ARTHRITIS & RHEUMATISM Vol. 48, No. 6, June 2003, pp 1484–1492 DOI 10.1002/art.10947 © 2003, American College of Rheumatology REVIEW Rituximab Therapy and Autoimmune Disorders Prospects for Anti–B Cell Therapy Gregg J. Silverman and Stuart Weisman tural homologies, CD20 has been postulated to function as a calcium channel subunit (for review, see ref. 4), which may explain the finding that ligation of CD20 can affect B cell activation, differentiation, and cell cycle progression from the G1 to the S phase (5,6). Important for its use as a therapeutic target, binding of CD20 does not modulate its expression or result in substantial internalization. CD20 is also not shed, and there are no other known membrane or secreted analogs to interfere with its use for B cell targeting (7). In recent reports, the properties of different monoclonal antibodies against human CD20 have been investigated (8). The clinical potential of CD20-targeted therapy derives, in large part, from the unexpected finding that treatment with an antibody to CD20 induces the death of B lymphocytes, even without the need to conjugate the antibody to a toxin. The best-explored mechanism has been the properties of rituximab (trade name Rituxan), a chimeric monoclonal antibody specific for human CD20, comprising the variable regions of a murine anti-human CD20 B cell hybridoma fused to human IgG- and -constant regions (9) (see Figure 1). Rituximab has a binding affinity for human CD20 of ⬃8 nM. The clearance of B cells is, in part, mediated through induction of complement-mediated activities and triggering of antibody-dependent cellular cytotoxicity. This latter activity is dependent on interactions with cellular receptors for the IgG1-constant regions (i.e., Fc␥ receptors), especially Fc␥ receptor type IIIa, which is expressed on a variety of cells including phagocytic cells (10). Rituximab has also been shown to directly trigger intracellular pathways for apoptotic B cell death that involve the activation of phospholipase C␥, interruption of the signal transducer and activator of transcription 3/interleukin-6 pathway, down-regulation of c-myc, and up-regulation of the proapoptosis molecule Bax, a member of the Bcl-2 family (11,12). This sequence of molecular events results in the activation of Introduction In recent years, advances in our understanding of the regulation of the immune system have enabled the identification of cellular and molecular targets that could potentially affect the pathogenesis of many autoimmune diseases. In particular, the demonstration that B lymphocytes could play a central role in pathogenesis suggests that their elimination may be a highly beneficial therapeutic goal in a variety of diseases. Hybridoma antibody technology has been applied as a first step toward developing such specific agents. One of the initial applications of this technology was the characterization of the surface molecules on lymphocytes, to enable the discrimination of each type of lymphocyte. These early studies identified CD20 as a specific marker for B cells (1). CD20 has been found to be highly expressed on the surface of pre–B lymphocytes as well as on both resting and activated mature B lymphocytes, whereas it is not expressed by hematopoietic stem cells, pro–B cells, or other normal tissues (Figure 1). However, despite extensive investigation, the role of CD20 in B cell physiology has remained a mystery; there is no known natural ligand, and CD20-knockout mice are without a discernible immunologic phenotype (2). From the characterization of its encoding gene, CD20 has been predicted to be a 33–37-kd membraneassociated phosphoprotein, with a structure of 4 transmembrane regions, a 44–amino acid extracellular loop, and cytoplasmic N- and C-termini (3). Based on strucSupported by grants AI-40305, AR-47360, and AI-46637 from the NIH, and a grant from the Alliance for Lupus Research. Gregg J. Silverman, MD, Stuart Weisman, MD: University of California, San Diego. Address correspondence and reprint requests to Gregg J. Silverman, MD, Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663. E-mail: email@example.com. Submitted for publication September 16, 2002; accepted in revised form January 28, 2003. 1484 RITUXIMAB THERAPY AND AUTOIMMUNE DISORDERS 1485 Figure 1. Rituximab (trade name Rituxan) is a chimeric monoclonal antibody specific for human CD20 (9). An IgG antibody molecule is composed of 2 heavy chains and 2 light chains connected by disulfide bonds. The variable regions determine the antigen binding specificity, while the constant regions are highly conserved between different antibodies and determine antibody effector functions within the body. Because the treatment of patients with mouse antibodies can result in severe allergic responses, the mouse antibodies are often engineered so that the murine portions are replaced by human protein sequences that do not adversely affect antibody functional capacities. One approach is to generate a chimeric antibody in which the variable-region domains from a mouse antibody are grafted by protein engineering techniques to human constant-region domains. To generate rituximab, the variable regions of a murine anti-human CD20 B cell hybridoma were fused to the human IgG- and -constant regions. the mitogen-activated protein kinase family members p44 and p42, and induction of activator protein 1 (13), ending in the activation of caspases, a special set of cysteine proteases that degrade cellular proteins. These final stages of the process are associated with nuclear condensation, DNA degradation, and cytoplasmic membrane changes that enable the efficient clearance of these dying cells by adjacent cells, including phagocytes, without the induction of inflammation or damage to neighboring cells or tissues. The importance of each of these mechanisms for in vivo B cell deletion is uncertain. Rituximab for B cell lymphoma In late 1997, rituximab became the first therapeutic monoclonal antibody approved by the Food and Drug Administration for the treatment of cancer (14), and since its introduction, there have been more than 300,000 treatments with this agent administered to patients with relapsed, low-grade, follicular non-Hodgkin’s B cell lymphoma (NHL) (for review, see refs. 15 and 16). Rituximab is administered as an intravenous infusion with a recommended dosage of 375 mg/m2 given once weekly for 4 weeks. Shortly after treatment, in more than 70% of these patients, the mature and malignant B cells become greatly diminished in the blood and bone marrow and generally remain greatly depressed for a median of 9–12 months. Similarly, in patients with relapsed NHL, responses to this single-agent therapy are usually of limited duration, lasting a median of 1 year. However, treatment does not affect stem cells or plasma cells, 1486 SILVERMAN AND WEISMAN Figure 2. A simplified view of the surface phenotype of B lymphocytes undergoing differentiation, from pro–B cells to plasma cells. Differentiation to the transitional B cell stage is associated with emigration from the bone marrow into the periphery, where a small proportion of recently emergent lymphocytes enter the recirculating pool of mature B cells. Following antigen (Ag) encounter and clonal selection in peripheral germinal centers (GC), certain progeny differentiate into antibody (Ab)–secreting cells and plasma cells that may return to the bone marrow or are selected into the long-lived memory B cell pool. During differentiation, the surface phenotype of these cells changes, and CD20 is expressed only at intermediate stages and not on plasma cells. The level of expression of CD20 on memory B cells has not yet been determined. which are the terminally differentiated B cell lineage cells that are the major source of antibodies in the body (Figure 2) and which have down-regulated or absent CD20 (9). The limited capacity to affect plasma cells may explain why there is little or no effect on the levels of Ig in these patients, and perhaps may explain why treatment is associated with a low incidence of opportunistic infections. Infections with common bacterial pathogens are also not significantly increased. Rituximab is generally well tolerated in lymphoma patients, and serious adverse effects are uncommon (for review, see ref. 17). However, during the first infusion, many patients will experience limited, infusionrelated effects such as hypotension, fever, and rigors, but these symptoms generally subside with temporary cessation of the infusion and subsequent use of a slower infusion rate. These reactions tend not to recur after subsequent infusions. Of note, in patients with lymphoma who have undergone treatment with rituximab, the occurrence of antichimeric antibodies is low (18). With any antibody-based therapy, the potential for the induction of human antichimeric antibodies (HACA) is a topic of great concern, because sensitization may lead to more serious allergic adverse events, and upon retreatment, the effectiveness of the agent may become greatly diminished. For the treatment of more advanced lymphoma, investigators have also begun to evaluate the use of RITUXIMAB THERAPY AND AUTOIMMUNE DISORDERS rituximab in combination with chemotherapeutic agents. In a recent study, a French cooperative group reported the outcome of a clinical trial involving elderly patients with diffuse large B cell lymphoma who received a standard combination regimen (i.e., cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP]) with or without rituximab. At a median followup of 2 years, event-free and overall survival were significantly higher in the CHOP-rituximab group (P ⬍ 0.001 and P ⫽ 0.007, respectively) without a clinically significant increase in toxicity. Moreover, although tumor expression of bcl-2, an antiapoptotic protein, is generally associated with a worse prognosis, the addition of rituximab was also shown to prevent treatment failure in bcl-2–positive patients (19). Rituximab for autoimmune thrombocytopenia Based on the success of rituximab for the treatment of lymphoma, it was inevitable that it would soon be used to treat patients with a broader range of diseases involving B cells (for review, see refs. 20 and 21). Autoimmune thrombocytopenia (also known as idiopathic thrombocytopenic purpura [ITP]), the most common hematologic autoimmune disease, has been useful for the development of innovative clinical trials, in part because of its perceived simple pathogenetic process; high titers of autoantibodies directed against platelet glycoproteins lead to depressed platelet counts and a bleeding diathesis. Moreover, it has been reported that up to 16% of patients presenting with autoimmune thrombocytopenia will later develop systemic lupus erythematosus (SLE) (22), suggesting that a subset of patients with these autoimmune conditions share the common features of genetic predisposition and pathogenesis. In a prospective, dose-escalation, phase I/II trial of chronic ITP, adult patients with or without a history of splenectomy who, despite corticosteroid therapy, still had platelet counts of ⬍75,000/mm3 were studied (23). The first group received 50 mg/m2 for the first infusion, followed by 150 mg/m2 on each of 3 subsequent weeks. However, although no significant toxicity was observed, none of these 3 patients had a clinical response. The second group received an initial dose at 150 mg/m2, followed by a weekly infusion of 375 mg/m2 for 3 weeks. A third group received 375 mg/m2 at each of the 4 weekly treatments. Of the 10 patients in these latter groups, 2 had partial responses lasting at least 3 months, as demonstrated by a platelet count of ⬎100,000/mm3 without additional therapy, while 1 patient had a com- 1487 plete response that was characterized by normalization of the platelet count to ⬎150,000/mm3 without additional therapy, lasting for more than 6 months. Notably, although pretreatment levels of platelet-associated IgG antibodies were significantly increased in 6 of these patients, only 1 of the responders displayed a posttreatment decline in these autoantibody levels (23). In another study, 25 adult patients with chronic ITP who had demonstrated resistance to conventional treatment regimens received the full 4-week regimen of rituximab at 375 mg/m2 (24). Of these 25 patients, 5 had increases in their platelet counts to ⬎100,000/mm3, and 5 had increases in their platelet counts to the range of 50,000–100,000/mm3. An additional 3 patients displayed minor responses, with stabilization of their platelet counts at ⬍50,000/mm3, without the need for continued treatment. Several patients had reductions to subnormal levels in their serum IgG, IgM, or both. In general, the infusions were well tolerated in these studies of ITP patients, since no major toxic effects and only minor infusion-related fevers and chills were reported (23,24). Rituximab for SLE At the 2001 and 2002 annual meetings of the American College of Rheumatology (ACR), interim results were reported on a therapeutic trial of SLE patients with clinically active disease but without severe organ-threatening disease activity (i.e., SLE Disease Activity Measure score of ⬍6) (25,26). In this singledose escalation study, infusions of either 100 or 375 mg/m2 of rituximab often resulted in significant depletions in the levels of peripheral B cells, which correlated with improvements in disease manifestations such as rash, arthritis, and fatigue. However, during the 6 months of followup, the levels of autoantibodies to native DNA and complement were not affected. Although these treatments were well tolerated, inductions of antichimeric antibodies were noted in 3 of the 12 patients studied. In another recently reported open study, 6 female patients with SLE who were resistant to standard immunosuppressive therapy received 2 infusions of 500 mg of rituximab in combination with 2 infusions of 750 mg of cyclophosphamide and oral prednisolone (27). These patients had active disease manifestations, including renal disease in 3 patients and central nervous system disease in 1 patient, in addition to Raynaud’s phenomenon, arthritis, lymphopenia, anemia, and cutaneous vasculitis. All of these patients had elevated levels of anti–native DNA antibodies and depressed levels of 1488 complement. After treatment, the levels of B lymphocytes were depleted in the peripheral blood for at least 3–16 months. During followup periods of 6–18 months, most patients demonstrated significant improvement, with mean British Isles Lupus Assessment Group global scores of 15.3 at the start to a mean of 8.3 at 6 months. In general, fatigue, arthralgia/arthritis, serositis, and skin vasculitis exhibited good responses, while there was improvement of renal involvement in 2 of 3 patients. C3 levels normalized, at least transiently, in most patients. Although modest decreases in the levels of serum Ig were common (IgA decreased a mean of 0.2 gm/liter, range 0–0.6; IgG decreased a mean of 3.9 gm/liter, range 0–6.3; IgM decreased a mean of 0.5 gm/liter, range 0–1.6), these levels remained within the normal range, and no consistent trends in anti–native DNA antibody levels were demonstrated. For some patients, treatment appeared to provide benefits that extended beyond the period of B lymphocyte depletion, enabling decreases in the maintenance doses of corticosteroids, although disease activity subsequently flared in most patients. In general, treatment was safe and well tolerated, with only minor infections reported. Rituximab for rheumatoid arthritis (RA) In 2001, Edwards and Cambridge reported their initial experience in an open study of 5 patients with classic erosive RA that was refractory to at least 5 disease-modifying agents (i.e., disease-modifying antirheumatic drugs) (28). These patients received the standard dosing regimen of rituximab in conjunction with 2 intravenous doses of 750 mg of cyclophosphamide and oral prednisolone at 60 mg per day for 11–22 days, followed by tapering of each dosage. All patients exhibited significant reductions in synovitis, and at 6 months, 3 patients displayed an ACR 70% improvement (ACR70) response (29), while the other 2 achieved an ACR50 response, and clinical benefits persisted for at least 1 year in most patients. In all patients, blood B cells were undetectable shortly after the infusions and remained depressed for at least 6 months. Even though total Ig levels displayed only modest declines (IgA decreased a mean of 0.7 gm/liter, range 0–3; IgG decreased a mean of 3.1 gm/liter, range 0–8.2; IgM decreased a mean of 0.5 gm/liter, range 0–1.5) (30), primarily in the first 10 weeks, the levels of rheumatoid factor remained depressed for at least 3–6 months in all patients (28). These regimens were well tolerated and lacked major infusion-related events, since there were only 2 episodes of limited respiratory infections and a SILVERMAN AND WEISMAN case of minor thrombocytopenia that resolved without complication (28). In a followup report, interim results were reported on a total of 22 patients who received different regimens that included lower and higher doses of rituximab (30,31). However, among these patients, clinical responses correlated with the use of higher doses of rituximab in conjunction with cyclophosphamide and oral corticosteroids, and several patients required retreatment. At the 2002 ACR annual meeting, the first results were presented from a randomized, double-blind, placebo-controlled trial involving patients with seropositive RA. According to these interim results, 4 groups of 30–31 patients in each treatment arm received either methotrexate alone, methotrexate plus rituximab, rituximab alone, or rituximab plus cyclophosphamide. After 24 weeks of followup, each regimen was well tolerated, although responses with rituximab alone surpassed those with the current clinical standard, methotrexate. However, rituximab in combination with methotrexate or cyclophosphamide produced the highest ACR20 (32), ACR50, and ACR70 responses. Specifically, only 10% of the patients receiving methotrexate alone (⬎10 mg/ week) achieved an ACR50 response, whereas this level of response was noted in 32% of patients receiving rituximab alone, with comparable responses seen in 45% of patients receiving rituximab and cyclophosphamide (at the above-described doses), and in 50% of those receiving rituximab and methotrexate (33). In another recent study, 5 female patients with RA that was refractory to remittive agents received the standard 4-week rituximab regimen (i.e., 375 mg/m2), and only low-dose oral prednisone, nonsteroidal antiinflammatory drugs, and antimalarial agents were allowed (34). At followup, improvements were confirmed by radiographic and ultrasonographic imaging studies, and 1 patient had an ACR70 response, while a second patient had an ACR50 response, each of which lasted at least 10 months. Two additional patients had ACR20 responses that were more limited in duration. The fifth patient displayed no benefit. All responders demonstrated a significant reduction or normalization of the levels of C-reactive protein and serum rheumatoid factor. All patients had significant depletion of peripheral B cells, and 4 of 5 patients demonstrated 30–50% drops in their serum IgM levels, but these remained in the normal range. These patients also tolerated the treatment regimen, since no major adverse effects were noted, and 2 patients experienced uncomplicated lower urinary tract infections. RITUXIMAB THERAPY AND AUTOIMMUNE DISORDERS Table 1. Rituximab treatment of autoimmune diseases Table 3. Refs. Autoimmune thrombocytopenia Systemic lupus erythematosus Rheumatoid arthritis Autoimmune hemolytic anemia Cold agglutinin disease Mixed cryoglobulinemia Neuropathies associated with autoantibodies Myasthenia gravis Wegener’s granulomatosis Dermatomyositis 23,24,50 25–27 28,31,33,34 51–54 55–58 59,60 61 62 63 64 Mechanisms of autoimmunity and rituximab Following the dramatic successes with the treatment of lymphoma, rituximab has become an appealing candidate for the treatment of nonmalignant diseases involving B cells (Table 1). For these disorders, the effective targeting of B cells may also affect pathogenesis by removing the many functional contributions of B lymphocytes to the cell-to-cell interactions that drive the disease process (35) (Tables 2 and 3). The clinical responsiveness of these disorders to rituximab treatment will also reflect the inherent differences between the B cells involved in neoplasias and those involved in autoimmune diseases. Specifically, the pathogenesis of B cell lymphoma is largely a reflection of the accumulation of neoplastic clonal sets of B cells, which are directly susceptible to anti-CD20–mediated induction of cell death. In contrast, the B cells in different autoimmune diseases can play several different roles that support the underlying pathologic process (Table 3). For example, B cells can act as highly efficient antigen-presenting cells (APCs), supporting the activation and autoreactivity of T cells involved in the process. In fact, by virtue of the high affinity of a specific membrane-associated Ig for an antigen, an antigenspecific B cell can take up, process, and present peptides from nominal antigen with ⱖ1,000-fold more efficiency than a “professional” APC. In addition, activated B cells can also be the Table 2. Immunobiologic functions of B lymphocytes in health* 1. Provide cognate help for T cells 2. Produce cytokines (i.e., IL-4 and IL-10) that support other mononuclear cells 3. Antigen uptake via surface Ig for processing and presentation 4. Antigen-induced production of Ig/antibodies 5. Constitutive production of Ig/antibodies by plasma cells 6. Memory cell (semidormant) awaiting antigen re-exposure * IL-4 ⫽ interleukin-4. 1489 Immunobiologic functions of B lymphocytes in disease* 1. Provide cognate help for autoreactive T cells 2. Produce cytokines (i.e., IL-4 and IL-10) that support other mononuclear cells 3. Autoantigen uptake via surface Ig for processing and presentation 4. Autoantigen-induced production of autoantibodies that are directly or indirectly (e.g., immune complex formation) destructive 5. Constitutive production of autoantibodies by plasma cells 6. Autoreactive memory cell awaiting (sequestered) autoantigen reexposure 7. Disease-associated uncontrolled clonal proliferation (or prolonged lifespan) 8. Direct infiltration of end organs (e.g., the kidneys in SLE, the joints in RA, the liver in mixed cryoglobulinemia) * IL-4 ⫽ interleukin-4; SLE ⫽ systemic lupus erythematosus; RA ⫽ rheumatoid arthritis. source of cytokines and membrane-associated molecules that provide cognate help and enlist and support the activities of autoreactive T cells. For instance, studies of human synovium–SCID mouse chimeras have confirmed that in RA, the T cell activation is dependent on B cells, and APCs other than B cells can not substitute for the maintenance of T cell activation (36). Moreover, the B cells involved in an autoimmune disease may also be less accessible to the effects of rituximab. In fact, data from nonhuman primates with cross-reactive CD20 indicate that treatment with certain anti-CD20 antibodies may be much less effective at depleting B cells in lymph nodes (37), but published data on this topic are currently limited. Presumably, these findings are also relevant to the effects at sites of tissue infiltration (e.g., joints), where disease-associated lymphocytes may, in fact, preferentially localize in different disorders (9). Available clinical experience also suggests that rituximab as a single agent may not be adequate for the treatment of diseases resulting from the production of pathogenic autoantibodies, since it is likely that the dominant cellular source of disease-associated autoantibodies, especially IgG antibodies, are plasma cells that do not bear CD20 (Figure 2). Moreover, at least a fraction of plasma cells are reported to turn over and be replenished from precursor B cells only very slowly (38,39). Thus, even after a course of rituximab has depleted susceptible mature B cells, plasma cells may still continue to produce disease-causing autoantibodies for months or even years (39). For this reason, the optimal treatment of diseases that have autoantibodymediated pathology may require a regimen that also affects plasma cells. In addition, evolving therapeutic regimens should also take into consideration the elusive 1490 lymphoid cells that are committed to retaining the immunologic memory of the autoantigen (i.e., autoreactive memory B cells). These cells may remain relatively dormant for prolonged periods of time, only to be reactivated months, or even decades, later to be engaged in (auto)antigen-specific responses. Therefore, to induce a durable remission and reestablish immunologic selftolerance in these patients, the long-term goal of therapy should be to eliminate all components of the diseaseassociated autoimmune process, including the offending autoreactive B cells, plasma cells, and memory cells. The efficacy of rituximab as a single agent may also be diminished when the pathogenesis of the condition provides B cells with additional stimulatory and prosurvival signals. It has been speculated that lessons from the “two signal” model of lymphocyte activation may be applicable to understanding the potentials and limitations of rituximab therapy. Although the functional role of CD20 in B cells remains controversial, in vitro treatment of human B cell lines with rituximab has been reported to induce death signaling pathways that have many similarities with those for B cell receptor (BCR)–mediated induction of apoptotic death, but this has been a controversial topic (13). Therefore, the in vivo susceptibility to rituximab of B cells in autoimmune diseases such as SLE and RA may be affected by the activities and influences of mononuclear cells, including local activated T cells, since cytokine and cognate “second signals” will potentially oppose the proapoptotic effects of rituximab. Although, in theory, as long as a B cell expresses CD20, it should be affected by complement-dependent and antibody-dependent cytotoxicity mechanisms, interim results from the controlled RA study indicate that clinical response was improved by combination regimens with either methotrexate or cyclophosphamide (33), presumably due to enhanced deletion of B cells. Prosurvival effects may also be associated with the dysregulated overproduction of stimulatory factors, such as interferon-␣ (40), which is currently believed to contribute to the accelerated B cell differentiation into plasma cells seen in pediatric lupus patients (41). Similarly, tumor necrosis factor ␣ and stem cell–derived growth factor 1 may play somewhat similar roles in supporting the erosive synovitis of RA (42,43). Although, in the limited studies that have been reported to date, rituximab has been generally well tolerated, it remains a concern that patients with autoimmune diseases may be especially predisposed to untoward effects associated with rituximab infusions. For example, based on their apparent predisposition to drug SILVERMAN AND WEISMAN allergies and greater incidence of HACA after infusions, lupus patients may benefit from the inclusion of premedication with agents such as an antihistamine (e.g., benadryl) and acetaminophen, also commonly used before transfusions, and cotreatment with corticosteroids, to decrease the induction of infusion reactions and raised HACA titers. Moreover, with regard to patients receiving immunosuppressive therapy, those who have recently received rituximab would not be expected to respond to prophylactic vaccinations. Although the tendency to develop serious or opportunistic infections has not been observed in such patients, they should still be managed with a heightened vigilance for signs and symptoms of infection. The recent reports of combination regimen trials in SLE and RA suggest that the efficacy of rituximab treatments for patients with autoimmune diseases may be improved by the addition of second agents, such as conventional chemotherapeutic drugs. Alternatively, it is likely that future studies will also evaluate cotreatments with specific biologic agents to interfere with T cell helper signals, such as blocking antibodies to CD40– CD40 ligand (44,45) or the use of CTLA4-Ig (46). As an alternative approach, it may be desirable to utilize agents that block the recently discovered BLyS/BAFF/ zTNF4 system (for review, see ref. 47), to interfere with these potent survival signals directed toward membraneassociated receptors on peripheral B cells. It is also likely that the apoptotic threshold of B cells may be selectively lowered by cotreatment with an agent such as staphylococcal protein A (SpA) (48,49), which forms proapoptotic BCR complexes on targeted B cells, and which likely represents the mechanism of action of SpApheresis therapy. In conclusion, we believe that before rituximab will come into routine usage by rheumatologists, the therapeutic regimens will likely need to be tailored to the inherent differences in the pathogenesis of autoimmune diseases. In particular, it is now generally accepted that the varied functional capabilities of B cells involved in autoimmune diseases are intimately intertwined with those of other immunocytes and inflammatory cells that are recruited into the pathologic responses. 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