IgG subclass antibodies to human and bacterial HSP60 are not associated with disease activity and progression over time in axial spondyloarthritis

Introduction

Spondyloarthritis (SpA), an interrelated group of rheumatic diseases, has been suggested to be triggered by bacterial infections prior to the development of an autoimmune response that causes inflammation of the spinal and peripheral joints. Because human heat shock protein 60 (HSP60), recently renamed HSPD1, and bacterial HSP60 are highly homologous, immunological cross-reactivity has been proposed as a mechanism of disease initiation. However, previous investigations of the humoral immune response to HSP60 in SpA patients have lacked determination of immunoglobulin G (IgG) subclasses and patient follow-up. In this study, we have focused on these parameters in a cohort of axial SpA patients with a well-established set of clinical characteristics, including MRI changes and human leukocyte antigen B27.

Methods

IgG subclass antibodies (IgG1, IgG2, IgG3 and IgG4) against recombinant HSP60 of three reactive arthritis-related bacteria; human HSP60; and the microorganisms Chlamydia trachomatis and C. pneumoniae were determined by ELISA. Serum samples collected from 2004 to 2006 and in 2010 and 2011 from 39 axial SpA patients were analyzed and compared with samples from 39 healthy controls. The Mann-Whitney U test and Wilcoxon matched pairs test were used to compare the antibody levels in different and paired groups, respectively. P < 0.01 was considered significant. The Spearman nonparametric correlation was used to determine correlation between antibody levels and between antibody levels and the disease parameters.

Results

Elevated levels of IgG1 and IgG3 to human HSP60 and IgG1 to HSP60 of Salmonella enterica Enteritidis were observed in SpA patients compared with healthy controls at both time points. The antibody levels were almost constant over time for IgG1, whereas high levels of IgG3 to human HSP60 tended to decrease over time. The antibody response to human HSP60 was predominantly of the IgG3 subclass, and patients with high levels of IgG3 to this antigen had low levels of IgG1, indicating an inverse association. Different IgG subclasses were produced against bacterial and human HSP60 in the same serum sample, IgG1 and IgG3, respectively, indicating that there was no cross-reaction.

Conclusions

A significant association was observed between axial SpA and the presence of IgG1/IgG3 antibodies to human HSP60 and of IgG1 to S. enterica Enteritidis and C. trachomatis. Generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60. No association was observed between clinical and MRI changes with antibodies over time. Altogether, such antibodies do not reflect the disease activity in these patients.This study has been approved by the Regional Research Ethics Committee of Central Jutland, Denmark. Trial registration numbers: 20050046 and 20100083     

Efficacy of anakinra in gouty arthritis: a retrospective study of 40 cases

Introduction

Gout is a common arthritis that occurs particularly in patients who frequently have associated comorbidities that limit the use of conventional therapies. The main mechanism of crystal-induced inflammation is interleukin-1 production by activation of the inflammasome. We aimed to evaluate the efficacy and tolerance of anakinra in gouty patients.

Methods

We conducted a multicenter retrospective review of patients receiving anakinra for gouty arthritis. We reviewed the response to treatment, adverse events and relapses.

Results

We examined data for 40 gouty patients (32 men; mean age 60.0 ± 13.9 years) receiving anakinra. Mean disease duration was 8.7 ± 8.7 years. All patients showed contraindications to and/or failure of at least two conventional therapies. Most (36; 90%) demonstrated good response to anakinra. Median pain on a 100-mm visual analog scale was rapidly decreased (73.5 (70.0 to 80.0) to 25.0 (20.0 to 32.5) mm, P <0.0001), as was median C-reactive protein (CRP) level (130.5 (55.8 to 238.8) to 16.0 (5.0 to 29.5) mg/l, P <0.0001). After a median follow-up of 7.0 (2.0 to 13.0) months, relapse occurred in 13 patients after a median delay of 15.0 (10.0 to 70.0) days. Seven infectious events, mainly with long-term use of anakinra, were noted.

Conclusions

Anakinra may be efficient in gouty arthritis, is relatively well tolerated with short-term use, and could be a relevant option in managing gouty arthritis when conventional therapies are ineffective or contraindicated. Its long-term use could be limited by infectious complications.     

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

A Monoclonal Antibody that Specifically Recognizes m'A Nucleoside

Abstract

A hybridoma against the nucleoside m⁶A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m⁶A. The competition assays showed that the monoclonal antibody was highly specific for m⁶A nucleoside.     

Synthesis of 2,2′-Anhydro-2-Hydroxy- and 6,2′-Anhydro-6-Hydroxy-1-β-D-Arabindfuranosylnicotinamide as Conformationally Restricted Nicotinamide Nucleoside Analogs1

Abstract

1-(β-D-Ribofuranosy1)-2(1H)-pyridone-3-carboxamide (6a) and the 6(1H)-pyridone derivative (6b) were prepared by condensation of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3) with 2- and 6-hydroxynicotinic acid, respectively, to 4a and 4b, followed by conversion of the carboxylic acid function of 4a,b into their corresponding carboxamides 5, and then deprotection of 5. Bath 6a and 6b were then treated with 1,3-dichlom-1,1,3,3-tetraisopropyldisiloxane to give the corresponding 3′,5′-O-TPDS derivatives, 7a and 7b. Mesylation of 7a,b with mesyl chloride in pyridine afforded the stable, protected mesylates 8a,b. Upon de-O-silylation of 8a,b with ET₃NHF gave a mixture of unprotectd mesylates 9a,b and 2,2-anhydro- and 6,2′-anhydronucleosides, 1a and 1b. Upon storage of 9a,b at man temperature, they are quantitatively converted into 1a,b. Mild alkaline hydrolysis of 1a,b afforded their corresponding arabino nucleosides 10a,b.     

A New Method of Synthesis of Fluorescently Labelled Oligonucleotides and Their Application in DNA Sequencing

Abstract

A new approach to the chemical synthesis of oligonucleotides bearing reporter functional groups based on the modification of cytosine residues during the final deprotection step is reported. The application of the fluorescently labelled primer for the automated DNA sequencing is shown.     

Phosphitylation of Primary Carboxamides. Synthesis of Peptide-Oligonucleotide Conjugates with Acylphosphoramidate Linkages

Abstract

N-acylphosphoramidates can be obtained from the reaction of phosphitylated primary carboxamides and an alcohol in the presence of an acid catalyst such as tetrazole and subsequent oxidation. The reaction is useful for the preparation of peptide-oligonucleotide conjugates.     

Note: A Convenient Method for the Preparation of N2, N2-Dimethylguanosine

Abstract

The preparation of N², N²-dimethylguanosine is described. The use of the 2-(p-nitrophenyl)ethyl group instead of the benzyl protecting group for the O⁶ position of the guanine ring resulted in better yields and shorter protocols.     

The Cytotoxicity of Anti-PAI-I Oligonucleotides and Their Conjugates

Abstract

The cytotoxicity of anti-PAI-5 hexadecanucleotides (phosphodiesters and phosphorothioates) and their conjugates with lipophilic alcohols was tested in EA.hy 926 hybrid endothelial cells. Some cytotoxicity was found for cholesteryl and bornyl conjugates at concentrations higher than those used for antisense inhibition experiments.     

Use of a Base-Labile Protected Derivative of 6-Mercaptohexanol for the Preparation of Oligonucleotides Containing a Thiol Group at the 5′-End

Abstract

The preparation of a base-labile (Dnpe) protected derivative of 6-mercaptohexanol is described. The use of the phosphoramidite derivative of this compound improves both yields and the time needed for the preparation of oligonucleotides containing a thiol group at the 5′-end.