepared using standardized centrifugation protocol. Antimicrobial activity was examined on standard strains of Pg and Aa using well diffusion method.Statistical Analysis Used:Means for the width of zones of inhibition were calculated along with standard deviations, and the comparison was made using Wilcoxon signed-rank test.Results:In case of Pg, I-PRF had the widest zone of inhibition which was significantly wider as compared to PRF. Furthermore, PRP had significantly wider zone of inhibition against PRF. In case of Aa, PRP had wider zone of inhibition which was significantly wider as compared to that of PRF and I-PRF.Conclusions:All the three platelet concentrates PRP, PRF, and I-PRF have antibacterial activity, but PRP and I-PRF are more active as compared to PRF. I-PRF being autologous and easy to prepare can be a very useful adjunct to the surgical therapy in bringing down the bacterial count helping in wound healing and regeneration.

Preanalytic processing of rat plasma influences thrombin generation and fibrinolysis assays.

Thrombin generation assays (TGA) have potential applications as measures of hemostatic balance in animal models. However, variations in plasma processing greatly influence human TGA, and may also impact on the translational value of TGA in animal studies. The purpose of the study was to compare the performance characteristics of Sprague-Dawley rat plasma prepared by single vs double centrifugation protocols in TGA and fibrinolysis assays. Platelet-poor plasma (PPP) from adult rats (n = 20 males; 20 females) was prepared by centrifugation at 1200g × 12 min, or 2 sequential centrifugations of 2500g × 15 min. Plasma aliquots were assayed fresh and after freeze-thaw in a commercial fluorogenic TGA (Technothrombin TGA, Technoclone) using 2 different trigger reagents containing approximately 7 pM human tissue factor. In addition to TGA variables (lag time, peak thrombin, endogenous thrombin potential), we compared clotting time test and fibrinogen concentration, residual platelet and platelet-derived microparticle (PMP) counts measured by flow cytometry, and variables of fibrin clot formation and lysis measured in turbidimetric assays. Single-centrifugation PPP demonstrated significantly greater thrombin-generating potential regardless of trigger reagent, yielded higher residual platelet and procoagulant PMP counts, and more stable fibrin clot profiles. The influence of a freeze-thaw cycle on TGA varied depending on trigger reagent, and male sex was associated with an overall "procoagulant" phenotype. Preanalytic processing and sex have significant effects on many functional measures of hemostasis in rats. A standardized double centrifugation protocol to prepare PPP is recommended for future studies.

Concentration of Plasmodium falciparum gametocytes in whole blood samples by magnetic cell sorting enhances parasite infection rates in mosquito feeding assays

BackgroundMosquito-feeding assays are important tools to guide the development and support the evaluation of transmission-blocking interventions. These functional bioassays measure the sporogonic development of gametocytes in blood-fed mosquitoes. Measuring the infectivity of low gametocyte densities has become increasingly important in malaria elimination scenarios. This will pose challenges to the sensitivity and throughput of existing mosquito-feeding assay protocols. Here, different gametocyte concentration methods of blood samples were explored to optimize conditions for detection of positive mosquito infections.MethodsMature gametocytes of Plasmodium falciparum were diluted into whole blood samples of malaria-naïve volunteers. Standard centrifugation, Percoll gradient, magnetic cell sorting (MACS) enrichment were compared using starting blood volumes larger than the control (direct) feed.ResultsMACS gametocyte enrichment resulted in the highest infection intensity with statistically significant increases in mean oocyst density in 2 of 3 experiments (p = 0.0003; p ≤ 0.0001; p = 0.2348). The Percoll gradient and standard centrifugation procedures resulted in variable infectivity. A significant increase in the proportion of infected mosquitoes and oocyst density was found when larger volumes of gametocyte-infected blood were used with the MACS procedure.ConclusionsThe current study demonstrates that concentration methods of P. falciparum gametocyte-infected whole blood samples can enhance transmission in mosquito-feeding assays. Gametocyte purification by MACS was the most efficient method, allowing the assessment of gametocyte infectivity in low-density gametocyte infections, as can be expected in natural or experimental conditions.

Exosomes derived from bone marrow stromal cells decrease the sensitivity of leukemic cells to etoposide.

The aim of the study was to investigate the effect of exosomes derived from bone marrow stromal cells (BM-SCs) on the chemoresistant characteristics of nalm-6 cells treated with etoposide (VP16). The present study isolated exosomes from BM-SC-conditioned medium by using standard differential centrifugation steps and detected the expression of 70 kilodalton heat shock proteins (HSP70) and lysosomal-associated membrane protein 3 (CD63) in exosomes by western blot analysis. Nalm-6 cells were co-cultured with exosomes in the presence of VP16. Cell viability and apoptosis were then detected using the Cell Counting Kit-8 method and Annexin-V/propidium iodide, respectively. Finally, protein levels of B-cell lymphoma 2 (BCL-2), BCL-2-like protein 4 (BAX), caspase-3, and poly ADP-ribose polymerase (PARP) were examined by western blot analysis. Exosomes were successfully isolated from the conditioned medium and confirmed by the expression of HSP70 and CD63. BM-SC-derived exosomes increased the viability of nalm-6 cells in the presence of VP16 and inhibited the apoptosis induced by VP16. Western blot analysis results showed that exosomes can block the significant reduction of BCL-2, full-length caspase-3 and full-length PARP, while preventing the increase of BAX, cleaved caspase-3 and cleaved PARP induced by VP16. Exosomes derived from BM-SCs can protect nalm-6 cells from VP16-induced apoptosis to maintain their survival and induce resistance to VP16. In addition, BCL-2/BAX, caspase-3, and PARP may be involved in the mechanism of exosome-induced drug resistance.

Functional outcomes of platelet rich plasma injections in plantar fascitis

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; The study was conducted to analyse the clinical outcomes following a single injection of platelet rich plasma, produced using autologous blood by standard double centrifugation technique in patients with plantar fasciitis who have not had relief using conservative methods.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; Between June 2013 and June 2015, 26 consecutive patients (Mean age 45.92) (11 males/15 females) who had symptoms for a minimum of 3 months and had failed conservative management options such as rest, anti-inflammatories and physiotherapy were given a single injection of PRP in the medial calcaneal tubercle.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; The outcomes were measured using Visual analogue scale and Foot function index at pre injection, and at 1, 6 and 12 months post injection and results tabulated. All patients reported an improvement in VAS and FFI scores consistently. One superficial infection was encountered.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Single injection of platelet rich plasma improves pain and function in the foot in patients suffering from plantar fasciitis when conservative management has failed. The synergism between various autologous growth factors may play a role in tendon healing. &lt;/p&gt;

High Throughput Cell-Free Extraction of Plasma by an Integrated Microfluidic Device Combining Inertial Focusing and Membrane

Plasma is a host of numerous analytes such as proteins, metabolites, circulating nucleic acids (CNAs), and pathogens, and it contains massive information about the functioning of the whole body, which is of great importance for the clinical diagnosis. Plasma needs to be completely cell-free for effective detection of these analytes. The key process of plasma extraction is to eliminate the contamination from blood cells. Centrifugation, a golden standard method for blood separation, is generally lab-intensive, time consuming, and even dangerous to some extent, and needs to be operated by well-trained staffs. Membrane filtration can filter cells very effectively according to its pore size, but it is prone to clogging by dense particle concentration and suffers from limited capacity of filtration. Frequent rinse is lab-intensive and undesirable. In this work, we proposed and fabricated an integrated microfluidic device that combined particle inertial focusing and membrane filter for high efficient blood plasma separation. The integrated microfluidic device was evaluated by the diluted (×1/10, ×1/20) whole blood, and the quality of the extracted blood plasma was measured and compared with that from the standard centrifugation. We found that the quality of the extracted blood plasma from the proposed device can be equivalent to that from the standard centrifugation. This study demonstrates a significant progress toward the practical application of inertial microfluidics with membrane filter for high-throughput and highly efficient blood plasma extraction.

Abstract WP255: Rapid Coagulation Tests for Acute Ischemic Stroke

Background and objectives: The results of coagulation tests are important for the decision of thrombolysis in acute ischemic stroke patients who are taking oral anticoagulants. In coagulation tests, the preparation of blood specimens is a major bottleneck in the workflow. Clinical and Laboratory Standard Institute (CLSI) guidelines require that routine coagulation tests are performed with platelet-poor plasma (&lt;10,000 platelets per microliter). We investigated whether high acceleration centrifugation can reduce the time of coagulation tests without breaking the guidelines. Methods: We compared the high acceleration centrifugation condition (3 minutes, 8000 rpm) with the standard centrifugation condition (15 minutes, 3000 rpm) in 32 patients. The pre- and post-centrifugation platelet counts were measured and compared between the groups. Results: Sixteen samples were centrifuged in high acceleration condition and another 16 samples in standard condition. The pre-centrifugation platelet counts were not different (median, 165,000 (interquartile range, 146,750-185,500) per microliter vs. 166,500 (128,000-193,250), p=0.675) between the groups. Although the post-centrifugation platelet counts were higher in the high acceleration group than the standard group (4,500 (2,250-6,000) vs. 1,000 (0-2,000), p&lt;0.001), all the samples in the high acceleration group met the requirements of platelet-poor plasma. Using the high acceleration centrifugation method, the reporting time of coagulation tests decreased from 24 (23-25) to 12 (12-13.5) minutes. Conclusion: High acceleration centrifugation can reduce the time of coagulation tests for 12 minutes without breaking the guidelines. This method may help to shorten the door-to-needle time in acute ischemic stroke patients who are taking oral anticoagulants.

Comparative evaluation of platelet count and antimicrobial efficacy of injectable platelet-rich fibrin with other platelet concentrates: An in vitro study.

Background:Platelet concentrates are used in various medical procedures to promote soft- and hard-tissue regeneration. In recent times, their antimicrobial efficacy is also explored. However, various platelet concentrates have evolved which differ in the centrifugation protocols. One such recently introduced platelet concentrate is injectable platelet-rich fibrin (i-PRF) concentrate. Hence, the aim was to evaluate the antimicrobial property, and platelet count of i-PRF in comparison to other platelet concentrates, i.e., PRF, platelet-rich plasma (PRP), and control (whole blood).Materials and Methods:Blood samples were obtained from 10 chronic generalized marginal gingivitis patients. Platelet concentrates were prepared using standardized centrifugation protocol. Platelet count was evaluated by manual counting method using smear preparation of each sample. Subsequently, antimicrobial activity against oral bacteria was examined on blood agar using disc diffusion method to quantify the inhibitory effects.Results:Statistical significance was analyzed by one-way analysis of variance (ANOVA). P <0.05 was considered statistically significant. Mean zone of inhibition around i-PRF (P < 0.01) and PRF (P < 0.05) showed statistical significance. Although a distinct zone of inhibition was seen with PRP, it was not statistically significant (P > 0.05). i-PRF showed statistically significant difference (P < 0.001) in platelet count when compared to control. It was also significant when compared to PRP (P < 0.01), PRF (P < 0.001).Conclusion:i-PRF has maximum antimicrobial efficacy and higher platelet count in comparison to other platelet concentrates, thereby indicating to have a better regenerative potential then others.

Analysis of Lipoasiprated Following Centrifugation

The success of lipotransfer strongly depends on the harvesting, processing, and placement of the lipoaspirated samples. This study was designed to assess the histomorphometric characteristics and viability of fat harvested using different techniques (wet and dry) following centrifugation, as described by Coleman. The study enrolled 85 consecutive, nonrandomized, healthy patients from March 2010 to December 2014 (45 males and 40 females). The mean age was 40 years (range, 18-59 years), and the mean body mass index was 25.8 (range, 24-32). The authors performed a histological analysis (hematoxylin/eosin), morphometry (ImageJ 1.33 free-share image analysis software), and a viability assessment (Trypan Blue exclusion test; Sigma-Aldrich, Milan, Italy) of the lipoaspirated samples. The hematoxylin and eosin-stained sections exhibited similar features; in particular, clear-cut morphological signs of adipocyte disruption, apoptosis, or necrosis were not detected in the examined samples. Morphometry confirmed the visual findings, and the values of the mean surface area of the adipocyte vacuoles were not significantly different. Additionally, the adipocyte viability was not significantly different in the analyzed fat tissue samples. The results from this study showed, for the first time, that there is not a reduction in the viability of fat grafts harvested with the dry or wet technique following centrifugation according to Coleman technique. Both methods of fat harvesting collect viable cells, which are not influenced by standard centrifugation. The fat grafts harvested and processed by this technique could be used in clinical settings without increasing the reabsorption rate. V.

In vivo study of hepatic oxidative stress and mitochondrial function in rabbits with severe hypotension after propofol prolonged infusion.

In humans, prolonged sedations with propofol or using high doses have been associated with propofol infusion syndrome. The main objective of this study was to evaluate the effects of prolonged high-dose administration of a specific propofol emulsion (Propofol Lipuro) and an improved lipid formulation (SMOFlipid) in liver mitochondrial bioenergetics and oxidative stress of rabbits, comparatively to a saline control. Twenty-one male New Zealand white rabbits were randomly allocated in three groups that were continuously treated for 20 h. Each group of seven animals received separately: NaCl 0.9 % (saline), SMOFlipid (lipid-based emulsion without propofol) and Lipuro 2 % (propofol lipid emulsion). An intravenous propofol bolus of 20 mg kg−1 was given to the propofol Lipuro group to allow blind orotracheal intubation and mechanical ventilation. Anesthesia was maintained using infusion rates of: 20, 30, 40, 50 and 60 mg kg−1 h−1, according to the clinical scale of anesthetic depth and the index of consciousness values. The SMOFlipid and saline groups received the same infusion rate as the propofol Lipuro group, which were infused during 20 consecutive hours. At the end, the animals were euthanized, livers collected and mitochondria isolated by standard differential centrifugation. Mitochondrial respiration, membrane potential, swelling and oxidative stress were evaluated. Data were processed using one-way ANOVA (p < 0.05). The animals revealed a significant decrease in cardiovascular parameters showing bradycardia and severe hypotension. No statistical differences were observed when using pyruvate as substrate, however, when using succinate as respiratory substrate, significant decrease in ADP-stimulated respiration rate was observed for SMOFlipid group (p = 0.002). Lipid peroxides (p < 0.01) and protein carbonyls (p = 0.01) showed a statistically significant difference between propofol Lipuro and the SMOFlipid groups. These results suggest that lipid-based emulsions can be involved in the regulation of different pathways that ultimately lead to a decrease of state 3 mitochondrial respiration rate. The infusion of propofol Lipuro during prolonged periods, in addition to marked hypotension and hypoperfusion, also showed to have higher anti-oxidant activity and lower impairment of the mitochondrial function comparatively to the improved lipid formulation, SMOFlipid, using the rabbit as animal model.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2970-2) contains supplementary material, which is available to authorized users.