Dear Editor,

I read with interest the recent study describing clinical characteristics associated with antibodies that bind fragments of the Mi-2 beta antigen1 and it raises the question of how to best define autoantibodies. For economic reasons, ELISAs are taking over much of the immunology testing in rheumatology practice today. ELISAs are being used despite their often limited validation and the many concerns regarding the sensitivity, specificity and reproducibility of these approaches.2 There is considerable evidence that ELISA assays may not be measuring the same antibody populations that have traditionally been detected by immunodiffusion, indirect immunofluorescence or immunoprecipitation methods – the classic approaches that originally defined the clinical usefulness of most autoantibodies in systemic rheumatic conditions. Many groups have recognized this problem, and coordinated efforts similar to those of the Diabetes Antibody Standardisation Program to improve the sensitivity, specificity and comparability of the measurement of autoantibodies associated with type 1 diabetes (http://www.idsoc.org/committees/antibody/dasphome.html) should be encouraged in other fields.

Important examples in the literature also show that antibodies raised against a given antigen may not interact with the target protein in the same way that spontaneously occurring autoantibodies do, and it is possible that such differences have important implications regarding the origin and pathologic significance of human autoantibodies. Prime examples are anti-Jo-1 autoantibodies directed against histidyl-tRNA synthetase. These myositis-specific autoantibodies were originally defined by immunodiffusion and immunoprecipitation of the target protein in a complex with its cognate tRNA and were found in all cases to inhibit the function of the target antigen.3 Later studies in mice suggested that antibodies could easily be induced that bind histidyl-tRNA synthetase, however, they were completely different in their disease association, epitope specificity, capacity to immunoprecipitate the target antigen with its cognate tRNA, and their ability to inhibit the function of the antigenic enzyme.4 Should such different antibodies also be called anti-Jo-1 autoantibodies? I think not and suggest that they should rather be referred to simply as antibodies that bind histidyl-tRNA synthetase or its peptide fragments by the methodology used.

I believe that this issue is a widespread one and a clarification of such antibody nomenclature would enhance our understanding and appreciation of the limitations that different methodologies impose. Until and unless data are generated to demonstrate that a new method of measuring a particular autoantibody gives equivalent results to a previously validated approach, I believe that it would be desirable that the “new antibody” should be given a new name in conjunction with the methodology used to detect that antibody. Such nomenclature would potentially minimize confusion in the literature regarding the sensitivity, specificity, phenotypic associations and clinical use of such antibodies.

This brings us back to the study by Hengstman et al.1 Is it possible that the differences in clinical associations noted in this paper, as compared to those found in previous studies5, are due to the detection of different antibody populations by different methods? Antibodies that bind to fragments of the chromodomain helicase DNA binding protein 4 (the Mi-2 beta antigen) by ELISA may be quiet different than those which are positive by immunodiffusion or which immunoprecipitate the Mi-2 antigen in conjunction with the rest of the nucleosome remodeling and histone deacetylase complex - the original ways that anti-Mi-2 autoantibodies and their clinical associations were defined. It would have been useful for the authors to have compared these methods directly in this study to address this possibility. Until this possible methodologic difference is resolved, however, perhaps it would be best to not define such ELISA- binding antibodies as anti-Mi-2 autoantibodies, but rather as antibodies which bind fragments of the chromodomain helicase DNA binding protein 4 by ELISA.

References

(1) Hengstman GJ, Vree Egberts WT, Seelig HP et al. Clinical characteristics of patients with myositis and autoantibodies to different fragments of the Mi-2 beta antigen. Ann Rheum Dis 2006; 65(2):242-245.

(2) Fritzler MJ, Wiik A, Tan EM et al. A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. III. Comparative performance characteristics of academic and manufacturers' laboratories. J Rheumatol 2003; 30(11):2374- 2381.

(3) Mathews MB, Bernstein RM. Myositis autoantibody inhibits histidyl-tRNA synthetase: a model for autoimmunity. Nature 1983; 304:177- 179.

(4) Miller FW, Waite KA, Biswas T, Plotz PH. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity. Proc Natl Acad Sci U S A 1990; 87(24):9933-9937.

(5) Love LA, Leff RL, Fraser DD et al. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine (Baltimore) 1991; 70(6):360-374.

Dear Editor,

We read with great interest the recently published article by Otero et al. [1] where they report significantly increased levels of leptin, adiponectin and visfatin plasma levels in patients with rheumatoid arthritis compared with controls. Their finding concerning the level of leptin in rheumatoid arthritis contradicts the results from other studies including our own (Ljung et al, submitted).[2][3] Our concern in this matter is that the relation of male vs. female subjects differs in the patient group and the control group, with 71% females among the patients and only 56% females among controls.

It is well known that serum leptin levels are considerably higher in females than in males, and the mean levels presented in females are more than double the levels in males. [4][5] The same goes for adiponectin, although not as dramatically different. [5] Thus, there is a risk that this influences the results. It is tempting to conclude, that the significance levels (p < 0.05) obtained in the statistical analyses of leptin and adiponectin reflect the lack of gender stratification. We would be interested to see the groups divided by sex to appreciate the value of the results.

Lotta Ljung, MD Solbritt Rantapää Dahlqvist, MD, Professor.
Department of Public Health and Clinical Medicine, Rheumatology, University Hospital, 901 85 Umeå, Sweden.

References

1 Otero M, Lago R, Gomez R, Lago F, Dieguez C, Gomez-Reino JJ, et al. Changes in fat-derived hormones plasma concentrations: adiponectin, leptin, resistin, and visfatin in rheumatoid arthritis subjects. Ann Rheum Dis 2006 Jan 13;[Epub ahead of print]

2 Popa C, Netea MG, Radstake TR, van Riel PL, Barrera P, van der Meer JW. Markers of inflammation are negatively correlated with serum leptin in rheumatoid arthritis. Ann Rheum Dis 2005;64:1195-8

3 Anders HJ, Rihl M, Heufelder A, Loch O, Schattenkirchner M. Leptin serum levels are not correlated with disease activity in patients with rheumatoid arthritis. Metabolism 1999 Jun;48:745-8.

4 Hickey MS, Israel RG, Gardiner SN, Considine RV, McCammon MR, Tyndall GL, et al. Gender differences in serum leptin levels in humans. Biochem Mol Med 1996;59:1-6.

5 Cnop M, Havel PJ, Utzschneider KM, Carr DB, Sinha MK, Boyko EJ, et al. Relationship of adiponectin to body fat distribution, insulin sensitivity and plasma lipoproteins: evidence for independent roles of age and sex. Diabetologia 2003;46:459-69.

Dear Editor,

We read with interest an article on EULAR/PRES Endorsed Consensus Criteria for the Classification of Childhood Vasculitides under review by the ACR by Ozen et al.[1] The new classification criteria for Henoch-Schonlein purpura (HSP) by the EULAR deleted the age at onset, included predominant IgA deposition, and added arthritis and renal involvement to the group of criteria. Nevertheless, only diffuse abdominal pain was included in the existing ACR and new EULAR criteria despite the variety of gastrointestinal manifestations in HSP, which include not only diffuse abdominal pain but also gastrointestinal bleeding (such as hematemesis, melena, hematochezia, or positive occult blood in stool) and vomiting, etc.[2] Although these symptoms may coexist, isolated presentation of other gastrointestinal symptoms, such as silent gastrointestinal bleeding, can also occur. Therefore, we suggest that gastrointestinal involvement would be a more appropriate term rather than diffuse abdominal pain or bowel angina.

Also, it would be necessary to discuss this problem if the third survey is possible. We speculate that this new EULAR criteria for HSP could be helpful for clinicians to diagnose HSP more accurately and to predict the prognosis of this disease. In the future, further studies should be performed to elucidate the epidemiology and natural course of HSP based on this new EULAR criteria as well as the usefulness of the criteria.

Competing interest statement: None of the authors have any competing interest.

References

1. Ozen S, Ruperto N, Dillon M, et al. EULAR/PRES Endorsed Consensus Criteria for the Classification of Childhood Vasculitides under review by the ACR. Ann Rheum Dis 2005 Dec 1; [in press].

2. Chang WL, Yang YH, Lin YT, Chiang BL. Gastrointestinal manifestations in Henoch-Schonlein purpura: a review of 261 patients. Acta Paediatr 2004;93:1427-1431.

Dear Editor,

We read with interest the recent article “Smoking is a risk factor for anti-CCP antibodies only in RA patients that carry HLA-DRB1 Shared Epitope alleles” by Dr. Linn-Rasker and colleagues in Leiden.[1] The question of whether true gene-environment interaction exists between genotype (HLA-DRB1 shared epitope, SE) and an environmental exposure, cigarette smoking in this case, both of which are known to greatly increase the risk of developing RA, is central to our understanding of how these factors predispose to RA. Does the risk associated with having both of these risk factors exceed what would be expected if these risks were independent and additive, and how so? Case-only analyses, such as this, are an efficient and sound method for screening for gene-environment interactions, under the assumption of independence of exposure and genotype in the population.[2,3]

In a recent editorial, Ahlbom and Alfredsson explained the concepts of biological versus statistical interaction.[4,5] Biological interaction is seen when two risk factors are involved in the same pathway to development of disease; statistically this is seen as a departure from additivity of disease rates. For example, Padyukov and colleagues in Sweden have demonstrated that a greater than additive interaction exists for the effects of cigarette smoking and the presence of 0, 1 or 2 copies of the SE.[6] To do so, they calculated the attributable proportion (AP), the proportion of RA among those with both exposures that is attributable to an additive interaction between these risk factors.[7] They found that among current smokers with a double copy of the SE, the AP is 0.7, or 70%, (95% CI 0.4, 0.9).[6] Another way of statistically testing for gene-environment interaction is to create a cross-classified variable, or interaction term, in a logistic regression model, and test it for significance, pointing to a greater than multiplicative interaction that is significant.

In their study, Linn-Rasker and colleagues employed a case-only analysis of patients in the Leiden Early RA clinic to evaluate the interaction between smoking and HLA-SE in predicting the risk of Anti-CCP antibodies. Their findings are very interesting and suggest an interaction between the two exposures, but it does not appear that the presence of a gene-environment interaction was statistically tested. We have employed their data to test statistically for the presence of a gene-environment interaction, both greater than additive and greater than multiplicative. Using the data presented in table 2 of their article, the AP, or the proportion of RA cases positive for anti-CCP antibodies which is attributable to an interaction between cigarette smoking and the presence of the SE, is 0.5, or 50%. (Given our limited access to the data, we are not able to use a re-sampling technique to obtain a 95% confidence interval.) When a cross-classified variable (smoking x HLA-SE) is created and employed in a logistic regression model using the data in Table 2, the term is not significant for a multiplicative interaction (p=0.19). Employing the terminology suggested by Ahlbom and Alfredsson,[4,5] the fact that an additive, but not multiplicative, interaction exists is important and suggests that cigarette smoking and HLA SE lie on the same causal pathway to the development of anti-CCP antibodies and RA. Presenting such statistical analyses promotes translational research between epidemiologists and basic scientists.

Karen H. Costenbader, MD, MPH (A)
Lori B. Chibnik, MPH (A)
Lisa Mandl, MD, MPH (B)
Elizabeth W. Karlson, MD (A)

(A) Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
(B) Hospital for Special Surgery, Weill Cornell Medical College, New York, NY

References

1. Linn-Rasker SP, van der Helm-van Mil AH, Van Gaalen FA, Kloppenburg M, de Vries R, le Cessie S, Breedveld FC, Toes RE, Huizinga TW. Smoking is a risk factor for anti-CCP antibodies only in RA patients that carry HLA-DRB1 Shared Epitope alleles. Ann Rheum Dis 2005.

2. Botto LD, Khoury MJ. Commentary: facing the challenge of gene- environment interaction: the two-by-four table and beyond. Am J Epidemiol 2001; 153:1016-20.

3. Gatto NM, Campbell UB, Rundle AG, Ahsan H. Further development of the case-only design for assessing gene-environment interaction: evaluation of and adjustment for bias. Int J Epidemiol 2004; 33:1014-24. 4.

Andersson T, Alfredsson L, Kallberg H, Zdravkovic S, Ahlbom A. Calculating measures of biological interaction. Eur J Epidemiol 2005; 20:575-9.

5. Ahlbom A, Alfredsson L. Interaction: A word with two meanings creates confusion. Eur J Epidemiol 2005; 20:563-4.

6. Padyukov L, Silva C, Stolt P, Alfredsson L, Klareskog L. A gene- environment interaction between smoking and shared epitope genes in HLA-DR provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum 2004; 50:3085-92.

7. Rothman KJ, Greenland S, Walker AM. Concepts of interaction. Am J Epidemiol 1980; 112:467-70.