Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and ß-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.

Relative quantitation of multi-antennary N-glycan classes: combining PGC-LC-ESI-MS with exoglycosidase digestion.

In the search for N-glycan disease biomarkers current glycoanalytical methods may not be revealing a complete picture of precious samples, and we may be missing valuable structural information that fall outside analysis windows. We report a targeted strategy combining PGC-LC-ESI-MS with exoglycosidases to improve the relative quantitation of tri and tetra-antennary glycan classes.

Acute exercise increases hippocampal TNF-α, Caspase-3 and Caspase-7 expression in healthy young and older mice.

AIM: Regular exercise may protect against cognitive decline by preventing central inflammation. The effect of an acute exercise bout on central cytokine and apoptotic protein expression is not known. The brain may be protected from transient oxidative stress such as that induced by acute-exercise. The purpose of this exploratory study was to determine the effect of a single bout of intense exercise on hippocampal expression of inflammatory mediators (TNF-α) and apoptotic proteins (caspase-3, caspase-7), and to evaluate any potential age-related differences. METHODS: Using a C57BL/6 mouse model (N.=98), we evaluated the effect of an acute exercise bout (90 minutes of treadmill running: 10 min warm-up, 30 min at 22 m.min⁻¹, 30 min at 25 m.min⁻¹, and 30 min at 28 m.min⁻¹, 2° slope) on hippocampal inflammation in young (3-4 months), middle-aged (13-14 months) and older (16-17 months) C57BL/6 mice. RESULTS: Our results show post-exercise increases in hippocampal TNF-α and caspase-3/7 in each age group (main effect of acute exercise, P<0.05). Older mice displayed higher TNF-α (main effect of age, P<0.05) expression compared with younger animals at baseline. Young mice demonstrated greater increases in caspase-7 following acute exercise, compared to older mice (interaction effect for caspase-7, P<0.05). CONCLUSION: Given the relationship between aging, inflammation and neurodegenerative disease, and the protective effects of exercise, we cautiously propose that acute-exercise induced inflammation may be a normal physiologic response that elicits a favorable (anti-inflammatory) hippocampal environment.

The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration.

BACKGROUND: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. METHODS: An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. RESULTS: Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. CONCLUSIONS: This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Apoptotic and inflammatory cytokine protein expression in intestinal lymphocytes after acute treadmill exercise in young and old mice.

AIM: Gastrointestinal disturbances are common in athletes following intense exercise. Variations in apoptotic protein expression and cell death may contribute to acute exercise-induced intestinal inflammation. The effect of age on apoptotic protein response in the intestinal compartment in response to exercise is not known. Using a mouse model, we examined the effects of a single bout of treadmill running in young and old mice on intestinal lymphocyte (IL) expression of the apoptosis-inducing cytokine TNF-α, the pro-apoptotic proteins caspase-3 and 7, the anti-apoptotic protein Bcl-2, and IL apoptotic status (% AnnexinV+). METHODS: Young (3-4 months, N.=44) and old (13-14 months, N.=45) female C57Bl/6 mice were randomized to treadmill exercise (10 min warm-up, 20 min at 22 m min-1, 30 min at 25 m min-1, 30 min at 28 m min-1, 2º slope) with sacrifice immediately (IMM) or 2hr after (2Hr), or to a non-exercised control (SED). IL were removed and prepared for analysis of % apoptosis (flow cytometry) and determination of apoptotic protein and cytokine expression (Western blotting). Plasma corticosterone and 8-iso-PGF2α were measured by EIA. RESULTS: Exercise was associated with a higher IL expression of caspase-3 in IMM and 2Hr groups vs. SED (P<0.001), a higher expression of TNF-α in the IMM group vs. SED (P<0.001), and a lower Bcl-2 expression in the IMM and 2Hr groups vs. SED (P<0.01). There was a trend (P=0.07) for increased caspase-7 expression after exercise. IL caspase-3 and 7 and TNF-α expression did not differ by age whereas Bcl-2 expression was lower (P<0.001) and % Annexin V+ IL was higher (P<0.05) in old vs. young mice. Plasma corticosterone and 8-iso-PGF2α were higher (P<0.001 and P<0.05) in IMM vs. SED mice but did not differ by age. CONCLUSION: The expression of the pro-apoptotic proteins, caspase-3 and caspase-7, and the apoptosis-inducing cytokine, TNF-α, in IL did not differ by age in this animal model in response to a single intense exercise challenge. However, Old mice had lower expression of the 'protective' anti-apoptotic protein Bcl-2 and a higher percentage of early apoptotic IL. Whether repeated exercise results in less IL resiliency in elderly individuals remains to be determined.

FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy.

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

Freewheel training decreases pro- and increases anti-inflammatory cytokine expression in mouse intestinal lymphocytes.

Intestinal inflammation and inflammatory bowel diseases (IBD) may occur due to imbalances in pro- and anti-inflammatory cytokines. Long-term exercise reduces the risk for IBD. The purpose of this study was to determine the effect of long-term wheel running in healthy mice on intestinal lymphocyte (IL) expression of pro- and anti-inflammatory cytokine proteins. In addition, pro- and anti-apoptotic proteins and the percentage of early apoptotic, late apoptotic, and dead IL were measured with wheel running and following acute aerobic exercise. Female C57BL/6 mice were given 16 weeks of wheel running (WR) or a control condition (No WR) and at the end of training were assigned to a single acute treadmill exercise session with sacrifice immediately, 2h after, or 24h after completion of exercise, or were not run (sedentary) with respect to the acute treadmill exercise. Intestinal lymphocytes were assessed for pro-(TNF-α, IL-17) and anti-(IL-10) inflammatory, and pleiotropic (IL-6) cytokines, and pro-(caspase 3 and 7, AIF) and anti-(Bcl-2) apoptotic protein expression. The percent of early (Annexin(+)) and late (Annexin(+)PI(+)) apoptotic, and dead (PI(+)) IL was determined. WR mice had lower TNF-α and caspase 7, and higher IL-10 and IL-6 expression in IL than No WR mice. A single exposure to intense aerobic treadmill exercise increased pro-(TNF-α) and anti-(IL-10) inflammatory cytokine and pro-apoptotic protein (caspase 3) expression in IL. The percent of early and late apoptotic, and dead IL were higher after acute exercise. Although long-term voluntary wheel running did not protect against acute exercise-induced changes in IL cytokine expression or apoptosis, there was an overall 'anti-inflammatory' effect observed as a result of wheel running in healthy mice.

Does exercise protect from cognitive decline by altering brain cytokine and apoptotic protein levels? A systematic review of the literature.

Regular exercise is thought to provide protection against age-related cognitive decline and possibly reduce risk of dementias. The mechanisms for the exercise protective effects are not known although changes in inflammatory cytokine levels may be involved. We conducted a systematic review of the literature to assess (1) the effects of exercise on cytokines in the brain, (2) the methodological rigour of studies which have examined these exercise effects and (3) the potential role of regular exercise in reducing the pro-inflammatory cytokine milieu that may contribute to dementia. We also reviewed the effects of exercise on concurrent pro and anti-apoptotic protein expression in the brain as related to cytokine changes. Five databases were searched until January 2010 with an initial 630 articles identified; 61 articles were retrieved of which 10 met study inclusion criteria. Investigations of both acute and chronic (training) exercise were assessed for methodological quality using a modified PEDro scale. Two studies were carried out with human participants and eight with mouse or rat models; studies differed markedly in design and methodological rigour; the types, intensities and durations of exercise, the cytokine and apoptotic proteins measured, and the regions of the brain (or proxy compartments) sampled. Despite variations in design, specific cytokine outcomes, and exercise type, the 10 studies provide limited evidence that acute strenuous exercise increases and exercise training decreases pro-inflammatory cytokines centrally. Two animal studies relate training associated decreases in pro-inflammatory cytokines with improved cognitive function using behavioural assessments such as the Morris maze. Recommendations for the design of future research on exercise, central cytokines, and cognition are offered.

Does physical activity affect quality of life, disease symptoms and immune measures in patients with inflammatory bowel disease? A systematic review.

Inflammatory bowel diseases (IBD) are a group of chronic, episodic inflammatory conditions of the large and small intestines. Individuals with IBD have been reported to use physical activity (PA) as a complementary therapy although the effectiveness of PA for reducing disease burden in patients with IBD is not known. The review objective is to evaluate published studies on physical activity and IBD focusing on quality of life, disease burden markers and immunological outcomes. A literature search was carried out using MEDLINE, WEB OF SCIENCE, CINHAL, and SCOPUS (to December 2008). Studies were included if they 1) were provided in English; 2) dealt with IBD in humans; 3) focused on the outcome measures of health related quality of life, clinical disease indicators or immune function; and 4) included PA as a primary intervention for IBD cases. In total, 7 studies were included in this systematic review: 5 were on PA and quality of life measures and inflammatory disease markers, and 2 on PA and immune measures. Four studies showed that PA significantly increased quality of life for IBD patients as assessed by various questionnaires. PA was also associated with decreased disease activity. There was no evidence that PA affected immune outcomes in patients with IBD. The role of PA as an adjunctive therapy for patients with IBD has not been well characterized in the literature. However, there is some evidence that PA may improve quality of life and reduce disease activity in patients with IBD.

GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources.

GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco-protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O:-linked oligosaccharides from the last 50 years and most N:-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono-isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N:- or O:-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at http://www.glycosuite.com.

Molecular analysis of the phospholipids of Escherichia coli k12.

Phospholipids from Escherichia coli K12 were converted to 1,2-diacylglycerols with phospholipase C from Bacillus cereus. High-pressure liquid chromatography of 1,2-diacylglycerol p-methoxybenzoates on LiChrosorb RP-18 using 2-propanol/acetonitrile (35:65) as eluant permitted separation of 14 molecular species. The main combinations of fatty acids were 1-16:0-2-16:1, 1-16:0-2-cyclo-17:0 and 1-16:0-2-18:1. Positional isomers were not present. The 1,2-di-16:0 compound was present at a significant level (7-10 mol%). Proportions of molecular species varied between phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phospholipid from the outer membrane of E. coli K12 contained a lower level of molecules with two unsaturated chains than was present in the cytoplasmic membrane. The method is sensitive, has good resolving power and employs readily available equipment.

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.

Desialylated LDL uptake in human and mouse macrophages can be mediated by a lectin receptor.

We have compared the uptake of desialylated low density lipoprotein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was also effectively competed by asialofetuin. Surprisingly, 125I-labelled NT-LDL degradation was also effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.

Protein phosphorylation: technologies for the identification of phosphoamino acids.

Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.

Identification and quantitation of cysteine in proteins separated by gel electrophoresis.

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 37 degrees C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

The relationship between the structures of the O polysaccharides from Escherichia coli O17 and O16.

The chemical structure of the O16 antigen from the lipopolysaccharide of Escherichia coli strain P4 has been determined. Comparison with the structures of other O16 antigens and that of the O17 antigen explains the previously reported cross-reaction of O antigen from the O16 strain K-12 with anti-O17 antibody [D. Liu and P.R. Reeves, Microbiology, 140 (1994) 49-57].

Operative treatment of chronic ruptures of the rotator cuff of the shoulder.

The results of 63 operative repairs of chronic tears of the rotator cuff in 61 patients are reviewed retrospectively; the mean follow-up was 32.7 months. Fifty-four patients presented with symptoms of persistent pain and seven patients with gross loss of movement. All the patients had failed to respond to conservative treatment. Results were assessed in terms of relief of pain, restoration of movement, the patients' ability to return to work and whether they were satisfied with the results. Overall, a good result in terms of relief of pain was achieved in 40 shoulders. In 31 shoulders (30 with pain and one without pain) the operation included particular measures to decompress the subacromial space; 26 of the patients achieved relief of pain which was significantly better than in those patients whose operation did not include a decompression. The complications and failures are discussed. It is suggested that operative repair of the chronically torn rotator cuff of the shoulder is a worthwhile operation and that the operation should include an adequate decompression of the subacromial space.

Arthrography of the shoulder after operative repair of the torn rotator cuff.

Double-contrast shoulder arthrograms were performed in 20 patients at an average of 30 months after operative repair of a torn rotator cuff. In 18 out of 20 shoulders the contrast medium leaked into the subacromial bursa indicating a defect in the rotator cuff. Despite this, 17 patients had complete relief of pain and 15 had a full range of shoulder elevation. The results suggest that a completely watertight closure is not essential for a good functional result, and that arthrography may not be helpful in the investigation of failure of repair.

Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis.

Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding. We report a means of determining monosaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel. The sensitivity of the method depends on the degree of glycosylation of the protein. We show that bovine fetuin can be analyzed and identified at the level of 100 pmol. 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences. Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized pH gradient. Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis. The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.

Waiting times. Bones of contention.

A slot system allocating a set number of outpatient orthopaedic appointments to general practices in North West Anglia HA means all patients referred are seen in eight weeks. The system has provoked criticism from GPs on the grounds that the burden of waiting lists has shifted onto them, and it limits access to services. The scheme has improved communication between GPs and orthopaedic consultants.