A highly efficient method for the production and purification of recombinant human CXCL8.

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.

A Simple and Efficient System for Producing Recombinant Human CXCL8 in Escherichia coli.

Multifunctional pro-inflammatory cytokine CXCL8 is a small peptide of 8-10 kDa in size and it functions as a monomer or dimer. CXCL8 harbors 2 disulfide bonds for its stability. Although production of the CXCL8 protein in a large quantity in both mammalian and bacterial systems has been reported, the processes are complicated and lengthy. Here, we develop a new bacterial expression system for recombinant CXCL8 and simplify the purification system to yield a high amount of protein quickly. The purified CXCL8 protein from our new system develops a crystal structure that is identical to that produced through the mammalian expression system. Thus, we have established a simple and efficient recombinant CXCL8-producing system, which can be easily operated and is suitable to those requiring a large quantity of CXCL8.

Function of hesperidin alleviating inflammation and oxidative stress responses in COPD mice might be related to SIRT1/PGC-1α/NF-κB signaling axis.

Purpose: Hesperidin has anti-inflammatory and anti-oxidant stress effects, but its functions in chronic obstructive pulmonary disease (COPD) remains unknown. This study analyzed the role of hesperidin in COPD mice, aiming to provide a basis for the hesperidin application.Materials and methods: Mice were injected with cigarette smoke extract (CSE) to construct COPD models and then treated with budesonide or hesperidin. Hematoxylin-eosin (HE) and TUNEL assays were used to observe the pathological changes and cell death of lung tissue. The levels of interleukin (IL)-6, IL-8, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in bronchoalveolar lavage fluid (BLAF), as well as myeloperoxidase (MPO) content in lung tissues were confirmed. The expression levels of SIRT1, PGC-1α, and p65 proteins were measured by western blotting (WB) analysis.Results: CSE induced inflammatory cell infiltration and cell death in the lung tissues of mice, whereas budesonide and hesperidin effectively alleviated these pathological changes. The levels of IL-6, IL-8, and MDA in BLAF and pulmonary MPO content in the COPD mice were effectively increased, while the levels of SOD and CAT in BLAF were decreased, which could be reversed by budesonide and hesperidin. Moreover, the addition of budesonide or hesperidin reliably accelerated the expression levels of PGC-1α and SIRT1 but suppressed the phosphorylation of p65 in COPD mice. In general, high-dose hesperidin had a stronger regulatory effect on COPD mice.Conclusions: Hesperidin alleviated inflammation and oxidative stress responses in CES-induced COPD mice, associated with SIRT1/PGC-1α/NF-κB signaling axis, which might become a new direction for COPD treatment.

Plasmonic droplet screen for single-cell secretion analysis.

Single-cell secretion analysis technologies are needed to elucidate the heterogeneity of cellular functionalities. Although ligand binding assays in microwells provide a promising approach for measuring single-cell secretions, their throughput is limited. Recently, droplet assays have been developed for high-throughput single-cell screening. However, because washing steps are difficult to perform with droplets, there are still challenges in measuring secretions using droplet assays. In this study, a plasmonic droplet screen approach is developed for one-step washing-free multiplex detection of single-cell secretions. Individual cells are encapsulated with antibody-conjugated gold nanorods (AuNRs) in droplets to evaluate their secretion levels. The shift in the plasmon resonance peak reflects the amount of secreted protein without needing additional indicator and washing steps. The plasmonic signals from a continuous flow of single-cell droplets are collected by dark-field spectroscopy (∼100-150 cells min-1). This platform is tested by screening interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) secreted from suspended leukemia cells and adherent breast cancer cells. Overall, this novel strategy shows the potential and flexibility of high-efficiency multiplex single-cell secretion analysis.

Improving the Sensitivity of Fluorescence-Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads.

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre- and post-photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high-sensitivity LX-200 system using photobleached magnetic beads, human interleukin-8 is detected with a threefold improvement in detection limit and signal-to-noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence-based assays.

A dual-functional microfluidic chip for on-line detection of interleukin-8 based on rolling circle amplification.

Interleukin 8 (IL-8), also known as C-X-C motif ligand 8(CXCL8), is a proinflammatory chemokine functioned in neutrophil chemotaxis and activation. And it plays an important role in the process of glioma stem-like cell vascularization in the latest research. Herein, a dual-function microfluidic biosensor based on rolling circle amplification (RCA) was fabricated for cell culture and online IL-8 detection. A microfluidic chip was designed with two high passages connected by the vertical channels. One of the channels with immobilized capture antibody was prepared for IL-8 detection and another channel for cell culture. Immunoassays were achieved by a sandwich structure consisting of antibodies, IL-8, and aptamers. Signal amplification was mainly due to RCA and biotin-streptavidin linkage. The linear range for IL-8 was 7.5 -120pgmL-1 in this assay. Moreover, the developed method was successfully applied to detect the IL-8 in tumor-derived endothelial cells (TDEC) and Human Umbilical Vein Endothelial cells (HUVEC) under chemical hypoxia condition. Semi-quantitative detection of IL-8 consumption in HUVEC cells in low oxygen condition was also achieved. These results were in statistical agreement with those obtained by commercial assay of enzyme-linked immunoassay kit (ELISA). The microfluidic chip based biosensor reported hereby has a large prospect in the basic research and clinical diagnosis of cancer stem cell.

Characterization of Adsorbents for Cytokine Removal from Blood in an In Vitro Model.

INTRODUCTION: Cytokines are basic targets that have to be removed effectively in order to improve the patient's health status in treating severe inflammation, sepsis, and septic shock. Although there are different adsorbents commercially available, the success of their clinical use is limited. Here, we tested different adsorbents for their effective removal of cytokines from plasma and the resulting effect on endothelial cell activation. METHODS: The three polystyrene divinylbenzene (PS-DVB) based adsorbents Amberchrom CG161c and CG300m and a clinically approved haemoperfusion adsorbent (HAC) were studied with regard to cytokine removal in human blood. To induce cytokine release from leucocytes, human blood cells were stimulated with 1 ng/ml LPS for 4 hours. Plasma was separated and adsorption experiments in a dynamic model were performed. The effect of cytokine removal on endothelial cell activation was evaluated using a HUVEC-based cell culture model. The beneficial outcome was assessed by measuring ICAM-1, E-selectin, and secreted cytokines IL-8 and IL-6. Additionally the threshold concentration for HUVEC activation by TNF-α and IL-1ß was determined using this cell culture model. RESULTS: CG161c showed promising results in removing the investigated cytokines. Due to its pore size the adsorbent efficiently removed the key factor TNF-α, outperforming the commercially available adsorbents. The CG161c treatment reduced cytokine secretion and expression of cell adhesion molecules by HUVEC which underlines the importance of effective removal of TNF-α in inflammatory diseases. CONCLUSION: These results confirm the hypothesis that cytokine removal from the blood should approach physiological levels in order to reduce endothelial cell activation.

Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

Western analysis of intracellular interleukin-8 in human mononuclear leukocytes.

Most cytokines are stored in the cytoplasm until their release into the extracellular environment; however, some cytokines have been reported to localize in the nucleus. Traditional whole cell extract preparation does not provide information about the intracellular localization of cytokines. Here, we describe how to prepare cytoplasmic and nuclear extracts that can be analyzed by immunoblotting. While in this chapter we use this method to analyze intracellular localization of interleukin-8 (IL-8) in human mononuclear leukocytes, this protocol is adaptable to any cell type or protein of interest.

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies.

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.

Expression of recombinant human interleukin-8 and its purification using a single buffer system.

Chemokines, a class of small secreted proteins, direct immune cells to their target sites and play an important role in chronic inflammations and allergies. To study their interactions with their cellular receptors or potential inhibitors large quantities of chemokines are required. Here we present a fast and efficient strategy to purify the human chemokine interleukin-8 (IL-8, CXCL8). The chemokine is expressed with a pelB-leader peptide that is cleaved off its N-terminus by an endogenous bacterial peptidase. This yields wild-type 72aa IL-8 with a serine at its N-terminus. IL-8 is recovered in the soluble fraction after lysis while pelB-IL8 fusion protein remains in the pellet. Interleukin-8 is purified via cation exchange chromatography and heparin affinity chromatography using a single inexpensive buffer system. No dialysis or membrane filtration steps are required and the final protein fractions may be used without any desalting steps. The use of 0.5% Triton X-114 in the lysis buffer leads to low endotoxin levels in the resulting protein. The protein can be eluted from the gel filtration column with a variety of buffers and is ready to be used in binding assays and activity assays.

Treatment of severe sepsis and septic shock by CHDF using a PMMA membrane hemofilter as a cytokine modulator.

It has been reported that various types of blood purification intended for the removal of humoral mediators, such as cytokines, were performed in patients with severe sepsis/septic shock. While high-volume hemofiltration, hemofiltration using high cut-off membrane filters, and direct hemoperfusion with a polymyxin-B immobilized column are widely used in the treatment of severe sepsis/septic shock, we perform continuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter (PMMA-CHDF), which shows an excellent cytokine-adsorbing capacity, for the treatment of severe sepsis/septic shock. In our previous study, it was found that PMMA-CHDF could efficiently remove various pro-inflammatory cytokines such as TNFalpha, IL-6 and IL-8 from the bloodstream, resulting in early recovery from septic shock. Furthermore, PMMA-CHDF could remove anti-inflammatory cytokines such as IL-10 from bloodstream, suggesting that it might improve immunoparalysis as well. These findings suggest that PMMA-CHDF is useful for the treatment of patients with severe sepsis/septic shock as a cytokine modulator.

Practitioner empathy and the duration of the common cold.

OBJECTIVE: This study's objective was to assess the relationship of empathy in medical office visits to subsequent outcomes of the common cold. METHODS: A total of 350 subjects ? 12 years of age received either a standard or enhanced physician visit as part of a randomized controlled trial. Enhanced visits emphasized empathy on the part of the physician. The patient-scored Consultation and Relational Empathy (CARE) questionnaire assessed practitioner-patient interaction, especially empathy. Cold severity and duration were assessed from twice-daily symptom reports. Nasal wash was performed to measure the immune cytokine interleukin-8 (IL-8). RESULTS: Eighty-four individuals reported perfect (score of 50) CARE scores. They tended to be older with less education but reported similar health status, quality of life, and levels of optimism. In those with perfect CARE scores, cold duration was shorter (mean 7.10 days versus 8.01 days), and there was a trend toward reduced severity (mean area under receiver-operator characteristics curve 240.40 versus 284.49). After accounting for possible confounding variables, cold severity and duration were significantly lower in those reporting perfect CARE scores. In these models, a perfect score also correlated with a larger increase in IL-8 levels. CONCLUSIONS: Clinician empathy, as perceived by patients with the common cold, significantly predicts subsequent duration and severity of illness and is associated with immune system changes.

A new protocol for high-yield purification of recombinant human CXCL8((3-72))K11R/G31P expressed in Escherichia coli.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8, CXCL8((3-72))K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both CXCR1 and CXCR2. To obtain large quantities of hG31P, we have successfully constructed and expressed hG31P in Escherichia coli. Moreover, we have developed a new protocol for high-yield purification of hG31P and for the removal of lipopolysaccharide (LPS, endotoxin) associated with hG31P due to the expression in E. coli. The purity of hG31P is more than 95% and the final yield is 9.7mg hG31P per gram of cell paste. The purified hG31P was tested by various biological assays. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that this purification protocol is very simple and easy to amplify at a large scale. The results of this study will provide an effective route to produce enough hG31P for future clinical studies.

The biological effects of rainbow trout (Oncorhynchus mykiss) recombinant interleukin-8.

In this report, recombinant interleukin-8 (rIL-8) was produced and its activity tested for the first time in fish. The rainbow trout rIL-8 was produced in Escherichia coli and purified using a 6xHis tag at the N-terminus. The rIL-8 induced a dose-dependent migration of head kidney leukocytes at concentrations from 0.1 to 10 ng/ml, with a peak response at 1 ng/ml. Trout rIL-8 also had a significant effect on superoxide production by head kidney cells, with maximal activity at 0.1 and 1 ng/ml. When injected intraperitoneally into trout, rIL-8 had a clear effect on total leukocyte number in the peritoneal cavity, with increasing doses (up to 5 microg) eliciting more cells. Of three leukocyte types distinguished, neutrophils were the dominant cell type, especially at higher rIL-8 concentrations. In contrast, the proportion of macrophages and lymphocytes decreased with rIL-8 administration, suggesting that they were not attracted at the same rate as neutrophils.

Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus).

This paper reports the cloning and sequencing of two pro-inflammatory cytokines, interleukin (IL)-1beta and IL-8, in haddock (Melanogrammus aeglefinus) by homology cloning. The complete transcript of the haddock IL-1beta was sequenced and contained 1043 bp, including a 762 bp open reading frame. The 3' end of the gene includes a polyadenylation signal 13 bp upstream of the poly(A) tail, along with 10 instability motifs. The predicted protein of 253aa revealed the presence of the IL-1 family signature and the absence of an ICE cut site. The cDNA of the chemokine IL-8 was sequenced in haddock and contained 903 bp of which 306 bp are the open reading frame. Interestingly, the predicted protein sequence of 101aa, contains an ELR motif preceding the CXC signature, common in all vertebrate IL-8 molecules but absent in all teleost genes sequenced to date. The expression of both haddock cytokines was studied in four different tissues: head kidney, spleen, liver and gill. Tissues were obtained from both healthy fish and fish stimulated in vivo with four commercial serotypes of LPS, namely Escherichia coli 026:B6, 055:B5, 0111:B4 and 0127:B8 and PMA. Haddock IL-1beta was not constitutively expressed and expression was only observed following stimulation. However, this expression was stimulant dependent and only PMA and LPS 026:B6 induced high levels of expression in the head kidney. The haddock IL-8 gene on the other hand, showed a constitutive expression, that could be up or down-regulated depending on the immunostimulant used, although to a lesser extent than IL-1beta.

Super high flux hemodialysis at high dialysate flows: an ex vivo assessment.

BACKGROUND AND OBJECTIVES: The removal of cytokines by standard hemofiltration is limited. Super high flux membranes may significantly improve removal even when used in dialysis mode. We sought to measure cytokine clearance using a large surface super high-flux membrane and a standard hemodialysis setting. SETTING: ICU laboratory of a tertiary institution. SUBJECTS: Six healthy volunteers. METHODS: Blood form healthy volunteers was incubated for 4 hours with E. coli endotoxin to stimulate cytokine production. Cytokine containing blood was then circulated through a dialysis circuit at 3 different dialysate flow rates. Blood and dialysate were sampled for cytokine and albumin measurements and calculation of clearances. RESULTS: Super high-flux dialysis achieved high median cytokine clearances (IL-1 clearance of 106 ml/min, IL-6 clearance of 66.8 ml/min, IL-8 clearance of 61.7 ml/min and TNF clearance of 36.1 ml/min). Increasing dialysate flow rate from 300 to 500 ml/min did not significantly increase cytokine clearances. Albumin clearances however were between 2.7 and 5.4 ml/min. CONCLUSIONS: Cytokine dialysis is feasible at high dialysate flow rates yielding high cytokine clearances. Albumin loss, however, is appreciable and may require separate supplementation in the clinical setting.

In vitro Bartonella quintana infection modulates the programmed cell death and inflammatory reaction of endothelial cells.

Bartonella quintana is an epicellular bacterium, which in vivo as well as in vitro, invades endothelial cells and develops within them inducing proliferative effects that play a pivotal role in neovascular manifestation of this disease. We investigated the effect of live Bartonella quintana and its LPS on apoptosis and inflammatory response in HUVEC-C, an endothelial cell line. The kinetics of the programmed cell death of Bartonella quintana-infected HUVEC-C showed a peculiar course. Even if early during infection apoptosis reached a peak after 6 h, later on apoptosis was inhibited. Such apoptosis inhibition was not observed during Bartonella quintana lipopolysaccharide treatment because LPS-stimulated HUVEC-C did progress to cell death. Evaluation of multiple cell signal transduction pathways revealed an overexpression of Apaf 1 and caspase 8 in HUVEC-C after 2 h of infection, and of bcl-2 starting from 10 h post Bartonella quintana infection. Moreover, Bartonella quintana and its LPS showed a different effect on the activation of genes involved in inflammatory response as revealed by molecular analysis of host cells. Bartonella quintana appears to be able to inhibit programmed cell death, inducing intracellular signals leading to survival and proliferation through the bcl-2 gene, despite the early increase of inflammatory status induced in endothelial cells. This mechanism, together with a poor endotoxin ability to stimulate strong inflammatory response, could contribute to the capability of the bacteria to persist intracellularly, causing chronic disease and producing neovascular manifestations.