Simultaneous detection of two growth factors from human single-embryo culture medium by a bead-based digital microfluidic chip.

The measurement of growth factors released in a culture medium is considered to be an attractive non-invasive approach, apart from the embryo morphology, to identify the condition of an embryo development after fertilization in vitro (IVF), but the available embryo culture medium in the current method is only a few microlitres. This small sample volume, also of small concentration, makes difficult the application of a conventional detection method, such as an enzyme-linked immunosorbent assay (ELISA). A reliable detection of the growth factor from each embryo culture medium of such a small concentration hence remains a challenge. Here for the first time we report the results of measurement of not just one, but two, growth factors, human IL-1ß and human TNF-α, from an individual droplet of embryo culture medium with a bead-based digital microfluidic chip. The required sample volume for a single measurement is only 520 nL; the total duration of the on-chip process is less than 40 min. Using the culture media of human embryos with normal morphologic features, we found that the concentrations of TNF-α change little from day 3 to day 5-6, but the concentrations of IL-1ß for some embryos might double from day 3 to day 5-6. For other embryos even with similar normal morphologic features, some growth factors, such as IL-1ß, might exhibit different expressions during the culture period. Those growth factors could serve to distinguish the development conditions of each embryo, not merely from an observation of embryo morphology.

Pro-inflammatory cytokines and growth factors in human milk: an exploratory analysis of racial differences to inform breast cancer etiology.

BACKGROUND: Analysis of cytokines and growth factors in human milk offers a noninvasive approach for studying the microenvironment of the postpartum breast, which may better reflect tissue levels than testing blood samples. Given that Black women have a higher incidence of early-onset breast cancers than White women, we hypothesized that milk of the former contains higher levels of pro-inflammatory cytokines, adipokines, and growth factors. METHODS: Participants included 130 Black and 162 White women without a history of a breast biopsy who completed a health assessment questionnaire and donated milk for research. Concentrations of 15 analytes in milk were examined using two multiplex and 4 single-analyte electrochemiluminescent sandwich assays to measure pro-inflammatory cytokines, angiogenesis factors, and adipokines. Mixed-effects ordinal logistic regression was used to identify determinants of analyte levels and to compare results by race, with adjustment for confounders. Factor analysis was used to examine covariation among analytes. RESULTS: Thirteen of 15 analytes were detected in ≥ 25% of the human milk specimens. In multivariable models, elevated BMI was significantly associated with increased concentrations of 5 cytokines: IL-1ß, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1ß, controlling for BMI. Factor analysis of analyte levels identified two factors related to inflammation and growth factor pathways. CONCLUSION: This exploratory study demonstrated the feasibility of measuring pro-inflammatory cytokines, adipokines, and angiogenesis factors in human milk, and revealed higher levels of some pro-inflammatory factors, as well as increased leptin levels, among Black as compared with White women.

A fully integrated electrochemical biosensor platform fabrication process for cytokines detection.

Interleukin-1b (IL-1b) and interleukin-10 (IL-10) biomarkers are one of many antigens that are secreted in acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). In the present study, we have developed a fully integrated electrochemical biosensor platform for cytokine detection at minute concentrations. Using eight gold working microelectrodes (WEs) the design will increase the sensitivity of detection, decrease the time of measurements, and allow a simultaneous detection of varying cytokine biomarkers. The biosensor platform was fabricated onto silicon substrates using silicon technology. Monoclonal antibodies (mAb) of anti-human IL-1b and anti-human IL-10 were electroaddressed onto the gold WEs through functionalization with 4-carboxymethyl aryl diazonium (CMA). Cyclic voltammetry (CV) was applied during the WE functionalization process to characterize the gold WE surface properties. Finally, electrochemical impedance spectroscopy (EIS) characterized the modified gold WE. The biosensor platform was highly sensitive to the corresponding cytokines and no interference with other cytokines was observed. Both cytokines: IL-10 and IL-1b were detected within the range of 1pgmL-1 to 15pgmL-1. The present electrochemical biosensor platform is very promising for multi-detection of biomolecules which can dramatically decrease the time of analysis. This can provide data to clinicians and doctors concerning cytokines secretion at minute concentrations and the prediction of the first signs of inflammation after LVAD implantation.

High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris.

As an important pro-inflammatory cytokine, interleukin-1beta (IL-1ß) participates in a variety of physiological and pathological responses. In order to obtain higher yielded recombinant human interleukin-1 beta (rhIL-1ß), we cloned hIL-1ß cDNA sequences based on the coding sequence of human mature IL-1ß. After recombinant pPICZαA/hIL-1ß was separated and sequenced, we transformed recombinant pPICZαA/hIL-1ß into Pichia pastoris GS115, SMD1168 and X-33 strain via electroporation. The results showed that recombinant pPICZαA/ hIL-1ß had the highest expression level in X-33 Pichia pastoris. Subsequently, rhIL-1ß was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and identified by Western blot. Then the fermentation process was optimized to increase product yield. Under the fermentation conditions of the absorption value of fermentation liquor before induction of 600, oxygen concentration of 20%, methanol concentration of 0.25% with pH 5.0, the yield of rhIL-1ß reached to 250 mg/L after 72 h induction at 26°C. After aqueous two-phase extraction combined with chromatography, the purity of rhIL-1ß was 95% and the yield was up to 85%. The biological activity of rhIL-1ß was detected by MTT assay, and the result showed that rhIL-1ß significantly inhibited the growth and proliferation of B16 melanoma cells.

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.

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1ß production: optimization, modeling, and physiological aspects.

Saccharomyces cerevisiae CEN.PK113-5D, a strain auxotrophic for uracil belonging to the CEN.PK family of the yeast S. cerevisiae, was cultured in aerated fed-batch reactor as such and once transformed to express human interleukin-1ß (IL-1ß), aiming at obtaining high cell densities and optimizing IL-1ß production. Three different exponentially increasing glucose feeding profiles were tested, all of them "in theory" promoting respiratory metabolism to obtain high biomass/product yield. A non-structured non-segregated model was developed to describe the performance of S. cerevisiae CEN.PK113-5D during the fed-batch process and, in particular, its capability to metabolize simultaneously glucose and ethanol which derived from the precedent batch growth. Our study showed that the proliferative capacity of the yeast population declined along the fed-batch run, as shown by the exponentially decreasing specific growth rates on glucose. Further, a shift towards fermentative metabolism occurred. This shift took place earlier the higher was the feed rate and was more pronounced in the case of the recombinant strain. Determination of some physiological markers (acetate production, intracellular ROS accumulation, catalase activity and cell viability) showed that neither poor oxygenation nor oxidative stress was responsible for the decreased specific growth rate, nor for the shift to fermentative metabolism.

The concentration of IL-1B in saliva of children with oral lesions associated to histiocytosis / Concentracion de il-1β en saliva de niños con lesiones bucales asociadas a histiocitosis

La Histiocitosis de células de Langerhans(HCL) es una enfermedad de etiología y patogenia aún desconocidas. Afecta diferentes órganos y tejidos en los que produce lesiones de distinta gravedad. La histopatología de las lesiones y la clínica sugieren la participación de citoquinas en su patogenia. La IL-1β podría tener un rol importante en el desarrollo de la enfermedad. El objetivo de este estudio fue determinar las concentracionesde IL-1β de las salivas de pacientes pediátricos con diagnóstico de Histiocitosis de Célula de Langerhans con y sin manifestaciones bucales (grupos 1 y 2 respectivamente), en relación a un grupo control (grupo 3), de pacientes pediátricos que no presentaron antecedentes médicos ni lesiones bucales. Fueron estudiadas las salivas de 20 pacientes con la enfer -medad de HCL, en relación a un grupo control de 11 pacientes pediátricos que no presentaron antecedentes médicos. Los niños con Histiocitosis cuyas edades oscilaban entre 4 meses y 16 años fueron derivados del servicio de Oncohematología del Hospital Garrahan y Hospital de Clínicas, a la Cátedra de Odontología Integral Niños de la Facultad de Odontología de la Universidad de Buenos Aires. Se determinaron las concentraciones de IL-1β en los diferentes grupos, y se utilizó el Enzyme Inmune Assay Kit (Cayman, MI, USA), se expresó en pg/ml. El análisis de los resultados se realizó según el test de Kruskall Wallis, se obtuvieron diferencias significativas entre los tres grupos (H = 20,36; P<0,001). Luego se realizó el análisis de comparaciones múltiples de Dunn que mostró diferencias estadísticamente significativas entre los grupos 1 y 2 y entre los grupos 1 y 3 (p < 0,05). Se observaron valores más elevados de IL-1β en los pacientes con Histiocitosis con manifestaciones bucales (grupo 1), en relación con el grupo sin manifestaciones bucales (grupo 2) y con el grupo control (grupo 3).

Langerhans Cell Histiocytosis (LCH) is a disease whosetiology and pathogenesis are still unknown. It affects several organs and tissues, producing lesions of different severity. Its histopathology and clinical picture suggest the participation of cytokines in its pathogenesis. IL-1β might have an important role in its development. The purpose of this study was to determine the concentrations of IL-1β in saliva of pediatric patients diagnosed with LCH, with and without oral manifestations (Groups 1 and 2respectively) compared to a Control Group (Group 3) of pediatric patients without medical antecedents or oral lesions.The saliva of twenty patients with LCH was studied and compared to a Control Group consisting of eleven pediatric patients without medical antecedents. The children with histiocytosis, aged four months to sixteen years, were referred by the Onco haematology Service at Garrahan Hospital and Hospital de Clínicas, to the Department of Comprehensive Children’s Dentistry, School of Dentistry, University of BuenosAires (UBA).The concentrations of IL-1β in the different groups were determined using the Enzyme Immune Assay Kit (Cayman MI,USA) and expressed in pg/ml. Results were analyzed by the Kruskall Wallis test. Significant differences between the three cohorts were found, (H = 20.36, P< 0.001). Dunn ́s multiple comparison analysis was performed, which showed significant differences between Groups 1 and 2, and between Groups 1 and 3 (P < 0.05). Higher values of IL-1βwere found in the patients with histiocytosis with oral manifestations (Group 1) than in patients without manifestations (Group 2) and patients in the Control Group (Group 3).

Preparation of Soluble Proteins from Escherichia coli.

Purification of human IL-1ß is used in this unit as an example of the preparation of a soluble protein from E. coli. Bacteria containing IL-1ß are lysed, and IL-1 ß in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation, and cation-exchange chromatography, and then concentrated. Finally, the IL-1 ß protein is applied to a gel-filtration column to separate it from remaining higher- and lower-molecular-weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. In addition, the purification procedure serves as an example of how to use classical protein purifications methods, which may also be used in conjunction with the affinity-based methods now more commonly used.

[A method of quantitative assessment of cytokine gene mRNA expression levels using real-time PCR in homogenous low cell number populations].

We propose a method of quantitative functional activity assessment in cells isolated via sorting on a flow cytometer. We show that cell populations vary in the mRNA expression of cytokine genes immediately after isolation and sorting, while the maintenance of homogenous populations in culture without stimulation results in an increase in these gene mRNA expression. Using the original system, it is now possible to detect mRNA cytokine genes with high sensitivity, starting from 90 cells per specimen. This approach permits genetic and immunogenetic analysis of gene expression with the goal of determining their functions in the in vitro studies.

One target-two different binding modes: structural insights into gevokizumab and canakinumab interactions to interleukin-1ß.

Interleukin-1ß (IL-1ß) is a key orchestrator in inflammatory and several immune responses. IL-1ß exerts its effects through interleukin-1 receptor type I (IL-1RI) and interleukin-1 receptor accessory protein (IL-1RAcP), which together form a heterotrimeric signaling-competent complex. Canakinumab and gevokizumab are highly specific IL-1ß monoclonal antibodies. Canakinumab is known to neutralize IL-1ß by competing for binding to IL-1R and therefore blocking signaling by the antigen:antibody complex. Gevokizumab is claimed to be a regulatory therapeutic antibody that modulates IL-1ß bioactivity by reducing the affinity for its IL-1RI:IL-1RAcP signaling complex. How IL-1ß signaling is affected by both canakinumab and gevokizumab was not yet experimentally determined. We have analyzed the crystal structures of canakinumab and gevokizumab antibody binding fragment (Fab) as well as of their binary complexes with IL-1ß. Furthermore, we characterized the epitopes on IL-1ß employed by the antibodies by NMR epitope mapping studies. The direct comparison of NMR and X-ray data shows that the epitope defined by the crystal structure encompasses predominantly those residues whose NMR resonances are severely perturbed upon complex formation. The antigen:Fab co-structures confirm the previously identified key contact residues on IL-1ß and provide insight into the mechanisms leading to their distinct modulation of IL-1ß signaling. A significant steric overlap of the binding interfaces of IL-1R and canakinumab on IL-1ß causes competitive inhibition of the association of IL-1ß and its receptor. In contrast, gevokizumab occupies an allosteric site on IL-1ß and complex formation results in a minor reduction of binding affinity to IL-1RI. This suggests two different mechanisms of IL-1ß pathway attenuation.

Evidence for sustained elevation of IL-6 in the CNS as a key contributor of depressive-like phenotypes.

There is compelling clinical literature implicating a role for cytokines in the pathophysiology of major depressive disorder (MDD). Interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) are pleiotropic inflammatory cytokines that have been reported to be elevated in patients with MDD. The present studies were undertaken to investigate the relationship between IL-6 and IL-1ß in animal models of depressive-like behavior. Analysis of brain tissue homogenates in the cortex of rats subjected to chronic stress paradigms revealed elevated levels of IL-6 protein in the absence of elevations in IL-1ß. Central administration of recombinant mouse IL-6 produced depressive-like phenotypes in mice, which were not accompanied by IL-1ß-induced increases in the brain tissue or IL-1ß-related sickness behavior typical of a general central nervous system inflammatory response. Systemic administration of fluoxetine in the presence of centrally administered IL-6 failed to produce the expected antidepressant-like response in mice relative to sham-infused controls. Further, administration of fluoxetine to mice with endogenous overexpression of brain IL-6 (MRL/MpJ-Fas(LPR/LPR) (LPR mice)) failed to produce the expected antidepressant-like effect relative to fluoxetine-treated control mice (MRL/MpJ(+/+)). Interestingly, blockade of IL-6 trans-signaling by coadministration of a gp130/Fc monomer or an anti-mouse IL-6 antibody with IL-6 prevented the IL-6-induced increases in immobility time as well as attenuated IL-6-induced increases of protein in the cortex. Taken together, these data indicate that elevations in IL-6 may have a pathophysiological role underlying depression and more specifically resistance to current classes of antidepressant medications and suggest that modulation of the IL-6 signaling pathway may have therapeutic potential for treatment-resistant depression.

Device for continuous extracorporeal blood purification using target-specific metal nanomagnets.

BACKGROUND: The present work illustrates how magnetic separation-based blood purification using ultra-strong iron nanomagnets can be implemented into an extracorporeal blood purification circuit. By this promising technique, today's blood purification may be extended to specifically filter high-molecular compounds without being limited by filter cut-offs or column surface saturation. METHODS: Blood spiked with digoxin (small molecule drug) and interleukin-1ß (inflammatory protein) was circulated ex vivo through a device composed of approved blood transfusion lines. Target-specific nanomagnets were continuously injected and subsequently recovered with the aid of a magnetic separator before recirculating the blood. RESULTS: Magnetic blood purification was successfully carried out under flow conditions: already in single-pass experiments, removal efficiencies reached values of 75 and 40% for digoxin and interleukin-1ß, respectively. Circulating 0.5 L of digoxin-intoxicated blood in a closed loop, digoxin concentration was decreased from initially toxic to therapeutic concentrations within 30 min and purification extents of 90% were achieved after 1.5 h. CONCLUSIONS: Magnetic separation can be successfully implemented into an extracorporeal blood purification device. Simultaneous and specific filtering of high-molecular compounds may offer promising new therapeutic tools for the future treatment of complex diseases, such as sepsis and autoimmune disorders.

Interleukin-1 beta levels in gingival crevicular fluid and serum under naturally occurring and experimentally induced gingivitis.

AIMS: To evaluate the interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid (GCF) and serum in either naturally occurring (N-O) or experimentally induced (E-I) plaque-associated gingivitis. MATERIAL AND METHODS: Thirty-seven periodontally healthy subjects were evaluated in real life conditions (N-O gingivitis) as well as after 21 days of experimental gingivitis trial (E-I gingivitis). During the experimental gingivitis trial, in one maxillary quadrant (test quadrant), gingival inflammation was induced by oral hygiene abstention, while in the contralateral (control) quadrant, oral hygiene was routinely continued. IL-1 beta concentrations in N-O and E-I gingivitis were investigated for IL-1B(+3954) and IL-1B(-511) gene polymorphisms. RESULTS: (i) GCF IL-1 beta concentrations in E-I gingivitis were significantly higher compared with N-O gingivitis; (ii) an intra-individual correlation between GCF concentrations of IL-1 beta detected in N-O and E-I gingivitis was observed in control quadrants, but not in test quadrants; (iii) IL-1 beta concentration in GCF was associated with IL-1B(+3954) genotype only at test quadrants; (iv) IL-1 beta was detectable in serum only at low levels in a limited number of subjects, without difference between gingivitis conditions. CONCLUSIONS: Aspects of the bacterial challenge to the gingival tissues, such as the amount of plaque deposits and plaque accumulation rate, appear to affect the IL-1 beta levels in GCF in subjects with a specific IL-1B genotype.

Mesoporous carbide-derived carbon for cytokine removal from blood plasma.

Porous carbons can be used for purification of bio-fluids due to their excellent biocompatibility with blood. Since the ability to adsorb a range of inflammatory cytokines within the shortest possible time is crucial to stop the progression of sepsis, the improvement of the adsorption rate is a key factor to achieving efficient removal of cytokines. Here, we demonstrate the effect of synthesis temperatures (from 600 degrees C to 1200 degrees C), carbon particle sizes (from below 35 microm to 300 microm), and annealing conditions (Ar, NH(3), H(2), Cl(2), and vacuum annealing) that determine the surface chemistry, on the ability of carbide-derived carbons (CDCs) to remove cytokines TNF-alpha, IL-6, and IL-1 beta from blood plasma. Optimization of CDC processing and structure leads to up to two orders of magnitude increase in the adsorption rate. Mesoporous CDCs that were produced at 800 degrees C from Ti(2)AlC with the precursor particle size of <35 microm and annealed in NH(3), displayed complete removal of large molecules of TNF-alpha in less than an hour, with >85% and >95% TNF-alpha removal in 5 and 30 min, respectively. This is a very significant improvement compared to the previously published results for CDC (90% TNF-alpha removal after 1h) and activated carbons. Smaller interleukin IL-6 and IL-1 beta molecules can be completely removed within 5 min. These differences in adsorption rates show that carbons with controlled porosity can also be used for separation of protein molecules.

Molecular characterization, recombinant expression and bioactivity analysis of the interleukin-1 beta from the yellowfin sea bream, Acanthopagrus latus (Houttuyn).

Interleukin-1 beta (IL-1 beta) is an important inflammatory mediator and has also the potential as an immunoadjuvant. Here we describe the isolation and characterization of yellowfin sea bream IL-1 beta (sbIL-1 beta) cDNA and gene. The sbIL-1 beta cDNA contains a 121-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 762bp that translated into a 253 amino acid protein, a 342-bp 3' UTR with six cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail. The organization of the genomic IL-1 beta appears to be five exons and four introns, the intron and exon boundaries all follow the GT-AG consensus. The analysis of the expression pattern showed that sbIL-1 beta was expressed weakly in the kidney, spleen, gill and intestine, but not in the liver, heart and muscle. After injection with 150 microg LPS, the expression analysis in vivo showed that sbIL-1 beta was induced in the kidney and spleen and expression level was maximal after 4h. The predicted 253 amino acid sequence shares 23.5-88.5% identity and 43.9-93.3% similarity to known IL-1 beta. Thus, IL-1 beta is very conserved in fish of the same family. No interleukin-converting enzyme (ICE) cut site is found in sbIL-1 beta, but the alignment of the amino acid sequence with other species showed a possible cut site between Tyr87 and Thr88 that would give rise to a 166-amino-acid mature peptide. The putative mature peptide was expressed in E. coli, and the recombinant sbIL-1 beta could induce the transcription of sbIL-1 beta in a dose-dependent manner and the expression levels were almost equal in the samples treated with 50 ng/ml recombinant sbIL-1 beta and 5 microg/ml LPS, which showed recombinant sbIL-1 beta was biologically active and had the potential as an immunoadjuvant.

Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.

We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protein preparation was apparently free of host cell proteins, endotoxins, protease, and aggregates. The N-terminal amino acid sequence of IL-1beta was in full agreement with the natural mature form of IL-1beta. The homogeneity of the product was further shown by X-ray structure determination which confirmed the previously solved structure of the protein. We propose the applied workflow as a strategy for industrial production of protein-based biopharmaceuticals.

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.

Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal.

Interleukin 1beta (IL-1beta) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.

Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags.

Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1beta in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.