Primary Human Leukocytes Research Articles

A novel method for autophagy detection in primary cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8+ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.

Targeting the leukocyte activation cascade: Getting to the site of inflammation using microfabricated assays

This paper describes the use of microfabricated devices to study the leukocyte activation cascade (LAC). The devices consist of microchannels fabricated in polydimethylsiloxane using soft lithography. Microfluidics, used to generate physiologically relevant levels of shear flow, was achieved by the simple attachment of a syringe pump. Microchannel surfaces were modified by self-assembled monolayer (SAM) chemistries. The devices were adapted to standard 96-well tissue culture format with microchannels that could accommodate either a monolayer of endothelial cells or a SAM with immobilized chemokines. Chemotaxis was performed using linear gradients of chemokine set in a 3D matrix. Using this approach, we demonstrated robust chemotaxis of primary human leukocytes (PHLs) in response to a gradient of the chemokine CCL2. Rolling and adhesion assays performed under shear flow demonstrated that leukocyte recruitment to the substrate was highly sensitive to both biological and physical forces. CCL2 and CXCL12 treatment of PHLs dose dependently increased activation and adhesion. These actions could be inhibited by the use of peptide or small molecule antagonists. These devices provide a robust platform to perform LAC assays under in vivo-like conditions.

Identification and Characterisation of an Iron-Responsive Candidate Probiotic

BackgroundIron is an essential cofactor in almost all biological systems. The lactic acid bacteria (LAB), frequently employed as probiotics, are unusual in having little or no requirement for iron. Iron in the human body is sequestered by transferrins and lactoferrin, limiting bacterial growth. An increase in the availability of iron in the intestine by bleeding, surgery, or under stress leads to an increase in the growth and virulence of many pathogens. Under these high iron conditions, LAB are rapidly out-competed; for the levels of probiotic bacteria to be maintained under high iron conditions they must be able to respond by increasing growth rate to compete with the normal flora. Despite this, iron-responsive genera are poorly characterised as probiotics.Methodology/Principal FindingsHere, we show that a panel of probiotics are not able to respond to increased iron availability, and identify an isolate of Streptococcus thermophilus that can increase growth rate in response to increased iron availability. The isolate of S. thermophilus selected was able to reduce epithelial cell death as well as NF-κB signalling and IL-8 production triggered by pathogens. It was capable of crossing an epithelial cell barrier in conjunction with E. coli and downregulating Th1 and Th17 responses in primary human intestinal leukocytes.Conclusions/SignificanceWe propose that an inability to compete with potential pathogens under conditions of high iron availability such as stress and trauma may contribute to the lack of efficacy of many LAB-based probiotics in treating disease. Therefore, we offer an alternative paradigm which considers that probiotics should be able to be competitive during periods of intestinal bleeding, trauma or stress.

Micropatterned aptasensors for continuous monitoring of cytokine release from human leukocytes.

We report the development of a microdevice for detecting local interferon gamma (IFN-γ) release from primary human leukocytes in real time. Our microdevice makes use of miniature aptamer-modified electrodes integrated with microfluidics to monitor cellular production of IFN-γ. The aptamer species consists of a DNA hairpin molecule with thiol groups on the 3'-end for self-assembly onto Au electrodes. A redox reporter is covalently attached at the 5'-end for electrochemical sensing. This aptasensor has excellent sensitivity for IFN-γ (<60 pM detection limit) and responds to the target analyte in real time without additional washing or labeling steps. Aptamer-functionalized electrode arrays are fabricated on glass slides containing poly(ethylene glycol) (PEG) hydrogel patterns designed to expose glass regions adjacent to electrodes while protecting the remainder of the surface from nonspecific adsorption. The micropatterned substrates are integrated with PDMS microfluidic channels and incubated with T-cell-specific antibodies (Ab) (anti-CD4). Upon injection of blood, leukocytes are bound to Ab-modified glass regions in proximity to aptasensors. Cytokine release from captured cells is triggered by mitogenic activation and detected at the aptamer-modified electrodes using square wave voltammetry (SWV). The IFN-γ signal is monitored in real time with signal appearing as early as 15 min poststimulation from as few as 90 T cells. The observed IFN-γ release profiles are used to calculate an initial IFN-γ production rate of 0.0079 pg cell(-1) h(-1) upon activation. The work described here represents an important step toward development of aptasensors for immune cell analysis and blood-based diagnostics.

SHARPIN is an endogenous inhibitor of β1-integrin activation

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.

The Fc receptor-cytoskeleton complex from human neutrophils

The Fc receptor complex and its associated phagocytic cytoskeleton machinery were captured from the surface of live cells by IgG coated microbeads and identified by mass spectrometry. The random and independently sampled intensity values of peptides were similar in the control and IgG samples. After log transformation, the parent and fragment intensity values showed a normal distribution where ≥99.9% of the data was well above the background noise. Some proteins showed significant differences in intensity between the IgG and control samples by ANOVA followed by the Tukey-Kramer honestly significant difference test. However many proteins were specific to the IgG beads or the control beads. The set of detected cytoskeleton proteins, binding proteins and enzymes detected on the IgG beads were used to predict the network of actin-associated regulatory factors. Signaling factors/proteins such as PIK3, PLC, GTPases (such CDC42, Rho GAPs/GEFs), annexins and inositol triphosphate receptors were all identified as being specific to the activated receptor complex by mass spectrometry. In addition, the tyrosine kinase Fak was detected with the IgG coated beads. Hence, an activated receptor cytoskeleton complex and its associated regulatory proteins were captured from the surface of live human primary leukocytes.

Specific Inhibition of Syk Suppresses Leukocyte Immune Function and Alleviates Inflammation In Rodent Models of Rheumatoid Arthritis

AbstractAbstract 1727Genetic ablation of Syk in hematopoietic cells blocks various leukocyte immune functions, and protects mice from immune-complex mediated inflammation. These data have helped to identify Syk as an important therapeutic target for immune-mediated diseases. The next step is to test the hypothesis that low level, and specific, pharmacological inhibition of Syk retains the immunomodulatory potential observed with Syk genetic deficiency. With this goal in mind, we provide an update on the development of P505-15, a highly specific and potent small molecule Syk inhibitor which suppresses signaling and activation of primary human and rodent leukocyte immune function. The specificity of P505-15 was tested in a panel of 270 independent purified kinase assays at 300nM. At this concentration, Syk and 8 other kinases were inhibited by ≥ 80%. Subsequent analysis demonstrated a Syk IC50 of 1nM, whereas the next most potently inhibited kinase required an IC50 of 81nM. In a variety of cellular assays we observed potent inhibition of B cell receptor (BCR) induced Syk signaling, but not of Lyn, phorbol 12-myristate 13-acetate (PMA) induced protein kinase C, T cell receptor induced Zap70, or cytokine induced JAK1 (IL6), JAK2 (GM-CSF), or JAK1/3 (IL4) dependent STAT phosphorylation. Consistently, in Ba/F3 cell lines transformed by various kinases, P505-15 only inhibited proliferation of those cells transformed by Syk (IC50 = 0.12μM), and not by Zap70 or JAK family members (IC50 > 6μM). In human whole blood, P505-15 suppressed BCR-induced Syk signaling and cellular activation with IC50's of 0.383μM and 0.362μM, respectively. FceR1-induced basophil degranulation was similarly suppressed with an IC50 of 0.171μM. Importantly, Syk-independent signaling and cellular activation in human whole blood via PMA (B cell assays) or fMLP (basophil degranulation) was unaffected by this compound at 4μM and 1μM, respectively (the highest concentrations tested), again demonstrating its specificity of action. Oral administration of P505-15 in mice led to a reversible inhibition of Syk, with an IC50 of 0.282μM as determined by an ex vivo whole blood BCR stimulation assay. Finally, we tested the immunomodulatory potential of specific Syk inhibition in vivo using rodent models of rheumatoid arthritis. Oral administration of P505-15 resulted in statistically significant and dose-dependent anti-inflammatory activity in both the mouse collagen antibody-induced arthritis and rat collagen induced arthritis models. In each case, anti-inflammatory effects were achieved at sub-micromolar plasma concentrations in which Syk specificity was maintained. These data support the hypothesis that specific Syk inhibition can modulate immune function in vivo, and provide a therapeutic strategy for the treatment of human inflammatory disease by inhibition of this kinase. P505-15 is currently being evaluated in phase I clinical trials. Disclosures:Coffey:Portola Pharmaceuticals: Employment. Francis:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Pak:Portola Pharmaceuticals: Employment. Delaney:Portola Pharmaceuticals: Employment. Betz:Portola Pharmaceuticals: Employment. Jia:Portola Pharmaceuticals: Employment. Xu:Portola Pharmaceuticals: Employment. Bauer:Portola Pharmaceuticals: Employment. Song:Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment. Baker:Portola Pharmaceuticals: Employment. Hollenbach:Portola Pharmaceuticals: Employment. Phillips:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals: Employment.

Major Differences in the Responses of Primary Human Leukocyte Subsets to IFN-β

Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-β and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-β, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-β activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-β increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.

Ubiquitin receptor binding and signaling in primary human leukocytes

Utilizing the human monocyte/macrophage cell line THP1, we recently identified extracellular ubiquitin as an endogenous agonist of the G protein-coupled receptor CXC chemokine receptor (CXCR) 4. Because receptor binding and signaling properties of extracellular ubiquitin have not been evaluated in primary human leukocytes, we analyzed its binding characteristics and subsequent Ca2+ signaling in freshly isolated human B-cells, T-cells and monocytes. Ubiquitin binding shows typical receptor binding characteristics and promotes intracellular Ca2+ flux within seconds in all three cell populations. The Kd for the ubiquitin receptor interaction in freshly isolated human monocytes is consistent with the affinity of the ubiquitin CXCR4 interaction that we reported for THP1 cells. As detected in THP1 cells previously, the ubiquitin induced Ca2+ flux can be attenuated with a phospholipase C inhibitor in all primary leukocyte cultures. Our observations further support the finding that ubiquitin is a CXCR4 agonist and demonstrate that extracellular ubiquitin induces physiological relevant signaling events in primary human leukocytes. Although the exact mechanism of the ubiquitin CXCR4 interaction, its receptor selectivity and subsequent signaling events remain to be determined, our findings identify a novel and unexpected biological role of extracellular ubiquitin as an endogenous immune modulator.

Cooperative and Synergistic Activities of the Epithelial Cell Derived Cytokines TSLP, IL-25, and IL-33 on Primary Leukocyte Cultures

RATIONALE: The cytokines thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 individually have been shown to have a profound influence on the development of allergic inflammatory responses. The common epithelial cell origin of these cytokines and overlapping target cell populations suggest that in an appropriate inflammatory state the opportunity for cooperative activities between TSLP, IL-25, and IL-33 may exist. Utilizing a variety of primary human and mouse leukocyte cultures and readouts we examined the individual and combined effects of TSLP, IL-25, and IL-33.

Exploring the Human Leukocyte Phosphoproteome Using a Microfluidic Reversed-Phase−TiO2−Reversed-Phase High-Performance Liquid Chromatography Phosphochip Coupled to a Quadrupole Time-of-Flight Mass Spectrometer

The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

BackgroundHerpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A) infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked.MethodsCell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation.ResultsU83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4.ConclusionU83A diverts human chemokines from signalling, but not regulatory or scavenger, receptors facilitating their clearance, while occupying signalling receptors at the cell surface. This can enhance virus specific inflammation, facilitating dissemination to replication sensitive leukocytes while evading clearance; this has implications for linked neuro-inflammatory pathologies.

GTS-21 inhibits pro-inflammatory cytokine release independent of the Toll-like receptor stimulated via a transcriptional mechanism involving JAK2 activation

The vagus nerve can limit inflammation via the α7 nicotinic acetylcholine receptor (α7nAChR). Selective pharmacological stimulation of the α7nAChR may have therapeutic potential for the treatment of inflammatory conditions. We determined the anti-inflammatory potential of GTS-21, an α7nAChR-selective partial agonist, on primary human leukocytes and compared it with nicotine, the nAChR agonist widely used for research into the anti-inflammatory effects of α7nAChR stimulation. Furthermore, we investigated whether the effects of both nicotinic agonists were restricted to specific Toll-like receptors (TLRs) stimulated and explored the mechanism behind the anti-inflammatory effect of GTS-21. GTS-21 and nicotine inhibited the release of pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs), monocytes and whole blood independent of the TLR stimulated, with higher potency/efficacy for GTS-21 compared to nicotine. The anti-inflammatory cytokine IL-10 was relatively unaffected by both nicotinic agonists. The effects of GTS-21 and nicotine could not be reversed by nAChR antagonists, while the JAK2 inhibitor AG490 abolished the anti-inflammatory effects. GTS-21 downregulated monocyte cell-surface expression of TLR2, TLR4 and CD14. qPCR analysis demonstrated that the anti-inflammatory effect of GTS-21 is mediated at the transcriptional level and involves JAK2-STAT3 activation. In conclusion, GTS-21 has a profound anti-inflammatory effect in human leukocytes and that GTS-21 is more potent/efficacious than nicotine. The absence of a blocking effect of nAChR antagonists in human leukocytes might indicate different pharmacological properties of the α7nAChR in human leukocytes compared to other cell types. GTS-21 may be promising from a therapeutic perspective because of its suitability for human use.

Primary Leukocyte Screens for Innate Immune Agonists

The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor. The authors describe 2 assays for the identification of immunostimulants that employ primary human leukocytes cocultured with hepatitis C virus (HCV) replicon-expressing cells. In these assays, stimulation of innate immune pathways in leukocytes induces interferon (IFN) expression, which acts to inhibit HCV replication, providing a high-throughput and low-cost readout for leukocyte activation. These assays are highly sensitive and provide a physiologically relevant system for the identification of a broad range of immunostimulant agents.

Assessing biological variation and protein processing in primary human leukocytes by automated multiplex stable isotope labeling coupled to 2 dimensional peptide separation

Determining the relative abundances of proteins in biological systems is an important aspect of proteomics. Quantitation provides the possibility to unravel the often subtle molecular differences that regulate biological processes in cells and organisms. A common method to analyze differences in protein expression in complex samples is differential stable isotopic labeling combined with 2D-LC-MS separation. In such experiments, proteins or peptides from different samples are labeled with different stable isotopes and their relative amounts are determined from the peptide ion intensities using mass spectrometry. When human tissue samples are investigated, chemical stable isotope labeling strategies instead of metabolic labeling strategies are required. However, biological variation in protein expression between individuals is a key concern.Here we describe a method that allows for fully automated quantitative proteome analysis; involving desalting, triplex stable isotopic dimethyl labeling and multi-dimensional strong cation exchange/reversed phase separation of peptides prior to mass spectrometric analysis that can be applied to complex samples such as human tissue lysates. We highlight the usability of the method by characterizing the extent of biological variation between the proteomes of primary human leukocytes from three healthy donors. Using our method we were able to quantify 967 proteins with a minimum of 2 peptides, revealing very limited biological variation between the donors. The discovery is noteworthy considering the presence of significant endogenous protease activity, originating primarily from the enzyme neutrophil elastase. This dataset represents the largest quantitative dataset for human leukocytes proteins, which was made possible by the use of an automated labeling strategy.

N‐Nonanoyl‐CCL14 (NNY‐CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2

Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca(2+)]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.

“Proteotyping”: Population Proteomics of Human Leukocytes Using Top Down Mass Spectrometry

Characterizing combinations of coding polymorphisms (cSNPs), alternative splicing and post-translational modifications (PTMs) on a single protein by standard peptide-based proteomics is challenging owing to <100% sequence coverage and the uncoupling effect of proteolysis on such variations >10-20 residues apart. Because top down MS measures the whole protein, combinations of all the variations affecting primary sequence can be detected as they occur in combination. The protein form generated by all types of variation is here termed the "proteotype", akin to a haplotype at the DNA level. Analysis of proteins from human primary leukocytes harvested from leukoreduction filters using a dual on-line/off-line top down MS strategy produced >600 unique intact masses, 133 of which were identified from 67 unique genes. Utilizing a two-dimensional platform, termed multidimensional protein characterization by automated top down (MudCAT), 108 of the above protein forms were subsequently identified in the absence of MS/MS in 4 days. Additionally, MudCAT enables the quantitation of allele ratios for heterozygotes and PTM occupancies for phosphorylated species. The diversity of the human proteome is embodied in the fact that 32 of the identified proteins harbored cSNPs, PTMs, or were detected as proteolysis products. Among the information were three partially phosphorylated proteins and three proteins heterozygous at known cSNP loci, with evidence for non-1:1 expression ratios obtained for different alleles.

Calcium, leukocyte cell death and the use of annexin V: fatal encounters

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane's outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results.

The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages

BackgroundBacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC) family, Anthrolysin O (ALO).ResultsIn the present studies we show that recombinant ALO (rALO) or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs), monocytes, monocyte-derived macrophages (MDMs), lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH) release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+), UT231 (ALO-), and a complemented strain expressing ALO, UT231 (pUTE544), and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%.ConclusionThe lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

Totipotency, cell differentiation and reprogramming in humans

Understanding the molecular mechanisms defining totipotency and cell differentiation in humans is a promising strategy in order to expand knowledge about reprogramming. Totipotency and the very first steps of cell differentiation can be studied well in early human embryos. Based on analysis of marker genes such as Oct-4 and β- HCG, blastomeres seem to differ in their potency and can be regarded as lineage-specific stem cells as early as the 4-cell stage. The allocation of these stem cells to specific fates might hereby follow a pattern reminiscent of animal and vegetal poles. On the opposite end of the developmental spectrum, differentiated human cells can be used as a means of studying nuclear reprogramming. Intact human 293T kidney cells and primary leukocytes were reprogrammed towards a more undifferentiated state by Xenopus laevis egg extract. Molecular screens identified the chromatin-remodelling ATPase BRG1 as a factor required for this process. Based on these results, more efficient reprogramming protocols allowing for the generation of fully differentiated or undifferentiated human cells for clinical application may be developed.