Sunanda V Kane

Dr Andrew Robinson
email: andrew.robinson@srft.nhs.uk

Understanding adherence to medication in ulcerative colitis: innovative thinking and evolving concepts

Keywords: Inflammatory bowel disease < Disease-based, Ulcerative colitis < Disease-based, X keyword Inflammatory bowel disease, ulcerative colitis, adherence, necessity-concerns framework

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SUMMARY Background

Non-adherence to medication is an important challenge in the treatment of ulcerative colitis (UC). Epidemiological studies have demonstrated that nonadherence affects 40-60% of UC patients, and has a substantial impact on the course of the disease. In one study, non-adherence was associated with a fivefold increase in the risk of relapse.

Aims

We review factors contributing to non-adherence to UC therapy, and emerging concepts in addressing the problem.

Results

Adherence is a complex, multifactorial issue, with factors varying between patients and changing over time. Identifying patients at risk of non-adherence is a key first step in targeting interventions to improve adherence. However, investigations of single factors that affect adherence have provided conflicting evidence. Evaluating physical and perceptual barriers to adherence, and acknowledging the role of patients' beliefs and concerns regarding treatment, provides valuable insights into the causes of non-adherence. This allows development of targeted interventions to improve adherence to UC therapy. Clinical tools to identify patients at risk of non-adherence are being developed to facilitate this approach.

Conclusions

To help patients adhere better to maintenance therapy for UC, it is vital to consider patients' beliefs and concerns, and to evaluate and address both physical and perceptual barriers to adherence.

Background

Non-adherence to medication is highly prevalent and is a particular challenge in the treatment of long-term conditions. Recent reports from the World Health Organization (WHO) and the National Institute for Health and Clinical Excellence (NICE) indicate that, in patients with chronic, long-term illnesses, as many as 30-50% are non-adherent. 1;2 Asymptomatic conditions, such as hypertension, have particularly low rates of adherence. 1;2 Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disease of the colon, [START_REF] Kane | Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis[END_REF] characterised by periods of acute, active disease interspersed with phases of asymptomatic remission. The onset of relapses is both unpredictable and associated with a marked reduction in quality of life. [START_REF] Han | Predictors of quality of life in ulcerative colitis: the importance of symptoms and illness representations[END_REF] For both the induction and maintenance of remission in mild to moderate UC, 5-aminosalicylic acid (5-ASA) is a key first line treatment. [START_REF] Carter | Guidelines for the management of inflammatory bowel disease in adults[END_REF] Studies of adherence in UC patients demonstrate that 40-60% of patients are not fully adherent (defined in these studies as taking less than 80% of their medication) to their prescribed 5-ASA regimens. 6;7 A number of terms have been used to describe the concept of non-adherence.

Compliance, adherence, concordance and persistence have all been commonly used to define instances in which a patient is not strictly following the prescribed regimen, yet each has a distinct meaning. -5 -differences between these terms in their 2006 report for the National Institute for Health Research (NIHR), and the definitions are summarised in Table 1. [START_REF] Horne | Concordance, adherence and complicance in medicine taking: a conceptual map and research priorities[END_REF] The definition of compliance implies that the patient is not involved in the decision making process, whereas adherence implies the need for agreement between patient and physician. Persistence is largely used when discussing long-term aspects of adherence in chronic illnesses, as adherence will often change over time. Consequently, and in line with the recommendations of the NICE and WHO reports, adherence is used as the term of choice in this article.

With non-adherence to medication being so prevalent, considerable efforts are being invested to elucidate the causative factors, and to develop successful interventions to promote adherence. However, adherence is a complicated issue for healthcare providers to successfully address. The term 'non-adherence' encompasses a very broad spectrum of behaviour, from a patient who misses an occasional dose, to a patient who does not take any of their medication. In addition, these patients may have very different reasons for not taking their medication, and will respond differently to measures to improve adherence. In this review, we outline the successes and shortcomings of traditional approaches to adherence in UC therapy, discuss the newly emerging concepts for dealing with this important issue, and elucidate how these may be translated into clinical practice.

F

Clinical impact of non-adherence to ulcerative colitis therapy

The reported levels of non-adherence in UC therapy indicate that as many as 40-60% of patients are not fully adherent to their medication. 6;7 This has a significant impact on the course of the disease. In a non-interventional study, Kane et al. followed a cohort of 99 UC patients who were in remission, and demonstrated that there was a strong correlation between non-adherence (defined as taking less than 80% of the prescribed medication) and disease flares. In total, 89% of adherent patients maintained remission over the 2-year period compared to only 39% of the non-adherent patients. [START_REF] Kane | Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis[END_REF] In addition, the authors calculated that patients in remission who are non-adherent are five times more likely to relapse than adherent patients (hazard ratio 5.5 [95% CI 2.3-13], p<0.001). [START_REF] Kane | Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis[END_REF] Furthermore, there is evidence to suggest that 5-ASA may have a chemoprotective effect against colorectal cancer. 9 It could, therefore, be postulated that adherence to UC therapy may also reduce the risk of patients developing this disease. [START_REF] Velayos | Effect of 5-aminosalicylate use on colorectal cancer and dysplasia risk: a systematic review and metaanalysis of observational studies[END_REF] Studies investigating the healthcare costs of UC patients suggest that nonadherence not only has a substantial impact on a patient's health, but also confers a significantly higher cost to the healthcare provider. A study of over 4000 UC patients receiving 5-ASA maintenance therapy demonstrated that nonadherence (defined as failure to refill a prescription) is strongly correlated with higher healthcare costs, with non-adherence being associated with a two-fold increase in inpatient costs, compared to adherent patients (p<0.01). [START_REF] Kane | Medication non-adherence is associated with increased medical health care costs[END_REF] In addition, the same study found that adherence to 5-ASA therapy for UC resulted in 12.5% lower total annual healthcare costs, compared to non-adherent patients, despite increased medication expenditure. [START_REF] Kane | Medication non-adherence is associated with increased medical health care costs[END_REF] These findings indicate that increased adherence not only provides benefits to patients, in terms of health and quality of life, but may also result in significant savings for healthcare providers.

Conventional approaches to identifying non-adherence

In order to effectively target interventions and initiatives to improve adherence, it is important to identify patients who are non-adherent. This is often difficult in routine clinical practice, and there is currently no widely accepted standard method for measuring adherence. In clinical trials, adherence can be measured based on assessments such as pill count, patient self-reporting or prescription refills. One commonly used measure is the Medication Possession Ratio (MPR), which is the ratio of the number of days' supply of medication obtained by a patient to the number of days in the study period. [START_REF] Sclar | Effect of health education in promoting prescription refill compliance among patients with hypertension[END_REF] However, in real-world situations, the use of such detailed measures of adherence is not feasible in everyday practice.

Therefore, the identification of factors that are predictive of non-adherence would be useful. However, adherence is a complex and multifactorial issue, in which a wide variety of factors play a role yet no single factor predominates (Figure 1A). [START_REF] Kane | The challenge of compliance and persistence: focus on ulcerative colitis[END_REF] Identifying demographic characteristics that are predictive of non-adherence represents an attractive possibility; however, demographic studies have provided -8 -contradictory evidence. 3;7;10;13;14 Conversely, disease-related factors do have a significant effect on adherence, with those patients who experience more frequent and more severe flares being more likely to have higher rates of adherence. 3;7;15 Indeed, adherence rates not only vary between patients, but also over time. Anecdotal observations have suggested that patients who adhere to therapy during acute exacerbations of UC may become less adherent during periods of quiescent disease. This is analogous to the very well known situation of patients who are prescribed antibiotics: while symptoms are present there is ample motivation to take the medication, yet when the symptoms subside many patients will not complete the prescribed course.

One factor that has commanded much attention in recent years, particularly relating to 5-ASA therapy, is the therapeutic regimen. A factor that stands out for many patients is that 5-ASA therapy may be delivered rectally, and in this case the discomfort and potential inconvenience of the therapy may affect adherence.

However, the true impact of rectal 5-ASA therapies on adherence remains poorly established, and the published literature provides conflicting evidence on the levels of adherence when comparing rectal and oral 5-ASA therapy. 13;14 More recently, there has been considerable debate regarding the complexity of oral 5-ASA regimens. Historically, 5-ASA was prescribed in divided doses, threeor four-times daily, but modern 5-ASA formulations have enabled the simplification of regimens to once-or twice-daily (OD or BD). A meta-analysis by Claxton et al., drawing data from several disease areas, has suggested that less frequent dosing is related to higher adherence. 16 However, significant differences were only demonstrated between once-daily and three-times daily (p=0.008), or four-times daily (p<0.001) dosing. 16 No significant difference was found between OD and BD regimens. 16 Similarly, studies specific to UC have failed to demonstrate a consistent significant relationship between dosing frequency and adherence. 7;17 Although one study reported higher adherence to once-daily than twice-or three-times daily regimens after 3 months, by 6 months there was no significant difference between the regimens. 17 In a recent systematic review, Jackson et al. noted that the number of daily doses is not consistently related to non-adherence, and none of the significant relationships that have been observed relate to OD dosing compared to BD. 18 Additionally, no significant differences in adherence have been found between the various oral 5-ASA formulations or different daily dosages. 7;10 Although there remains much discussion regarding the optimum dosage of 5-ASA, a fact that places additional context on patients' adherence decisions, the impact of such controversy on adherence has not been evaluated and so remains unclear.

Taken together, these data suggest that, while individual patients may be more adherent to certain regimens, or have preferences for different oral 5-ASA formulations, these factors are unable to provide a universal solution to nonadherence in UC patients. 7;10;13;16;17

Emerging concepts: anticipating non-adherence in ulcerative colitis

Traditional approaches to identifying and addressing non-adherence in UC, based on generalised strategies focussing on risk factors (such as demographics, regimen or formulation), have given disappointing results. [START_REF] Haynes | Interventions for enhancing medication adherence[END_REF] Consequently, leading experts on medicines adherence have proposed alternative approaches, whereby non-adherence is explained based on key patient-specific issues. [START_REF] Horne | Representations of medication and treatment: advances in theory and measurement[END_REF][START_REF] Horne | Treatment perceptions and self regulation[END_REF][START_REF] Horne | Patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease[END_REF] In this way, it adherence is approached not by investigating risk factors contributing to non-adherence across large populations, but rather, by switching the emphasis towards addressing the specific reasons behind non-adherence in individual patients.

The newly emerging approaches to non-adherence are summarised in Figure 1B. These approaches combine a number of models, in particular the practicalities and perceptions approach and the necessity-concerns framework. [START_REF] Horne | Representations of medication and treatment: advances in theory and measurement[END_REF][START_REF] Horne | Treatment perceptions and self regulation[END_REF] Non-adherent patients can be divided into two classifications: unintentional and intentional (Figure 1B). [START_REF] Horne | Adherence to medication: a review of existing research[END_REF] In the former case, patients are unable to take their medication for reasons beyond their control. Conversely, intentionally nonadherent patients make a conscious decision not to take their medication.

According to the perceptions and practicalities approach, for both the intentional and unintentional groups, there may be a combination of practical and perceptual In contrast, perceptual barriers are more complex, and are based on an internal negotiation between the perceived necessity of the treatment and any concerns relating to it (Figure 1B).

To illustrate, an intentionally non-adherent patient may choose not to take their medication for a variety of perceptual reasons, such as a fear of side effects or a belief that it isn't necessary, but may also be influenced by practical factors such as cost. Equally, one of the most common reasons cited by patients for nonadherence is forgetfulness, which represents an important element of unintentional non-adherence, influenced by both practical (e.g. complex dosing regimens) and perceptual factors (e.g. lack of belief in the necessity of the medication). One confounding factor in this case is that forgetfulness may be misreported by patients; for example, a patient may believe that their doctor has certain expectations of them, and feel obliged to live up to them. for low uptake of free tuberculosis check-ups. [START_REF] Rosenstock | Historical origins of the health belief model[END_REF] A further development to the necessity-concerns framework is the addition of attitudinal analysis. [START_REF] Horne | Patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease[END_REF] Quantitative assessment of patients' beliefs and concerns, using the BMQ, allows patients to be categorised into four "attitudinal groups"accepting, ambivalent, indifferent and sceptical (Figure 2). [START_REF] Horne | Patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease[END_REF] Patients who report that they believe in the necessity of their medication, for example those that agree with the statement in the BMQ "My life would be impossible without this medication", would gain high scores for necessity. They would, therefore, be categorised as accepting or ambivalent (Figure 2). Similarly, patients with strong concerns about their medication (e.g. those who agree with the BMQ statement "Having to take this medicine worries me"), would be classed as sceptical or ambivalent. It would seem logical that these attitudinal groups might have a -13 -relationship to adherence. Indeed, this hypothesis has been borne out by experimental observations. A large survey was conducted, collecting data from 1871 patients suffering from inflammatory bowel disease. Analysis of these data demonstrates that significant relationships exist between adherence rates and attitudinal grouping. [START_REF] Horne | Patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease[END_REF] Patients categorised into the accepting group, who believe their medication is necessary and have few concerns about their medication, have the highest levels of adherence. In contrast, those in the sceptical group, who are more concerned and less convinced of the necessity of their medication, have the lowest levels of adherence (Figure 3). [START_REF] Horne | Patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease[END_REF] The necessity-concerns framework and attitudinal analysis provide methods of quantifying the complex balance of factors that affect adherence as a whole. This allows non-adherence to be anticipated, and patients at risk of non-adherence can be identified.

Addressing non-adherence: new clinical approaches

Evaluation of patients' attitudes and beliefs enables the identification of the root causes of non-adherence, and, therefore, provides potential targets to assist in improving adherence. This approach makes it possible to establish a framework that allows specific interventions to be developed for individual patients, thereby maximising the chance of successfully improving adherence. To facilitate such approaches, it would be beneficial to develop tools to identify patients' beliefs and concerns. These tools could be used in a clinical setting to screen patients and identify those at risk of non-adherence, while simultaneously identifying the probable causes of non-adherence, should it manifest. To date, no standardised approach has been adopted. However, several clinics have piloted short questionnaires, based on the more detailed BMQ and MARS tools, to be completed by patients in the clinic -while waiting to see their doctor, for example. These can be quickly evaluated to identify potentially non-adherent patients and rapidly identify their reasons, allowing specific issues to be addressed.

Given that patients' beliefs and concerns play a key role in adherence, [START_REF] Horne | Patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease[END_REF] they should, therefore, be taken into account during consultations. In line with the guidelines from NICE, treatment decisions should be reached following an informed dialogue between patient and physician. [START_REF]CG76: Medicines adherence: involving patients in decisions about prescribed medicines and supporting adherence[END_REF] To illustrate with a previous example, a patient who is involved in their treatment decisions, and believes that the treatment is appropriate and effective, may be more motivated to take their medication. Such a patient may also be less likely to forget to take their medication. In this way, informed discussion may positively influence both intentional and unintentional non-adherence.

Closely linked to this is the concept of health literacy. Patients may not fully understand their condition or the purpose of their medication, so an element of -15 -patient education could be included in consultations. There are a number of sources of information and support available throughout the wider healthcare system. In particular, it may be beneficial to direct patients to relevant patient groups, which can provide valuable information and support. A well-informed patient may be better equipped to make reasoned, logical decisions regarding their treatment. Additionally, patient education could help to reinforce the necessity of maintenance therapy. For example, patients should be reminded of the evidence that there is a far higher risk of relapse if they do not adhere to their medication. [START_REF] Kane | Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis[END_REF] In addition, it may be beneficial to discuss the potential chemoprotective effects of 5-ASA. [START_REF] Velayos | Effect of 5-aminosalicylate use on colorectal cancer and dysplasia risk: a systematic review and metaanalysis of observational studies[END_REF] Evidence suggests that adherence varies with time, with patients often becoming less adherent with increasing duration of treatment. 13 Maintaining adherence for extended periods of time is defined as persistence. A study investigating the prescription refill data from more than 3500 UC patients over a 2-year period demonstrated that there is a marked decrease in adherence over time, with 57% of patients adherent at 3 months, but only 55% of those patients were still adherent after 12 months. 13 This suggests that patients may need some reinforcement of the importance of their medication at certain time points. This is the premise behind a concept which could be termed "interval empathy", whereby patients are contacted at certain times when the risk of non-adherence is judged to be greater. For example, after 3 months, a patient is likely to be in remission but may be at increased risk of non-adherence. The patient could be -16 -contacted to reinforce the message that continuing with their treatment is beneficial to their health. This could also give patients an opportunity to voice any new concerns that may have arisen since their last consultation. Various approaches are being piloted, with doctors or specialist nurses contacting patients by means such as telephone, text message or email.

Whilst predictive tools to identify patients at risk of non-adherence are likely to be very useful, it is still important to monitor adherence between consultations and identify, at the earliest opportunity, those patients who slip through the net and do still become non-adherent. One approach that could be developed in the future might be the adoption of a more integrated approach, whereby pharmacies and primary care could coordinate with secondary care clinicians to identify signs that a patient is not adhering to their medication. This may assist clinicians who, when encountering relapsing patients, may wish to take steps to ensure that the prescribed regimen is being adhered to before starting a new course of therapy.

Conclusions

Non-adherence to medication is a significant issue in the treatment of chronic conditions. Indeed, it has even been suggested that, due to the broad applicability of any interventional measures, a successful strategy to improve medication adherence may have a greater benefit to the health of the general population than improvements in medical therapies. [START_REF] Haynes | Interventions for enhancing medication adherence[END_REF] In the treatment of UC, Assessment of adherence could form a key element of consultations with relapsing patients, to ensure that the prescribed regimen is being adhered to.

There is also a need for improved health literacy and patient education, permitting an informed dialogue between patient and physician. This could facilitate a better agreement on the proposed treatment regimen, and potentially lead to increased adherence.

Further studies are needed to optimise the application of the necessity-concerns framework in the clinic, but the potential benefits of such an approach could provide significant improvements, for both patients and healthcare providers, in the management of UC.

Authors' declaration of personal interests:

Dr Kane Compliance is defined as "the extent to which the patient's behaviour matches the prescriber's recommendations".

Adherence is defined as "the extent to which the patient's behaviour matches agreed recommendations from the prescriber".

Concordance is a relatively new concept, predominantly applied in the UK, defined as a two-way relationship between patient and physician, where treatment decisions are discussed and the treatment of choice is the one most acceptable to both parties.

Persistence is defined as the continued adherence over time to the prescribed medication.

  that affect adherence. Practical barriers may include the size of the pills, the cost of prescriptions, the route of delivery (e.g. rectal or oral), or side effects.

  The necessity-concerns framework aims to further understand perceptual barriers to adherence. Although practical barriers to adherence can be relatively simple to evaluate, whereas perceptual barriers, based on patients' beliefs and concerns, are often far more complex. The concept of a balance between beliefs and concerns affecting health decisions was first espoused by researchers at the US Public Health Service in the 1960s, in an attempt to understand the reasons

  Horne et al. took this concept, applied it to the study of non-adherence in chronic illness, and developed the necessity-concerns framework, 25 which describes how adherence is affected by the interplay between a patient's belief in the necessity of their medication and their concerns relating to it. To develop the necessity-concerns framework, Horne et al.developed standardised questionnaires (the Beliefs about Medicines Questionnaire [BMQ] 26 and the Medication Adherence Report Scale [MARS] 27 ) to allow quantitative analyses of patients' perceptions and levels of adherence to be performed. The necessity-concerns framework allows a detailed insight to be gained into the root causes of non-adherence, thus providing possible factors to be targeted to encourage adherence.

  clinical and financial impacts correlated with low adherence to maintenance therapy, resulting from a substantially increased risk of relapse.3;10 Therefore, addressing non-adherence represents a priority issue for UC therapy. However, adherence is a complex issue, with multiple factors all contributing to overall levels. This has made it difficult to target effective interventions based on the traditional approach of employing generalised strategies based on specific risk factors. For example, no clear evidence has demonstrated that once-daily dosing is superior to twice-daily, in terms of adherence. Evaluating the complex combination of barriers to adherence is an essential step towards improving adherence rates in UC.Levels of adherence are determined by a mixture of both physical and perceptual factors, based on a subtle balance of an individual patient's beliefs and concerns.The necessity-concerns framework and attitudinal analysis provide a clear insight into patients' reasons for non-adherence. Furthermore, they also allow the balance between beliefs and concerns to be evaluated and related to the likelihood of non-adherence. Using this framework as a basis, clinical tools can be developed to facilitate the rapid identification of patients at increased risk of non-adherence and enable specific interventions to be targeted to individual patients. non-adherent patients is also important in addressing the issue.

Figure 1 A

 1 Figure 1

Figure 2 Figure 3 FFigure 1 .Figure 1 .Figure 2 .Figure 3 .

 231123 Figure 2

  Horne et al. clearly defined the

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Table 1 .

 1 has served as a consultant to Warner Chilcott UK Ltd, Ferring Pharmaceuticals and Shire Pharmaceuticals Group plc, and has received research funding from Shire Pharmaceuticals Group plc. Writing support was provided by David Finch and Ian Watson of Acumen Healthcare Communications Ltd, funded by Warner Chilcott UK Ltd. Attitudinal analysis. Based on their beliefs and concerns, patients can be classified into four distinct attitudinal groups. 22 Repinted with permission of John Wiley & Sons, Inc. Copyright © 2009, The Crohn's and Colitis Foundation of Definitions of the key terms commonly used to describe the concept of non-adherence 8

	Dr Robinson has served as a speaker, a consultant or an advisory board
	member for Procter & Gamble Pharmaceuticals Ltd, Warner Chilcott UK Ltd,
	Ferring Pharmaceuticals Ltd, Dr Falk Pharma GmbH, Abbott Laboratories Ltd,
	and Chiesi Ltd.

(18) Jackson CA, Clatworthy J, Robinson A, Horne R. Factors associated with non-adherence to oral medication for inflammatory bowel disease: a systematic review. Am J Gastroenterol 2010;105(3):525-39.


Adnan Alahmad

Klaus-Dieter Preuss

Joachim-F ; Schenk

Wolfgang Füreder

Hubert Schrezenmeier

Nikolaus Müller-Lantzsch

Michael Pfreundschuh

Jörg Schubert
email: jschubert@evkhamm.de

Prof. Dr. med Jörg Schubert

Desmoplakin and MPP1 as target antigens in patients with Paroxysmal nocturnal Hemoglobinuria

Keywords: Paroxysmal nocturnal Hemoglobinuria (PNH), M-phase phosphoprotein Paroxysmal nocturnal Hemoglobinuria (PNH), M-phase phosphoprotein 1 (MPP1), desmoplakin, aplastic Anaemia, antigens

Paroxysmal nocturnal Hemoglobinuria (PNH) is a unique disorder characterized by chronic intravascular hemolysis. The characteristic biochemical feature in PNH is a lack of glycosyl phosphatidyl inositol anchored proteins (GPI-APs) due to an incomplete assembly of the GPI anchor. PIGA genes are involved in the biosynthesis of the GPIanchor and the binding of the anchor to proteins in the endoplasmic reticulum (ER). A PIGA gene has been cloned, and found to be mutated in all cases of PNH reported to date (Miyata et al, 1993;Ostendorf et al, 1995).

In PNH, one or a few abnormal clones expand to replace almost the entire haematopoietic stem cells pool. These clones do not appear to be "malignant", but underlie a normal cellular regulation. In addition, GPI-deficient cells with PIGA mutations occur very frequently at low frequency in normal individuals but do not expand in competition with normal hematopoietic cells. In order to develop PNH, two events are required: first, the occurrence of a GPI-deficient clone arising in a multipotent hematopoietic stem cell, second an additional event that enables the expansion of the PNH clone over the residual normal hematopoiesis (Hillmen et al, 2006). The clonal expansion was proposed to be a consequence of a relative growth advantage for the PNH cells. Because PIGA mutant cells have no intrinsic growth advantage over normal cells, a second event responsible for clonal expansion is mandatory (Araten et al, 1999).

At least two hypotheses have been suggested for the explanation of the clonal expansion in PNH. According to the first hypothesis, clonal selection eliminates normal hematopoietic stem cells, but spares PIGA mutant clones due to their non-expression of the GPI anchor. The second hypothetical model comprises a second internal event within the mutant cells such as a second somatic mutation leading to a predominant proliferation of PIGA mutant stem cells. The close association of PNH with aplastic anaemia suggests that the clonal selection pressure might be immune-mediated. According to this hypothesis, normal hematopoiesis is suppressed by the immune system, presumably, F o r P e e r R e v i e w 4 either directly or indirectly through one or more GPI-linked antigens, and therefore this attack spares the GPI-deficient clone (Inoue et al, 2006).

The study of Horikawa (1997) provides evidence, that blood cells obtained from patients with PNH are less susceptible to both spontaneous and ligand-induced apoptosis in vitro than those from healthy volunteers (Horikawa et al, 1997). In other study, it was shown that leukemic cells with PIGA mutations (GPI -cells) are less susceptible to killing by natural killer (NK) cells in vitro than the control counterparts (GPI + cells), which in turn supports the selective survival advantage theory. The study also implied that neither CD55 nor CD59 would be target antigens for such NK-mediated killing (Nagakura et al, 2002). Murakami et al (2002) provided the first experimental evidence that supports the immunologic selection hypothesis for the clonal expansion of PNH cells. They showed that GPI hematopoietic cells become dominant in a mouse model due to selection by allogeneic CD4 + T cells. In addition, GPI antigen-presenting cells do not stimulate antigen-specific and allogeneic CD4 + T cells efficiently, which suggests that some GPIanchored proteins on APCs act as a ligands for co-stimulatory molecules on T cells (Murakami et al, 2002).

We hypothesized that normal blood cells might present antigens which are targets for the immune system attack induce an autoimmune response that eventually results in the expansion of the PNH clone either by no expression of such antigens or by less susceptibility against cytotoxic attack of autoimmune T lymphocytes. So far, the significance of autoantibodies against hematopoietic antigens in PNH sera is unclear. On the one hand, it might confirm the role of T lymphocytes in the pathophysiology of PNH; on the other hand, the identification of such antigens might demonstrate that such immune reactions in PNH could recognize mutated antigens and thus demonstrate the presence of altered antigens in PNH.

Isolation of positive clones

The same amount was taken from each of the 10 PNH sera and mixed together. These mixed sera were diluted 1:100 in 1×TBS containing 0.5% (wt/vol) low-fat milk. For preservation, 0.01% NaN 3 was added. These diluted sera were stored at +4°C and used for SEREX screening to identify candidate target auto-antigens in PNH, SEREX was F o r P e e r R e v i e w 6 performed as described previously (Sahin et al, 1997). We screened a human fetal liver cDNA library (Takara Bio Europe, Saint-Germain-en-Laye, France) known to contain a high proportion of cells derived from hematopoietic stem/progenitor cell HSPC. Briefly, XL1-Blue Escherichia coli (Takara Bio Europe, Saint-Germain-en-Laye, France) was transfected with recombinant phages, plated on agar plates, and cultured at 37°C. Expression of recombinant proteins was induced by incubating the bacterial lawns with isopropyl β-D-thiogalactoside (IPTG). The released proteins were transferred to nitrocellulose filters by incubation at 37° C for 9 minutes. Filters were then washed in TBST (10 mM Tris [tris (hydroxymethyl) amino methane], 150 mM NaCl, 0.05% Tween 20, pH 8.0) and blocked overnight with blocking buffer (5% wt/vol non-fat dry milk in TBST). Filters were then incubated at 4˚C overnight with diluted patients' sera. Specific binding of antibody to recombinant protein was detected by incubation with alkaline phosphatase-conjugated goat antihuman IgG antibody (Dianova, Hamburg, Germany) diluted at 1:7500. Visualization of the antigen-antibody complex was accomplished by staining with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Roth, Karlsruhe, Germany). Complementary DNA inserts from positive clones were subcloned, purified, and in vivo excised to plasmid forms (Takara Bio Europe, Saint-Germain-en-Laye, France) according to the manufacturer's instructions. The DNA inserts were subsequently sequenced with appropriate sequencing primers.

Phage assay for detecting serum antibodies

Positive clones detected in the library screening were mixed with non-reactive phages of the cDNA library as an internal negative control at a ratio of 1:10, and used to transfect bacteria as described above. Each PNH serum was diluted 1:100 and used individually to detect antibodies against the positive clones, the PNH pool was used as a control positive serum.

Hematopoietic progenitor cell assay

CD34 + cells were purified from bone marrow of PNH-1 and a non-PNH patient (as control) using magnetic cells sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) F o r P e e r R e v i e w 7 according to the manufacturer's instructions. Total RNA was isolated using Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was constructed using oligodt18 primer and SuperScript™II (Invitrogen, Carlsbad, NM, USA).

Expression pattern of mRNA

To evaluate the mRNA expression pattern of identified clones, RT-PCR was performed by amplification of the cDNA from normal and PNH CD34 + cells. The gene-specific primers used in RT-PCR reactions were designed to amplify coding sequences of corresponding serologically defined antigens and synthesized commercially (MWG, Biotech, Ebersberg, Germany). 0.8 µl of first-strand cDNA was amplified using 0.2 U AmpliTaq Gold (Perkin Elmer, Rodgau, Germany), 1 µl of dNTP (10 mMol of each dATP, dTTP, dCTP, dGTP), 1 µl of each primer and 3 µl of PCR buffer in a reaction volume of 30 µl. The amplification was performed with 35 cycles at a denaturation temperature of 94°C (1 min/cycle); an annealing temperature of 60°C for Desmoplakin and 51°C for MPP1(1 min/cycle); and an extension temperature of 72°C (1 min/1kb).

The PCR products were analyzed by agarose gel electrophoresis with 1% agarose gel. We used GAPDH as housekeeping gene, and testis cDNA as DNA positive control. The PCR products were excised from the gel, purified by gel band purification kit (GE Healthcare, Piscataway, NJ, USA) and sequenced.

Fusion Protein Construction and Expression in HEK293 Cells:

Using primers specific for desmoplakin and MPP1, we isolated cDNA inserts from positive phages and subcloned them in frame into a pSectag/FRT/V5-His Topo TA expression vector (Invitrogen, Carlsbad, NM, USA). and pSfi-Express-HA vector, respectively. The pSfi-Express-HA is a derivative of pEGFP-C1 vector (Takara Bio Europe, Saint-Germain-en-Laye, France); the plasmid has a neomycin resistance gene for selection in mammalian cells. We used human embryonic kidney 293 (Hek 293) cells for transfection. The induction and affinity purification of fusion proteins was performed according to the manufacturer's instructions. Western blot analysis was applied to confirm size and specificity using a (1:3000) mouse anti-His monoclonal antibody (Sigma, St Louis, MO, USA) and a (1: 500) MPP1-specific mouse polyclonal antibody

Western blot analysis

Purified proteins were prepared in sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of protein were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, Mass, USA), and incubated with blocking buffer overnight. Immunoblots were performed at 1:500 dilution of patient's serum (if not otherwise indicated), mouse anti-His monoclonal antibody, or MPP1 mouse polyclonal antibody (Abnova, Taipei City, Taiwan) and desmoplakin 1&2 mouse monoclonal antibody, respectively. Immunodetection was performed by incubation with (1:10.000) horseradish peroxidase-conjugated antihuman IgG (Promega, Madison, WI, USA) or (1:5000) anti-mouse IgG secondary antibody (Promega, Madison, WI, USA) as indicated by the host origin of the primary antibody and developed by chemiluminescence (NEN Life Science Products, Boston, USA).

Titration of antibodies by ELISA

Enzyme-linked immunosorbent assay (ELISA) plates were coated with mouse anti MPP1 antibody for MPP1 and rabbit anti mouse antibody followed by mouse anti-desmoplakin antibody for desmoplakin. Plates were washed and blocked overnight at 4°C with 5% non-fat dry milk in TBST. Patients' sera were added to a final dilution of 1:1000 to 1:5000 and incubated at room temperature. After washing, the plates were incubated with (1:7500) alkaline phosphatase-conjugated goat antihuman IgG antibody (Promega, Madison, WI, USA) at room temperature. Finally, the plates were washed and incubated with p-nitrophenyl phosphate (PNPP) substrate (Pierce, Rockford, IL, USA) at room temperature, and the optic density (OD) at 405 nm was determined. A positive reaction was defined as an absorbance value exceeding the mean OD absorbance value of sera from healthy donors by three standard deviations.

Results

The SEREX approach was applied to identify auto-antigens in the sera of patients with PNH. We screened a human fetal liver cDNA library because a high proportion of fetal liver cells share characteristics with pluripotent HSPC. By screening more than 1 x 10 6 phage plaques with 1/100 diluted sera from 10 PNH patients we identified the products of two genes that were recognized by sera of PNH patients. Lineage-specific expression was determined by searching the National Centre for Biotechnology Information (NCBI) UniGene database (http://www.ncbi.nlm.nih.gov/UniGene/). The first antigen was Mphase phosphoprotein 1 (MPP1). MPP1 is a member of a set of mitotic phosphoproteins specifically recognized by the MPM2 antibody at the G2/M transition. The second antigen is encoded by the desmoplakin gene. Desmoplakin is an obligate component of functional desmosomes that anchors intermediate filaments to desmosomal plaques. There was no sufficient information about the tissue expression of desmoplakin, but it is known that desmosomes are major sites of intercellular contact found in a variety of cells such as epithelial cells, cardiac myocytes, arachnoidal cells of meanings, and dendritic cells of germinal centres in lymph nodes (Virata et al, 1992).

In addition, individual sera from 10 PNH patients and 20 healthy controls were analyzed for immune-reactivity against MPP1 and desmoplakin clones. Three and four of the 10 PNH sera showed a positive reaction against MPP1 and desmoplakin, respectively.

Antibodies against MPP1 were detected in PNH sera 1, 5, and 9, that is in three of the four sera with the most elevated LDH. Antibodies against desmoplakin were detected in one 1/20 sera from healthy controls and in four patients (5,6,7,10), without any obvious correlation to the clinical characteristics of the desmoplakin-antibody positive patients (Tab. 1).

Detection of IgG antibodies against desmoplakin and MPP1 by ELISA

In order to determine the titres of antibodies against the two gene products in the sera of patients with PNH and healthy controls, sera were tested for reactivity against desmoplakin and MPP1 by ELISA. A serum was considered positive if its absorbance value exceeded the mean absorbance value of sera from healthy donors by 3 standard deviations. The results of ELISA for MPP1 are shown in figure 1a. Three out of 10 PNH sera exhibited an optic density higher than the cut-off in the MPP1 ELISA, but none of the five aplastic anaemia sera was positive. Desmoplakin reactivity was detected in 4 of the 10 PNH sera, but in none of the aplastic anaemia sera. A positive reaction against desmoplakin was detected in one normal serum but the OD of all positive PNH Sera (OD = 1.22 ± 0.07) was significantly higher than that of the normal serum (OD= 0.186 ± 0.09). ELISA results of 10 PNH sera, 20 healthy donors and 5 aplastic anaemia sera against desmoplakin are depicted in Figure 1b.

Expression of desmoplakin and MPP1 in CD34 + cells

We analyzed the expression of desmoplakin and MPP1 genes by PCR using cDNAs derived from testis and the CD34 + cells from one PNH patient and one healthy control.

Sequence analysis of Desmoplakin and MPP1

Sequencing revealed no differences between desmoplakin and MPP1 derived from PNH CD34 + cells and from a healthy donor.

Size of the GPI -deficient Granulocytes and erythrocytes in PNH patients

The size of PNH clone in granulocytes and erythrocytes were determined in 8 PNH patients using flow cytometry (Tab.1). For the remaining two patients, flow cytometric analyses were performed to establish the diagnosis of PNH, but no quantitative results are available.

Discussion

The pathophysiology of the clonal expansion in PNH is still a matter of debate. On one hand, there is a pathogenetic link between AA and PNH suggesting an immune-mediated selection process (Bessler et al, 1994;Luzzatto et al, 1997). According to this immune selection/immune escape theory, GPI-deficient cells would be able to escape autoimmunity as has been shown for AA, conferring a growth advantage to PNH clone(s) that results in their relative expansion in the aplastic marrow. The alternative hypothesis includes internal alterations within clonal cells such as mutations in the genes regulating cell proliferation and apoptosis recognized by immune system. For either hypothesis, it would be important to identify gene products that are recognized by the immune system, and induce autoimmunity similar to what has been shown in patients with aplastic anaemia.

We therefore applied the SEREX approach to screen a human fetal liver cDNA library, because fetal liver is known to be highly enriched for CD34 + cells. Using sera from 10 PNH patients, we found two auto immunogenic structures, MPP1, and desmoplakin. MPP1 is a member of a set of mitotic phosphoproteins specifically recognized by antimitotic protein monoclonal antibody (MPM2) at the G2/M transition. It had been demonstrated that there is no expression of the protein by adult liver tissue (MatsumotoTaniura et al, 1996). MPP1 is a slow plus-end-directed kinesin-related protein (KRP) that plays critical roles in cytokinesis. MPP1 has a specific pattern of localization and expression during the cell cycle, being mostly nuclear in interphase cells with a sharp increase in expression in G 2 and diffuse pattern in metaphase cells, with subsequent association to the central spindle and the midbody at the end of mitosis. The work of Abaza (2003) showed that MPP1 is a novel KRP, the activity of which is required for proper progression of cytokinesis in human cells (Abaza et al, 2003).

Desmoplakin is a protein associated with desmosomes, which are intercellular junctions that tightly link adjacent cells. Desmoplakin is an obligate component of functional desmosomes that anchors intermediate filaments to desmosomal plaques. The N-terminus of desmoplakin is required for localization to the desmosomes and interacts with the Nterminal region of plakophilin 1 and plakoglobin. The C-terminus of desmoplakin binds to intermediate filaments. In the mid-region of desmoplakin, a coiled-coiled rod domain is responsible for homodimerization. Mutations in this gene are the cause of several cardiomyopathies and keratodermas as well as the autoimmunity-mediated paraneoplastic pemphigus (Jiao & Bystryn, 1998). In this study, we demonstrate that both MPP1 and desmoplakin are expressed by CD34 + cells of PNH patients (Figure 2).

Using ELISA, we found IgG antibodies against MPP1 and desmoplakin in a significant number of PNH patients (30% and 40%, respectively). No antibody response agasint MPP1 was detected in 5 aplastic anaemia patients and 20 healthy volunteers. Our results support the hypothesis that humoral immune responses to antigens expressed by haematopoietic cells might be might be involved in the pathophysiology of PNH. The serological reactivity against MPP1 and desmoplakin were shown both in patients who did not have a history of transfusion (patients number 1 and 7 who exhibited reactivity against MPP1 and desmoplakin respectively) and in patients who had received blood transfusions (patient number 5). Therefore, it is unlikely that the patients developed the antibodies because of repeat blood transfusions. The fact that not all patients with PNH had antibodies against MPP1 and desmoplakin in their serum might reflect the broad heterogeneity of the disease. With respect to MPP1, it is remarkable that three of four PNH patients with the highest LDH developed anti-MPP1 antibodies and that in two of these patients the fraction of GPI-deficient granulocytes was > 94% (with no data for the third anti-MPP1 positive patient being available), but no obvious association with clinical characteristics was found in the four patients with antidesmoplakin antibodies. While desmoplakin expressed by intact cells might be accessible for antibodies, MPP1 is not expressed on the cell surface, and a direct role of MPP1 antibodies in the pathogenesis of PNH is unlikely. Because anti-MPP1 antibodies were preferentially found in the sera with high LDH, the detection of MPP1 antibodies appears to be rather the result than the reason for increased cell destruction. Since MPP1 is mostly located in the nucleus, cellular destruction would be the prerequisite for the binding of the antibodies to MPP1. The fact that anti-MPP1 antibodies were high-titred IgG antibodies implies that cognate T-cell help is needed for their production. Thus, the identification of MPP1 by high-titred IgG antibodies now enables the definition of MHC-I and MHC-II restricted antigenic peptides of MPP1 in an approach of "reverse T-cell immunology", which has been successfully employed to define T-cell epitopes of antigens detected by SEREX (Neumann et al, 2005;Neumann et al, 2009;Neumann et al, 2004). Once such epitopes are identified it would be straightforward to compare the susceptibility or resistance of PNH + and normal haematopoietic cells of the identical autologous patients against CD8 + and CD4 + responses.

The elucidation of such T-cell responses should give us more insight into the role of antidesmoplakin and anti-MPP1 responses in the pathophysiology of PNH. Oligoclonal T cell repertoire has recently been described in PNH patients supporting a role of T cells in the pathogenesis of PNH (Karadimitris et al, 2000). The analysis of GPI-defective lymphocyte functions in PNH patients revealed severe defects in TCR-dependent proliferation, CD25, CD54, and HLA-DR surface expression, while these parameters were increased in the GPI + T cells of PNH patients compared to healthy donors (Terrazzano et al, 2005). It will now be interesting to check whether the PNH patients with antibodies to MPP1 and/or desmoplakin have a skewed T cell repertoire and to While MPP1 and desmoplakin are expressed by CD34 + cells in both PNH patients and healthy individuals, the conditions that render the two structures immunogenic in patients with PNH, but not in healthy controls, must be determined. According to one model, the presentation of non-altered molecules in the context of "danger" breaks down autotolerance (Matzinger, 2002). Alternatively, mutations of the two antigens in the CD34 + cells of PNH patients or posttranslational modifications like differential glycosylations or phosphorylations could render desmoplakin and MPP1 auto-immunogenic in PNH patients, as has recently been shown for antigenic targets of paraproteins in the sera of patients with multiple myeloma (Grass et al, 2009). We determined the size of PNH clones in granulocytes and erythrocytes of eight out of ten PNH patients, but we found no correlation between the proportion of PNH cells and the production of autoantibodies in patients.

Our results can only be a first step to a better understanding of the pathophysiology of clonal expansionin PNH. So far, it can not be completely excluded that these antibodies may arise by chance. However, the titres obtained from the patients compared to the controls are quite striking. Similarly, it might be argued that the antibodies are not consistently present in all PNH patients. However, with the clinical heterogeneity of PNH, one would hardly expect that a single immune phenomenon to explain the pathophysiology of all cases of PNH. So far, the immunological mechanism underlying clonal expansion is PNH remains unknown. It may be that the immunological basis of such an immunological recognition is quite heterogeneous and consists of multiple antigen recognition mechanisms. It is, of course, very challenging to assume a pathophysiological role for the antigens detected in this study. The occurrence of antibodies against these antigens might indicate increased cellular destruction (as seems to be the case for MPP1); on the other hand, auto-antibodies could serve as a guide to deregulated genes in affected cells. The specificity of desmoplakin and MPP1 as autoantigens for PNH must be further investigated by studying other types of autoimmune diseases. Until then, desmoplakin and MPP1 remain the most promising proteins in the immuno-pathophysiology of PNH. PIGA gene has been cloned, and found to be mutated in all cases of PNH reported to date (Miyata et al, 1993;Ostendorf et al, 1995).

In PNH, one or a few abnormal clones expand to replace almost the entire haematopoietic stem cells pool. These clones do not appear to be "malignant", but underlie a normal cellular regulation. In addition, GPI-deficient cells with PIGA mutations occur very frequently at low frequency in normal individuals but do not expand in competition with normal hematopoietic cells. In order to develop PNH, two events are required: first, the occurrence of a GPI-deficient clone arising in a multipotent hematopoietic stem cell, second an additional event that enables the expansion of the PNH clone over the residual normal hematopoiesis (Hillmen et al, 2006). The clonal expansion was proposed to be a consequence of a relative growth advantage for the PNH cells. Because PIGA mutant cells have no intrinsic growth advantage over normal cells, a second event responsible for clonal expansion is mandatory (Araten et al, 1999).

At least two hypotheses have been suggested for the explanation of the clonal expansion The study of Horikawa (1997) provides evidence, that blood cells obtained from patients with PNH are less susceptible to both spontaneous and ligand-induced apoptosis in vitro than those from healthy volunteers (Horikawa et al, 1997). In other study, it was shown that leukemic cells with PIGA mutations (GPI -cells) are less susceptible to killing by natural killer (NK) cells in vitro than the control counterparts (GPI + cells), which in turn supports the selective survival advantage theory. The study also implied that neither CD55 nor CD59 would be target antigens for such NK-mediated killing (Nagakura et al, 2002). Murakami et al (2002) provided the first experimental evidence that supports the immunologic selection hypothesis for the clonal expansion of PNH cells. They showed that GPI hematopoietic cells become dominant in a mouse model due to selection by allogeneic CD4 + T cells. In addition, GPI antigen-presenting cells do not stimulate antigen-specific and allogeneic CD4 + T cells efficiently, which suggests that some GPIanchored proteins on APCs act as a ligands for co-stimulatory molecules on T cells (Murakami et al, 2002).

We hypothesized that normal blood cells might present antigens which are targets for the immune system attack induce an autoimmune response that eventually results in the expansion of the PNH clone either by no expression of such antigens or by less susceptibility against cytotoxic attack of autoimmune T lymphocytes. So far, the significance of autoantibodies against hematopoietic antigens in PNH sera is unclear. On

Isolation of positive clones

The same amount was taken from each of the 10 PNH sera and mixed together. These mixed sera were diluted 1:100 in 1×TBS containing 0.5% (wt/vol) low-fat milk. For preservation, 0.01% NaN 3 was added. These diluted sera were stored at +4°C and used for SEREX screening to identify candidate target auto-antigens in PNH, SEREX was (Sahin et al, 1997). We screened a human fetal liver cDNA library (Takara Bio Europe, Saint-Germain-en-Laye, France) known to contain a high proportion of cells derived from hematopoietic stem/progenitor cell HSPC. Briefly, XL1-Blue Escherichia coli (Takara Bio Europe, Saint-Germain-en-Laye, France) was transfected with recombinant phages, plated on agar plates, and cultured at 37°C. Expression of recombinant proteins was induced by incubating the bacterial lawns with isopropyl β-D-thiogalactoside (IPTG). The released proteins were transferred to nitrocellulose filters by incubation at 37° C for 9 minutes. Filters were then washed in TBST (10 mM Tris [tris (hydroxymethyl) amino methane], 150 mM NaCl, 0.05% Tween 20, pH 8.0) and blocked overnight with blocking buffer (5% wt/vol non-fat dry milk in TBST). Filters were then incubated at 4˚C overnight with diluted patients' sera. Specific binding of antibody to recombinant protein was detected by incubation with alkaline phosphatase-conjugated goat antihuman IgG antibody (Dianova, Hamburg, Germany) diluted at 1:7500. Visualization of the antigen-antibody complex was accomplished by staining with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Roth, Karlsruhe, Germany). Complementary DNA inserts from positive clones were subcloned, purified, and in vivo excised to plasmid forms (Takara Bio Europe, Saint-Germain-en-Laye, France) according to the manufacturer's instructions. The DNA inserts were subsequently sequenced with appropriate sequencing primers.

Phage assay for detecting serum antibodies

Positive clones detected in the library screening were mixed with non-reactive phages of the cDNA library as an internal negative control at a ratio of 1:10, and used to transfect bacteria as described above. Each PNH serum was diluted 1:100 and used individually to detect antibodies against the positive clones, the PNH pool was used as a control positive serum.

Hematopoietic progenitor cell assay CD34 + cells were purified from bone marrow of PNH-1 and a non-PNH patient (as control) using magnetic cells sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) F o r P e e r R e v i e w 7 according to the manufacturer's instructions. Total RNA was isolated using Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was constructed using oligodt18 primer and SuperScript™II (Invitrogen, Carlsbad, NM, USA).

Expression pattern of mRNA

To evaluate the mRNA expression pattern of identified clones, RT-PCR was performed by amplification of the cDNA from normal and PNH CD34 + cells. The gene-specific primers used in RT-PCR reactions were designed to amplify coding sequences of corresponding serologically defined antigens and synthesized commercially (MWG, Biotech, Ebersberg, Germany). 0.8 µl of first-strand cDNA was amplified using 0.2 U AmpliTaq Gold (Perkin Elmer, Rodgau, Germany), 1 µl of dNTP (10 mMol of each dATP, dTTP, dCTP, dGTP), 1 µl of each primer and 3 µl of PCR buffer in a reaction volume of 30 µl. The amplification was performed with 35 cycles at a denaturation temperature of 94°C (1 min/cycle); an annealing temperature of 60°C for Desmoplakin and 51°C for MPP1(1 min/cycle); and an extension temperature of 72°C (1 min/1kb).

The PCR products were analyzed by agarose gel electrophoresis with 1% agarose gel. We used GAPDH as housekeeping gene, and testis cDNA as DNA positive control. The PCR products were excised from the gel, purified by gel band purification kit (GE Healthcare, Piscataway, NJ, USA) and sequenced.

Fusion Protein Construction and Expression in HEK293 Cells:

Using primers specific for desmoplakin and MPP1, we isolated cDNA inserts from positive phages and subcloned them in frame into a pSectag/FRT/V5-His Topo TA expression vector (Invitrogen, Carlsbad, NM, USA). and pSfi-Express-HA vector, respectively. The pSfi-Express-HA is a derivative of pEGFP-C1 vector (Takara Bio Europe, Saint-Germain-en-Laye, France); the plasmid has a neomycin resistance gene for selection in mammalian cells. We used human embryonic kidney 293 (Hek 293) cells for transfection. The induction and affinity purification of fusion proteins was performed according to the manufacturer's instructions. Western blot analysis was applied to confirm size and specificity using a (1:3000) mouse anti-His monoclonal antibody (Sigma, St Louis, MO, USA) and a (1: 500) MPP1-specific mouse polyclonal antibody

Western blot analysis

Purified proteins were prepared in sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of protein were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, Mass, USA), and incubated with blocking buffer overnight. Immunoblots were performed at 1:500 dilution of patient's serum (if not otherwise indicated), mouse anti-His monoclonal antibody, or MPP1 mouse polyclonal antibody (Abnova, Taipei City, Taiwan) and desmoplakin 1&2 mouse monoclonal antibody, respectively. Immunodetection was performed by incubation with (1:10.000) horseradish peroxidase-conjugated antihuman IgG (Promega, Madison, WI, USA) or (1:5000) anti-mouse IgG secondary antibody (Promega, Madison, WI, USA) as indicated by the host origin of the primary antibody and developed by chemiluminescence (NEN Life Science Products, Boston, USA).

Titration of antibodies by ELISA

Enzyme-linked immunosorbent assay (ELISA) plates were coated with mouse anti MPP1 antibody for MPP1 and rabbit anti mouse antibody followed by mouse anti-desmoplakin antibody for desmoplakin. Plates were washed and blocked overnight at 4°C with 5% non-fat dry milk in TBST. Patients' sera were added to a final dilution of 1:1000 to 1:5000 and incubated at room temperature. After washing, the plates were incubated with (1:7500) alkaline phosphatase-conjugated goat antihuman IgG antibody (Promega, Madison, WI, USA) at room temperature. Finally, the plates were washed and incubated with p-nitrophenyl phosphate (PNPP) substrate (Pierce, Rockford, IL, USA) at room temperature, and the optic density (OD) at 405 nm was determined. A positive reaction was defined as an absorbance value exceeding the mean OD absorbance value of sera from healthy donors by three standard deviations.

Results

The SEREX approach was applied to identify auto-antigens in the sera of patients with PNH. We screened a human fetal liver cDNA library because a high proportion of fetal liver cells share characteristics with pluripotent HSPC. By screening more than 1 x 10 6 phage plaques with 1/100 diluted sera from 10 PNH patients we identified the products of two genes that were recognized by sera of PNH patients. Lineage-specific expression was determined by searching the National Centre for Biotechnology Information (NCBI) UniGene database (http://www.ncbi.nlm.nih.gov/UniGene/). The first antigen was Mphase phosphoprotein 1 (MPP1). MPP1 is a member of a set of mitotic phosphoproteins There was no sufficient information about the tissue expression of desmoplakin, but it is known that desmosomes are major sites of intercellular contact found in a variety of cells such as epithelial cells, cardiac myocytes, arachnoidal cells of meanings, and dendritic cells of germinal centres in lymph nodes (Virata et al, 1992).

In addition, individual sera from 10 PNH patients and 20 healthy controls were analyzed for immune-reactivity against MPP1 and desmoplakin clones. Three and four of the 10 PNH sera showed a positive reaction against MPP1 and desmoplakin, respectively.

Antibodies against MPP1 were detected in PNH sera 1, 5, and 9, that is in three of the four sera with the most elevated LDH. Antibodies against desmoplakin were detected in one 1/20 sera from healthy controls and in four patients (5,6,7,10), without any obvious correlation to the clinical characteristics of the desmoplakin-antibody positive patients (Tab. 1).

Detection of IgG antibodies against desmoplakin and MPP1 by ELISA

In order to determine the titres of antibodies against the two gene products in the sera of patients with PNH and healthy controls, sera were tested for reactivity against desmoplakin and MPP1 by ELISA. A serum was considered positive if its absorbance value exceeded the mean absorbance value of sera from healthy donors by 3 standard deviations. The results of ELISA for MPP1 are shown in figure 1a. Three out of 10 PNH sera exhibited an optic density higher than the cut-off in the MPP1 ELISA, but none of the five aplastic anaemia sera was positive. Desmoplakin reactivity was detected in 4 of the 10 PNH sera, but in none of the aplastic anaemia sera. A positive reaction against desmoplakin was detected in one normal serum but the OD of all positive PNH Sera (OD = 1.22 ± 0.07) was significantly higher than that of the normal serum (OD= 0.186 ± 0.09). ELISA results of 10 PNH sera, 20 healthy donors and 5 aplastic anaemia sera against desmoplakin are depicted in Figure 1b.

Expression of desmoplakin and MPP1 in CD34 + cells

We analyzed the expression of desmoplakin and MPP1 genes by PCR using cDNAs derived from testis and the CD34 + cells from one PNH patient and one healthy control.

Sequence analysis of Desmoplakin and MPP1

Sequencing revealed no differences between desmoplakin and MPP1 derived from PNH CD34 + cells and from a healthy donor.

Size of the GPI -deficient Granulocytes and erythrocytes in PNH patients

The size of PNH clone in granulocytes and erythrocytes were determined in 8 PNH patients using flow cytometry (Tab.1). For the remaining two patients, flow cytometric analyses were performed to establish the diagnosis of PNH, but no quantitative results are available.

Discussion

The pathophysiology of the clonal expansion in PNH is still a matter of debate. On one hand, there is a pathogenetic link between AA and PNH suggesting an immune-mediated selection process (Bessler et al, 1994;Luzzatto et al, 1997). According to this immune selection/immune escape theory, GPI-deficient cells would be able to escape autoimmunity as has been shown for AA, conferring a growth advantage to PNH clone(s) that results in their relative expansion in the aplastic marrow. The alternative hypothesis includes internal alterations within clonal cells such as mutations in the genes regulating cell proliferation and apoptosis recognized by immune system. For either hypothesis, it would be important to identify gene products that are recognized by the immune system, and induce autoimmunity similar to what has been shown in patients with aplastic anaemia.

We therefore applied the SEREX approach to screen a human fetal liver cDNA library, because fetal liver is known to be highly enriched for CD34 + cells. Using sera from 10 PNH patients, we found two auto immunogenic structures, MPP1, and desmoplakin. MPP1 is a member of a set of mitotic phosphoproteins specifically recognized by antimitotic protein monoclonal antibody (MPM2) at the G2/M transition. It had been demonstrated that there is no expression of the protein by adult liver tissue (MatsumotoTaniura et al, 1996). MPP1 is a slow plus-end-directed kinesin-related protein (KRP) that plays critical roles in cytokinesis. MPP1 has a specific pattern of localization and expression during the cell cycle, being mostly nuclear in interphase cells with a sharp increase in expression in G 2 and diffuse pattern in metaphase cells, with subsequent association to the central spindle and the midbody at the end of mitosis. The work of Abaza (2003) showed that MPP1 is a novel KRP, the activity of which is required for proper progression of cytokinesis in human cells (Abaza et al, 2003).

Desmoplakin is a protein associated with desmosomes, which are intercellular junctions that tightly link adjacent cells. Desmoplakin is an obligate component of functional desmosomes that anchors intermediate filaments to desmosomal plaques. The N-terminus of desmoplakin is required for localization to the desmosomes and interacts with the Nterminal region of plakophilin 1 and plakoglobin. The C-terminus of desmoplakin binds to intermediate filaments. In the mid-region of desmoplakin, a coiled-coiled rod domain is responsible for homodimerization. Mutations in this gene are the cause of several cardiomyopathies and keratodermas as well as the autoimmunity-mediated paraneoplastic pemphigus (Jiao & Bystryn, 1998). In this study, we demonstrate that both MPP1 and desmoplakin are expressed by CD34 + cells of PNH patients (Figure 2).

Using ELISA, we found IgG antibodies against MPP1 and desmoplakin in a significant number of PNH patients (30% and 40%, respectively). No antibody response agasint MPP1 was detected in 5 aplastic anaemia patients and 20 healthy volunteers. Our results support the hypothesis that humoral immune responses to antigens expressed by haematopoietic cells might be might be involved in the pathophysiology of PNH. The serological reactivity against MPP1 and desmoplakin were shown both in patients who did not have a history of transfusion (patients number 1 and 7 who exhibited reactivity against MPP1 and desmoplakin respectively) and in patients who had received blood transfusions (patient number 5). Therefore, it is unlikely that the patients developed the antibodies because of repeat blood transfusions. The fact that not all patients with PNH had antibodies against MPP1 and desmoplakin in their serum might reflect the broad heterogeneity of the disease. With respect to MPP1, it is remarkable that three of four PNH patients with the highest LDH developed anti-MPP1 antibodies and that in two of these patients the fraction of GPI-deficient granulocytes was > 94% (with no data for the third anti-MPP1 positive patient being available), but no obvious association with clinical characteristics was found in the four patients with antidesmoplakin antibodies. While desmoplakin expressed by intact cells might be accessible for antibodies, MPP1 is not expressed on the cell surface, and a direct role of MPP1 antibodies in the pathogenesis of PNH is unlikely. Because anti-MPP1 antibodies were preferentially found in the sera with high LDH, the detection of MPP1 antibodies appears to be rather the result than the reason for increased cell destruction. Since MPP1 is mostly located in the nucleus, cellular destruction would be the prerequisite for the binding of the antibodies to MPP1. The fact that anti-MPP1 antibodies were high-titred IgG antibodies implies that cognate T-cell help is needed for their production. Thus, the identification of MPP1 by high-titred IgG antibodies now enables the definition of MHC-I and MHC-II restricted antigenic peptides of MPP1 in an approach of "reverse T-cell immunology", which has been successfully employed to define T-cell epitopes of antigens detected by SEREX (Neumann et al, 2005;Neumann et al, 2009;Neumann et al, 2004). Once such epitopes are identified it would be straightforward to compare the susceptibility or resistance of PNH + and normal haematopoietic cells of the identical autologous patients against CD8 + and CD4 + responses.

The elucidation of such T-cell responses should give us more insight into the role of antidesmoplakin and anti-MPP1 responses in the pathophysiology of PNH. Oligoclonal T cell repertoire has recently been described in PNH patients supporting a role of T cells in the pathogenesis of PNH (Karadimitris et al, 2000). The analysis of GPI-defective lymphocyte functions in PNH patients revealed severe defects in TCR-dependent proliferation, CD25, CD54, and HLA-DR surface expression, while these parameters were increased in the GPI + T cells of PNH patients compared to healthy donors (Terrazzano et al, 2005). It will now be interesting to check whether the PNH patients with antibodies to MPP1 and/or desmoplakin have a skewed T cell repertoire and to While MPP1 and desmoplakin are expressed by CD34 + cells in both PNH patients and healthy individuals, the conditions that render the two structures immunogenic in patients with PNH, but not in healthy controls, must be determined. According to one model, the presentation of non-altered molecules in the context of "danger" breaks down autotolerance (Matzinger, 2002). Alternatively, mutations of the two antigens in the CD34 + cells of PNH patients or posttranslational modifications like differential glycosylations or phosphorylations could render desmoplakin and MPP1 auto-immunogenic in PNH patients, as has recently been shown for antigenic targets of paraproteins in the sera of patients with multiple myeloma (Grass et al, 2009). We determined the size of PNH clones in granulocytes and erythrocytes of eight out of ten PNH patients, but we found no correlation between the proportion of PNH cells and the production of autoantibodies in patients.

Our results can only be a first step to a better understanding of the pathophysiology of clonal expansionin PNH. So far, it can not be completely excluded that these antibodies may arise by chance. However, the titres obtained from the patients compared to the controls are quite striking. Similarly, it might be argued that the antibodies are not consistently present in all PNH patients. However, with the clinical heterogeneity of PNH, one would hardly expect that a single immune phenomenon to explain the pathophysiology of all cases of PNH. So far, the immunological mechanism underlying clonal expansion is PNH remains unknown. It may be that the immunological basis of such an immunological recognition is quite heterogeneous and consists of multiple antigen recognition mechanisms. It is, of course, very challenging to assume a pathophysiological role for the antigens detected in this study. The occurrence of antibodies against these antigens might indicate increased cellular destruction (as seems to be the case for MPP1); on the other hand, auto-antibodies could serve as a guide to deregulated genes in affected cells. The specificity of desmoplakin and MPP1 as autoantigens for PNH must be further investigated by studying other types of autoimmune diseases. Until then, desmoplakin and MPP1 remain the most promising proteins in the immuno-pathophysiology of PNH.

  The study had been approved by the local ethics review committee (Ethikkommission der Ärztekammer des Saarlandes) and was done in accordance with the Declaration of Helsinki. Recombinant DNA work was done with the permission and according to the regulations of the local authorities (Regierung des Saarlandes). Sera from 10 patients with PNH were obtained during routine diagnosis or follow-up at the Department of Internal Medicine Ι, Saarland University Medical School (Homburg, Germany) and the Department of Haematology and Haemostaseology, University of Vienna (Vienna, Austria). Table I describes the characteristics of the PNH patients included in our study. Diagnosis of PNH had been established by flow cytometry. All patients gave informed consent for blood analysis. Sera from five patients with newly diagnosed aplastic anaemia were frozen before starting immunosuppressive therapy at the Institute for Transfusion Medicine and Immunogenetic clinics for Aplastic Anaemia in Ulm, Germany. Twenty healthy individuals donating blood at the Department of Transfusion Medicine, University of Saarland (Homburg Germany) were used as a control group. All sera were stored at -80° C until use for screening as described below.

  Taipei City, Taiwan) for MPP1 protein, and (1:2000) desmoplakin 1&2 mouse monoclonal antibody (Biodesign, Saco, USA) for desmoplakin.

  Three mL of peripheral blood was collected from antecubital vein to a tube with EDTA anticoagulant. 100 µL blood samples were immunostained in tubes with the following two-colour combinations of monoclonal antibodies (Becton Dickinson Diagnostic Systems, Heidelberg, Germany): isotype control mAbs and anti-CD45 FITC/CD14 PE, anti-CD3 FITC/CD19 PE, anti-CD4 FITC/CD8 PE, anti-CD3 FITC/CD16+56 PE mAbs for determination of main subpopulations of leukocytes. For the detection of PNH-type granulocytes, monocytes and lymphocytes we used fluorescein-isothiocyanate (FITC)labeled anti-CD55 (clone IA10) and phycoerythrin (PE)-labeled anti-CD59 (clone p282, H19) monoclonal antibodies (Becton Dickinson). The cells were incubated with antibodies for 15 min at room temperature in the dark. After that, a 2-mL volume of FACS-lysing solution (Becton Dickinson) diluted 1:10 was added to each tube, gently mixed, and incubated for further 10 min in the dark. The tubes were washed twice with PBS and centrifuged at 1200 × g for 4 min. The white cell pellets were resuspended in 0.5 mL PBS and analyzed by flow cytometry using a FACScan flow analyzer and CellQuest software (Becton Dickinson). Expression of CD55 and CD59 molecules was quantified by the determination of mean fluorescence intensity (MFI).

  as housekeeping gene. The gel electrophoresis of the PCR products is shown in figure2: both desmoplakin and MPP1 were expressed in CD34 + cells of the PNH patient and normal individuals.

  underlying the activation of the T cells responsive to desmoplakin and MPP1.

  Figure LegendsFigure1: ELISA results of all sera against the isolated antigens; A. MPP1: Three out of 10 PNH sera exhibited an optic density higher than the cut-off in the MPP1 ELISA, but none of the five aplastic anaemia sera was positive. B. Desmoplakin: reactivity against desmoplakin was detected in 4 of the 10 PNH sera, in none of the aplastic anaemia sera. in one normal serum (number 6).

Figure 2 :

 2 Figure 2: Gel electrophoresis analysis for cDNA from CD34 + cells; A. Desmoplakin, B.MPP1, C. GAPDH. 1: Negative control, 2: Testis cDNA, 3: Normal CD34+, 4: PNH CD34+. We can see the expression of Desmoplakin by normal (A3) and PNH patient (A4). MPP1 expression in CD34 + from normal (B3) and PNH (B4) patient.

  been proposed to explain the mechanism of clonal expansion of mutant cells in paroxysmal nocturnal Hemoglobinuria (PNH). One hypothesis assumes an immune escape mechanism and the other proposes an intrinsic second mutational event within clonal cells. We hypothesized that autoantibodies detected in PNH patients could identify antigens that might play a role in the pathophysiology of this disease and screened a human fetal liver cDNA library for serologic reactivity against hematopoietic stem/ progenitor cells antigens using the SEREX approach. Two antigens were identified which are constitutively expressed in CD34 + cells. Three and four of 10 PNH patients showed antibody responses against M-phase phosphoprotein 1 (MPP1) and desmoplakin (DSP) respectively. We also found an antibody response in one of 20 healthy volunteers against desmoplakin, yet at a much lower titre than in PNH patients. No response to MPP1 or desmoplakin was detected in five patients with aplastic anaemia without a GPIdeficient clone. We conclude that MPP1 and desmoplakin are the first auto-antigens shown to be recognized by the immune system of patients with PNH. The analysis of the mechanisms underlying the autoimmune response might contribute to our understanding of the clonal expansion in PNH.Keywords: Paroxysmal nocturnal Hemoglobinuria (PNH), M-phase phosphoprotein 1 (MPP1), desmoplakin, aplastic Anaemia, antigens.Paroxysmal nocturnal Hemoglobinuria (PNH) is a unique disorder characterized by chronic intravascular hemolysis. The characteristic biochemical feature in PNH is a lack of glycosyl phosphatidyl inositol anchored proteins (GPI-APs) due to an incomplete assembly of the GPI anchor. PIGA genes are involved in the biosynthesis of the GPIanchor and the binding of the anchor to proteins in the endoplasmic reticulum (ER). A

  in PNH. According to the first hypothesis, clonal selection eliminates normal hematopoietic stem cells, but spares PIGA mutant clones due to their non-expression of the GPI anchor. The second hypothetical model comprises a second internal event within the mutant cells such as a second somatic mutation leading to a predominant proliferation of PIGA mutant stem cells. The close association of PNH with aplastic anaemia suggests that the clonal selection pressure might be immune-mediated. According to this hypothesis, normal hematopoiesis is suppressed by the immune system, presumably, indirectly through one or more GPI-linked antigens, and therefore this attack spares the GPI-deficient clone(Inoue et al, 2006).

  the one hand, it might confirm the role of T lymphocytes in the pathophysiology of PNH; on the other hand, the identification of such antigens might demonstrate that such immune reactions in PNH could recognize mutated antigens and thus demonstrate the presence of altered antigens in PNH. The study had been approved by the local ethics review committee (Ethikkommission der Ärztekammer des Saarlandes) and was done in accordance with the Declaration of Helsinki. Recombinant DNA work was done with the permission and according to the regulations of the local authorities (Regierung des Saarlandes). Sera from 10 patients with PNH were obtained during routine diagnosis or follow-up at the Department of Internal Medicine Ι, Saarland University Medical School (Homburg, Germany) and the Department of Haematology and Haemostaseology, University of Vienna (Vienna, Austria). Table I describes the characteristics of the PNH patients included in our study. Diagnosis of PNH had been established by flow cytometry. All patients gave informed consent for blood analysis. Sera from five patients with newly diagnosed aplastic anaemia were frozen before starting immunosuppressive therapy at the Institute for Transfusion Medicine and Immunogenetic clinics for Aplastic Anaemia in Ulm, Germany. Twenty healthy individuals donating blood at the Department of Transfusion Medicine, University of Saarland (Homburg Germany) were used as a control group. All sera were stored at -80° C until use for screening as described below.

  Taipei City, Taiwan) for MPP1 protein, and (1:2000) desmoplakin 1&2 mouse monoclonal antibody (Biodesign, Saco, USA) for desmoplakin.

  Three mL of peripheral blood was collected from antecubital vein to a tube with EDTA anticoagulant. 100 µL blood samples were immunostained in tubes with the following two-colour combinations of monoclonal antibodies (Becton Dickinson Diagnostic Systems, Heidelberg, Germany): isotype control mAbs and anti-CD45 FITC/CD14 PE, anti-CD3 FITC/CD19 PE, anti-CD4 FITC/CD8 PE, anti-CD3 FITC/CD16+56 PE mAbs for determination of main subpopulations of leukocytes. For the detection of PNH-type granulocytes, monocytes and lymphocytes we used fluorescein-isothiocyanate (FITC)labeled anti-CD55 (clone IA10) and phycoerythrin (PE)-labeled anti-CD59 (clone p282, H19) monoclonal antibodies (Becton Dickinson). The cells were incubated with antibodies for 15 min at room temperature in the dark. After that, a 2-mL volume of FACS-lysing solution (Becton Dickinson) diluted 1:10 was added to each tube, gently mixed, and incubated for further 10 min in the dark. The tubes were washed twice with PBS and centrifuged at 1200 × g for 4 min. The white cell pellets were resuspended in 0.5 mL PBS and analyzed by flow cytometry using a FACScan flow analyzer and CellQuest software (Becton Dickinson). Expression of CD55 and CD59 molecules was quantified by the determination of mean fluorescence intensity (MFI).

  specifically recognized by the MPM2 antibody at the G2/M transition. The second antigen is encoded by the desmoplakin gene. Desmoplakin is an obligate component of functional desmosomes that anchors intermediate filaments to desmosomal plaques.

  as housekeeping gene. The gel electrophoresis of the PCR products is shown in figure2: both desmoplakin and MPP1 were expressed in CD34 + cells of the PNH patient and normal individuals.

  underlying the activation of the T cells responsive to desmoplakin and MPP1.

  Figure LegendsFigure1: ELISA results of all sera against the isolated antigens; A. MPP1: Three out of 10 PNH sera exhibited an optic density higher than the cut-off in the MPP1 ELISA, but none of the five aplastic anaemia sera was positive. B. Desmoplakin: reactivity against desmoplakin was detected in 4 of the 10 PNH sera, in none of the aplastic anaemia sera. in one normal serum (number 6).

Figure 2 :

 2 Figure 2: Gel electrophoresis analysis for cDNA from CD34 + cells; A. Desmoplakin, B.MPP1, C. GAPDH. 1: Negative control, 2: Testis cDNA, 3: Normal CD34+, 4: PNH CD34+. We can see the expression of Desmoplakin by normal (A3) and PNH patient (A4). MPP1 expression in CD34 + from normal (B3) and PNH (B4) patient.

British Journal of Haematology

Acknowledgement

We thank Maria Kemele, Evi Regitz, and Natalie Fadle for the excellent technical assistance.

Acknowledgement

We thank Maria Kemele, Evi Regitz, and Natalie Fadle for the excellent technical assistance.

Authorship:

Contribution: Adnan Alahmad and Klaus-Dieter Preuss performed experiments; Adnan Alahmad, Michael Pfreundschuh, and Jörg Schubert analyzed results and wrote the manuscript. Joachim-F Schenk, Wolfgang Füreder, Hubert Schrezenmeier, and Nikolaus Müller-Lantzsch helped in designing the study protocol, collecting the data, and contributed patients to the study.

Conflict of interest statement:

We declare that we have no conflict of interest.

Authorship:

Contribution: Adnan Alahmad and Klaus-Dieter Preuss performed experiments; Adnan Alahmad, Michael Pfreundschuh, and Jörg Schubert analyzed results and wrote the manuscript. Joachim-F Schenk, Wolfgang Füreder, Hubert Schrezenmeier, and Nikolaus Müller-Lantzsch helped in designing the study protocol, collecting the data, and contributed patients to the study.

Conflict of interest statement:

We declare that we have no conflict of interest.