John G. Routsias

Blood Leptin and Adiponectin as Possible Mediators of the Relation Between Fat Mass and BMD in Perimenopausal Women

Fat mass is a predictor of BMD; however, the mechanisms involved remain uncertain. Two adipokines, leptin and adiponectin, were examined as potential mediators of this relation in 80 perimenopausal women. Adiponectin did not exert any effect on BMD, whereas leptin exerted a negative one, with insulin acting as a confounder to this relation.

Autoimmune response and target autoantigens in Sjogren's syndrome

Eur J Clin Invest 2010; 40 (11): 1026–1036

Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen, enhancing recognition by anti-Ro60 kD autoantibodies.

Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide–calreticulin is recognized specifically by anti‐Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi‐step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175–184aa(10p) and 216–232aa(17p). The interaction of the peptide–calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty‐eight anti‐Ro60 KD positive and 23 anti‐Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS) were tested. All anti‐Ro60 kD positive sera bound the complex calreticulin‐17p, while 95% of the same sera had activity against the complex calreticulin − 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti‐Ro60 kD positive sera. Anti‐Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10ρ and 17ρ or with the complexes calreticulin − 10p and calreticulin‐17p (<5%). These results suggest that calreticulin can induce conformation‐dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.

Autoantibodies to intracellular autoantigens and their B-cell epitopes: Molecular Probes to Study the Autoimmune Response

A common laboratory finding in systemic autoimmune diseases is the presence of autoantibodies against intracellular autoantigens. Although their pathogenesis is not fully understood, autoantibodies are important tools for establishing diagnosis, classification, and prognosis of autoimmune diseases. Autoantibodies mainly target multicomponent complexes containing both protein antigens and (ribo)-nucleic acid(s), such as the spliceosome or Ro/La RNPs. In this review, we address the main characteristics and the clinical value of the main autoantibody types with respect to their disease association, and we describe the corresponding autoantigens, their biologic function, and their B-cell antigenic determinants (epitopes). The structural characteristics and clinical associations of these epitopes, and their utility as tools to investigate the autoimmune response, are discussed in detail. New insights into the pathogenetic role of epitopes in systemic autoimmunity are also examined. In this regard, using the defined structures of the B-cell antigenic epitopes, complementary epitopes can be designed according to the “molecular recognition” theory. These complementary epitopes can be used as probes to study pathogenetic and immunoregulatory aspects of the anti-idiotypic response. The origin of humoral autoimmunity and the spreading of the epitopes in systemic lupus erythematosus are also discussed. Finally, the ability of post-translational modifications to induce autoreactive immune attack via the generation of neo-epitopes is summarized.

Peripheral neuropathies in Sjögren syndrome: a new reappraisal

Background The prevalence of peripheral neuropathy in patients with Sjögren syndrome remains unclear owing to conflicting results in the published series, with numbers ranging from 2% to over 60% of Sjögren syndrome patients. Whether peripheral neuropathy is a feature of the systemic or glandular disease or whether it is related to a circulating antineuronal antibody remains also uncertain. Methods The authors reviewed the records of patients with primary Sjögren syndrome (pSS), fulfilling the Revised European—American Classification Criteria, seen in their department from 1992 to 2009. The patients with previously recorded neuropathic features were re-examined clinically and electrophysiologically. Other causes of polyneuropathy were excluded. The authors also searched for circulating antineural antibodies using immunofluorescence and western blot and for antibodies against muscarinic and nicotinic acetylcholine receptors as potential biomarkers. Results 509 cases met the diagnostic criteria for pSS. Among these, 44 patients were recorded as having neuropathic symptoms. After completing the evaluation, however, only nine (1.8%) had polyneuropathy with objective clinical signs and abnormal electrophysiological findings. The neuropathy was axonal in all, in five pure sensory and in four sensorimotor. The patients with peripheral neuropathy had extraglandular manifestations such as palpable purpura and vasculitis. No evidence of antineural autoimmunity was found, and no candidate biomarkers were identified. Conclusion Polyneuropathy is a rare manifestation of pSS occurring in 1.8% of patients. In the majority of patients, it is a late event and frequently associated with systemic disease or risk factors for lymphoma development.

B-cell epitopes of the intracellular autoantigens Ro/SSA and La/SSB: Tools to study the regulation of the autoimmune response

A common serologic finding in systemic autoimmune diseases is the presence of autoantibodies against intracellular autoantigens. Although their pathogenesis is not fully understood, autoantibodies are important tools for establishing diagnosis, classification and prognosis of autoimmune diseases. In Systemic Lupus Erythematosus (SLE) and Sjögrens syndrome (SS) autoantibodies mainly target multicomponent ribonucleoprotein complex Ro/La RNP. The last years, the main characteristics, the clinical significance of the anti-Ro/SSA and anti-La/SSB autoantibodies, their biologic function, as well as their B-cell antigenic determinants (epitopes) have been addressed. More specifically, the structural characteristics and clinical associations of epitopes along with their utility as tools to investigate the autoimmune response have been investigated in detail. New insights for the pathogenetic role of epitopes in initiation, propagation and regulation of systemic autoimmunity have been emerged. In this regard, the role of epitope spreading in the diversification of autoimmune response and the anti-idiotypic antibodies in the regulation of autoantibodies (idiotypic) response are addressed.

Cross‐reaction between antibodies to the major epitope of Ro60 kD autoantigen and a homologous peptide of Coxsackie virus 2B protein

Coxsackie virus RNA has recently been detected in biopsy specimens of minor salivary glands from patients with primary Sjögrens Syndrome (pSS). A peptide derived from Coxsackie virus 2B protein (pepCoxs) presents 87% sequence homology with the 222–229 region of the major linear B‐cell epitope of  Ro60 kD  autoantigen  (pep216–232).  Synthetic  peptides  corresponding to pep216–232: 216KALSVETEKLLKYLEAV232 and pepCoxs: 31MVTSTITEKL LKNLVKI47, were prepared. Sera from 42 patients with pSS and 43 patients with systemic lupus erythematosus (SLE) as well as sera from 27 healthy individuals (normal controls) and sera from 30 patients with rheumatoid arthritis (disease controls) were tested against the two homologous peptides. Twenty‐five percent of SLE sera and 33·3% of pSS sera reacted against pep216–232, whereas 28% of SLE sera and 37% of pSS sera recognized the pepCoxs. The sera reacting with pep216–232 were apparently the same as those reacting with pepCoxs. Normal sera and disease control sera presented only a limited reactivity against both peptides (ranging from 3·7% to 10%). Both peptides reacted more prominently with anti‐Ro/La (+) sera from pSS patients. Thus, pep216–232 was recognized by 17% of the anti‐Ro (+) sera and by 42% of the anti‐Ro/La (+) sera, whereas pepCoxs was recognized by 28·5% and 38% of the a‐Ro(+) and a‐Ro/La(+) sera, respectively. Purified anti‐pep216–232 antibodies readily reacted with both peptides while inhibition experiments revealed the specificity of this reaction. These results suggest a possible cross‐reaction between antibodies to the major linear B‐cell epitope of Ro60 kD autoantigen and the homologous pepCoxs in pSS patients. This cross‐reaction might potentially play a role in autoantibody formation and the perpetuation of the autoimmune response against Ro/SSA and La/SSB.

Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious diseases.

Toll‐like receptors (TLRs) are the best‐studied family of pattern‐recognition receptors (PRRs), whose task is to rapidly recognize evolutionarily conserved structures on the invading microorganisms. Through binding to these patterns, TLRs trigger a number of proinflammatory and anti‐microbial responses, playing a key role in the first line of defence against the pathogens also promoting adaptive immunity responses. Growing amounts of data suggest that single nucleotide polymorphisms (SNPs) on the various human TLR proteins are associated with altered susceptibility to infection. This review summarizes the role of TLRs in innate immunity, their ligands and signalling and focuses on the TLR SNPs which have been linked to infectious disease susceptibility.

Idiotype, anti-idiotype network of autoantibodies: pathogenetic considerations and clinical application.

A common serologic finding in autoimmune diseases is the presence of autoantibodies against intracellular autoantigens. Recent data suggest that an anti-idiotypic network exists in these diseases, regulating the production of autoantibodies (idiotypic response). The anti-idiotypic antibodies can be monitored using complementary epitopes, designed according to the “molecular recognition” theory. The role of antiidiotypic antibodies in neonatal lupus and type 1 diabetes are discussed. In neonatal lupus, mothers with high anti-idiotypic antibody activity against anti-La autoantibodies are at lower risk of giving birth to an un-healthy child, as compared with mothers without anti-idiotypic antibodies. Similarly,the lack of particular anti-idiotypic antibodies against anti-GAD65 autoantibodies predispose in type 1 diabetes. These findings imply that antiidiotypic antibodies may confer protection from the harmful effect of autoantibodies in certain autoimmune diseases [corrected].

Association of the idiotype:antiidiotype antibody ratio with the efficacy of intravenous immunoglobulin treatment for the prevention of recurrent autoimmune‐associated congenital heart block

OBJECTIVE Congenital heart block (CHB), a manifestation of neonatal lupus, is associated with maternal anti-Ro/SSA and anti-La/SSB autoantibodies and recurs in ∼18% of subsequent pregnancies. This study was undertaken to investigate the effect of the idiotype:antiidiotype (Id:anti-Id) antibody ratio in the ability of intravenous immunoglobulin (IVIG) administered during subsequent pregnancies to prevent CHB. METHODS We studied 16 anti-Ro/SSA and anti-La/SSB-positive pregnant women from the Preventive IVIG Therapy for Congenital Heart Block study who had previously given birth to a child with neonatal lupus. In 3 of the mothers, the study pregnancy resulted in the birth of a child with neonatal lupus (2 with CHB and 1 with rash). Sequential serum samples were obtained from all mothers immediately before the administration of IVIG during pregnancy and were evaluated for antibodies against the major B cell epitope 349-364aa of La/SSB (idiotype) and its antiidiotypic antibodies. RESULTS Following IVIG treatment, serum titers of anti-La(349-364) (Id antibodies) decreased in 80% of the mothers, and in 60% an increase in anti-Id antibodies against anti-La(349-364) was observed. The Id:anti-Id ratio was significantly higher in mothers whose offspring developed neonatal lupus compared to mothers who gave birth to a healthy child (P<0.0001). Removal of anti-Id antibodies substantially increased the reactivity against La(349-364) in sera from 5 of 7 mothers tested. All IVIG preparations were examined for Id and anti-Id antibody activity. IVIG from batches administered to mothers who gave birth to a healthy child had an Id:anti-Id activity ratio of <1, in contrast to that given to mothers who gave birth to a child with neonatal lupus. Addition of the IVIG preparations to the maternal sera further enhanced antiidiotypic activity (by up to 4.7-fold) in 11 of 13 patients studied. CONCLUSION This is the first study in humans to demonstrate that IVIG influences the Id-anti-Id network of a specific pathogenic autoantibody. Specifically, we showed that IVIG enhanced the anti-Id antibody response in pregnant women with anti-La/SSB antibodies. A high Id:anti-Id ratio in both the IVIG preparation and the maternal serum may explain the absence of an effect of IVIG in preventing recurrent neonatal lupus in some cases.