Bioassays for TSH-receptor autoantibodies: An update

Autoimmunity Reviews 10 (2010) 116–122

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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev

Review

Bioassays for TSH-receptor autoantibodies: An update☆ Simon D. Lytton ⁎, George J. Kahaly Thyroid Research Laboratory, Department of Medicine I, Gutenberg University Medical Center, Mainz 55101, Germany

a r t i c l e

i n f o

Article history: Accepted 16 August 2010 Available online 31 August 2010

a b s t r a c t Immunoglobulins in patients with Graves' disease (GD) that modulate the thyroid stimulating hormone receptor (TSH-R) do so via stimulating cAMP dependent signals (TSI), blocking TSH or inhibition of TSHreceptor activation (TBI) or inducing apoptotic signals. These functional immunoglobulins represent powerful biomarkers of anti-self reactivity in the thyroid and systemic tissues that harbor TSH-R expressing target cells. TSI on thyrocytes induce hyperthyroidism, and TSI on TSH-R fibroblasts of orbital muscles, skin and heart provoke the release of cytokines and antigen-specific T-cell responses leading to systemic inflammation. Bioassays of anti-TSH-R autoantibodies provide decisive information on GD activity. This review examines the past and present bioassays in GD. The critical goal of cell-based anti-TSH-R autoantibody bioassays, to identify the pathogenic immunoglobulins in GD under robust and standardized conditions suitable for routine clinical laboratory practice, is discussed. © 2010 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rationale for TSH-R bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TSH-R autoantibodies and immune pathology of Graves' disease. . . . . . . . . . . . . . . . . 3.1. TSI dominate the immune pathology . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. TSH-R autoantigen depot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. History of TSH-R bioassays: patrolling the immunoglobulin flotilla for functional autoantibodies in 4.1. The animal model bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Cell-based bioassays — laborious beginnings . . . . . . . . . . . . . . . . . . . . . . . 4.3. The cAMP endpoint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. TSH-R luciferase reporter bioassay — the age of light technology . . . . . . . . . . . . . 5. Clinical evaluations of TSH-R bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Future of the TSI bioassay: from bioassay to bedside . . . . . . . . . . . . . . . . . . . . . . Take-home . messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction TSH-receptor (TSH-R) function, the center stage of thyroid health and illness, is the outcome of G-protein coupled cAMP dependent signaling influenced by genetic, environmental, hormonal and immunological inputs [1–6]. In Graves' disease (GD), the most Abbreviations: TSH-R, thyroid stimulating hormone receptor; rLH-R, rat luteinizing hormone receptor; TSI, thyroid stimulating immunoglobulins; GD, Graves' disease; HT, Hashimoto's thyroiditis. ☆ Disclosure: GJK consults for Diagnostic Hybrids, Inc., Athens, OH, USA. SDL has nothing to disclose. ⁎ Corresponding author. Tel.: + 49 6131 17 6950; fax: + 49 6131 17 3460. E-mail address: [email protected] (S.D. Lytton). 1568-9972/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2010.08.018

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116 117 117 117 117 118 118 118 118 119 120 121 121 121

prevalent of autoimmune conditions with prevalence of 1%, the TSI on TSH-R expressing target cells of the thyroid and extra-thyroid tissues provoke the immune pathology that is mediated by autoreactive T-cells and cytokines [7–11]. Previous reviews have focused on TSH-R autoantibodies and TSH-R epitope structures [1,4,5,12], the history of commercial assays of TSH-R autoantibody binding (TRAb) [13], the role of B-cell stimulatory factors [2] and TSH-R autoantibodies [1–4,11,12] in autoimmune thyroid disease. To date no systematic surveys are available on the bioassays of functional TSH-R autoantibodies. The current bioassays of TSH-R autoantibodies assess the cAMP-inducible luciferase activity triggered by TSI action on fully functional receptor of live cells [14–23]. The bioassays have also been adapted to detect the antagonistic immunoglobulins with

S.D. Lytton, G.J. Kahaly / Autoimmunity Reviews 10 (2010) 116–122

blocking/inhibitory activity and to distinguish between the TBI and the non-signaling neutral binding immunoglobulins [15,16,19,21,24– 27]. The purpose of the review is to evaluate the rationale for TSH-R bioassays in GD and to highlight the advantages and disadvantages of past and present bioassays. 2. Rationale for TSH-R bioassays TSH-R bioassays are functional cell-based tests that directly assess the bio-active immunoglobulins having either stimulating or inhibitory input on the TSH-R cAMP dependent signaling (Fig. 1). TSI evoke metabolic changes and/or cytokine responses within TSH-R expressing target cells [7–10]. The routine application of TSH-R bioassays in clinical laboratory practice has potential to better predict relapsed patients or GD remission. High persistent TSI levels are associated with active and severe systemic manifestations with poor responses to therapy [22,28]. In contrast low TSI levels associate with patients in remission [19,28]. Thyroid blocking immunoglobulins (TBI) have been found to coexist with TSI in the same patient serum sample [27,29]. The monitoring of TBI may help to optimize the dosage and timing of anti-thyroid drugs during pregnancy and in mothers postpartum who experience alternating episodes of hyper- and hypothyroidism [1,5]. Approximately one third of patients with GD have blocking antibodies [27]. The switch from TSI to TBI in euthyroid GD patients may lead to the development of Hashimoto's thyroiditis [5,25,27,29]. Finally, the determination of TSI levels and TSI titers in GD patients undergoing treatments with anti-thyroid medications and immune suppressive drugs is expected to provide insight into disease activity and severity and substantially improve the management of TSH-R autoreactivity in individual patients [22,27–30]. 3. TSH-R autoantibodies and immune pathology of Graves' disease 3.1. TSI dominate the immune pathology The etiology of autoimmune thyroid disease is a story of genes and environment [3,6,11,30]. Personalized risk factors such as smoking, contribute to the immune pathology in GD but the anti-TSH-R

A

117

autoantibodies and their direct action on TSH-R target cells are the main provoker of TSH-R autoreactivity [1,4,7,8,11]. Anti-TSH-R autoantibodies act in a similar way to anti-receptor autoantibodies found in other autoimmune conditions [31,32]. Rather than destroying the target cells per se the functional fraction of immunoglobulins in GD hijack the endocrine metabolism by mimicry of the receptor's natural ligand; either stimulating (TSI) cAMP dependent signal transduction or antagonizing TSH-R by either blocking TSH binding or interacting with TSH-R epitopes that inhibit cAMP production (TBI). TSI on thyrocytes induce increased output of the thyroid hormones fT3 and fT4 with hyperthyroidism [20,23]. TSI on TSH-R positive target cells of the orbital tissues [7,8,33], heart [1,4] and skin [10] are associated with orbitopathy, cardiomyopathy and dermatopathy, respectively. The TSI-induced pathogenesis of Graves' orbitopathy (GO) is mediated by TSI/TSH-R and Il-6/IL-6R signaling in orbital fibroblasts [7–9,33,34]. Autoantigens other than TSH-R have been identified [7] but the autoantibodies and T-cell proliferative responses associated with nonTSH-R autoantigens are a side-product of inflammation and likely have a minor impact only as prognostic indicators of GD [2,7,11].

3.2. TSH-R autoantigen depot The autoantigenic depot in GD immune pathology is comprised of the TSH-R in the thyroid [1,4] and extra-thyroid tissues [7,8,10,33,34], the TSH-R/insulin growth factor-1 receptor (IGF-1R) complex expressed in fibroblasts and thyrocytes [7,8,34], and the IGF-1R upregulated in activated lymphocytes of GD patients [35]. TSH-R positive orbital tissue fibroblasts in GO patients express IGF-1R at 4-fold higher levels than normal orbital fibroblasts [34]. TSH-R and IGF-1R form a tight and stable complex in membranes of thyrocytes and fibroblasts as evident by co-immunoprecipitation experiments with IGF-1R specific antibodies [34]. Furthermore, the two autoantigens colocalize in immunofluorescence staining of orbital fibroblasts. Serum of GD patients that contain anti-IGF-1R immunoglobulins ligate to IGF-1R on fibroblasts [34], B-cells and T-cells [35] causing profound alteration in cytokines and chemotactic factors in vitro. Another important depot of TSH-R autoantigen in GD pathogenesis is the soluble alpha subunit that is shed into the blood circulation

B

wt hTSH-R Mc4hTSH-R wt hTSH-R Mc4hTSH-R wt hTSH-R Mc4hTSH-R wt hTSH-R Mc4hTSH-R wt hTSH-R Mc4hTSH-R wt hTSH-R Mc4hTSH-R

MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLI 60 ETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPD 120 ALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETL 180 TLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTA 240

LPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLM TLPSKEKFTSLLVATLTYPSHCCAFSNLPKKE------CNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQE ------- --QNFSFSIFEN--------------FSKQCESTVRKADNETLYSAIFEE-*************** wt hTSH-R DEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYKFLRIV Mc4hTSH-R NELSGW-D ***

300 293 360 327 420

Fig. 1. Model of TSH-R bioassay with modified human TSH-R sequences. A. The human TSH-R alpha subunit 10-repeat leucine rich domain (LRD) a concave surface containing the TSI-M22-specific contact residues, E34− E35− D36− T56− Y82− E107− K129− N208− Q235− N256− R255 of resolved crystal structure [12] (3G04 Protein Data Bank), is depicted with a schematic diagram of the beta subunit as a fully functional holoreceptor. The incoming positive TSI activate the TSH-R cAMP dependent signaling in a highly sensitive luciferase reporter bioassay. CRE = cyclic AMP response elements. TBI inhibit the TSH-R cAMP either by blocking TSH or TSI binding sites within the LRD or via inhibitory epitopes [27]. B. Amino acid sequence alignment of wt human and chimeric Mc4TSH-R shows identical (white) and disparate (shaded) amino acids. In the Mc4 TSH-R underlined amino acids 262– 368 of wt hTSH-R, containing the cleavage sites [1,30] and TBI epitopes [27], are replaced with 73 amino acids 262–334 of the rLH-R highlighted in bold [27,63]. The amino acids of wt and Mc4 TSH-R are identical within LRD (boxed region) and the amino acids 421–764 (not shown) corresponding to the 7 transmembrane helices and cytosolic C-terminus.

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during the course of GD [1,4,30]. Immunoglobulins that recognize specific epitopes in the cleavage sites of native TSH holoreceptor have been identified in the serum of treated GD patients or relapsed patients and may inhibit the TSH-induced protease cleavage [1,4]. The reduction of the exposed di-sulfide bonded cysteine residues by reducing agents leads to the shedding of the alpha ectodomain and an abundance of cleaved TSH-R β subunit remains in the plasma membrane of thyrocytes [1,30]. TSI monoclonal antibodies derived from patients with GD bind to epitopes of the leucine repeat domain (LRD) presented on soluble TSH-R alpha subunit ectodomain and the functionally intact TSH holoreceptor of CHO cells (Fig. 1). The intramolecular cleavage and shedding of the TSH-R, a natural physiological process presenting a depot of soluble TSH-R ectodomain to the immune system, has been described as the major mechanism that drives affinity maturation of TSI causing GD [36]. 4. History of TSH-R bioassays: patrolling the immunoglobulin flotilla for functional autoantibodies in GD 4.1. The animal model bioassays The bioassays of functional anti-TSH-R autoantibodies in GD have an over 50 years of history. In 1956 Adams and Purves discovered an abnormal long acting thyroid stimulator in the serum of patients suffering thyrotoxicosis named LATS. The nature of LATS, a slow inducer of thyrotoxicosis, 9–24 h after intravenous injection of serum, compared with 1.5–3 h after injection of animals with TSH [37–39], was later identified as an autoantibody against thyroid follicular cell thyrotropin receptors [40–44]. Since the discovery of LATS [37,38] the condition of GD hyperthyroidism first given clinical description by Graves in 1835, has been classified as an autoimmune disease [1,11]. The bioassays of LATS (Table 1), in vivo experiments first performed in guinea pigs [37] and later in mice [38] showed strong correlations between the LATS levels in sera and the increased thyroid activity and thyroid mass of patients with GD, myxedema, hyperthyroid pregnant women, infants suffering neonatal thyrotoxicosis and severe exophthalmic ophthalmoplegia. Control sera of euthyroid normal donors and patients with thyroid adenomas tested negative for LATS. LATS activity was assessed in animals fed an iodide deplete diet and injected with thyroxine and 131I to load-up their thyroid glands for high specific radioactivity under suppressed endogenous TSH. The amount of 131I thyroid hormones released from the thyroid gland into blood circulation was measured with a scintillation counter at regular time intervals between 2 and 24 h following a single intravenous injection of up to 0.5 mL of the test serum [38]. The early chemical characterization of LATS by starch block electrophoresis or ion exchange chromatography on DEAE was unsuccessful with considerable loss of activity in the eluted fractions [37]. Precipitation

of serum with either cold ethanol or ammonium sulfate gave concentrated immunoglobulin preparations with 10 fold higher LATS activity per mg protein compared with the original serum. Proteolytic digests of the LATS-IgG preparations, diminished the characteristic 9 hour 131I peak in the mouse bioassay. The identification and purification of LATS-IgG enabled quantification of the LATS levels and the determination of LATS titer in patient serum samples using known LATS-IgG reference standards [38]. In precision testing of 24 mice with two doses of thyrotopic standard and six observation time points, the error of the mouse bioassay was approximately 23% [38]. 4.2. Cell-based bioassays — laborious beginnings The interaction of TSH with thyroid issue in vitro, first assessed by estimates of 131I release from mouse thyroid slices maintained in tissue culture and incubated with serum [39,40] was modified [41] and later improved by direct Na 125I labeling of porcine thyroid cells [42]. Porcine thyroid cells exhibited excellent dose–response uptake of 125I during an 8 hour treatment with TSH in the range of 0.5–5 ng/ mL. However, the TSH and LATS dose–response effects could not be distinguished on the basis of 131I release nor 125I uptake [42]. The hypothesis that LATS and TSH ligand recognize and exert their biological effects via activation of cAMP signaling of a common thyrotropic receptor in thyroid tissue was supported by the following experimental evidence. Human 125I TSH of high purity, prepared from fresh isolated human pituitary, were used to assess the specific binding of TSH to human thyroid plasma membranes. The TSH binding was displaced by LATS IgG of GD patients but not by IgG of control serum [43,44]. Additionally, the actions of TSH and dibutyrylcAMP on isolated bovine thyroid cells showed identical biphasic kinetics of 131I incorporation into thyroglobulin [45]. These insightful early observations did not immediately lead to improved TSI bioassays. Two technical advances were necessary before effective use of the cAMP endpoint in the in vitro TSI bioassay became feasible. First, preliminary studies on the in vitro action of the intracellular second messenger cyclic 3′5′ adenosine monophosphate (cAMP) in stimulated human thyroid tissues and the measurement of its accumulation in thyrocytes [46–50], second, molecular cloning of the human TSH-R [51]. 4.3. The cAMP endpoint The first TSI bioassay with cAMP endpoint assessed the formation of colloidal droplets and measured the accumulation of 125I-labeled cAMP in the follicles of incubated human thyroid slices within several hours after the direct additions of either sera of untreated hyperthyroid GD or purified LATS IgG to slices of thyroid tissue [46]. The

Table 1 Bioassays of functional TSH-R autoantibodies — animal models and primary cells. TSH-R source Animal models Guinea pig Mouse

TSH-R primary cells Mouse thyroid

Endpoint

Analyte/Assay time

Pros

Cons

Chronology (years)

Reference

131

LATS/weeks LATS/days

Serum factor causes thyrotoxicosis, LATS, 9–16 hour 131I peak versus 2 h TSH-induced 131I peak

High standard error Fractionated serum inconsistent results, LATS levels sensitive to mouse adrenalin and stress

1956 1958

[37] [38]

LATS IgG/weeks

first in vitro bioassay LATS is IgG

Thyrotropic target unknown

1967

[37,39,40]

TSI serum/days TSH, LATS IgG/4 days

Human origin Thyroid follicle formation with polarity and cAMP uptake Robust cells, reproducible assay

Variable TSI from surgical specimens Tissue and primary cell preparation

1973–1988 1973–1975

[44,46–50] [42,46]

Extensive cell culture

1983–1994

[52,53,56]

131

I T3,T4 I T3,T4

131

Human thyroid Porcine thyroid

I T3,T4 chromatography RIA cAMP RIA cAMP, 125I uptake

FRTL-5 rat thyrocytes

RIA cAMP,

125

I uptake

TSI and TBI/2–3 days

S.D. Lytton, G.J. Kahaly / Autoimmunity Reviews 10 (2010) 116–122

measurements of accumulated intracellular cAMP and the cAMP-LATS dose responses in thyroid slices of a variety of different species identified the human thyroid tissue as the most physiologically relevant source material to screen patient sera for LATS-IgG activity (Table 1). Due to the inconvenience of procuring human tissues and the variable TSI responses obtained with different batches of surgical specimens there was an urgent need to develop animal cell culture bioassays to circumvent these troubles. The rat thyrocyte cell line FRTL-5, showed excellent recovery from cryopreservation and gave consistent cAMP levels upon stimulation with GD serum [52,53]. For this reason the FRTL-5 initially emerged as the lead and favorite bioassay of choice. FRTL-5 cell culture requires approximately 2 days in complete medium to reach subconfluence followed by 5 days in medium lacking bovine TSH. Stimulation requires 5 h using precipitated patient serum and the radioactive cAMP endpoint involves capturing 3[H]-cAMP on Dowex columns and scintillation counting [52]. Advances in molecular cloning and antibiotic selection of genetically modified cell lines quickly led to the successful transfection of CHO cell line with a fully functional and stable wild type human TSH-R [54,55] or chimeric TSH-R constructs [24–26] (Table 2) and the replacement of the human thyrocyte and FRTL-5 TSI bioassays [56] (Table 1). The most important advantage of using animal cells transfected with human TSH-R is the constitutive activation of cAMP by the G-coupled receptor of physiologically relevant species and the significant increased accumulation of cAMP in response to specific ligands TSH or TSI [54–58]. The TSI bioassay with JP09 CHO cell line shortened the time course of cell culture to two days, and achieved efficient stimulation within 30 min after additions of unmodified patient serum diluted into hypotonic sodium chloride free buffer [54– 56]. The interaction of serum with human TSH-R cell cultures normally requires polyethylene glycol or ammonium sulfate to precipitate and concentrate the bulk immunoglobulins and to decorate the TSH-Rs expressed on the cell surface with specific antiTSH-R IgG [59]. The reaction buffers used to dilute TSI containing human serum samples avoid inclusion of monovalent cations and physiological salt solution which were found to interfere with TSI activity [48,52]. Radioactive measurements with commercial cAMP RIA kits were introduced in the mid 1990s to standardize the quantification of intracellular cAMP. However, the endpoints of radioactive cAMP were perceived as inconvenient and the attempt to reintroduce the FRTL-5 thyrocytes with improved metaphase index endpoint on microtiter plate [53] was deemed not suitable for routine use in a clinical laboratory. 4.4. TSH-R luciferase reporter bioassay — the age of light technology Three major breakthroughs led to the use of TSH-R bioassays of simplified and efficient cell culture protocols with chemiluminescent

119

cAMP endpoint: first, the identification of cyclic AMP-inducible response elements, CRE, and characterization of their potent effect on CAT promoter activity [14]; second, the molecular cloning of the human G-coupled glycoprotein hormone receptor that constitutively activates adenylate cyclase [58]; and third, the transfection of CHO cells with luciferase reporter gene [60]. The first TSH-R luciferase reporter bioassay, the C6–13 CHO cell line [15] of stable and constitutive TSH-R expression with the firefly luciferase gene controlled by cAMP-inducible promoter [14], was the prototype for subsequent development of the JP26 CHO cell line [16]. The later proved to be a highly efficient bioassay (Table 2). TSH-R density of 2000 human (h) TSH-R in JP26 CHO cells seeded at 20,000 cells per reaction exhibited a 2–3 fold greater stimulation index (maximum bTSH-stimulated cAMP production/cAMP production stimulated by pooled normal serum without bTSH) than the JP09 cell line seeded at 40,000 cells per well and 90,000 hTSH-R per cell. The learned lesson: use of CHO cells of lower hTSH-R density, within the range of 1000–10,000 TSH-R found on human thyroid epithelial cells, likely reduces the negative cooperativity of G-coupled receptors known to occur with receptors expressed at high density on CHO cells [16]. The increased sensitivity and the simple operation of the luciferase reporter bioassay compared with cAMP RIA (Table 2) quickly ignited interest in using the TSH-R luciferase reporter bioassays to detect the thyroid blocking immunoglobulins (TBI or TSBAb) [18–20]. The feasibility of inhibiting cAMP luciferase production with TBI was demonstrated using CHO K1 lu/lu cell line [21]. Further testing of the TBI assay with serum of 12 patients from Singapore who initially presented with GD and later became hypothyroid with high TBI and with a panel of 6 inhibitory monoclonal antibodies [18] revealed potent antagonistic effects; up to 70% inhibition, upon mixing the test sample with the stimulator either TSH or serum of GD patients containing TSI of high stimulation index. A recent report of monoclonal antibodies to the TSH-R, one with stimulating activity and one with blocking activity, obtained from the same blood sample, confirms the assumption that immunoglobulins of stimulating and blocking activity can coexist in the same patient serum [27,29]. The pioneering structure–activity studies of the wild type human TSH-R and various chimeric TSH-R constructs in CHO cells, revealed a loss of responsiveness to the inhibitory immunoglobulins present in the serum of Hashimoto's patients and patients with idiopathic myxedema evaluated on a chimeric TSH-R denoted Mc4 [24,26]. Taking into account this finding, the TSH-R luciferase reporter bioassay was vastly improved by expression of a derivatized Mc4 TSH-R, amino acid residues 262–368 of the wild type (wt) human TSH-R replaced with amino acids 262–334 of the rat luteinizing hormone receptor (rLH-R) (Fig. 1B), under constitutive promoter in newly bio-engineered CHO K1 cell line with CRE/CREB cAMP-induced luciferase reporter gene. The TSI bioassay developed from a clone of the CHO K1 showed striking sensitivity to TSI in GD [22,23].

Table 2 Bioassays of functional TSH-receptor autoantibodies — human TSH-R CHO cell lines. Source CHO cell line/hTSH-R type

Endpoint

Analyte/Assay time

Pros

Cons

Chronology (years)

Ref

JP14, JP26, JP28, JP209/wt

RIA

3 H-cAMP, DNA synthesis/3–4 days

1st stable expression of human TSH-R in animal cells TSH-cAMP dose responses of different clones

TSI dose responses not optimized

1990–1993

[54,55]

JP09/wt K1/wt and chimerics C6–13/wt JPO9 and JP26/wt

RIA cAMP RIA cAMP cAMP luciferase cAMP luciferase

TSI/2 days TSI and TBI/20 h TSI/24 h TSI and TBI/26 h

Purified IgG, serum free culture required TSH-R expression not optimized, serum free cell culture

1994–1998 1997 1998 1999

[25,56] [24] [15] [16]

K1/wt

cAMP luciferase

TSI and TBI/24 h

No clinical evaluation

2001–2006

[17,18,20]

1st luminescent assay TSH-R expression optimized, high sensitivity TSI and TBI MAb screening, serum starved culture not required

120

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The LRD structure of the alpha subunit, known to comprise the major TSI epitopes (Fig. 1A) [4,13], is not altered in the chimeric construct as predicted by the identical LRD amino acid sequence to wild type TSH-R (Fig. 1B). The ß subunit of chimeric Mc4 TSH-R no longer has cleavage sites, amino acids 316–366 flanking the cysteine hinge residues. The physiological importance of hTSH-R cleavage in GD has been intensively reviewed [1,4,30] and more recently implicated in the affinity maturation of TSI causing GD [36]. The cleavage of wt TSH-R, excision of amino acids 316–366, is assumed not to occur in the chimeric Mc4-TSH-R. However, it has yet to be proven if the conservative substitution with sequences of rLH-R renders the stable and intact chimeric Mc4-holoreceptor resistant to cleavage by TSH or by patient serum known to bind at cleavage-site epitopes and to inhibit the shedding [4,12,30]. The enhanced sensitivity of Mc4 TSH-R bioassay for detection of TSI compared with wt TSH-R bioassay [22] indicates a promising future role for this bioassay in GD diagnostics. 5. Clinical evaluations of TSH-R bioassay The early evaluations of human thyrocyte TSI bioassay with sera of GD, Hashimoto's, non-thyroidal autoimmune diseases and controls confirmed that the cell-based assay of functional immunoglobulins is of clinical importance [47–49]. Cryo-preserved human goiter tissue, the most biologically relevant source to assess TSI accumulated 3– 18 pmol 125I-cAMP/2.5 × 104 cells in hypotonic cell lysates after 4 h stimulation with 1.5–9 mg/mL of GD IgG [49,50]. The TSI levels, accumulated cAMP induced by patient test serum as a percentage of the accumulated cAMP in the presence of normal IgG, distributed into two clusters among patients with hyperthyroid GD patients (n = 60); mean 350%, range 180–500% and mean 1500%, range 900–3000%. TSI of the euthyroid GD patients in remission (n = 25) and normal control subjects (n = 20) were all below the cut-off b160%. High clinical specificity of the human thyrocyte bioassay was underscored by the low TSI positivity found among patients with non-autoimmune thyroid disorders (1 of 37, 2.7%), Hashimotos' thyroditis (4 of 33, 12%) and atrophic thyroiditis (3 of 17, 18%) [50]. Retrospective evaluation of the human thyrocyte TSI bioassay, sensitivity 96% and specificity 100%, reported 31% TSI positivity among confirmed GD patients (n = 140) at 18 months after treat-

ments with daily carbimazole [61]. The prevalence of the TSI positivity significantly increased among 40 relapsed patients at 18–36 months compared with patients who remained in remission three years after withdrawal of treatment, (55% versus 13%). This study found similar prevalence of positive TSH-R autoantibodies among GD patients in remission and relapsed patients using TRAb methods. However, GD patients first rendered euthyroid and then randomized to treatment with methimazole alone or methimazole plus anti-CD20 (Rituximab) therapy showed a significant diminution of TSI that was associated with Rituximab and remission [62], a finding that is consistent with earlier work showing greater predictive value of TSI compared with radioreceptor binding for relapsed GD [19]. The human TSH-R CHO JP09 bioassay of radioactive cAMP endpoint reported TSI levels that correlated well with TSI levels obtained in FTRL-5 (r = 0.87) [55,56]. Furthermore, positive TSI was detected in 24 of 29 newly diagnosed GD, 0 of 42 controls and 4 of 40 non-thyroid autoimmune patients [52]. These findings sparked interest to perform follow-up comparisons of the CHO JPO9 with FRTL-5 [56] and to pursue the first complete clinical evaluations of a TSI bioassay [57], Table 3. The most striking outcome was the superior sensitivity of the JP09 cAMP RIA compared with FRTL-5 cAMP RIA (92% versus 75%, n = 51 GD and n = 23 controls) [56]. The ROC analysis of JP09 cAMP RIA in a second study with optimal decision threshold cut-off of 1.18 stimulation index (SI) resulted in a sensitivity of 85% and a specificity of 98% (n = 126 GD and n = 100 controls) [19,25] (Table 3). The capacity of different wt TSH-R CHO clones to distinguish between the stimulating and blocking immunoglobulins in patients undergoing treatments with anti-thyroid medications became the major focus for subsequent clinical evaluations of the hTSH-R reporter bioassay [16–18]. Among 197 GD patients treated with anti-thyroid drugs, 111 (56%) were TSI positive and 34 of the 86 (40%) TSI negative were positive for TBI as assessed using the JP26 CHO cell line. In contrast, only 10 of the 34 TBI could be detected with the JP09 CHO luciferase reporter bioassay. Additionally, TSI values but not anti-TSHR binding assessed by displacement of radiolabeled TSH bound to purified porcine TSH-R alpha domain immobilized on solid supports (TRAb assays) were found to positively associate with fT4 levels [20]. The Mc4–TSI bioassay, the first luciferase bioassay validated using an FDA-cleared protocol [22,63], detects the cAMP-induced luciferase

Table 3 TSH-receptor bioassays with clinical evaluations. Source CHO cell line/hTSH-R type

Endpoint

Analyte/assay time

N

JP09/wt

cAMP RIA

TSI

JPO9/wt

cAMP RIA

TSI, TBI/4 d

JP09, JP26/wt

cAMP RIA

TSI, TRAb/2–3 d

K1/wt

cAMP luciferase

TSI/36 h

K1/chimerica

cAMP luciferase

TSI/20 h

51 40 21 126 40 64 100 97 100 45 155 40 54 180 62 85 103 110 99

K1 chimeric Thyretain™

cAMP luciferase

Disease group

GD Nodular goiter Controls GD-Untreated HT NT AID Controls GD-Treated Controls GD-Treated GO-Treated Controls GD-Ut Controls HT NT AID GD-untreated Controls HT

% Pos

92 0 0 85 10 1 2 ND ND 44 95 2,5 0 2 0 95 0 4

ROC Analysis

Cut-off

CV % Intra/ inter

Chronology

Ref

96

NA

NA

1997

[57]

85%

98%

SI = 1.18

b 12/7.3–20

1998–2005

[19,25]

ND

ND

N 3 SD

b 15/b15

1999

[16]

85

95

140 SRR%

7.3 ± 5/23

2010

[22]

80 96

100 99

140 SRR% 140 SRR%

5.8 ± 5/5 5/12

2010 2010

[22] [63]

95

96

128 SRR%

4.2/10

2010

[23]

Sens

Spec

92

Pub med search with keywords TSH-R bioassay or TSI bioassay or TSAb bioassay retrieved a total of 100 hits for period January 1, 1975–July 27, 2010. TBI bioassay gave additional 44 hits for this time period. The references listed are representative of clinical evaluations. SI = stimulation index, sens = sensitivity, spec = specificity, HT = Hashimoto's thyroiditis, NT AID = non-thyroidal autoimmune disease, NA = data not available. a 1st FDA-cleared TSI bioassay.

S.D. Lytton, G.J. Kahaly / Autoimmunity Reviews 10 (2010) 116–122

activity and reports on the percent ratio of the mean relative light units (RLU) of triplicate wells containing the test serum specimen to the mean RLU of triplicate wells containing the bovine TSH reference standard (SRR%, Table 3). The ROC analysis using a cut-off of SRR% 140 showed a sensitivity of 95% in 103 untreated GD and 80% in 155 treated GD with specificity 100% and 96% in two independent studies involving n = 40 and n = 100 euthyroid control sera, respectively. All sera of patients with non-thyroid autoimmune diseases tested negative below the cut-off; 0/16 and 0/85, 0% positive. In Hashimoto's thyroiditis the Mc4–TSI percent positivity was 1/62 (0%), [63] and 4/ 99 (4%) [23]; the intra-day assay precision of the Mc4–TSI bioassay CV of 2–16% for SRR% b140 and CV of 2–12% for SRR% in the range of 140–840. The inter-day precision showed CV of 5–7.7%. The most striking feature of the Mc4–TSI bioassay is its greater sensitivity to TSI of Graves' eye disease (GO) patients compared with treated GD patients. High TSI levels were detected especially among GO patients that were either heavily smoking, non-responsive to antithyroid drugs and/or suffering from systemic manifestations [22]. Moreover, the TSI levels of the TSI bioassays but not the TRAb assays strongly correlated with the indices of GO clinical activity r = 0.7 (wt TSH-R) and r = 0.87 (Mc4 TSH-R) versus r = 0.17–0.54 (TRAb assays) and GO clinical severity; r = 0.72 (wt TSH-R) and r = 0.86 (Mc4 TSH-R) versus r = 0.15–0.51 (TRAb assays) [22]. 6. Future of the TSI bioassay: from bioassay to bedside The view of TSH-R bioassays, important bio-tool of TSH-R autoantibodies that activate fully functional TSH-R on live cells, but with cumbersome and time consuming procedures not suitable for routine use in GD diagnostics, is about to change. The TSI-Mc4 bioassay, amenable to automation, shows requisite clinical sensitivity and high specificity with robust performance [22,23,63]. The rapid 20 hour total assay time, from direct reconstitution and plating of the cryo-preserved CHO K1 cell line to data acquisition in 96 well microtiter plate reader luminometer, gives consistent and high precision [22]. For each plate run the mean SRR% values, derived from the RLU measurements of triplicate wells, are determined for 12 test sera, positive sera, bTSH reference and normal serum. Major challenges and issues must be resolved before a new generation of TSH-R bioassays become an integral part of the multidisciplinary approaches to the management and care of patients with thyroid autoimmunity and GO pathogenesis [64]. Introduction of the bioassay into the theatre of routine GD diagnostics requires coordination between multiple needs; pricing by manufacturers, reimbursement policies of national health insurance, and clear demonstration of clinical added value and cost-effectiveness compared with existing TSH-R binding assays. Prospective studies of TSI levels and TSI titers at baseline and at regular time intervals during treatment are in progress to determine if the TSI biomarker has utility to optimize patient responses to therapy and for prediction of relapse and remission. The impact of TSI bioassay on reducing the need for follow-up thyroid scans and expensive imaging techniques is currently under evaluation. Take-home messages TSH-R bioassays show outstanding features: • Biological activity of specific immunoglobulins is directly assessed on a fully functional TSH-R holoreceptor expressed on intact live cells, a platform that is easily adaptable and tailored to detect autoantibodies of specific function. The TSH-R protein structure can be bio-engineered and stably expressed in cell lines with protocols optimized for detection of TSI or TBI. • Autoreactivity of an individual patient is revealed with added clinical value. The bioassay of TSH-R autoantibodies measures the autoantibody function that is highly correlated with GD activity.

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• Bioassays are the best option to improve the personalized management of GD patients. The monitoring of TSI levels and TSI titers add another dimension to the assessment of GD activity with potential to predict relapse or remission of individual patient. • A new Mc4 TSH-R chimeric TSI bioassay with a standardized protocol shows enhanced sensitivity for TSI that either cause and/or influence the course of GD.

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IVIg in pemphigus Intravenous immunoglobulin (IVIg) has been used as treatment for several autoimmune disorders. In a recent paper, Aoyama Y (J Dermatol 2010;37:239-45) did a review on IVIG mechanisms of action on pemphigus. The authors discusses that the first multicenter, randomized, doubleblind, placebo controlled study was done in Japan and could demonstrate the efficacy in pemphigus. The authors reviews that one important mechanism is the decrease of pathogenic autoantibodies in this dermatological condition proved by experimental models and clinical studies. In summary, this article does an extensive review on IVIg in an important bullous dermatological disease – pemphigus.

Parvovirus B19 and its relationship with lupus Parvovirus B19 is the etiological agent of the Fifth disease in children and has been linked as a trigger of various autoimmune disorders. In a review article, Pavlovic et al. (Lupus 2010;19:783-92) reported the studies on parvovirus B19 and systemic lupus erythematosus as clinical as molecular mechanisms. Furthermore, the authors purpose a model that consists in the presence of hydrolyzing anti-ssDNA antibodies that may also hydrolyze viral B19 ssDNA in fluids, blood and intracellular environmental. According to the authors, this mechanism may contribute to maintenance of a “vicious cycle” and the presence of flares in lupus.