PEPOP 2.0: new approaches to mimic non-continuous epitopes.

BACKGROUND: Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits in replacing a protein which is difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitopes predicted by bioinformatics tools are commonly used and these sequential epitopes are used as is in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools have been proposed so far to predict peptide sequences: Superficial and PEPOP 2.0. PEPOP 2.0 can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. RESULTS: We have developed an improved version of PEPOP (PEPOP 2.0) dedicated to answer to experimentalists' need for a tool able to handle proteins and to turn them into peptides. The PEPOP 2.0 web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP 2.0 proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature. CONCLUSION: Through the presentation of its user-friendly, well-structured new web site conceived in close proximity to experimentalists, we report original methods that show how PEPOP 2.0 can assist biologists in dealing with discontinuous epitopes.

Treatment of giant-cell arteritis, a literature review.

Giant-cell arteritis (GCA) is the most common vasculitis in people aged more than 50 years. Despite the frequency of this disease, there is currently no international consensus on its therapeutic modalities. The aim of this study was to conduct a review on an international literature about the treatment of GCA, whatever the clinical pattern might be. Oral corticosteroids remain the cornerstone treatment, possibly preceded by intravenous bolus in complicated forms. In cases of glucocorticoid (GC) dependence or GC-related side effects, a GC-sparing agent may be necessary. Methotrexate is one of the most used treatments despite its low level of evidence and mild efficacy. Cyclophosphamide and tocilizumab look promising but require validation in further studies. The results for TNF-α blockers and azathioprine are disappointing. Preventing complications of prolonged corticosteroid therapy is a world challenge and the management of GC-induced osteoporosis is not the same from one country to another. There is a significant risk of arterial thrombosis, mainly at treatment onset, which may encourage to associate an antiplatelet therapy, especially in patients with other cardiovascular risk factors. Place of statins in the treatment of the disease is uncertain.

Osteitis in Systemic Sclerosis: A Nationwide Case-Control Retrospective Study.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by skin fibrosis, vasculopathy, and dysimmunity. Data regarding osteitis in SSc are scarce. METHODS: We performed a nationwide multicenter, retrospective, case-control study including patients with SSc, according to the 2013 American College of Rheumatology/European Alliance of Associations for Rheumatology classification, with a diagnosis of osteitis. The objectives of the study were to describe, to characterize, and to identify associated factors for osteitis in patients with SSc. RESULTS: Forty-eight patients were included. Twenty-six patients (54.1%) had osteitis beneath digital tip ulcers. Physical symptoms included pain (36 of 48, 75%), erythema (35 of 48, 73%), and local warmth (35 of 48, 73%). Thirty-one (65%) patients had median (interquartile range) C-reactive protein levels >2 mg/liter of 8 (2.7-44.3) mg/liter. On radiography, computed tomography, or magnetic resonance imaging, osteitis was characterized by swelling or abscess of soft tissues, with acro-osteolysis or lysis in 28 patients (58%). Microbiological sampling was performed in 45 (94%) patients. Most pathogens were Staphylococcus aureus (43.8%), anaerobes and Enterobacteriaceae (29.1%), and Pseudomonas aeruginosa (10.4%). Management comprised antibiotics in 37 (77.1%) patients and/or surgery in 26 (54.2%). Fluoroquinolones were used in 22 (45.8%) patients, and amoxicillin plus ß-lactamase inhibitor in 7 (14.6%). Six (12.6%) patients relapsed, 6 (12.6%) patients had osteitis recurrence, 15 (32%) sequelae, and 2 patients had septic shock and died. CONCLUSION: This study confirmed digital tip ulcers as an associated factor for osteitis and revealed a high rate of functional sequelae. Antimicrobial therapy with oral fluoroquinolone or intravenous amoxicillin and ß-lactamase inhibitor are used as first-line antibiotic therapy in SSc patients with osteitis.

Erratum to "characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris".

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexa-histidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni2+-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement, respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with, respectively, a diameter of 34 and 28 nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect antibodies to HBcAg (anti-HBc) in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris.

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexahistidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

CXC ligand 5 is an adipose-tissue derived factor that links obesity to insulin resistance.

We show here high levels of expression and secretion of the chemokine CXC ligand 5 (CXCL5) in the macrophage fraction of white adipose tissue (WAT). Moreover, we find that CXCL5 is dramatically increased in serum of human obese compared to lean subjects. Conversely, CXCL5 concentration is decreased in obese subjects after a weight reduction program, or in obese non-insulin-resistant, compared to insulin-resistant, subjects. Most importantly we demonstrate that treatment with recombinant CXCL5 blocks insulin-stimulated glucose uptake in muscle in mice. CXCL5 blocks insulin signaling by activating the Jak2/STAT5/SOCS2 pathway. Finally, by treating obese, insulin-resistant mice with either anti-CXCL5 neutralizing antibodies or antagonists of CXCR2, which is the CXCL5 receptor, we demonstrate that CXCL5 mediates insulin resistance. Furthermore CXCR2-/- mice are protected against obesity-induced insulin resistance. Taken together, these results show that secretion of CXCL5 by WAT resident macrophages represents a link between obesity, inflammation, and insulin resistance.

Monoclonal antibody 76F distinguishes IA-2 from IA-2beta and overlaps an autoantibody epitope.

IA-2 and IA-2beta are highly related proteins that are autoantigens in type 1 diabetes, and provide a model for developing reagents and assays that distinguish similar proteins with unique autoantibody epitopes. Monoclonal antibodies (mAb) to IA-2 and IA-2beta were prepared and tested for their ability to bind to the related proteins and their ability to compete for specific autoantibody epitope binding by sera from patients with type 1 diabetes. Monoclonal antibodies that specifically bound IA-2 (76F) or bound both IA-2 and IA-2beta (A9) were isolated and characterized. 76F mAb recognized IA-2 of human, rat and mouse origin in native and denatured forms and had an epitope specificity for residues 626-630 (FEYQD) which are found in the juxtamembrane (JM) region of human and mouse IA-2, but not IA-2beta. This region overlaps with the autoantibody epitope JM2. Binding to the 76F monoclonal antibody was specifically inhibited by sera with antibodies to the JM2 epitope but not with antibodies to the adjacent JM1 epitope, indicating that unique epitopes can be distinguished by this approach. 76F mAb has the unique property to distinguish between the two closely related autoantigens IA-2 and IA-2beta by targeting an IA-2 specific epitope of the juxtamembrane region. The findings define an approach to develop assays for specific antibody epitope measurements which may be relevant for disease prognosis and monitoring intervention therapies.