AUTOIMMUNITY

Stratification on the basis of patient-reported symptoms of patients with primary
Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses
to immunomodulatory treatments.

J Intern Med. 2019 Mar 20. doi: 10.1111/joim.12888.[Epub ahead of print]...

Previous studies and own clinical observations of patients with systemic lupus erythematosus (SLE) suggest that SLE harbors distinct immunophenotypes. This heterogeneity might result in differences in response to treatment in different subgroups and obstruct clinical trials.

Sjögren’s syndrome (SS) is characterised by B cell abnormalities and immune-mediated destruction of exocrine glands, primarily the salivary and lacrimal glands.1 2 Among the reported genetic polymorphisms associated with primary SS (pSS), the FAM167A-BLK locus distinguishes itself as an...

One of the more perplexing questions in biomedical research is—why does the body's protective shield against infections, the immune system, attack its own vital cells, organs, and tissues? The answer to this question is central to understanding an array of autoimmune diseases, such as rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus, and Sjogren's syndrome. When some of the body's cellular proteins are recognized as "foreign" by immune cells called T lymphocytes, a destructive cascade of inflammation is set in place. Current therapies to combat these cases of cellular mistaken identity dampen the body's immune response and leave patients vulnerable to life-threatening infections. Research on stem cells is now providing new approaches to strategically remove the misguided immune cells and restore normal immune cells to the body. Presented here are some of the basic research investigations that are being guided by adult and embryonic stem cell discoveries. Introduction The body's main line of defense against invasion by infectious organisms is the immune system. To succeed, an immune system must distinguish the many cellular components of its own body (self) from the cells or components of invading organisms (nonself). "Nonself" should be attacked while "self" should not. Therefore, two general types of errors can be made by the immune system. If the immune system fails to quickly detect and destroy an invading organism, an infection will result. However, if the immune system fails to recognize self cells or components and mistakenly attacks them, the result is known as an autoimmune disease. Common autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosis (lupus), type 1 diabetes, multiple sclerosis, Sjogren's syndrome and inflammatory bowel disease. Although each of these diseases has different symptoms, they share the unfortunate reality that, for some reason, the body's immune system has turned against itself (see Box 6.1. Immune System Components: Common Terms and Definitions). How Does the Immune System Normally Keep Us Healthy? The "soldiers" of the immune system are white blood cells, including T and B lymphocytes, which originate in the bone marrow from hematopoietic stem cells. Every day the body comes into contact with many organisms such as bacteria, viruses, and parasites. Unopposed, these organisms have the potential to cause serious infections, such as pneumonia or AIDS. When a healthy individual is infected, the body responds by activating a variety of immune cells. Initially, invading bacteria or viruses are engulfed by an antigen presenting cell (APC), and their component proteins (antigens) are cut into pieces and displayed on the cell's surface. Pieces of the foreign protein (antigen) bind to the major histocompatibility complex (MHC) proteins, also known as human leukocyte antigen (HLA) molecules, on the surface of the APCs (see Figure 6.1 Immune Response to Self or Foreign Antigens). This complex, formed by a foreign protein and an MHC protein, then binds to a T cell receptor on the surface of another type of immune cell, the CD4 helper T cell. They are so named because they "help" immune responses proceed and have a protein called CD4 on their surface. This complex enables these T cells to focus the immune response to a specific invading organism. The antigen-specific CD4 helper T cells divide and multiply while secreting substances called cytokines, which cause inflammation and help activate other immune cells. The particular cytokines secreted by the CD4 helper T cells act on cells known as the CD8 "cytotoxic" T cells (because they can kill the cells that are infected by the invading organism and have the CD8 protein on their surface). The helper T cells can also activate antigen-specific B cells to produce antibodies, which can neutralize and help eliminate bacteria and viruses from the body. Some of the antigen-specific T and B cells that are activated to rid the body of infectious organisms become long-lived "memory" cells. Memory cells have the capacity to act quickly when confronted with the same infectious organism at later times. It is the memory cells that cause us to become "immune" from later reinfections with the same organism. Figure 6.1. Immune Response to Self or Foreign Antigens. (© 2001 Terese Winslow) How Do the Immune Cells of the Body Know What to Attack and What Not To? All immune and blood cells develop from multipotent hematopoietic stem cells that originate in the bone marrow. Upon their departure from the bone marrow, immature T cells undergo a final maturation process in the thymus, a small organ located in the upper chest, before being dispersed to the body with the rest of the immune cells (e.g., B cells). Within the thymus, T cells undergo an important process that "educates" them to distinguish between self (the proteins of their own body) and nonself (the invading organism's) antigens. Here, the T cells are selected for their ability to bind to the particular MHC proteins expressed by the individual. The particular array of MHCs varies slightly between individuals, and this variation is the basis of the immune response when a transplanted organ is rejected. MHCs and other less easily characterized molecules called minor histocompatibility antigens are genetically determined and this is the reason why donor organs from relatives of the recipient are preferred over unrelated donors. In the bone marrow, a highly diverse and random array of T cells is produced. Collectively, these T cells are capable of recognizing an almost unlimited number of antigens. Because the process of generating a T cell's antigen specificity is a random one, many immature T cells have the potential to react with the body's own (self) proteins. To avoid this potential disaster, the thymus provides an environment where T cells that recognize self-antigens (autoreactive or self-reactive T cells) are deleted or inactivated in a process called tolerance induction. Tolerance usually ensures that T cells do not attack the "autoantigens" (self-proteins) of the body. Given the importance of this task, it is not surprising that there are multiple checkpoints for destroying or inactivating T cells that might react to auto-antigens. Autoimmune diseases arise when this intricate system for the induction and maintenance of immune tolerance fails. These diseases result in cell and tissue destruction by antigen-specific CD8 cytotoxic T cells or autoantibodies (antibodies to self-proteins) and the accompanying inflammatory process. These mechanisms can lead to the destruction of the joints in rheumatoid arthritis, the destruction of the insulinproducing beta cells of the pancreas in type 1 diabetes, or damage to the kidneys in lupus. The reasons for the failure to induce or maintain tolerance are enigmatic. However, genetic factors, along with environmental and hormonal influences and certain infections, may contribute to tolerance and the development of autoimmune disease [4, 7]. Hematopoietic Stem Cell Therapy for Autoimmune Diseases The current treatments for many autoimmune diseases include the systemic use of anti-inflammatory drugs and potent immunosuppressive and immunomodulatory agents (i.e., steroids and inhibitor proteins that block the action of inflammatory cytokines). However, despite their profound effect on immune responses, these therapies are unable to induce clinically significant remissions in certain patients. In recent years, researchers have contemplated the use of stem cells to treat autoimmune disorders. Discussed here is some of the rationale for this approach, with a focus on experimental stem cell therapies for lupus, rheumatoid arthritis, and type 1 diabetes. The immune-mediated injury in autoimmune diseases can be organ-specific, such as type 1 diabetes which is the consequence of the destruction of the pancreatic beta islet cells or multiple sclerosis which results from the breakdown of the myelin covering of nerves. These autoimmune diseases are amenable to treatments involving the repair or replacement of damaged or destroyed cells or tissue (see Chapter 7. Stem Cells and Diabetes and Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). In contrast, non-organ-specific autoimmune diseases, such as lupus, are characterized by widespread injury due to immune reactions against many different organs and tissues. One approach is being evaluated in early clinical trials of patients with poorly responsive, life-threatening lupus. This is a severe disease affecting multiple organs in the body including muscles, skin, joints, and kidneys as well as the brain and nerves. Over 239,000 Americans, of which more than 90 percent are women, suffer from lupus. In addition, lupus disproportionately afflicts African-American and Hispanic women [11]. A major obstacle in the treatment of non-organ-specific autoimmune diseases such as lupus is the lack of a single specific target for the application of therapy. The objective of hematopoietic stem cell therapy for lupus is to destroy the mature, long-lived, and auto-reactive immune cells and to generate a new, properly functioning immune system. In most of these trials, the patient's own stem cells have been used in a procedure known as autologous (from "one's self") hematopoietic stem cell transplantation. First, patients receive injections of a growth factor, which coaxes large numbers of hematopoietic stem cells to be released from the bone marrow into the blood stream. These cells are harvested from the blood, purified away from mature immune cells, and stored. After sufficient quantities of these cells are obtained, the patient undergoes a regimen of cytotoxic (cell-killing) drug and/or radiation therapy, which eliminates the mature immune cells. Then, the hematopoietic stem cells are returned to the patient via a blood transfusion into the circulation where they migrate to the bone marrow and begin to differentiate to become mature immune cells. The body's immune system is then restored. Nonetheless, the recovery phase, until the immune system is reconstituted represents a period of dramatically increased susceptibility to bacterial, fungal, and viral infection, making this a high-risk therapy. Recent reports suggest that this replacement therapy may fundamentally alter the patient's immune system. Richard Burt and his colleagues [18] conducted a long-term follow-up (one to three years) of seven lupus patients who underwent this procedure and found that they remained free from active lupus and improved continuously after transplantation, without the need for immunosuppressive medications. One of the hallmarks of lupus is that during the natural progression of disease, the normally diverse repertoire of T cells become limited in the number of different antigens they recognize, suggesting that an increasing proportion of the patient's T cells are autoreactive. Burt and colleagues found that following hematopoietic stem cell transplantation, levels of T cell diversity were restored to those of healthy individuals. This finding provides evidence that stem cell replacement may be beneficial in reestablishing tolerance in T cells, thereby decreasing the likelihood of disease reoccurrence. Development of Hematopoietic Stem Cell Lines for Transplantation The ability to generate and propagate unlimited numbers of hematopoietic stem cells outside the body—whether from adult, umbilical cord blood, fetal, or embryonic sources—would have a major impact on the safety, cost, and availability of stem cells for transplantation. The current approach of isolating hematopoietic stem cells from a patient's own peripheral blood places the patient at risk for a flare-up of their autoimmune disease. This is a potential consequence of repeated administration of the stem cell growth factors needed to mobilize hematopoietic stem cells from the bone marrow to the blood stream in numbers sufficient for transplantation. In addition, contamination of the purified hematopoietic stem cells with the patient's mature autoreactive T and B cells could affect the success of the treatment in some patients. Propagation of pure cell lines in the laboratory would avoid these potential drawbacks and increase the numbers of stem cells available to each patient, thus shortening the at-risk interval before full immune reconstitution. Whether embryonic stem cells will provide advantages over stem cells derived from cord blood or adult bone marrow hematopoietic stem cells remains to be determined. However, hematopoietic stem cells, whether from umbilical cord blood or bone marrow, have a more limited potential for self-renewal than do pluripotent embryonic stem cells. Although new information will be needed to direct the differentiation of embryonic stem cells into hematopoietic stem cells, hematopoietic cells are present in differentiated cultures from human embryonic stem cells [9] and from human fetal-derived embryonic germ stem cells [17]. One potential advantage of using hematopoietic stem cell lines for transplantation in patients with autoimmune diseases is that these cells could be generated from unaffected individuals or, as predisposing genetic factors are defined, from embryonic stem cells lacking these genetic influences. In addition, use of genetically selected or genetically engineered cell types may further limit the possibility of disease progression or reemergence. One risk of using nonself hematopoietic stem cells is of immune rejection of the transplanted cells. Immune rejection is caused by MHC protein differences between the donor and the patient (recipient). In this scenario, the transplanted hematopoietic stem cells and their progeny are rejected by the patient's own T cells, which are originating from the patient's surviving bone marrow hematopoietic stem cells. In this regard, embryonic stem cell-derived hematopoietic stem cells may offer distinct advantages over cord blood and bone marrow hematopoietic stem cell lines in avoiding rejection of the transplant. Theoretically, banks of embryonic stem cells expressing various combinations of the three most critical MHC proteins could be generated to allow close matching to the recipient's MHC composition. Additionally, there is evidence that embryonic stem cells are considerably more receptive to genetic manipulation than are hematopoietic stem cells (see Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). This characteristic means that embryonic stem cells could be useful in strategies that could prevent their recognition by the patient's surviving immune cells. For example, it may be possible to introduce the recipient's MHC proteins into embryonic stem cells through targeted gene transfer. Alternatively, it is theoretically possible to generate a universal donor embryonic stem cell line by genetic alteration or removal of the MHC proteins. Researchers have accomplished this by genetically altering a mouse so that it has little or no surface expression of MHC molecules on any of the cells or tissues. There is no rejection of pancreatic beta islet cells from these genetically altered mice when the cells are transplanted into completely MHC-mismatched mice [13]. Additional research will be needed to determine the feasibility of these alternative strategies for prevention of graft rejection in humans [6]. Jon Odorico and colleagues have shown that expression of MHC proteins on mouse embryonic stem cells and differentiated embryonic stem cell progeny is either absent or greatly decreased compared with MHC expression on adult cells [8]. These preliminary findings raise the intriguing possibility that lines derived from embryonic stem cells may be inherently less susceptible to rejection by the recipient's immune system than lines derived from adult cells. This could have important implications for the transplantation of cells other than hematopoietic stem cells. Another potential advantage of using pure populations of donor hematopoietic stem cells achieved through stem cell technologies would be a lower incidence and severity of graft-versus-host disease, a potentially fatal complication of bone marrow transplantation. Graft-versus-host disease results from the immune-mediated injury to recipient tissues that occurs when mature organ-donor T cells remain within the organ at the time of transplant. Such mature donor alloreactive T cells would be absent from pure populations of multipotent hematopoietic stem cells, and under ideal conditions of immune tolerance induction in the recipient's thymus, the donor-derived mature T cell population would be tolerant to the host. Gene Therapy and Stem Cell Approaches for the Treatment of Autoimmune Diseases Gene therapy is the genetic modification of cells to produce a therapeutic effect (see Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). In most investigational protocols, DNA containing the therapeutic gene is transferred into cultured cells, and these cells are subsequently administered to the animal or patient. DNA can also be injected directly, entering cells at the site of the injection or in the circulation. Under ideal conditions, cells take up the DNA and produce the therapeutic protein encoded by the gene. Currently, there is an extensive amount of gene therapy research being conducted in animal models of autoimmune disease. The goal is to modify the aberrant, inflammatory immune response that is characteristic of autoimmune diseases [15, 19]. Researchers most often use one of two general strategies to modulate the immune system. The first strategy is to block the actions of an inflammatory cytokine (secreted by certain activated immune cells and inflamed tissues) by transferring a gene into cells that encodes a "decoy" receptor for that cytokine. Alternatively, a gene is transferred that encodes an anti-inflammatory cytokine, redirecting the auto-inflammatory immune response to a more "tolerant" state. In many animal studies, promising results have been achieved by using these approaches, and the studies have advanced understanding of the disease processes and the particular inflammatory cytokines involved in disease progression [15, 19]. Serious obstacles to the development of effective gene therapies for humans remain, however. Foremost among these are the difficulty of reliably transferring genetic material into adult and slowly dividing cells (including hematopoietic stem cells) and of producing long-lasting expression of the intended protein at levels that can be tightly controlled in response to disease activity. Importantly, embryonic stem cells are substantially more permissive to gene transfer compared with adult cells, and embryonic cells sustain protein expression during extensive self-renewal. Whether adult-derived stem cells, other than hematopoietic stem cells, are similarly amenable to gene transfer has not yet been determined. Ultimately, stem cell gene therapy should allow the development of novel methods for immune modulation in autoimmune diseases. One example is the genetic modification of hematopoietic stem cells or differentiated tissue cells with a "decoy" receptor for the inflammatory cytokine interferon gamma to treat lupus. For example, in a lupus mouse model, gene transfer of the decoy receptor, via DNA injection, arrested disease progression [12]. Other investigators have used a related but distinct approach in a mouse model of type 1 diabetes. Interleukin-12 (IL-12), an inflammatory cytokine, plays a prominent role in the development of diabetes in these mice. The investigators transferred the gene for a modified form of IL-12, which blocks the activity of the natural IL-12, into pancreatic beta islet cells (the target of autoimmune injury in type 1 diabetes). The islet cell gene therapy prevented the onset of diabetes in these mice [20]. Theoretically, embryonic stem cells or adult stem cells could be genetically modified before or during differentiation into pancreatic beta islet cells to be used for transplantation. The resulting immune-modulating islet cells might diminish the occurrence of ongoing autoimmunity, increase the likelihood of long-term function of the transplanted cells, and eliminate the need for immunosuppressive therapy following transplantation. Researchers are exploring similar genetic approaches to prevent progressive joint destruction and loss of cartilage and to repair damaged joints in animal models of rheumatoid arthritis. Rheumatoid arthritis is a debilitating autoimmune disease characterized by acute and chronic inflammation, in which the immune system primarily attacks the joints of the body. In a recent study, investigators genetically transferred an anti-inflammatory cytokine, interleukin-4 (IL-4), into a specialized, highly efficient antigen-presenting cell called a dendritic cell, and then injected these IL-4-secreting cells into mice that can be induced to develop a form of arthritis similar to rheumatoid arthritis in humans. These IL-4-secreting dendritic cells are presumed to act on the CD4 helper T cells to reintroduce tolerance to self-proteins. Treated mice showed complete suppression of their disease and, in addition to its immune-modulatory properties, IL-4 blocked bone resorption (a serious complication of rheumatoid arthritis), making it a particularly attractive cytokine for this therapy [10]. However, one obstacle to this approach is that human dendritic cells are difficult to isolate in large numbers. Investigators have also directed the differentiation of dendritic cells from mouse embryonic stem cells, indicating that a stem cell-based approach might work in patients with rheumatoid arthritis [5]. Longer-term follow-up and further characterization will be needed in animal models before researchers proceed with the development of such an approach in humans. In similar studies, using other inhibitors of inflammatory cytokines such as a decoy receptor for tumor necrosis factor-α (a prominent inflammatory cytokine in inflamed joints), an inhibitor of nuclear factor-κB (a protein within cells that turns on the production of many inflammatory cytokines), and interleukin-13 (an anti-inflammatory cytokine), researchers have shown promising results in animal models of rheumatoid arthritis [19]. Because of the complexity and redundancy of immune system signaling networks, it is likely that a multifaceted approach involving inhibitors of several different inflammatory cytokines will be successful, whereas approaches targeting single cytokines might fail or produce only short-lived responses. In addition, other cell types may prove to be even better vehicles for the delivery of gene therapy in this disease. Chondrocytes, cells that build cartilage in joints, may provide another avenue for stem cell-based treatment of rheumatoid arthritis. These cells have been derived from human bone marrow stromal stem cells derived from human bone marrow [14]. Little is known about the intermediate cells that ultimately differentiate into chondrocytes. In addition to adult bone marrow as a source for stromal stem cells, human embryonic stem cells can differentiate into precursor cells believed to lead ultimately to the stromal stem cells [16]. However, extensive research is needed to reliably achieve the directed derivation of the stromal stem cells from embryonic stem cells and, subsequently, the differentiation of chondrocytes from these stromal stem cells. The ideal cell for optimum cartilage repair may be a more primitive cell than the chondrocyte, such as the stromal cell, or an intermediate cell in the pathway (e.g., a connective tissue precursor) leading to the chondrocyte. Stromal stem cells can generate new chondrocytes and facilitate cartilage repair in a rabbit model [3]. Such cells may also prove to be ideal targets for the delivery of immune-modulatory gene therapy. Like hematopoietic stem cells, stromal stem cells have been used in animal models for delivery of gene therapy [1]. For example, a recent study demonstrated that genetically engineered chondrocytes, expressing a growth factor, can enhance the function of transplanted chondrocytes [2]. Two obstacles to the use of adult stromal stem cells or chondrocytes are the limited numbers of these cells that can be harvested and the difficulties in propagating them in the laboratory. Embryonic stem cells, genetically modified and expanded before directed differentiation to a connective tissue stem cell, may be an attractive alternative. Collectively, these results illustrate the tremendous potential these cells may offer for the treatment of rheumatoid arthritis and other autoimmune diseases. Conclusion Stem cell-based therapies offer many exciting possibilities for the development of novel treatments, and perhaps even cures, for autoimmune diseases. A challenging research effort remains to fully realize this potential and to address the many remaining questions, which include how best to direct the differentiation of specific cell types and determine which particular type of stem cell will be optimum for each therapeutic approach. Gene therapy with cytokines or their inhibitors is still in its infancy, but stem cells or their progeny may provide one of the better avenues for future delivery of immune-based therapies. Ultimately, the potential to alleviate these devastating chronic diseases with the use of stem cell-based technologies is enormous. References Allay, J.A., Dennis, J.E., Haynesworth, S.E., Majumdar, M.K., Clapp, D.W., Shultz, L.D., Caplan, A.I., and Gerson, S.L. (1997). LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors. Hum. Gene Ther. 8, 1417–1427. Brower-Toland, B.D., Saxer, R.A., Goodrich, L.R., Mi, Z., Robbins, P.D., Evans, C.H., and Nixon, A.J. (2001). Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function. Hum. Gene Ther. 12, 117–129. Caplan, A.I., Elyaderani, M., Mochizuki, Y., Wakitani, S., and Goldberg, V.M. (1997). Principles of cartilage repair and regeneration. Clin. Orthop. 342, 254–269. Cooper, G.S., Dooley, M.A., Treadwell, E.L., St Clair, E.W., Parks, C.G., and Gilkeson, G.S. (1998). Hormonal, environmental, and infectious risk factors for developing systemic lupus erythematosus. Arthritis Rheum. 41, 1714–1724. Fairchild, P.J., Brook, F.A., Gardner, R.L., Graca, L., Strong, V., Tone, Y., Tone, M., Nolan, K.F., and Waldmann, H. (2000). Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr. Biol. 10, 1515–1518. Gearhart, J. (1998). New potential for human embryonic stem cells. Science. 282, 1061–1062. Grossman, J.M. and Tsao, B.P. (2000). Genetics and systemic lupus erythematosus. Curr. Rheumatol. Rep. 2, 13–18. Harley, C.B., Gearhart, J., Jaenisch, R., Rossant, J., and Thomson, J. (2001). Keystone Symposia. Pluripotent stem cells: biology and applications. Durango, CO. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. (2000). Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol. Med. 6, 88–95. Kim, S.H., Kim, S., Evans, C.H., Ghivizzani, S.C., Oligino, T., and Robbins, P.D. (2001). Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4. J. Immunol. 166, 3499–3505. Lawrence, R.C., Helmick, C.G., Arnett, F.C., Deyo, R.A., Felson, D.T., Giannini, E.H., Heyse, S.P., Hirsch, R., Hochberg, M.C., Hunder, G.G., Liang, M.H., Pillemer, S.R., Steen, V.D., and Wolfe, F. (1998). Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum. 41, 778–799. Lawson, B.R., Prud'homme, G.J., Chang, Y., Gardner, H.A., Kuan, J., Kono, D.H., and Theofilopoulos, A.N. (2000). Treatment of murine lupus with cDNA encoding IFN-gammaR/Fc. J. Clin. Invest. 106, 207–215. Osorio, R.W., Ascher, N.L., Jaenisch, R., Freise, C.E., Roberts, J.P., and Stock, P.G. (1993). Major histocompatibility complex class I deficiency prolongs islet allograft survival. Diabetes. 42, 1520–1527. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143–147. Prud'homme, G.J. (2000). Gene therapy of autoimmune diseases with vectors encoding regulatory cytokines or inflammatory cytokine inhibitors. J. Gene. Med. 2, 222–232. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D., and Benvenisty, N. (2000). Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 97, 11307–11312. Shamblott, M.J., Axelman, J., Littlefield, J.W., Blumenthal, P.D., Huggins, G.R., Cui, Y., Cheng, L., and Gearhart, J.D. (2000). Human embryonic germ cell derivatives express a broad range of develpmentally distinct markers and proliferate extensively in vitro. Proc. Natl. Acad. Sci. U. S. A. 98, 113–118. Traynor, A.E., Schroeder, J., Rosa, R.M., Cheng, D., Stefka, J., Mujais, S., Baker, S., and Burt, R.K. (2000). Treatment of severe systemic lupus erythematosus with high-dose chemotherapy and haemopoietic stem-cell transplantation: a phase I study. Lancet. 356, 701–707. Tsokos, G.C. and Nepom, G.T. (2000). Gene therapy in the treatment of autoimmune diseases. J. Clin. Invest. 106, 181–183. Yasuda, H., Nagata, M., Arisawa, K., Yoshida, R., Fujihira, K., Okamoto, N., Moriyama, H., Miki, M., Saito, I., Hamada, H., Yokono, K., and Kasuga, M. (1998). Local expression of immunoregulatory IL-12p40 gene prolonged syngeneic islet graft survival in diabetic NOD mice. J. Clin. Invest. 102, 1807–1814.  Chapter 5 | Table of Contents | Chapter 7  Historical content: June 17, 2001

Via Krishan Maggon

Journal of Immunology Research is a peer-reviewed, Open Access journal that provides a platform for scientists and clinicians working in different areas of immunology and therapy. The journal publishes research articles, review articles, as well as clinical studies related to classical immunology,...

Full text KEYWORDS Antiphospholipid syndrome, antiphospholipid antibodies, thrombosis, pregnancy morbidity, catastrophic antiphospholipid syndrome, treatment INTRODUCTION  The antiphospholipid syndrome (APS) is defined by the occurrence of venous and/or arterial thrombotic events and/or pregnancy-related morbidity (≥ 3 unexplained consecutive spontaneous abortions < 10 weeks with exclusion of chromosomal causes, foetal death or severe pre-eclampsia before 34th week of gestation), combined with the presence of circulating antiphospholipid antibodies (aPL) and/or a lupus anticoagulant (LAC), see table 1.1 Formally, LAC is the result of aPL binding to plasma proteins, mainly β2-glycoprotein, that have affinity for the negatively-charged phospholipids; therefore, the pathologic auto-antibodies are not directed against phospholipids. In this paper, the term ‘aPL’ refers to both LAC and anticardiolipin (aCL)/anti-β2-glycoprotein-I (anti-β2GPI) antibodies, following clinical practice and literature. APS is considered a primary autoimmune disease, but is often diagnosed as being secondary to other auto-immune diseases; 30-40% of patients with systemic lupus erythematodes (SLE) also have APS, and APS can be diagnosed secondary to rheumatoid arthritis, systemic sclerosis, dermatomyositis or other autoimmune diseases.2 Large, controlled, randomised clinical trials for interventions in APS are limited, and the quality of current data is not sufficient to develop a structured guideline or assess evidence-based management strategies. However, clinically-relevant questions about diagnostic criteria and treatment of APS patients arise on a daily basis. We aim to provide clinicians with an expert consensus on the management of APS, with a focus on classification, diagnostics, risk stratification, and treatment.  METHODS A literature review was performed (KdL and ML) with the following Pubmed search terms: ‘antiphospholipid syndrome’, "Antiphospholipid Syndrome"[Mesh], ‘antiphospholipid antibodies’, "Antibodies, Antiphospholipid"[Mesh], ‘obstetric antiphospholipid syndrome’, ‘catastrophic antiphospholipid syndrome’, ‘laboratory diagnostics’, ‘Clinical Laboratory Techniques"[Mesh] ‘diagnosis’, "Diagnosis"[Mesh], ‘treatment’ and "Therapeutics"[Mesh]; papers focusing on diagnostics and/or treatment for APS were included. Non-English papers and papers published before 2000 were excluded. The literature search was performed on April 3rd, 2018; papers published after that date but with high impact according to the writing committee were included until September 1st, 2018. Statements on APS diagnostics and treatment were extracted from the papers selected, and a first draft of the consensus paper was written (KdL and ML). This draft was circulated amongst all authors and comments were collected. On June 6th, 2018, a first consensus meeting was held in Utrecht, The Netherlands. Based on the outcomes of this meeting, a second draft of the paper was written (JS, ML) and circulated again for comments. On November 5th, 2018, a second consensus meeting was held in Utrecht, The Netherlands. Degree of consensus on statements was assessed by a Delphi procedure and a final report was written afterwards (see table 3). Diagnosis, classification, risk stratification Diagnosis of APS No diagnostic criteria for APS exist. If a patient meets the classification criteria – developed for research purposes – for APS (e.g., a thrombotic event and/or pregnancy morbidity, combined with repeated presence of antiphospholipid antibodies (aPL); see table 1), most likely a clinical diagnosis of APS will be made, although thrombotic and pregnancy complications are not necessarily causally related to circulating aPL. In addition to the thrombotic or pregnancy-related events listed in the classification criteria, APS may be associated with a variety of non-criteria manifestations, such as superficial vein thrombosis, thrombocytopenia, renal microangiopathy, heart valve disease, livedo reticularis or racemosa, migraine, chorea, seizures and myelitis; see table 2. As a result, a clinical diagnosis of APS can be made in patients who do not fulfil the classification criteria.3 An even more complicating factor is the concept of seronegative APS, a term coined to include patients with clinical (criteria and non-criteria) features suggestive of APS, but who are persistently negative for aPL.4 The existence of seronegative APS is a point of international discussion among experts. If the suspicion of seronegative APS arises, we suggest referral to an expert centre. Catastrophic antiphospholipid syndrome (CAPS) is the most extreme APS variant that includes simultaneous multiple organ thrombosis and develops in a short period of time with a high mortality rate. Although strongly associated with the presence of LAC, no other laboratory or clinical determinants are known to be associated with CAPS.2 Risk stratification in APS Several assessment tools to risk-stratify patients have been proposed, mainly focusing on presence and levels of aPL and/or on clinical parameters. For aPL, the presence of LAC is the strongest risk factor for both arterial and venous thrombosis in APS.5,6 For aCL and anti-β2GPI, the association between (levels of) antibodies and thrombosis is less clear. It has been suggested that anti-β2GPIdependent LAC has a strong association with thrombotic risk.7 In several studies, it has been demonstrated that the risk of arterial and venous thrombosis increases with the number of positive tests for aPL, with the highest risks in patients with both LAC, aCL and anti-β2GPI antibodies, so-called ‘triple positive patients’.6,8 In clinical practice, risk stratification does not affect treatment decisions in most situations. The antiphospholipid score (aPL-S) has been developed to predict the risk of APS-related clinical events in patients with APS and other autoimmune diseases (such as SLE, rheumatoid arthritis, and Sjögren’s syndrome) based on the presence of aPL.9 The global APS score (GAPSS) is another clinical score, including both aPL and conventional cardiovascular risk factors, predicting the risk of thrombotic events in patients with SLE.10 The GAPSS has been validated in a cohort of APS patients, and a correlation between higher GAPSS values and recurrence of thrombotic events was observed.11 However, these scores are not sufficient to design treatment strategies for the individual patient. We consider triple positive patients to be at highest risk for recurrent thrombosis. Laboratory diagnostics in APS The classification criteria for APS indicate three different antibody subsets of aPL. For two of these, the antigen is well-defined: aCL antibodies recognize the plasma glycoprotein β2GPI in complex with the anionic phospholipid cardiolipin, and anti-β2GPI antibodies recognize the protein β2GPI in the absence of cardiolipin. Both antibody subtypes can be detected with quantitative solid phase assays in which the antigen is immobilized on a surface. The third aPL subtype, known as LAC, is detected with a functional assay: these antibodies manifest as phospholipid-dependent inhibitors of in vitro coagulation. They are detected with phospholipid sensitive coagulation assays. Although the exact antigen to which LAC are directed is currently unclear, there is ample evidence that antibodies against β2GPI as well as antibodies against prothrombin can induce the LAC phenomenon.12,13 The diagnosis of APS is made in cases of persistent presence of aPL (titre > 99th percentile), assessed in two separate samples taken with an arbitrarily defined interval of at least twelve weeks. This is an important distinction, as several aPL occur transiently in relation to viral and bacterial infections and are of uncertain clinical relevance.14 Moreover, since aPL is prevalent (1-5%) in the general population,15 aPL status should only be tested in patients considered at risk of having APS, such as those < 50 years of age, unprovoked arterial or venous thrombosis, thrombosis at an unusual site, recurrent thrombosis, and thrombotic/pregnancy complications with or without association with a systemic autoimmune disease.16-18 Unfortunately, gold standards for aPL detection are lacking, although aCL assays based on pooled human serum have been in use for over 20 years.19 Reports have been published of new standards based on human(ized) monoclonal antibodies against β2GPI and purified patient-derived polyclonal antibody preparations, but these are not yet available.20 No such standards exist for LAC-positive plasmas. For further harmonization of results between diagnostic laboratories, centres performing these tests participate in diagnostic surveys as part of laboratory accreditation. Measurement of anticardiolipin and anti-β2-glycoprotein I antibodies The classification criteria for APS specify that both immunoglobin G (IgG) and IgM class immunoglobulins against cardiolipin or β2GPI should be measured. Several commercial entities supply kits to measure these antibodies and many laboratories have developed their own solid phase assays. To minimize the effect of the lack of gold standards and the large number of assays in use for detection of aPL on assay standardization, guidance on assay characteristics has been provided by the Scientific Standardization Committee of the International Society on Thrombosis and Haemostasis.16 Detection of aCL and β2GPI antibodies can be performed in both trisodium citrate anticoagulated plasma and in serum. Since aCL antibodies associated with APS are β2GPI-dependent, diagnostic laboratories should use assays in which cardiolipin is saturated with human β2GPI. Samples should be considered positive when the value obtained in these assays exceeds the 99th percentile of the normal population, rather than antibody levels exceeding 40 arbitrary units as indicated in the classification criteria, as this appears to be more specific for APS.21 We recommend that the laboratory report mentions both a cut-off value (< 99th or > 99th percentile, e.g., negative or positive) and a continuous numeric value. Detection of lupus anticoagulant LAC are phospholipid-dependent coagulation inhibitors and are detected with sensitive coagulation assays in trisodium citrate anticoagulated plasma. Plasma samples should be double centrifuged to minimize contamination with platelets, as they are a major source of phospholipid and might therefore interfere with LAC detection.17,18 LAC can be detected with any phospholipid-dependent coagulation assay, however, no gold standard for LAC testing exists. For this reason, it is warranted to perform two tests based on a different assay principle for LAC detection, preferably a dilute Russell’s viper venom time (dRVVT) and a LAC-sensitive activated partial thromboplastin time (APTT).17 In order to be deemed LAC-positive, a sample should have a prolonged clotting time when a reagent with a low phospholipid content is used (screening test), which should correct when a reagent with a high phospholipid content is used (confirmatory test), indicating phospholipid-dependence of the prolongation. The presence of a coagulation inhibitor as the cause of the prolongation should be shown with a mixing test, in which patient plasma is mixed with an equal volume of pooled normal plasma. This will normalise any coagulation factor deficiencies that are present, and any remaining prolongation of the clotting time is therefore caused by an inhibitor. Cut-off values for LAC should be determined locally in each diagnostic laboratory, based on the 99th percentile of the local normal population or alternatively, on the mean + 2 standard deviation (SD) of the clotting time of the normal population. The strength of the LAC should be expressed as a ratio between screen and confirm clotting times, preferably normalized on the mean of the normal population, according to the following equation:   Samples are deemed positive for LAC when one or both tests for LAC detection (APTT and dRVVT-based tests) indicate the presence of LAC. The use of anticoagulant drugs interferes with detection of LAC, possibly resulting in false-positive test results. Samples for LAC-detection should therefore be collected before treatment with anticoagulants has started, or sufficiently long after cessation of treatment to minimize confounding effects. However, it should be avoided to use samples obtained in the acute phase after a thrombotic event or during infection, as this is associated with high Factor VIII levels, which might interfere with LAC detection by APTT-based assays. Although LAC can be determined in samples from patients receiving vitamin K antagonists when they are mixed with an equal volume of pooled normal plasma, the outcome of LAC tests in samples with an INR within therapeutic range (INR 2-3) should be interpreted with caution and measurement of LAC in samples with an international normalised ratio (INR) > 3 is not recommended. Mixing antagonists with normal plasma dilutes the titre of LAC and thus reduces the sensitivity of the assay. On the other hand, the mixing test may not completely correct the clotting time for samples in the high INR range and may lead to false-positive interpretation. Temporary discontinuation of vitamin K antagonists (or co-administration of vitamin K and continuing vitamin K antagonists) and bridging with low molecular weight heparin is possible. Unfractionated heparin, however, is incompatible with LAC testing, as is the use of direct oral anticoagulants, even at trough levels of factor Xa or thrombin inhibitors, with factor Xa inhibitors producing false-positive LAC.22 No alternatives exist for LAC detection in samples containing direct thrombin inhibitors. A possible means to detect LAC in samples containing rivaroxaban is the Taipan snake venom time/Ecarin clotting time combination, as these tests are insensitive to factor Xa-inhibitors.23 More studies on the specificity and sensitivity for LAC of this test combination are required before these tests will be widely adopted for LAC detection. Non-criteria aPL There are several reports on the association between various non-criteria aPL subtypes and thrombosis. Amongst these are IgA anti-β2GPI antibodies,24-26 antibodies against the phospholipid phosphatidylethanolamine (PE),27 antibodies against the complex between the phospholipid phosphatidylserine, and the coagulation factor prothrombin (aPS/PT).28 Currently, however, there is insufficient evidence of their clinical relevance to warrant routine detection of these antibodies. Treatment Venous thrombosis in APS A first venous thrombotic event (VTE; amongst others including deep vein thrombosis and pulmonary embolism, abdominal vein thrombosis, cerebral vein thrombosis) should be treated according to the current guidelines for treatment of VTE; no routine testing for aPL is indicated in the general population. In cases of recurrent thrombosis (both provoked and unprovoked), or patients with a pre-existing autoimmune disease (and in particular, SLE), additional testing for aPL should be performed. Low molecular weight heparin (LMWH) in therapeutic doses and subsequent vitamin K antagonists (VKA) are first-line treatments for a first or recurrent APS-related venous thrombotic event (VTE). Treatment with direct oral anticoagulants (DOACs; see separate section below) is not recommended. For APS patients with a first VTE, life-long anticoagulation is recommended. After treatment with LMWH in the acute phase, treatment will be switched to VKA, with an INR target range of 2.0-3.0 for venous events.29,30 High-intensity treatment with an INR ≥ 3.0 after a first VTE is not recommended.31,32 Arterial thrombosis in APS Optimal long-term treatment for arterial thrombosis (other than cerebral arterial thrombosis; see below) is still a matter of debate; either anti-platelet therapy such as aspirin or clopidogrel, VKA with an INR target range of 2.5-3.5 or combined therapy with VKA with an INR target range of 2.0-3.0 and anti-platelet therapy has been recommended.30 As combined therapy, VKA and anti-platelet therapy has not been shown to be superior to anti-platelet therapy alone, and since more major bleeding complications were observed in the combination group,33 we do not recommend up-front combined treatment with both VKA and anti-platelet therapy. Based on expert opinion and in slight contrast with international recommendations, we prefer treatment with either clopidogrel or VKA with an INR target range of 2.0-3.0 in these patients.33,34 In patients with a cerebral ischemic event (transient ischemic attack (TIA) or ischemic stroke) as clinical manifestation, there is no evidence supporting one therapy over the other. A prospective, comparative study in aPL-positive stroke patients showed no benefit of warfarin over aspirin (325 mg/day) on recurrent events; more (minor) haemorrhagic complications in the warfarin group were observed. However, these patients did not necessarily fulfil APS criteria.35 A small (n = 20), randomized, controlled trial in APS patients with ischemic stroke compared VKA and low-dose aspirin with low-dose aspirin alone, and demonstrated less recurrent stroke in the combined-therapy group.36 In APS patients with a first ischemic stroke or TIA without any cause other than APS on work-up, we propose treatment with either VKA with an INR target range of 2.0-3.0 or clopidogrel (since this is superior to aspirin for stroke prevention in a general population suffering from atherosclerotic cerebrovascular disease).37 Position of direct oral anticoagulants in APS DOACs such as dabigatran, rivaroxaban, apixaban, and edoxaban, were shown to be non-inferior to VKAs for treatment and secondary prevention of venous thromboembolic events and prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.38 Furthermore, these drugs have some advantages compared to VKAs as it is a fixed dose, no monitoring is required, interaction is limited, and there is a lower risk of intracranial and other major bleeding. Nonetheless, few studies are published concerning the use of DOACs in known APS. A recent review summarised the available data, including one randomized controlled trial (RAPS trial) and case series.39 From all available case series and case reports, 122 patients were analysed.39 High-risk APS patients with triple positivity or with several clinical criteria for definite APS, developed recurrent thrombosis more frequently while on DOACs in comparison to warfarin. The RAPS trial, comparing 54 APS patients treated with rivaroxaban to 56 APS patients treated with warfarin, demonstrated that the in vitro anticoagulant effect of rivaroxaban may be inferior to that of warfarin.40 A randomized controlled clinical trial in triple positive patients, comparing VKA treatment with rivaroxaban, was terminated early due to more thrombotic events in the rivaroxaban-arm, with particularly more ischemic strokes in the DOAC group.41 At this moment VKAs, compared to DOACs, remain the standard of care in the treatment of APS, especially in high-risk APS patients. DOACs might be an alternativetreatment modality for only those patients with instable INR or poor adherence to INR monitoring, and we anticipate more data from future trials using DOACs in thrombotic APS. Recurrent venous thrombotic events while using anticoagulation therapy Recurrent thrombosis in APS patients, despite adequate anticoagulation therapy (INR target range, 2.0-3.0) is common and occurs in up to one-third of all patients.2 However, recurrent thrombosis is uncommon in APS patients with a higher INR target range of 2.5-3.5 and most patients treated with a VKA with recurrent thrombosis, appear to have an INR target < 3.0.42 Therefore, anticoagulation therapy should be intensified with a INR target range of 2.5-3.5 in case of a recurrent venous event despite adequate INR. When a recurrent venous thrombosis is diagnosed with a suboptimal INR (< 2.0), therapeutic LMWH should be given for a period of two weeks and the INR treatment range should not be changed. Note that the INR value can be underestimated due to interference of thromboplastin and LAC, although this is mostly problematic with higher INR values, i.e. > 4.0.43 If the target INR cannot be reached and/or maintained with VKA, referral to an expert centre is recommended. Based on expert opinion, in patients with recurrent venous thrombosis, despite an adequate INR target range of 2.5-3.5 and after two weeks of therapeutic LMWH, additional long-term use of aspirin can be  recommended,  or  intensifying of  VKA  therapy with to an INR  target  range  of 3-4. Alternatively, a permanent switch to therapeutic LMWH can be considered. Combining VKA with hydroxychloroquine may also decrease the risk of recurrent venous thrombosis, although randomized studies with hydroxychloroquine in APS are still lacking.44,45 Until now, no clinical support for the use of statins in patients with recurrent venous thrombosis despite anticoagulation exists. However, based on in vitro work and surrogate endpoints, a beneficial role of statins has been suggested.46,47 Recurrent arterial thrombotic events while using anticoagulation therapy No consensus on treatment of recurrent arterial thrombosis was reached at the last meeting of the APS task force.30 Optimal treatment strategies for recurrent arterial thrombotic events have not been studied. In recurrent arterial thrombosis, including TIA or ischemic stroke, despite treatment with clopidogrel, we suggest switching treatment to VKA with an INR target range of 2.0-3.0. If recurrent arterial thrombosis occurs despite adequate treatment with VKA, referral to an expert centre is recommended. We do not recommend to routinely perform brain magnetic resonance imaging (MRI) in APS patients with an ischemic stroke. However, some experts believe that if neurological symptoms, including migraine or cognitive performance, deteriorate, a repeat brain MRI is indicated and new white matter abnormalities may lead to intensifying treatment. The possible benefits of this strategy have not been confirmed in clinical studies, and for patients with deterioration of neurological symptoms, referral to an expert centre is recommended.  Catastrophic antiphospholipid syndrome (CAPS) CAPS is a rare complication of APS and occurs in approximately 1% of APS patients. CAPS is a severe condition, including massive (mostly arterial) thrombosis of small vessels, causing multi-organ failure. Mortality in CAPS is high, up to 50%. Adequate and fast treatment initiation slightly improves clinical outcomes (mortality 20-40%) and treatment should be carried out in an expert centre.48,49 A combination of anticoagulant drugs (mostly unfractionated heparin with APTT ratio 2-2.5), intravenous corticosteroids (methylprednisolone 500-1000 mg/day for 3-5 days), therapeutic plasma exchange (TPE) and/or intravenous immunoglobulins (IVIG) (1 g/kg, for a time period of 3 days) is associated with highest survival rates. Combination of heparin-corticosteroids-plasmapheresis with or without additional IVIG results in 69-78% patient survival.50-52 TPE should be started when the clinical suspicion of CAPS arises, within a minimum of 5 days.52 Clinical response dictates the duration of TPE and no single clinical or laboratory parameter is used to determine when to discontinue treatment. For patients with CAPS and underlying SLE, treatment with cyclophosphamide (750 mg/m2 monthly) has been proposed.48,53 Rituximab (anti-CD20) has been administered to patients with refractory CAPS and may be of adjunctive value in a selected population of patients. Eculizumab (anti-complement 5) has been reported in case reports as a last resort, and could be considered in those patients refractory to all other therapies.54,55 Pregnancy and antiphospholipid antibodies Pregnancy complications of APS include recurrent first trimester pregnancy loss, intrauterine growth restriction (IUGR), preeclampsia (PET), premature birth, and intrauterine death (IUD). Early miscarriages are reported in 26-35% and aPL-related PET, premature birth or foetal death are seen in 10–20% of APS pregnancies.2,56 Women with positive aPL do not all carry the same obstetric risk. The following parameters are associated with an increased risk: the presence of LAC, more than one aPL (especially triple positivity), IgG aPL (instead of IgM), previous thrombosis, previous pregnancy complications, associated autoimmune condition, and hypocomplementemia.57 At present, however, risk stratification does not direct different treatment strategies. The current standard treatment, based on low-dose aspirin and LMWH, increases the percentage of a successful pregnancy from 20% to 54-80%.58 In patients with obstetrical APS, aspirin and a prophylactic dose of LMWH should be administered. Depending on the context (concurrent SLE, maternal age, non-criteria clinical features), treatment of patients with fewer than three miscarriages, and therefore, formally not classified as having APS, can be considered. To prevent overtreatment in these cases, confirmation of miscarriage by ultrasound is recommended. During pregnancy, regular clinical pregnancy follow-up is sufficient; only in specified subpopulations (e.g. patients with SLE, chronic kidney disease, morbid obesity, triple positive antibody profile, and patients with a thrombotic event during pregnancy), follow-up (of foetal growth and hypertensive pregnancy complications) should be intensified. Platelets should be checked at least once every 10-14 days after starting LMWH to exclude heparin-induced thrombocytopenia. If pregnancy complications (foetal death or miscarriage) still occur despite combined treatment with low-dose aspirin and prophylactic dose of LMWH, the dose of LMWH can be increased to a therapeutic dose; reverting back to a prophylactic dose of LMWH after 36 weeks of gestation can be considered. Based on expert opinion, patients with APS who already use therapeutic anticoagulation before pregnancy, should be switched to therapeutic dose of LMWH and low-dose aspirin should be added. Anti-factor Xa levels should be periodically monitored (at least once every trimester) in patients receiving therapeutic doses of LMWH, depending on the context. If this strategy fails, further treatment should take place in an expert centre. The recommendations are again based on expert opinion and retrospective cohort studies and include, for example, the addition of hydroxychloroquine or intravenous immunoglobulins. This should however, be confirmed in randomized controlled trials.59-61 In the postpartum period there is an increased risk for thrombosis. Patients with APS with an indication of therapeutic anticoagulation before pregnancy, should be switched to VKA or continue with therapeutic LMWH. Obstetrical APS patients should receive prophylactic or intermediate doses (a randomized clinical trial to investigate optimal dosing is currently underway62) of LMWH during a period of six weeks postpartum.57 Withdrawal of antithrombotic treatment in APS As mentioned above it is recommended to treat thrombotic APS patients lifelong. However, as long-term anticoagulation therapy is associated with haemorrhagic complications, the question arises whether it is possible to withdraw this therapy in selected patients, especially those who have a long-term event free period or who no longer have positive aPL, also called seroconversion. Only a few (uncontrolled and small) studies have addressed this question. Criado-Garcia et al described the effects of anticoagulation withdrawal in six primary APS patients in whom aPL had disappeared.63 None of these patients experienced a thrombotic event during a follow-up of 21 ± 4.9 months. It should be mentioned, however, that these patients were at low risk for recurrence, as they only had a venous thrombosis history. Similar results were found by Coloma Bazan et al, who reported no thrombotic recurrences after anticoagulation withdrawal durign a median follow-up of 20 months in 11 primary APS patients (seven with history of venous thrombosis and four with obstetrical APS).64 However, in a more recent study, 30 APS patients with anticoagulation withdrawal were compared to a thrombotic APS control population without withdrawal. During a median follow-up of 51 months, anticoagulation withdrawal was associated with a higher risk of thrombotic relapse (HR 4.82). Predictive factors were male gender, anti-β2GPI-positivity and triple positivity at onset as well as persistence positivity over time. Predictive factors for low risk of relapse were aspirin prescription and aPL disappearance during follow-up.65 In conclusion, data is insufficient to draw firm conclusions concerning anticoagulation withdrawal in APS patients. The available data suggests that anticoagulation could be withdrawn in APS patients with a single provoked venous thrombotic event in the presence of a known transient precipitating risk factor (such as smoking, disease activity, oral contraceptive use) together with disappearance of aPL. In all other situations, the high risk of thrombotic relapse favours the continuance of anticoagulation treatment. Randomized studies with larger sample sizes are still needed to confirm these statements.66,67 The decision to withdraw anticoagulation in APS patients should be made in an expert centre. Position of rituximab, hydroxychloroquine, intravenous immunoglobulins Several immunomodulatory drugs are suggested for the treatment for patients with APS, although their exact use in the treatment of APS is unclear; these include. rituximab, hydroxychloroquine (HCQ), and IVIG. Most evidence is based on cohort studies, case series or expert opinion. Only a few randomised controlled trials (RCTs) are published. Furthermore, their application is strongly dependent on the different clinical situations. For example, in secondary APS in SLE patients, HCQ is strongly recommended, as it has been shown that HCQ has ‘thrombo-protective’ effects, resulting in fewer venous and arterial thrombotic events.68 In primary APS, limited data is available. One retrospective cohort study demonstrated strong reduction of aPL titres and a decrease in the incidence of arterial thrombosis recurrence by using HCQ.69,70 One RCT randomizing 40 APS patients to VKAs versus VKAs with HCQ showed a protective effect of adding HCQ upon venous thrombotic events.45 At this moment, HCQ is not included in standard care of primary APS, but refractory cases, including refractory obstetrical APS, might benefit from this treatment. Treatment with HCQ (200-400 mg per day) is safe during pregnancy and lactation. Rituximab and IVIG may be used in difficult-totreat APS patients, especially in those with CAPS or recurrent haematological non-criteria manifestations as thrombocytopenia, but not as standard of care of APS.71,72 Prophylaxis in aPL-positive patients without earlier events The question remains whether patients with obstetric APS or individuals with positive aPL without thrombotic events should be treated to prevent thrombosis (primary thromboprophylaxis). At present, there is insufficient evidence to support prophylactic treatment for all of these patients.8,73 However, in patients with more risk factors for thrombosis (such as obesity, smoking, higher age) and/ or high-risk aPL profile (e.g., triple positive), low dose aspirin might be beneficial. In any case, attention should be paid to avoid or to treat any associated cardiovascular risk factors, e.g. using antihypertensives or cholesterollowering agents and avoidance of smoking, etc. Also, the administration of oral contraceptives should be used with caution and with counselling.74 Lastly, prophylaxis of venous thrombosis using LMWH is required for patients in situations associated with increased risk of thrombosis, such as surgical procedures, plaster casts, and those requiring bed rest.  CONCLUSION  APS is a rare and heterogeneous disease and as a result, well-designed and well-conducted clinical trials are scarce and the development of a formal guideline is difficult. However, by combining data from completed clinical intervention trials together with observational data and data from research in other thrombotic and/ or inflammatory conditions, recommendations for clinical practice can be formulated. Current national and international initiatives – such as the Dutch Arthritis Research and Collaboration Hub (ARCH) and the European Reference Network on Rare and Complex Connective Tissue and Musculoskeletal Diseases (ERN-ReConnet) – are aiming to structure the care for APS patients to offer a unique future opportunity to collect longitudinal clinical data on APS treatment and outcomes. Until a formal guideline has been made, this consensus paper fills the gap between evidence-based medicine and daily clinical practice for the care of APS patients. ACKNOWLEDGEMENTS KdL and ML performed the literature search and wrote the first article draft. All authors reviewed and commented on the first article draft and attended the first consensus meeting. JS and ML wrote the second article draft. All authors reviewed and commented on the second article draft. ML wrote the final report. All authors read the final report and agreed with the text. DISCLOSURES All authors declare no conflicts of interest. FUNDING This work was supported by the Arthritis Research and Collaboration Hub (ARCH) Foundation. REFERENCES Myakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Hemost. 2006;4:295-306. Cervera R, Piette JC, Font J et al. Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients. Arthritis Rheum. 2002;46:1019-27. Abreu MM, Danowski A, Wahl DG, et al. The relevance of "non-criteria" clinical manifestations of antiphospholipid syndrome: 14th International Congress on Antiphospholipid Antibodies Technical Task Force Report on Antiphospholipid Syndrome Clinical Features. Autoimmun Rev. 2015;14:401-14. Hughes GRV, Khamashta MA. Seronegative antiphospholipid syndrome. Ann Rheum Dis. 2003;62:1127. Galli M, Luciani D, Bertolini G, Barbui T. 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PeerJ. 2019 Mar 19;7:e6737. doi: 10.7717/peerj.6737. eCollection 2019.

Journal of Immunology Research is a peer-reviewed, Open Access journal that provides a platform for scientists and clinicians working in different areas of immunology and therapy. The journal publishes research articles, review articles, as well as clinical studies related to classical immunology,...

Sj&ouml;gren&rsquo;s syndrome (SS) is characterized by extensive lymphocytic infiltration of the salivary and lacrimal gland (LG), resulting in acinar cell destruction and organ dysfunction. The underlying pathogenesis of SS remains largely unknown, and studies historically focus on defining...

RMD Open. 2018 Oct 18;4(Suppl 1):e000789. doi: 10.1136/rmdopen-2018-000789. eCollection 2018.

Various cytokines, including interferon (IFN)-&gamma; and IL-17, are augmented, and autoreactive T cells and B cells are activated in the immune pathogenesis of Sj&ouml;gren&rsquo;s syndrome (SS). In particular, IFNs are involved in both the early stages of innate immunity by high level of type I...