LETTERS 299

Restricted clonality of T cells in the joints of rheumatoid arthritis patients

To the Editor: The possibility that specific T cells directed against

autoantigens in the synovial joint are involved in the patho- genesis of rheumatoid arthritis (RA) is of considerable inter- est. One experimental approach to this question has been to determine whether restricted clonality is a characteristic feature of T cells in the synovia of RA patients. Molecular techniques (e.g., Southern blot analysis) have been used in an attempt to identify dominant rearrangements in the vari- able (V) region genes of T cell receptors (TCR).

A recent report by Keystone et al showed that the V, genes in T cells from 15 RA synovial fluid samples did not exhibit a dominant rearrangement pattern, which indicates a lack of restricted clonality (1). Using similar techniques, we had previously noted that 3 of 11 synovial fluid samples from RA patients showed dominant rearrangements of V, genes when compared with T cells from paired blood samples (2). Studies using monoclonal antibodies have demonstrated a larger percentage of T cells expressing V$ and V$ genes in synovial membrane T cells compared with peripheral blood T cells from the same patients. This result also suggests that some preferential expansion or recruitment of T cells ex- pressing particular V, chains occurs (3). Although Ramos et al were unable to detect dominant V, gene rearrangements in synovial fluid T cells from 10 RA patients, they did find dominant rearrangement of the TCRy genes in 3 of their patients (4).

An alternative and complementary experimental ap- proach is to expand those T cells that are already activated in vivo by culturing them with exogenous interleukin-2 (IL-2) in the absence of specific antigens. Using this proce- dure, we observed that 25% of the synovial fluid-derived T cell lines examined showed dominant TCRP chain gene rearrangements (2). Similar results were obtained in 13 of 14 IL-Z-expanded T cell infiltrates from dissociated synovia analyzed by Stamenkovic et a1 ( 5 ) .

Thus, there is evidence that T cells exhibit dominant gene rearrangement patterns of both TCR 2 (dp ) and TCR 1 (y/S) receptors in the peripheral joints of RA patients. Taken together, these data on TCR gene rearrangements indicate that in a minority of RA patients, there is proliferation of particular T cell clones within the synovial fluid or mem- brane. However, the low incidence of detectable clonality in these studies might indicate transient activation of specific clones directed against antigens present within the joint during disease progression. Longitudinal studies of synovial fluid T cells from RA patients would establish whether restricted T cell clonality is a feature in certain patients, or whether it reflects a transient in vivo proliferative response (presumably antigen-specific) that is occumng at the time of sampling.

The specificity of the dominant clones observed is unknown. However, we have obtained T cell lines from the synovial fluid of other RA patients, some of which react with the Fc region of IgG, type I1 collagen, or the mycobacterial 65-kd heat shock protein (Lydyard PM, Walker P, Tsoulfa G: unpublished observations). Londei et al (6) have also isolated type I1 collagen-specific T cells from a single RA patient on 3 separate occasions over a 3-year period. How-

ever, the recent observation that $&bearing T cells react with mycobacterial stress proteins (7) makes the presence of these cell types in the rheumatoid joint (refs. 4, 8, and Lydyard PM et al: unpublished observations) potentially important since it has been proposed that stress proteins may be involved in the pathogenesis of RA (9,lO).

Peter M. Lydyard, PhD Peter J. Delves, PhD Bernard Colaco, MB, MRCP(UK) University College and

Middlesex School of Medicine London, UK

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Keystone EC, Minden M, KIock R, Poplonski L , Zalcberg J , Takadera T, Mak TW: Structure of T cell antigen receptor p chain in synovial fluid cells from patients with rheumatoid arthritis. Arthritis Rheum 31: 1555-1557, 1988 Savill CM, Delves PJ, Kioussis D, Walker P, Lydyard PM, Colaco B, Shipley M, Roitt IM: A minority of patients with rheumatoid arthritis show a dominant rearrangement of T cell receptor p chain genes in synovial lymphocytes. Scand J Immunol 25:629435, 1987 Brennan FM, Allard S, Londei M, Savill C, Boylston A, Carrel S, Maini RN, Feldmann M: Heterogeneity of T cell receptor idiotypes in rheumatoid arthritis. Clin Exp Immunol73:417423, 1988 Ramos F, Bignon YJ, Sauvezie B: Molecular study of clonal dominance in synovial fluid lymphocytes (abstract). Eur J Clin Invest 19:A53, 1989 Stamenkovic I, Stegagno M, Wright KA, Krane SM, Amento EP, Colvin RB, Duquesnoy RJ, Kurnick JT: Clonal dominance among T lymphocyte infiltrates in arthritis. Proc Natl Acad Sci

Londei M, Savill CM, Verhoef A, Brennan F, Leech ZA, Duance V, Maini RN, Feldmann M: Persistence of collagen type 11-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis. Proc Natl Acad Sci USA 86:636-640, 1989 Holoshitz J, Koning F, Coligan JE, de Braun J, Strober S: Isolation of C D C CD8- mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339: 226229, 1989 Brennan FM, Londei M, Jackson AM, Hercend T, Brenner MB. Maini RN, Feldmann M: T cells expressing -yS receptors in rheumatoid arthritis. J Autoimmun 1:319-326, 1988 Tsoulfa G, Rook GAW, Bahr GM, Sattai MA, Behbehani K, Young DB, Mehlert A, van Embden JDA, Hay FC, Isenberg DA, Lydyard PM: Elevated IgG antibody levels to the myco- bacterial 65kDa heat shock protein are a characteristic of patients with rheumatoid arthritis. Scand J Immunol 30: 519-527, 1989 Res PCM, Schaar CG, Breedveld FC, van Eden W, van Embden JDA, Cohen IR, de Vries RRP: Synovial fluid T cell reactivity against 65kD heat shock protein of mycobacteria in early chronic arthritis. Lancet II:478480, 1988

USA 85:1179-1183, 1988

The prevalence of Lyme disease in an endemic region

To the Editor: Lyme disease is now the most common tick-borne

illness in the United States. Since its initial recognition in 1975 (l), both the incidence of the disease and the number of states reporting cases have increased dramatically (2). In the northern part of Westchester County, New York, where the number of cases has reached almost epidemic proportions,

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previous studies show that ticks infected with Borrelia burgdorferi are ubiquitous and can be found on well- manicured lawns as well as in the woods (3). Thus, casual activities around the house can result in tick exposure and subsequent infection, putting a large population at risk.

Previous reports suggest that there may be a signifi- cant number of asymptomatic cases of Lyme disease. One survey of an island population found almost equal numbers of symptomatic and asymptomatic infection in individuals who were seropositive (4); another survey revealed that 32% of asymptomatic farmers in an endemic area had positive serologic findings (5). Because a large percentage of patients with erythema chronicum migrans (ECM) who are not treated with antibiotics subsequently develop other symp- toms of Lyme disease (6), it is important to further assess the degree of asymptomatic infection in endemic regions.

We screened a large number of individuals in north- ern Westchester County for serologic evidence of Lyme disease. Between April 25, 1988 and June 30, 1988, all patients having blood studies performed at a centrally lo- cated medical facility in the endemic area were offered free testing for Lyme disease, if excess serum was available. We asked consenting patients if they had any previous history of Lyme disease. Those who responded affirmatively, as well as those who tested positively in our study, were evaluated further by a review of their charts or by telephone interview. Assays for Lyme disease were performed in our laboratory by an IgG-specific enzyme-linked immunosorbent assay (ELISA) technique as previously described (7), using refer- ence sera generously supplied by Dr. Allen Steere.

Of the 452 specimens assayed, all but 24 were from patients residing in the endemic zone. Forty-two percent of the samples were from men, and 58% were from women. The age distribution of the patients followed a bell-shaped curve, ranging from 7 to 94 years; 80% were between the ages of 20 and 70. All sera were initially screened at a dilution of 1:lOO by the ELISA technique, and titers were determined for the positive samples.

Six patients (1.3%) had positive serologic results in our assay, with titers ranging from 1:200 to 1:3,200. Two of these patients (both with titers of 1:1,600) had a definite history of Lyme disease, 1 with severe monarticular arthritis 1 year previously, and the other with multiple lesions typical of ECM 3 years previously. Both had had positive results on previous serologic tests and had been treated with antibiot- ics. Of the remaining 4 patients with positive assay results, 1 (titer 1 :200) had a history of a tick bite and subsequent Bell’s palsy (in spite of therapy with cephalexin) in 1981, although he had not been diagnosed as having Lyme disease. The remaining 3 patients (0.7%) had no history suggesting infec- tion. Two of these patients (titers 1:800 and 1:1,600) were asymptomatic, while the other (titer 1:3,200) had a history of active seropositive rheumatoid arthritis.

Nineteen other patients reporting previous Lyme disease had negative serologic findings. Ten of these 19 patients had histories strongly indicating the disease (1 month to 5 years before the current study), but in all cases it had been limited to an early stage and had been treated early with antibiotics. Since patients with ECM alone may have only a temporary antibody response, which may be com- pletely aborted by antibiotics, these results are not unex- pected and are similar to previous reports (4,8,9). The

diagnosis in the remaining 9 patients was either questionable or doubtful.

Our results show that approximately 3.5% of a sam- ple population (16 of 452) in an area endemic for Lyme disease had either serologic evidence of, or a history consis- tent with, the illness. Most of these patients (13 of 16) developed symptoms that led to antibiotic treatment after consultation with a physician, and they were not expected to develop further disease manifestations (9). However, only 3 of 6 patients with positive serologic results in the current study had any history suggestive of Lyme disease, which is similar to the findings previously reported by Steere et al(4). Although the percentage represented by these patients in our study is not high (3 of 452; 0.7%), extrapolation to the population at large in northern Westchester County yields a significant number of undiagnosed cases. Except for the possibility of yielding positive results for other spirochetal infections, the ELISA is highly specific for Lyme disease (lo), and except for the patient with a history of infection 7 years previously, all the titers in this study were strongly positive. Thus, these patients presumably had been infected with B burgdorferi in the past, but failed to develop, ob- serve, or remember Lyme disease manifestations.

Studies of patients with chronic forms of Lyme disease have shown a gradual expansion of the immune response against new antigens, which is consistent with persistent infection (8). Furthermore, patients with un- treated ECM and positive serologic findings have a high risk of developing further symptoms of Lyme disease, some of which can be very disabling (6). If this applies to asympto- matic individuals as well, it may be important to identify and treat them before the disease can progress. With this in mind, prospective studies should be applied to this group of subjects who are asymptomatic but have serologic evidence of Lyme disease.

Malcolm Brown, MD Forsyth Memorial Hospital Winston-Salem, NC Patricia Redecha, BS Charles Christian, MD The Hospital for Special Surgery New York, N Y

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Steere AC, Malawista SE, Snydman DR, Shope RE, Andiman WA, Ross M R , Steele FM: Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecti- cut communities. Arthritis Rheum 20:7-17, 1977 Update: Lyme disease and cases occurring during pregnancy: United States. MMWR 34:376, 1985 Falco RC, Fish D: Prevalence of Ixodes dammini near the homes of Lyme disease patients in Westchester County, New York. Am J Epidemiol 127:826-830, 1988 Steere AC, Taylor E, Wilson ML: Longitudinal assessment of the clinical and epidemiological features of Lyme disease in a defined population. J Infect Dis 154:295-300, 1986 Dlesk A, Bjarnason DF, Goldberg JW, Lee M, Marx J , Mitchell P, Washington WL: Lyme disease seropositivity by ELISA among farmers in an endemic area (abstract). Arthritis Rheum 30 (suppl) 4:S49, 1987 Steere AC, Schoen RT, Taylor E: The clinical evolution of Lyme arthritis. Ann Intern Med 107:725-731, 1987

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Wilkinson HW: lmmunodiagnostic tests for Lyme disease. Yale J Biol Med 57:567-572, 1984 Craft JE, Fischer DK, Shimamoto GT, Steere AC: Antigens of Borrelia burgdorferi recognized during Lyme disease: appear- ance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J Clin Invest 78:934-939, 1986 Shrestha BS, Grodzicki RL, Steere AC: Diagnosing early Lyme disease. Am J Med 78:235-240, 1985 Craft JE , Grodzicki RL, Steere AC: Antibody response in Lyme disease: evaluation of diagnostic tests. J Infect Dis 149:789-795, 19x4

Comment on the article by Duston et al

To the Editor: We read with interest the article by Duston et al

regarding abdominal fat aspiration for the diagnosis of amy- loidosis ( I ) . They studied samples from abdominal fat aspi- rates that were sent to them for diagnosis. Biopsy specimens taken from 2 sites were available in 17 patients, making it feasible to calculate the sensitivity and specificity of this method.

In the past 5 years, we have also biopsied abdominal subcutaneous fat to determine the presence of amyloidosis, by using the methods described by Westermark (2) and Libbey et al (3). In our first 12 patients, we could only obtain a few fat cells, which were not useful for diagnosis. For this reason, we decided to try using a Tru-Cut biopsy needle (Travenol, Maurepas, France) to obtain the subcutaneous abdominal fat. Using this technique. we prospectively stud- ied 23 patients, of whom 6 had proven amyloidosis, and 17 showed a suggestive clinical picture (4). We also performed a rectal biopsy on all 23 patients and used 10 autopsy cases as controls. Positive biopsy results werc obtained in 21 of the 23 patients. The abdominal fat biopsy results were positive in 19 patients (90.4%). and the rectal biopsy results were positive in 17 patients (80.9%). The sensitivity of the fat biopsy was 82.6% and the specificity was loo%, with a positive predictive value of 100% and a negative prcdictivc value of 85.7%.

These represent more encouraging results than those of Duston et al. By using a Tru-Cut biopsy needle, a larger tissue sample can be obtained, improving the possibility of detecting amyloid substance. This method is as safe and easy to perform as thc fat aspiration method.

C. Raul Ariza, MD Alberto Frati, M D Leonor Barile. MD Romeo Garcia. MD Hospital de Especialidades Mexico City, Mexico

Duston MA, Skinner M, Meenan RF. Cohen AS: Sensitivity, specificity, and predictive value of abdominal fat aspiration for the diagnosis of amyloidosis. Arthritis Rheum 32:82-85. 1989 Westermark P: Occurrence of amyloid deposits in the skin in secondary systemic amyloidosis. Acta Pathol Microbiol lmmunol Scand [A] 80:718-720, 1972 Libbey CA, Skinner M, Cohen AS: Use of abdominal fat tissue aspirate in the diagnosis of systemic amyloidosis. Arch Intern Med 143:1549-1552, 1982

4. Ariza CR, Frati AC, Heras-Martini R, Garcia-Torres R, Pulido MA: La biopsia con aguja de Tru-Cut de la grasa subcutanea abdominal para el diagnostico de amiloidosis. Rev Invest Clin 401347-351, 1988

Comment on the article by Duston et a1

To the Editor: We read with interest the article by Duston e t al

concerning abdominal fat aspiration for the diagnosis of amyloidosis ( I ) . We agree with their conclusions regarding abdominal subcutaneous tissue as a site for easy sampling to assess the diagnosis of secondary amyloidosis. However, we would like to comment on the procedure used.

In fat aspiration, fat droplets and subcutaneous fat tissue are aspirated through a needle mounted o n a dispos- able syringe while a vacuum is drawn. Droplets are blown onto glass slides and then allowed to air dry before being stained with Congo red. In our preliminary experience with this technique, false-negative results were frequently ob- served.

In order to improve the sensitivity of this technique, we developed a biopsy procedure (2). After cleaning the skin with a phenylmercury borate solution and giving a local anesthestic with 1% lidocaine, a small incision of the skin (2 mm) is performed with a bistoury. Through this incision, a fat tissue specimen is obtained by using a disposable, 14- gauge, 15.2-cm biopsy needle (Tru-Cut; Travenol, Maure- pas, France). This procedure is repeated 3 times and yields tissue samples 2-15 mm in length. The size of these frag- ments allows one to perform histopathologic studies using standard staining procedures (hematoxylin and eosin, sa- fran, and Congo red) as well as KMnO, treatment (3) and immunohistochemical typing of amyloid.

We have investigated the diagnostic value of subcu- taneous tissue biopsy in 10 patients with chronic inflamma- tory arthritis (5 with rheumatoid arthritis [KA], 1 with juvenile RA, 1 with adult Still’s disease, 2 with ankylosing spondylitis, and 1 with psoriatic arthritis) and amyloidosis revealed in other tissues. Six patients had amyloid deposits in fat tissue specimens. Congo red staining disappeared after KMnO, treatment in the 4 tested samples, suggesting that deposits contained AA (secondary) arnyloid. In contrast, this procedure could not demonstrate amyloid deposits in subcutaneous fat tissue of 4 other patients with gamrnopa- thies and proven A L (primary) amyloidosis. These negative findings are inconsistent with previous reports on the value of abdominal fat aspirates in the diagnosis of A L amyloidosis (4). Comparison between the biopsy and the plain aspiration techniques requires further investigation.

FrCdCric LiotC, MD Charlotte Cywiner-Golenzer, MD Thomas Bardin, MD Antoine Dryll, MD Daniel Kuntz, MD H6pital Lariboisiere Paris, France

1. Duston MA, Skinner M, Meenan KF, Cohen AS: Sensitivity, specificity, and predictive value of abdominal fat aspiration for the diagnosis of amyloidosis. Arthritis Rheum 3232-85, 1989