- Email: [email protected]
Increased Levels of the Bullous Pemphigoid BP180 Autoantibody Are Associated with More Severe Dementia in Alzheimer’s Disease
Accepted Manuscript Increased Levels of the Bullous Pemphigoid BP180 Autoantibody Are Associated with More Severe Dementia in Alzheimer’s Disease Nina Kokkonen, Sanna-Kaisa Herukka, Laura Huilaja, Merja Kokki, Anne M. Koivisto, Päivi Hartikainen, Anne M. Remes, Kaisa Tasanen PII:
S0022-202X(16)32451-4
DOI:
10.1016/j.jid.2016.09.010
Reference:
JID 531
To appear in:
The Journal of Investigative Dermatology
Received Date: 13 April 2016 Revised Date:
12 August 2016
Accepted Date: 2 September 2016
Please cite this article as: Kokkonen N, Herukka S-K, Huilaja L, Kokki M, Koivisto AM, Hartikainen P, Remes AM, Tasanen K, Increased Levels of the Bullous Pemphigoid BP180 Autoantibody Are Associated with More Severe Dementia in Alzheimer’s Disease, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Increased Levels of the Bullous Pemphigoid BP180 Autoantibody Are Associated with More Severe Dementia in Alzheimer’s Disease Nina Kokkonen1,4, Sanna-Kaisa Herukka2,4, Laura Huilaja1,4, Merja Kokki3, Anne M. Koivisto2, Päivi Hartikainen2, Anne M. Remes2, and Kaisa Tasanen1 Department of Dermatology, PEDEGO Research Unit, Medical Research Center Oulu, Oulu
University Hospital and University of Oulu, Oulu, Finland 2
RI PT
1
Institute of Clinical Medicine – Neurology, University of Eastern Finland and Department of
Neurology, Kuopio University Hospital, Kuopio, Finland
Department of Anesthesia and Operative Service, Kuopio University Hospital, Kuopio, Finland;
4
These authors contributed equally to this work.
M AN U
SC
3
Short title: Bullous Pemphigoid and Alzheimer’s disease
TE D
Keywords: BP230, collagen XVII, dementia, hemidesmosome, neurodegeneration
Abbreviations: bullous pemphigoid (BP), Alzheimer’s disease (AD), central nervous system (CNS), cerebrospinal fluid (CSF), immunofluorescence (IF), Mini-Mental State Examination
EP
(MMSE), non-collagenous 16A (NC16A), enzyme-linked immunosorbent assay (ELISA)
AC C
Correspondence:
Kaisa Tasanen, Department of Dermatology, Medical Research Center Oulu, University of Oulu, Aapistie 5A, FIN-90220 OULU, Finland; Tel: +358 400 613361; E-mail: [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
Bullous pemphigoid (BP) is a subepidermal blistering skin disease, which has shown a strong association with neurological diseases in epidemiological studies. The BP autoantigens BP180 and BP230 are expressed in the cutaneous basement membrane and the central nervous system. Using BP180 and BP230 ELISA assays and immunoblotting against BP180 we analyzed the IgG
RI PT
reactivity in the sera of 115 patients with Alzheimer’s disease (AD) and 40 neurologically healthy controls. BP180 autoantibodies were found in 18 % of AD patients, whereas only 3% of controls had positive results (P=0.019). BP230 values were higher and more often elevated in AD patients than controls, but not significantly. None of the positive AD sera that recognized the full-length
SC
human BP180 in immunoblotting reacted with the cutaneous basement membrane in indirect immunofluorescence analysis. Moreover, a retrospective evaluation of the hospital records of the AD patients revealed neither BP diagnosis nor BP-like symptoms. Interestingly, increased BP180-
M AN U
NC16A autoantibody values correlated with cognitive decline measured by Mini-Mental State Examination scores (MMSE), but not with the concentration of AD biomarkers in cerebrospinal fluid. Our findings further the understanding of the role of BP180 as a shared autoantigen in neuro-
AC C
EP
TE D
dermatological interactions and the association between BP and neurodegenerative diseases.
2
ACCEPTED MANUSCRIPT INTRODUCTION Bullous pemphigoid (BP) is the most common subepidermal autoimmune blistering disease with increasing incidence among elderly people (Langan et al., 2008; Joly et al., 2012; Schmidt and Zillikens, 2013; Försti et al., 2014). Typically BP patients have severe pruritus and blistering skin lesions, but in up to 20% of patients blisters are absent and only excoriations, eczematous or
RI PT
urticated lesions occur (della Torre et al., 2012; Di Zenzo et al., 2012;). At the molecular level BP autoantibodies target two cutaneous basement membrane proteins, BP180 and BP230 (Nishie, 2014). The main antigen BP180, also known as collagen XVII or BPAg2, is a transmembrane protein of hemidesmosomes linking basal keratinocytes to the underlying dermis (Van den Bergh
SC
and Giudice, 2003; Franzke et al., 2005). IgG antibodies of BP patients target the immunodominant non-collagenous 16A (NC16A) domain, which is located extracellularly close to the transmembrane
M AN U
domain of BP180 (Schmidt and Zillikens, 2013; Nishie, 2014). As well as in the skin, BP180 is also expressed in various other tissues (Claudepierre et al., 2005; Huilaja et al., 2008; Hurskainen et al., 2012) including the brain (Seppänen et al., 2006; Seppänen et al. 2007; Seppänen, 2013). The other autoantigen, BP230 (bullous pemphigoid antigen 1, BPAg1e), is an intracellular component of hemidesmosomes and a ligand of BP180. It is the epithelial isoform of dystonin, and its variants BPAg1a1 and BPAg1a2 are expressed in both the central and the peripheral nervous system (Powell
TE D
et al., 2005; Kunzli et al., 2016). BP230 is targeted by autoantibodies in approximately 60% of patients with BP (Kasperkiewicz et al., 2012), but it seems that autoantibodies against BP230 result from intermolecular epitope spreading and never proceed to anti-BP180-autoantibodies (Di Zenzo
EP
et al., 2011; Di Zenzo et al., 2012).
AC C
The diagnosis of BP is established by typical clinical presentation and the finding of linear IgG or complement 3 along the basement membrane zone in a direct immunofluorescence (IF) analysis of a perilesional biopsy (Di Zenzo et al., 2012; Schmidt and Zillikens, 2013). In addition, the detection of circulating autoantibodies by BP180-NC16A ELISA has high specificity and moderate sensitivity in diagnosing BP (Tampoia et al., 2012).
Several epidemiological studies have revealed an association of BP with a range of neurological diseases including dementia, cerebral stroke, Parkinson’s disease, multiple sclerosis and motor neuron disease (Stinco et al., 2005; Cordel et al., 2007; Langan et al., 2011; Bastuji-Garin et al., 2011; Ong et al., 2013; Brick et al., 2014). An existing neurological disorder appears to increase the 3
ACCEPTED MANUSCRIPT subsequent risk for BP (Langan et al., 2011; Brick et al., 2014), but conversely, increased risk for developing neurological diseases has also been reported following BP diagnosis (Yang et al., 2011; Brick et al., 2014).
The expression of BP autoantigens in the central nervous system (CNS) and the association between
RI PT
BP and various neurodegenerative disorders suggest that BP180 and BP230 could act as shared antigens in cutaneous and neurological diseases (Seppänen, 2013; Kunzli et al., 2016). To test this hypothesis we measured BP autoantibodies in well-characterized patients with Alzheimer’s disease (AD). We also investigated postulated associations between BP autoantibodies and age, gender and
SC
cognitive decline as well as with the presence of cerebrospinal fluid (CSF) markers of AD
AC C
EP
TE D
M AN U
pathology.
4
ACCEPTED MANUSCRIPT RESULTS BP180 and BP230 autoantibodies in patients with Alzheimer’s disease Sera from patients with AD (n=115) and neurologically healthy control subjects (n=40) were tested for BP180 and BP230 IgG autoantibodies using a well-established, commercially available ELISA analysis and all anti-BP180 positive sera (24 AD samples and 3 controls) were further tested by
RI PT
immunoblotting against human recombinant BP180. A significantly higher proportion of AD samples (20; 18%) than control samples (1; 3%) were positive for both IgG autoantibodies against BP180-NC16A (based on a cut-off value >9 U/ml; Table 1, Figure 1) and the full-length BP180 (Figure 2). Accordingly, BP180 autoantibody values were significantly higher in the AD group (2.4
SC
U/ml, range 0–47.7 U/ml) compared to those of control group (2.0 U/ml, range 0.5-12.3 U/ml; P=0.05; Table 1, Figure 1). We also tested 18 of the anti-BP180 positive AD samples and the one
M AN U
positive control sample using indirect IF analysis. Under the same conditions in which the sera from BP patients demonstrated a clear linear immunostaining on the cutaneous basement membrane zone, none of the tested AD sera nor the positive control sample showed any specific immunostaining either in normal or salt-split human skin (Supplementary Figure S1 online).
TE D
In the ELISA assay IgG autoantibodies against BP230 were also found in a higher proportion of AD patients than controls and BP230 autoantibody values were higher in the AD group than those of the controls, but neither of these differences was statistically significant (Table 1, Figure 1). Autoantibodies against only BP180 were detected in 16 AD patients, six AD patients had anti-
EP
BP230 positivity without having reactivity against BP180 and eight AD patients had autoantibodies
AC C
against both BP180 and BP230.
A retrospective evaluation of the hospital records of AD patients with increased anti-BP180 or BP230 values revealed neither diagnosis nor clinical symptoms of BP in any patient.
The association of BP180 and BP230 autoantibodies with age, gender, cognitive impairment and Alzheimer’s disease biomarkers BP180 and BP230 ELISA values did not correlate with age in either the whole study group or the group of AD patients. However, BP180 and BP230 ELISA values showed a statistically significant 5
ACCEPTED MANUSCRIPT correlation to each other in the whole study group (r=0.329, P<0.001). This overall correlation was driven by the correlation in the AD patients (r=0.367, P<0.001), as there was no such correlation when the control group was considered separately. In the AD group, autoantibodies against BP180NC16A were significantly more common in females than males (P=0.029) and the median BP180 ELISA value was significantly higher in females than males (P=0.037). In contrast, there was no
RI PT
significant difference between the genders for positivity or values of BP230 ELISA.
The severity of dementia of AD patients was evaluated using the Mini-Mental State Examination (MMSE) (Table 1). MMSE data were available for 89 patients with AD. Interestingly, there was a
SC
negative correlation between MMSE score and BP180 ELISA value (r=-0.287, P=0,007) i.e. patients with lower MMSE, denoting more severe dementia, had higher serum anti-BP180 levels.
M AN U
As expected, MMSE score showed negative correlation with age (r=-0.351, P=0.001). Regarding AD biomarkers there was no correlation between serum anti-BP180 values and beta-amyloid (Αβ1-
EP
TE D
total tau or hyperphosphorylated tau (p-tau181) in the CSF samples of patients with AD.
AC C
42),
6
ACCEPTED MANUSCRIPT DISCUSSION Based on the association of several neurological diseases with BP and the CNS expression of BP autoantigens it has been suggested that neuroinflammation could lead to a cross-reactive immunoresponse between neural and cutaneous antigens (Langan et al., 2011; Seppänen, 2013; Kunzli et al., 2016). This theory is supported by our current study, which revealed the presence of
RI PT
increased IgG autoantibodies against BP180 in 18 % of patients with AD. The prevalence of BP autoantibodies in our control samples was slightly higher than in previous studies, which report that IgG autoantibodies against BP180 and BP230 exist in 0–2.0% of the general population (see Prussmann et al., 2015). The association between BP autoantibodies and dementia has been
SC
previously shown in a French study in which the reactivity against BP180-NC16A was analyzed in 138 subjects aged over 69 years with no signs of BP (69 dementia patients and 60 non-dementia
M AN U
controls) (Foureur et al., 2006). That study found BP180 autoantibodies in 7% of subjects with dementia (MMSE≤24), but not in controls (MMSE>24). When compared to this previous study, the prevalence of increased BP180-NC16A autoantibodies in our study was clearly higher among AD patients. Differing ELISA results were reported in a recent study in which only one out of 26 patients with various dementia types had autoantibodies against BP180-NC16A (Messingham et al., 2016). However, six of the same patients showed IgG reactivity in immunoblotting against the
TE D
entire intra- or extracellular domain of BP180 (Messingham et al., 2016) which closely resembles our current result. In addition, nine patients with Parkinson’s disease (n=24) had antibodies to BP180, but not against the NC16A domain (Messingham et al., 2016). Another recent study failed to detect any autoantibodies against BP180 or BP230 in patients with Parkinson’s disease (n=50)
EP
and multiple sclerosis (n=50) despite the use of several antigens and diagnostic methods (Recke et al., 2016). The differing results of these studies may be explained by methodological differences or
AC C
differences between the study populations. The dementia patients in our study were diagnosed according to internationally accepted diagnostic criteria for AD including clinical and neuropsychological examination, brain imaging and testing for CSF AD biomarkers (McKhann et al., 1984), whereas the other studies do not describe in detail the criteria by which their dementia diagnoses were made (Foureur et al., 2006; Messingham et al., 2016).
Our current study shows that BP180 autoantibodies had a significant association with cognitive decline: the lower the MMSE score, the higher the value of the BP180 ELISA. This is in line with an epidemiological study, which showed that more severe dementia (MMSE ≤17) is associated with 2-fold increase in the risk of BP (Bastuji-Garin et al., 2011). In contrast, there was no correlation 7
ACCEPTED MANUSCRIPT between the level of BP autoantibodies and the concentration of AD biomarkers in CSF samples. Beta-amyloid (Αβ1−42) is a marker for the deposition of Αβ, and total tau and hyperphosphorylated tau (p-tau181) can be used to quantify neurofibrillary tangles and neuronal loss in the brain (Tapiola et al., 2009). Among these three markers, only a decrease in the concentration of p-tau181in CSF correlates weakly with cognitive decline measured by the MMSE (Seppälä et al., 2011). The
RI PT
negative correlation between the level of BP180-NC16A autoantibodies and MMSE values demonstrates the need for further studies to analyze whether BP180 ELISA could be used as a predictive serum marker of AD onset or prognosis. Surprisingly, we found that IgG antibodies against BP180 were significantly more common in women than men with AD. Although the
SC
incidence of BP is higher in women than in men (Langan et al., 2008; Joly et al., 2012) dementia
M AN U
seems to be equally associated with BP in both genders (Chen et al., 2011; Langan et al., 2011).
The expression of the neural isoform of BP230 in the brain has often been suggested to explain the association between BP and neurological disorders (Langan et al., 2011; Seppänen, 2013; Kunzli et al., 2016), but so far attempts to demonstrate a cross-reactive immune reaction between the CNS and skin variants have failed (Kunzli et al., 2016). The neural isoforms of BP230 are widely expressed in both the CNS and the peripheral nervous system (Kunzli et al., 2016), whereas BP180
TE D
is expressed in different anatomical regions of the human brain (Seppänen, 2013). Of note, particularly strong BP180 expression has been detected in pyramidal cells of the hippocampus and the ganglionic layer of the cortex, regions that are well-recognized predilection areas for AD-related
EP
lesions (Seppänen et al., 2006; Seppänen et al. 2007; Seppänen, 2013). Our current data suggest that an autoimmune reaction against BP180, rather than BP230, seems to be associated with neurodegenerative disorders. In fact, reactivity against BP180 is more relevant for the development
AC C
of clinical BP, since IgG recognition of the BP180 ectodomain is an early and crucial event in the pathogenesis of BP, which is followed by IgG reactivity against BP230 through epitope spreading events (Di Zenzo et al., 2011).
The detection of BP180 autoantibodies has been shown to have high sensitivity and moderate specificity for diagnosing BP and it has even suggested to be used in the diagnostic screening for BP (Sakuma-Oyama et al., 2004; Tampoia et al., 2012). Using immunoblotting we confirmed that the IgG autoantibodies of AD patients recognized a 180-kDa protein that was sensitive to collagenase digestion, and could therefore be identified as BP180. Therefore, it is intriguing that 8
ACCEPTED MANUSCRIPT sera from our AD patients showed no IgG reactivity against the cutaneous basement membrane zone in indirect IF analysis. However, our negative IF results reflect those of the aforementioned work of Messingham and co-workers. They reported that anti-BP180 positive sera from four patients with Parkinson’s disease lacked reactivity with skin in indirect IF, but instead recognized tyrosine-hydroxylase positive neurons in human and rat substantia nigra (Messingham et al., 2016).
RI PT
Viewed with our current data, these results suggest that autoantibodies found in patients with neurological diseases are not identical to those in BP patients. Perhaps the patients with AD or other neurological diseases who have positive BP180 antibodies represent a subgroup of “pre-disease state” BP patients. These patients first have neuronal immunoreactivity against BP180 and later on,
SC
perhaps due to some additional triggers, some also develop cutaneous autoimmunity. The time from initial symptoms to clinically typical BP may range from several weeks to more than 10 years (Schmidt et al., 2014), but currently the typical latency between the detection of IgG reactivity
M AN U
against BP180 and the appearance of clinical BP symptoms is unknown. After a diagnosis of dementia, people aged 65 or older survive a median of 3–8 years, but some live for as long as 20 years (see Winblad et al., 2016). Thus it is possible that the majority of AD and other dementia patients with BP180 autoantibodies die before they develop reactivity against the skin. This hypothesis is supported by epidemiological data: the prevalence of dementia is 5–7% in most
TE D
regions of the world and AD is estimated to account for 50–70% of all dementia cases (see Winblad et al., 2016). Since the incidence of BP varies between 0.25 and 4.28/100 000 per year (Langan et al., 2008; Joly et al., 2012; Schmidt and Zillikens, 2013; Försti et al., 2014), a crude estimate would indicate that indeed only a very small subgroup of anti-BP180 positive AD patients finally develop
EP
clinical BP. On the other hand, in our study, increased anti-BP180 values correlated with more severe dementia. If BP manifests as pruritus and non-specific symptoms such as erosions, excoriations, eczema or papules (Di Zenzo et al., 2012; Schmidt and Zillikens, 2013), its diagnosis
AC C
can be easily missed, especially in patients with severe AD whose ability to communicate is impaired and who are usually treated in geriatric units or nursing homes rather than hospitals.
We conclude that levels of IgG antibodies against BP180 are detected in almost 20 % of patients with AD, and that BP180-NC16A ELISA values are significantly associated with the severity of dementia. Future studies are required to assess the exact epitopes and IgG subtypes of BP180 antibodies in neurological patients and measure BP autoantibodies in CSF samples of AD patients, and thereby clarify the molecular mechanisms leading to failure of immunological tolerance against BP autoantigens in the human brain. The key question to be answered is why the AD patients’ 9
ACCEPTED MANUSCRIPT BP180 autoantibodies do not bind to the cutaneous basement membrane. This could be explored, for example, by comparing the immunoreaction in the human brain between specific BP180 antibodies and anti-BP180 positive samples from patients with various neurological diseases as well as by analyzing whether autoantibodies from patients with BP bind to neural tissue. Finally, large prospective studies with well-characterized study populations and long enough follow-up periods
AC C
EP
TE D
M AN U
SC
its association with AD and other neurological diseases.
RI PT
will help us better to understand predisposing factors and additional triggers of the onset of BP and
10
ACCEPTED MANUSCRIPT MATERIALS AND METHODS Patient samples The study was performed according to the principles of the Declaration of Helsinki. The research ethics committee of Kuopio University Hospital approved the collection of human AD and control samples. The collection of BP sera samples and control skin biopsies for indirect IF analysis was
RI PT
approved by the Ethical Committee of the Northern Ostrobothnia Hospital District. All samples were taken after written informed consent.
SC
Serum samples were collected at the Kuopio University Hospital from patients with dementia and from neurologically healthy control subjects. All dementia patients were diagnosed with probable
M AN U
AD according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria (McKhann et al., 1984). The diagnosis was based on clinical and neuropsychological examination, brain imaging and CSF AD biomarkers. All control samples were taken from patients attending the hospital for knee replacement operations. The mean age of the AD patients was slightly higher than that of controls. In both control and AD groups, there were equal female predominance (41/74 in
TE D
study group and 14/26 in control group) corresponding to that in patients with AD in general. The MMSE score at the time of the clinical examinations were obtained from patient records. We obtained CSF samples from both the dementia patients and the controls by lumbar puncture in the L3-4 or L4-5 interspace and stored at -80 °C until measurement. The CSF concentrations of AD
EP
markers Αβ1-42, p-tau181 and total tau were measured as previously described in detail (Herukka et al., 2005). The patient records of all patients with BP180 or BP230 ELISA positivity were checked
AC C
for skin diseases. A dermatologist did not check the cutaneous status of AD patients or controls.
BP180 and BP230 enzyme-linked immunosorbent analysis (ELISA) Circulating antibodies against BP180 and BP230 were analyzed from serum samples stored at -80 °C until measurement with commercially available kits (Medical and Biological Laboratories Co Ltd, Nagoya, Japan) according to manufacturer’s instructions. A cut-off ELISA value of 9 U/ml was used, with a value of >9 U/ml being a positive result. BP180 ELISA was performed twice for each sample and the mean value was used for further analysis.
11
ACCEPTED MANUSCRIPT Immunoblotting COS-7 cells were transiently transfected with a plasmid containing human Col17A1 cDNA (Franzke et al., 2004) and Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After a 24-h transfection period, the culture medium was changed and 50 µg/ml ascorbic acid added to the cells. Then, 24 h later the cells were lysed in a
RI PT
RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, Protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO, USA], 2 mM EDTA) and aliquots of cell extracts were treated with 40 U/mL highly purified bacterial collagenase (type III, Sigma-Aldrich) for 2 h at 37°C. Proteins were size-separated under denaturing conditions,
SC
electrotransferred onto a nitrocellulose membrane and detected with human serum samples as previously described (Hurskainen et al. 2015). Shortly, the study serum samples were used as 1:50
M AN U
dilutions and anti-human IgG-peroxidase (Sigma-Aldrich, Manassas, VA, USA) as a secondary antibody. ECL Prime substrates (GE Healthcare, Buckinghamshire, UK) with an LAS-3000 Image analyzer (Fuji, Tokyo, Japan) were used for visualization. Polyclonal rabbit anti-human NC16A (Schumann et al., 2000) and goat anti-rabbit IgG-peroxidase (Sigma-Aldrich) antibodies were used
TE D
to detect BP180-protein in immunoblots.
Indirect immunofluorescence (IF)
Indirect IF analysis was performed as previously described (Hurskainen et al., 2015). Briefly, 5-µm frozen non-fixed sections of human skin (treated or not with 1 M NaCl) were blocked (1% BSA in
EP
PBS) and human sera in 1:4 to 1:200 dilutions were used as primary antibodies. Rabbit anti-human IgG-FITC (DAKO, Glostrup, Denmark) was used as a secondary antibody. ImmuMount (Shandon,
AC C
Thermo Scientific, Middletown VA, USA) -embedded sections were photographed with an Olympus FluoView FV1000 confocal microscope using a x60 oil objective. Identical settings and exposure times were used to photograph all sections.
Statistical analysis Data were entered and statistical analyses were conducted using the IBM SPSS Statistics version 21.0 software for Windows (IBM, Chicago, IL, USA). The Kolmogorov-Smirnov test was used to test the normality of distribution of the variables. The data for continuous variables are presented as mean (SD) of median (range) when the assumption for normality was not met. Differences between 12
ACCEPTED MANUSCRIPT the groups were analyzed using either ANOVA for normally distributed data or the Mann-Whitney U test when the assumptions were not met. Pearson correlation or Spearman’s rho test were used to analyze correlations between continuous variables. For the nominal variables, the results are presented as the number (percentage) of subjects when appropriate and the χ2 test was used for testing statistical difference between groups. A two-sided P-value of 0.05 was considered as the
RI PT
limit of statistical significance.
CONFLICT OF INTEREST
SC
The authors state no conflict of interest.
M AN U
ACKNOWLEDGEMENTS
We thank Ms Anja Mattila and Ms Riitta Vuento for their expert technical assistance. This study was supported by the Academy of Finland grant to NK, Oulu University Hospital to KT, and
AC C
EP
TE D
Kuopio University Hospital to S-KH and AMR.
13
ACCEPTED MANUSCRIPT REFERENCES Bastuji-Garin S, Joly P, Lemordant P, Sparsa A, Bedane C, Delaporte E et al. Risk factors for bullous pemphigoid in the elderly: A prospective case-control study. J Invest Dermatol 2011;131: 637-43
RI PT
Brick KE, Weaver CH, Savica R, Lohse CM, Pittelkow MR, Boeve BF et al. A population-based study of the association between bullous pemphigoid and neurologic disorders. J Am Acad Dermatol 2014;71: 1191-7 Chen YJ, Wu CY, Lin MW, Chen TJ, Liao KK, Chen YC et al. Comorbidity profiles among patients with bullous pemphigoid: A nationwide population-based study. Br J Dermatol 2011;165: 593-9
SC
Claudepierre T, Manglapus MK, Marengi N, Radner S, Champliaud MF, Tasanen K et al. Collagen XVII and BPAG1 expression in the retina: Evidence for an anchoring complex in the central nervous system. J Comp Neurol 2005;487: 190-203
M AN U
Cordel N, Chosidow O, Hellot MF, Delaporte E, Lok C, Vaillant L et al. Neurological disorders in patients with bullous pemphigoid. Dermatology 2007;215: 187-91 della Torre R, Combescure C, Cortes B, Marazza G, Beltraminelli H, Naldi L et al. Clinical presentation and diagnostic delay in bullous pemphigoid: A prospective nationwide cohort. Br J Dermatol 2012;167: 1111-7 Di Zenzo G, Della Torre R, Zambruno G, Borradori L. Bullous pemphigoid: From the clinic to the bench. Clin Dermatol 2012;30: 3-16
TE D
Di Zenzo G, Thoma-Uszynski S, Calabresi V, Fontao L, Hofmann SC, Lacour JP et al. Demonstration of epitope-spreading phenomena in bullous pemphigoid: Results of a prospective multicenter study. J Invest Dermatol 2011;131: 2271-80 Försti AK, Jokelainen J, Timonen M, Tasanen K. Increasing incidence of bullous pemphigoid in northern Finland: A retrospective database study in Oulu university hospital. Br J Dermatol 2014;171: 1223-6
EP
Foureur N, Mignot S, Senet P, Verpillat P, Picard-Dahan C, Crickx B et al. Correlation between the presence of type-2 anti-pemphigoid antibodies and dementia in elderly subjects with no clinical signs of pemphigoid. Ann Dermatol Venereol 2006;133: 439-43
AC C
Franzke CW, Tasanen K, Borradori L, Huotari V, Bruckner-Tuderman L. Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface. J Biol Chem 2004;279: 245219 Franzke CW, Bruckner P, Bruckner-Tuderman L. Collagenous transmembrane proteins: Recent insights into biology and pathology. J Biol Chem 2005;280: 4005-8 Herukka SK, Hallikainen M, Soininen H, Pirttila T. CSF Abeta42 and tau or phosphorylated tau and prediction of progressive mild cognitive impairment. Neurology 2005;64: 1294-7 Huilaja L, Hurskainen T, Autio-Harmainen H, Hofmann SC, Sormunen R, Rasanen J et al. Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes. Matrix Biol 2008;27: 190-200
14
ACCEPTED MANUSCRIPT Hurskainen T, Moilanen J, Sormunen R, Franzke CW, Soininen R, Loeffek S et al. Transmembrane collagen XVII is a novel component of the glomerular filtration barrier. Cell Tissue Res 2012;348: 579-88. Hurskainen T, Kokkonen N, Sormunen R, Jackow J, Löffek S, Soininen R, et al. Deletion of the major bullous pemphigoid epitope region of collagen XVII induces blistering, autoimmunization, and itching in mice. J Invest Dermatol 2015;135:1303-10
RI PT
Joly P, Baricault S, Sparsa A, Bernard P, Bedane C, Duvert-Lehembre S et al. Incidence and mortality of bullous pemphigoid in France. J Invest Dermatol 2012;132: 1998-2004 Kasperkiewicz M, Zillikens D, Schmidt E. Pemphigoid diseases: Pathogenesis, diagnosis, and treatment. Autoimmunity 2012;45: 55-70 Kunzli K, Favre B, Chofflon M, Borradori L. One gene but different proteins and diseases: The complexity of dystonin and bullous pemphigoid antigen 1. Exp Dermatol 2016;25: 10-6
SC
Langan SM, Groves RW, West J. The relationship between neurological disease and bullous pemphigoid: A population-based case-control study. J Invest Dermatol 2011;131: 631-6
M AN U
Langan SM, Smeeth L, Hubbard R, Fleming KM, Smith CJ, West J. Bullous pemphigoid and pemphigus vulgaris--incidence and mortality in the UK: Population based cohort study. BMJ 2008;337: a180 McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of alzheimer's disease: Report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on alzheimer's disease. Neurology 1984;34: 939-44
TE D
Messingham KA, Aust S, Helfenberger J, Parker KL, Schultz S, McKillip J et al. Autoantibodies to collagen XVII are present in parkinson's disease and localize to tyrosine-hydroxylase positive neurons. J Invest Dermatol 2016;136: 721-3 Nishie W. Update on the pathogenesis of bullous pemphigoid: An autoantibody-mediated blistering disease targeting collagen XVII. J Dermatol Sci 2014;73: 179-86
EP
Ong EL, Goldacre R, Taghipour K. The relationship between motor neuron disease and bullous pemphigoid: An english cohort study. J Am Acad Dermatol 2013;69: 836-7
AC C
Powell AM, Sakuma-Oyama Y, Oyama N, Black MM. Collagen XVII/BP180: A collagenous transmembrane protein and component of the dermoepidermal anchoring complex. Clin Exp Dermatol 2005;30: 682-7 Prussmann W, Prussmann J, Koga H, Recke A, Iwata H, Juhl D et al. Prevalence of pemphigus and pemphigoid autoantibodies in the general population. Orphanet J Rare Dis 2015; doi:10: 63,0150278-x Recke A, Oei A, Hubner F, Fechner K, Graf J, Hagenah J et al. Parkinson's disease and multiple sclerosis are not associated with autoantibodies against structural proteins of the dermal-epidermal junction [e-pub ahead of print]. Br J Dermatol doi:10.1111/bjd.14538 Sakuma-Oyama Y, Powell AM, Oyama N, Albert S, Bhogal BS, Black MM. Evaluation of a BP180-NC16a enzyme-linked immunosorbent assay in the initial diagnosis of bullous pemphigoid. Br J Dermatol 2004;151: 126-31 Schmidt E, Zillikens D. Pemphigoid diseases. Lancet 2013;381: 320-32
15
ACCEPTED MANUSCRIPT Schmidt T, Sitaru C, Amber K, Hertl M. BP180- and BP230-specific IgG autoantibodies in pruritic disorders of the elderly: A preclinical stage of bullous pemphigoid? Br J Dermatol 2014; 171: 212-9 Schumann H, Baetge J, Tasanen K, Wojnarowska F, Schäcke H, Zillikens D, et al. The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases. Am J Pathol 2000;156:685-95.
RI PT
Seppälä TT, Koivisto AM, Hartikainen P, Helisalmi P, Soininen H, Herukka S-K. Longitudinal changes of CSF Biomarkers in Alzheimer’s disease. J Alzheimers Dis 2011; 25:583-94. Seppänen A. Collagen XVII: A shared antigen in neurodermatological interactions? Clin Dev Immunol 2013: http://dx.doi.org/10.1155/2013/240570 Seppänen A, Autio-Harmainen H, Alafuzoff I, Särkioja T, Veijola J, Hurskainen T et al. Collagen XVII is expressed in human CNS neurons. Matrix Biol 2006;25: 185-8
SC
Seppänen A, Suuronen T, Hofmann SC, Majamaa K, Alafuzoff I. Distribution of collagen XVII in the human brain. Brain Res 2007;1158: 50-6
M AN U
Stinco G, Codutti R, Scarbolo M, Valent F, Patrone P. A retrospective epidemiological study on the association of bullous pemphigoid and neurological diseases. Acta Derm Venereol 2005;85: 136-9 Tampoia M, Giavarina D, Di Giorgio C, Bizzaro N. Diagnostic accuracy of enzyme-linked immunosorbent assays (ELISA) to detect anti-skin autoantibodies in autoimmune blistering skin diseases: A systematic review and meta-analysis. Autoimmun Rev 2012;12: 121-6 Tapiola T, Alafuzoff I, Herukka SK, Parkkinen L, Hartikainen P, Soininen H et al. Cerebrospinal fluid beta-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain. Arch Neurol 2009;66: 382-9
TE D
Van den Bergh F, Giudice GJ. BP180 (type XVII collagen) and its role in cutaneous biology and disease. Adv Dermatol 2003;19: 37-71 Winblad B, Amouyel P, Andrieu S, Ballard C, Brayne C, Brodaty H et al. Defeating Alzheimer's disease and other dementias: A priority for European science and society. Lancet Neurol 2016; 15: 455-532
AC C
EP
Yang YW, Chen YH, Xirasagar S, Lin HC. Increased risk of stroke in patients with bullous pemphigoid: A population-based follow-up study. Stroke 2011;42: 319-23
16
ACCEPTED MANUSCRIPT Table 1. Characteristics, bullous pemphigoid autoantibodies and Alzheimer’s disease biomarkers of neurologically healthy controls and patients with Alzheimer’s disease AD patients
P-value
n
40
115
Sex M/F
14/26 (35/65 %)
41/74 (36/64 %)
ns.
Age1
66.8±7.0
72.0±7.8
<0.001
MMSE2
na.
20 (8–29)
BP180 U/ml3
2.0 (0.5–12.3)
2.4 (0–47.7)
BP180 -/+3
37/1 (97/3%)
91/20 (82/18%)
BP230 U/ml
1.35 (0–24.1)
2.0 (0–35.8)
BP230 -/+
37/3 (92.5/7.5%)
101/14 (88/12%)
ns.
Aβ1-42 pg/ml
728 (263–1078)4
443 (139–1294)
<0.001
Tau pg/ml
266 (90–582)
p-Tau181 pg/ml1
51±14
RI PT
Controls
0.05
0.019
M AN U
SC
ns.
554 (48–1994)
<0.001
90±35
<0.001
Data given as median (range) unless otherwise indicated. Abbreviations: ns, not significant; MMSE, Mini-Mental State Examination; na, not available; Αβ1-42, beta-amyloid; tau, total tau; p-Tau181, hyperphosphorylated tau. Data given as mean ± standard deviation
2
Data available for 89 patients with Alzheimer’s disease
3
Two controls and four patients with Alzheimer’s disease were excluded due to lack of reactivity
TE D
1
against BP180 in immunoblotting
EP
Due to a method change, the levels are 10 % higher than in AD group.
AC C
4
17
ACCEPTED MANUSCRIPT Figure legends Figure 1. BP180 and BP230 autoantibody values of control subjects and patients with Alzheimer’s disease. Sera from controls (circles) and patients with Alzheimer’s disease (AD) (squares) were tested for IgG reactivity against BP180 and BP230 using ELISA kits. BP180 ELISA assay was performed as duplicates for all samples. (a) Increased anti-BP180 values (cut-off value
RI PT
>9U/ml, dashed line) were detected in 3/40 control samples and in 24/115 AD samples. The increased anti-BP180 values varied between 9.2 and 47.7 U/ml. Two control samples and four AD samples were excluded since they did not recognize full-length BP180 in immunoblotting. (b) Increased anti-BP230 values (cut-off value >9U/ml, dashed line) were detected in 3/40 control
SC
samples and in 14/115 AD samples. The increased anti-BP230 values varied between 9.6 and 35.8
M AN U
U/ml.
Figure 2. Autoantibodies present in the sera of patients with Alzheimer’s disease detect BP180 in immunoblotting. Twenty-four Alzheimer’s disease (AD) samples and three control samples, all of which showed increased anti-BP180 levels in ELISA assay, were further analysed by immunoblotting using human recombinant full-length BP180 expressed in COS-7 cells as a target
TE D
antigen. Anti-human IgG was used as a secondary antibody. (a) Human polyclonal anti-BP180 (NC16A) antibody and sera taken from a patient with bullous pemphigoid (BP), a control patient and an AD patient all recognized a 180-kDa protein that was not detected in collagenase-treated cell extract. (b) Four examples of immunoblotting, which verified that anti-BP180 ELISA positive AD
AC C
full-length BP180.
EP
samples recognize the full-length BP180. The ELISA positive control sample did not recognize the
18
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0022-202X(16)32451-4
DOI:
10.1016/j.jid.2016.09.010
Reference:
JID 531
To appear in:
The Journal of Investigative Dermatology
Received Date: 13 April 2016 Revised Date:
12 August 2016
Accepted Date: 2 September 2016
Please cite this article as: Kokkonen N, Herukka S-K, Huilaja L, Kokki M, Koivisto AM, Hartikainen P, Remes AM, Tasanen K, Increased Levels of the Bullous Pemphigoid BP180 Autoantibody Are Associated with More Severe Dementia in Alzheimer’s Disease, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Increased Levels of the Bullous Pemphigoid BP180 Autoantibody Are Associated with More Severe Dementia in Alzheimer’s Disease Nina Kokkonen1,4, Sanna-Kaisa Herukka2,4, Laura Huilaja1,4, Merja Kokki3, Anne M. Koivisto2, Päivi Hartikainen2, Anne M. Remes2, and Kaisa Tasanen1 Department of Dermatology, PEDEGO Research Unit, Medical Research Center Oulu, Oulu
University Hospital and University of Oulu, Oulu, Finland 2
RI PT
1
Institute of Clinical Medicine – Neurology, University of Eastern Finland and Department of
Neurology, Kuopio University Hospital, Kuopio, Finland
Department of Anesthesia and Operative Service, Kuopio University Hospital, Kuopio, Finland;
4
These authors contributed equally to this work.
M AN U
SC
3
Short title: Bullous Pemphigoid and Alzheimer’s disease
TE D
Keywords: BP230, collagen XVII, dementia, hemidesmosome, neurodegeneration
Abbreviations: bullous pemphigoid (BP), Alzheimer’s disease (AD), central nervous system (CNS), cerebrospinal fluid (CSF), immunofluorescence (IF), Mini-Mental State Examination
EP
(MMSE), non-collagenous 16A (NC16A), enzyme-linked immunosorbent assay (ELISA)
AC C
Correspondence:
Kaisa Tasanen, Department of Dermatology, Medical Research Center Oulu, University of Oulu, Aapistie 5A, FIN-90220 OULU, Finland; Tel: +358 400 613361; E-mail: [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
Bullous pemphigoid (BP) is a subepidermal blistering skin disease, which has shown a strong association with neurological diseases in epidemiological studies. The BP autoantigens BP180 and BP230 are expressed in the cutaneous basement membrane and the central nervous system. Using BP180 and BP230 ELISA assays and immunoblotting against BP180 we analyzed the IgG
RI PT
reactivity in the sera of 115 patients with Alzheimer’s disease (AD) and 40 neurologically healthy controls. BP180 autoantibodies were found in 18 % of AD patients, whereas only 3% of controls had positive results (P=0.019). BP230 values were higher and more often elevated in AD patients than controls, but not significantly. None of the positive AD sera that recognized the full-length
SC
human BP180 in immunoblotting reacted with the cutaneous basement membrane in indirect immunofluorescence analysis. Moreover, a retrospective evaluation of the hospital records of the AD patients revealed neither BP diagnosis nor BP-like symptoms. Interestingly, increased BP180-
M AN U
NC16A autoantibody values correlated with cognitive decline measured by Mini-Mental State Examination scores (MMSE), but not with the concentration of AD biomarkers in cerebrospinal fluid. Our findings further the understanding of the role of BP180 as a shared autoantigen in neuro-
AC C
EP
TE D
dermatological interactions and the association between BP and neurodegenerative diseases.
2
ACCEPTED MANUSCRIPT INTRODUCTION Bullous pemphigoid (BP) is the most common subepidermal autoimmune blistering disease with increasing incidence among elderly people (Langan et al., 2008; Joly et al., 2012; Schmidt and Zillikens, 2013; Försti et al., 2014). Typically BP patients have severe pruritus and blistering skin lesions, but in up to 20% of patients blisters are absent and only excoriations, eczematous or
RI PT
urticated lesions occur (della Torre et al., 2012; Di Zenzo et al., 2012;). At the molecular level BP autoantibodies target two cutaneous basement membrane proteins, BP180 and BP230 (Nishie, 2014). The main antigen BP180, also known as collagen XVII or BPAg2, is a transmembrane protein of hemidesmosomes linking basal keratinocytes to the underlying dermis (Van den Bergh
SC
and Giudice, 2003; Franzke et al., 2005). IgG antibodies of BP patients target the immunodominant non-collagenous 16A (NC16A) domain, which is located extracellularly close to the transmembrane
M AN U
domain of BP180 (Schmidt and Zillikens, 2013; Nishie, 2014). As well as in the skin, BP180 is also expressed in various other tissues (Claudepierre et al., 2005; Huilaja et al., 2008; Hurskainen et al., 2012) including the brain (Seppänen et al., 2006; Seppänen et al. 2007; Seppänen, 2013). The other autoantigen, BP230 (bullous pemphigoid antigen 1, BPAg1e), is an intracellular component of hemidesmosomes and a ligand of BP180. It is the epithelial isoform of dystonin, and its variants BPAg1a1 and BPAg1a2 are expressed in both the central and the peripheral nervous system (Powell
TE D
et al., 2005; Kunzli et al., 2016). BP230 is targeted by autoantibodies in approximately 60% of patients with BP (Kasperkiewicz et al., 2012), but it seems that autoantibodies against BP230 result from intermolecular epitope spreading and never proceed to anti-BP180-autoantibodies (Di Zenzo
EP
et al., 2011; Di Zenzo et al., 2012).
AC C
The diagnosis of BP is established by typical clinical presentation and the finding of linear IgG or complement 3 along the basement membrane zone in a direct immunofluorescence (IF) analysis of a perilesional biopsy (Di Zenzo et al., 2012; Schmidt and Zillikens, 2013). In addition, the detection of circulating autoantibodies by BP180-NC16A ELISA has high specificity and moderate sensitivity in diagnosing BP (Tampoia et al., 2012).
Several epidemiological studies have revealed an association of BP with a range of neurological diseases including dementia, cerebral stroke, Parkinson’s disease, multiple sclerosis and motor neuron disease (Stinco et al., 2005; Cordel et al., 2007; Langan et al., 2011; Bastuji-Garin et al., 2011; Ong et al., 2013; Brick et al., 2014). An existing neurological disorder appears to increase the 3
ACCEPTED MANUSCRIPT subsequent risk for BP (Langan et al., 2011; Brick et al., 2014), but conversely, increased risk for developing neurological diseases has also been reported following BP diagnosis (Yang et al., 2011; Brick et al., 2014).
The expression of BP autoantigens in the central nervous system (CNS) and the association between
RI PT
BP and various neurodegenerative disorders suggest that BP180 and BP230 could act as shared antigens in cutaneous and neurological diseases (Seppänen, 2013; Kunzli et al., 2016). To test this hypothesis we measured BP autoantibodies in well-characterized patients with Alzheimer’s disease (AD). We also investigated postulated associations between BP autoantibodies and age, gender and
SC
cognitive decline as well as with the presence of cerebrospinal fluid (CSF) markers of AD
AC C
EP
TE D
M AN U
pathology.
4
ACCEPTED MANUSCRIPT RESULTS BP180 and BP230 autoantibodies in patients with Alzheimer’s disease Sera from patients with AD (n=115) and neurologically healthy control subjects (n=40) were tested for BP180 and BP230 IgG autoantibodies using a well-established, commercially available ELISA analysis and all anti-BP180 positive sera (24 AD samples and 3 controls) were further tested by
RI PT
immunoblotting against human recombinant BP180. A significantly higher proportion of AD samples (20; 18%) than control samples (1; 3%) were positive for both IgG autoantibodies against BP180-NC16A (based on a cut-off value >9 U/ml; Table 1, Figure 1) and the full-length BP180 (Figure 2). Accordingly, BP180 autoantibody values were significantly higher in the AD group (2.4
SC
U/ml, range 0–47.7 U/ml) compared to those of control group (2.0 U/ml, range 0.5-12.3 U/ml; P=0.05; Table 1, Figure 1). We also tested 18 of the anti-BP180 positive AD samples and the one
M AN U
positive control sample using indirect IF analysis. Under the same conditions in which the sera from BP patients demonstrated a clear linear immunostaining on the cutaneous basement membrane zone, none of the tested AD sera nor the positive control sample showed any specific immunostaining either in normal or salt-split human skin (Supplementary Figure S1 online).
TE D
In the ELISA assay IgG autoantibodies against BP230 were also found in a higher proportion of AD patients than controls and BP230 autoantibody values were higher in the AD group than those of the controls, but neither of these differences was statistically significant (Table 1, Figure 1). Autoantibodies against only BP180 were detected in 16 AD patients, six AD patients had anti-
EP
BP230 positivity without having reactivity against BP180 and eight AD patients had autoantibodies
AC C
against both BP180 and BP230.
A retrospective evaluation of the hospital records of AD patients with increased anti-BP180 or BP230 values revealed neither diagnosis nor clinical symptoms of BP in any patient.
The association of BP180 and BP230 autoantibodies with age, gender, cognitive impairment and Alzheimer’s disease biomarkers BP180 and BP230 ELISA values did not correlate with age in either the whole study group or the group of AD patients. However, BP180 and BP230 ELISA values showed a statistically significant 5
ACCEPTED MANUSCRIPT correlation to each other in the whole study group (r=0.329, P<0.001). This overall correlation was driven by the correlation in the AD patients (r=0.367, P<0.001), as there was no such correlation when the control group was considered separately. In the AD group, autoantibodies against BP180NC16A were significantly more common in females than males (P=0.029) and the median BP180 ELISA value was significantly higher in females than males (P=0.037). In contrast, there was no
RI PT
significant difference between the genders for positivity or values of BP230 ELISA.
The severity of dementia of AD patients was evaluated using the Mini-Mental State Examination (MMSE) (Table 1). MMSE data were available for 89 patients with AD. Interestingly, there was a
SC
negative correlation between MMSE score and BP180 ELISA value (r=-0.287, P=0,007) i.e. patients with lower MMSE, denoting more severe dementia, had higher serum anti-BP180 levels.
M AN U
As expected, MMSE score showed negative correlation with age (r=-0.351, P=0.001). Regarding AD biomarkers there was no correlation between serum anti-BP180 values and beta-amyloid (Αβ1-
EP
TE D
total tau or hyperphosphorylated tau (p-tau181) in the CSF samples of patients with AD.
AC C
42),
6
ACCEPTED MANUSCRIPT DISCUSSION Based on the association of several neurological diseases with BP and the CNS expression of BP autoantigens it has been suggested that neuroinflammation could lead to a cross-reactive immunoresponse between neural and cutaneous antigens (Langan et al., 2011; Seppänen, 2013; Kunzli et al., 2016). This theory is supported by our current study, which revealed the presence of
RI PT
increased IgG autoantibodies against BP180 in 18 % of patients with AD. The prevalence of BP autoantibodies in our control samples was slightly higher than in previous studies, which report that IgG autoantibodies against BP180 and BP230 exist in 0–2.0% of the general population (see Prussmann et al., 2015). The association between BP autoantibodies and dementia has been
SC
previously shown in a French study in which the reactivity against BP180-NC16A was analyzed in 138 subjects aged over 69 years with no signs of BP (69 dementia patients and 60 non-dementia
M AN U
controls) (Foureur et al., 2006). That study found BP180 autoantibodies in 7% of subjects with dementia (MMSE≤24), but not in controls (MMSE>24). When compared to this previous study, the prevalence of increased BP180-NC16A autoantibodies in our study was clearly higher among AD patients. Differing ELISA results were reported in a recent study in which only one out of 26 patients with various dementia types had autoantibodies against BP180-NC16A (Messingham et al., 2016). However, six of the same patients showed IgG reactivity in immunoblotting against the
TE D
entire intra- or extracellular domain of BP180 (Messingham et al., 2016) which closely resembles our current result. In addition, nine patients with Parkinson’s disease (n=24) had antibodies to BP180, but not against the NC16A domain (Messingham et al., 2016). Another recent study failed to detect any autoantibodies against BP180 or BP230 in patients with Parkinson’s disease (n=50)
EP
and multiple sclerosis (n=50) despite the use of several antigens and diagnostic methods (Recke et al., 2016). The differing results of these studies may be explained by methodological differences or
AC C
differences between the study populations. The dementia patients in our study were diagnosed according to internationally accepted diagnostic criteria for AD including clinical and neuropsychological examination, brain imaging and testing for CSF AD biomarkers (McKhann et al., 1984), whereas the other studies do not describe in detail the criteria by which their dementia diagnoses were made (Foureur et al., 2006; Messingham et al., 2016).
Our current study shows that BP180 autoantibodies had a significant association with cognitive decline: the lower the MMSE score, the higher the value of the BP180 ELISA. This is in line with an epidemiological study, which showed that more severe dementia (MMSE ≤17) is associated with 2-fold increase in the risk of BP (Bastuji-Garin et al., 2011). In contrast, there was no correlation 7
ACCEPTED MANUSCRIPT between the level of BP autoantibodies and the concentration of AD biomarkers in CSF samples. Beta-amyloid (Αβ1−42) is a marker for the deposition of Αβ, and total tau and hyperphosphorylated tau (p-tau181) can be used to quantify neurofibrillary tangles and neuronal loss in the brain (Tapiola et al., 2009). Among these three markers, only a decrease in the concentration of p-tau181in CSF correlates weakly with cognitive decline measured by the MMSE (Seppälä et al., 2011). The
RI PT
negative correlation between the level of BP180-NC16A autoantibodies and MMSE values demonstrates the need for further studies to analyze whether BP180 ELISA could be used as a predictive serum marker of AD onset or prognosis. Surprisingly, we found that IgG antibodies against BP180 were significantly more common in women than men with AD. Although the
SC
incidence of BP is higher in women than in men (Langan et al., 2008; Joly et al., 2012) dementia
M AN U
seems to be equally associated with BP in both genders (Chen et al., 2011; Langan et al., 2011).
The expression of the neural isoform of BP230 in the brain has often been suggested to explain the association between BP and neurological disorders (Langan et al., 2011; Seppänen, 2013; Kunzli et al., 2016), but so far attempts to demonstrate a cross-reactive immune reaction between the CNS and skin variants have failed (Kunzli et al., 2016). The neural isoforms of BP230 are widely expressed in both the CNS and the peripheral nervous system (Kunzli et al., 2016), whereas BP180
TE D
is expressed in different anatomical regions of the human brain (Seppänen, 2013). Of note, particularly strong BP180 expression has been detected in pyramidal cells of the hippocampus and the ganglionic layer of the cortex, regions that are well-recognized predilection areas for AD-related
EP
lesions (Seppänen et al., 2006; Seppänen et al. 2007; Seppänen, 2013). Our current data suggest that an autoimmune reaction against BP180, rather than BP230, seems to be associated with neurodegenerative disorders. In fact, reactivity against BP180 is more relevant for the development
AC C
of clinical BP, since IgG recognition of the BP180 ectodomain is an early and crucial event in the pathogenesis of BP, which is followed by IgG reactivity against BP230 through epitope spreading events (Di Zenzo et al., 2011).
The detection of BP180 autoantibodies has been shown to have high sensitivity and moderate specificity for diagnosing BP and it has even suggested to be used in the diagnostic screening for BP (Sakuma-Oyama et al., 2004; Tampoia et al., 2012). Using immunoblotting we confirmed that the IgG autoantibodies of AD patients recognized a 180-kDa protein that was sensitive to collagenase digestion, and could therefore be identified as BP180. Therefore, it is intriguing that 8
ACCEPTED MANUSCRIPT sera from our AD patients showed no IgG reactivity against the cutaneous basement membrane zone in indirect IF analysis. However, our negative IF results reflect those of the aforementioned work of Messingham and co-workers. They reported that anti-BP180 positive sera from four patients with Parkinson’s disease lacked reactivity with skin in indirect IF, but instead recognized tyrosine-hydroxylase positive neurons in human and rat substantia nigra (Messingham et al., 2016).
RI PT
Viewed with our current data, these results suggest that autoantibodies found in patients with neurological diseases are not identical to those in BP patients. Perhaps the patients with AD or other neurological diseases who have positive BP180 antibodies represent a subgroup of “pre-disease state” BP patients. These patients first have neuronal immunoreactivity against BP180 and later on,
SC
perhaps due to some additional triggers, some also develop cutaneous autoimmunity. The time from initial symptoms to clinically typical BP may range from several weeks to more than 10 years (Schmidt et al., 2014), but currently the typical latency between the detection of IgG reactivity
M AN U
against BP180 and the appearance of clinical BP symptoms is unknown. After a diagnosis of dementia, people aged 65 or older survive a median of 3–8 years, but some live for as long as 20 years (see Winblad et al., 2016). Thus it is possible that the majority of AD and other dementia patients with BP180 autoantibodies die before they develop reactivity against the skin. This hypothesis is supported by epidemiological data: the prevalence of dementia is 5–7% in most
TE D
regions of the world and AD is estimated to account for 50–70% of all dementia cases (see Winblad et al., 2016). Since the incidence of BP varies between 0.25 and 4.28/100 000 per year (Langan et al., 2008; Joly et al., 2012; Schmidt and Zillikens, 2013; Försti et al., 2014), a crude estimate would indicate that indeed only a very small subgroup of anti-BP180 positive AD patients finally develop
EP
clinical BP. On the other hand, in our study, increased anti-BP180 values correlated with more severe dementia. If BP manifests as pruritus and non-specific symptoms such as erosions, excoriations, eczema or papules (Di Zenzo et al., 2012; Schmidt and Zillikens, 2013), its diagnosis
AC C
can be easily missed, especially in patients with severe AD whose ability to communicate is impaired and who are usually treated in geriatric units or nursing homes rather than hospitals.
We conclude that levels of IgG antibodies against BP180 are detected in almost 20 % of patients with AD, and that BP180-NC16A ELISA values are significantly associated with the severity of dementia. Future studies are required to assess the exact epitopes and IgG subtypes of BP180 antibodies in neurological patients and measure BP autoantibodies in CSF samples of AD patients, and thereby clarify the molecular mechanisms leading to failure of immunological tolerance against BP autoantigens in the human brain. The key question to be answered is why the AD patients’ 9
ACCEPTED MANUSCRIPT BP180 autoantibodies do not bind to the cutaneous basement membrane. This could be explored, for example, by comparing the immunoreaction in the human brain between specific BP180 antibodies and anti-BP180 positive samples from patients with various neurological diseases as well as by analyzing whether autoantibodies from patients with BP bind to neural tissue. Finally, large prospective studies with well-characterized study populations and long enough follow-up periods
AC C
EP
TE D
M AN U
SC
its association with AD and other neurological diseases.
RI PT
will help us better to understand predisposing factors and additional triggers of the onset of BP and
10
ACCEPTED MANUSCRIPT MATERIALS AND METHODS Patient samples The study was performed according to the principles of the Declaration of Helsinki. The research ethics committee of Kuopio University Hospital approved the collection of human AD and control samples. The collection of BP sera samples and control skin biopsies for indirect IF analysis was
RI PT
approved by the Ethical Committee of the Northern Ostrobothnia Hospital District. All samples were taken after written informed consent.
SC
Serum samples were collected at the Kuopio University Hospital from patients with dementia and from neurologically healthy control subjects. All dementia patients were diagnosed with probable
M AN U
AD according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria (McKhann et al., 1984). The diagnosis was based on clinical and neuropsychological examination, brain imaging and CSF AD biomarkers. All control samples were taken from patients attending the hospital for knee replacement operations. The mean age of the AD patients was slightly higher than that of controls. In both control and AD groups, there were equal female predominance (41/74 in
TE D
study group and 14/26 in control group) corresponding to that in patients with AD in general. The MMSE score at the time of the clinical examinations were obtained from patient records. We obtained CSF samples from both the dementia patients and the controls by lumbar puncture in the L3-4 or L4-5 interspace and stored at -80 °C until measurement. The CSF concentrations of AD
EP
markers Αβ1-42, p-tau181 and total tau were measured as previously described in detail (Herukka et al., 2005). The patient records of all patients with BP180 or BP230 ELISA positivity were checked
AC C
for skin diseases. A dermatologist did not check the cutaneous status of AD patients or controls.
BP180 and BP230 enzyme-linked immunosorbent analysis (ELISA) Circulating antibodies against BP180 and BP230 were analyzed from serum samples stored at -80 °C until measurement with commercially available kits (Medical and Biological Laboratories Co Ltd, Nagoya, Japan) according to manufacturer’s instructions. A cut-off ELISA value of 9 U/ml was used, with a value of >9 U/ml being a positive result. BP180 ELISA was performed twice for each sample and the mean value was used for further analysis.
11
ACCEPTED MANUSCRIPT Immunoblotting COS-7 cells were transiently transfected with a plasmid containing human Col17A1 cDNA (Franzke et al., 2004) and Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After a 24-h transfection period, the culture medium was changed and 50 µg/ml ascorbic acid added to the cells. Then, 24 h later the cells were lysed in a
RI PT
RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, Protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO, USA], 2 mM EDTA) and aliquots of cell extracts were treated with 40 U/mL highly purified bacterial collagenase (type III, Sigma-Aldrich) for 2 h at 37°C. Proteins were size-separated under denaturing conditions,
SC
electrotransferred onto a nitrocellulose membrane and detected with human serum samples as previously described (Hurskainen et al. 2015). Shortly, the study serum samples were used as 1:50
M AN U
dilutions and anti-human IgG-peroxidase (Sigma-Aldrich, Manassas, VA, USA) as a secondary antibody. ECL Prime substrates (GE Healthcare, Buckinghamshire, UK) with an LAS-3000 Image analyzer (Fuji, Tokyo, Japan) were used for visualization. Polyclonal rabbit anti-human NC16A (Schumann et al., 2000) and goat anti-rabbit IgG-peroxidase (Sigma-Aldrich) antibodies were used
TE D
to detect BP180-protein in immunoblots.
Indirect immunofluorescence (IF)
Indirect IF analysis was performed as previously described (Hurskainen et al., 2015). Briefly, 5-µm frozen non-fixed sections of human skin (treated or not with 1 M NaCl) were blocked (1% BSA in
EP
PBS) and human sera in 1:4 to 1:200 dilutions were used as primary antibodies. Rabbit anti-human IgG-FITC (DAKO, Glostrup, Denmark) was used as a secondary antibody. ImmuMount (Shandon,
AC C
Thermo Scientific, Middletown VA, USA) -embedded sections were photographed with an Olympus FluoView FV1000 confocal microscope using a x60 oil objective. Identical settings and exposure times were used to photograph all sections.
Statistical analysis Data were entered and statistical analyses were conducted using the IBM SPSS Statistics version 21.0 software for Windows (IBM, Chicago, IL, USA). The Kolmogorov-Smirnov test was used to test the normality of distribution of the variables. The data for continuous variables are presented as mean (SD) of median (range) when the assumption for normality was not met. Differences between 12
ACCEPTED MANUSCRIPT the groups were analyzed using either ANOVA for normally distributed data or the Mann-Whitney U test when the assumptions were not met. Pearson correlation or Spearman’s rho test were used to analyze correlations between continuous variables. For the nominal variables, the results are presented as the number (percentage) of subjects when appropriate and the χ2 test was used for testing statistical difference between groups. A two-sided P-value of 0.05 was considered as the
RI PT
limit of statistical significance.
CONFLICT OF INTEREST
SC
The authors state no conflict of interest.
M AN U
ACKNOWLEDGEMENTS
We thank Ms Anja Mattila and Ms Riitta Vuento for their expert technical assistance. This study was supported by the Academy of Finland grant to NK, Oulu University Hospital to KT, and
AC C
EP
TE D
Kuopio University Hospital to S-KH and AMR.
13
ACCEPTED MANUSCRIPT REFERENCES Bastuji-Garin S, Joly P, Lemordant P, Sparsa A, Bedane C, Delaporte E et al. Risk factors for bullous pemphigoid in the elderly: A prospective case-control study. J Invest Dermatol 2011;131: 637-43
RI PT
Brick KE, Weaver CH, Savica R, Lohse CM, Pittelkow MR, Boeve BF et al. A population-based study of the association between bullous pemphigoid and neurologic disorders. J Am Acad Dermatol 2014;71: 1191-7 Chen YJ, Wu CY, Lin MW, Chen TJ, Liao KK, Chen YC et al. Comorbidity profiles among patients with bullous pemphigoid: A nationwide population-based study. Br J Dermatol 2011;165: 593-9
SC
Claudepierre T, Manglapus MK, Marengi N, Radner S, Champliaud MF, Tasanen K et al. Collagen XVII and BPAG1 expression in the retina: Evidence for an anchoring complex in the central nervous system. J Comp Neurol 2005;487: 190-203
M AN U
Cordel N, Chosidow O, Hellot MF, Delaporte E, Lok C, Vaillant L et al. Neurological disorders in patients with bullous pemphigoid. Dermatology 2007;215: 187-91 della Torre R, Combescure C, Cortes B, Marazza G, Beltraminelli H, Naldi L et al. Clinical presentation and diagnostic delay in bullous pemphigoid: A prospective nationwide cohort. Br J Dermatol 2012;167: 1111-7 Di Zenzo G, Della Torre R, Zambruno G, Borradori L. Bullous pemphigoid: From the clinic to the bench. Clin Dermatol 2012;30: 3-16
TE D
Di Zenzo G, Thoma-Uszynski S, Calabresi V, Fontao L, Hofmann SC, Lacour JP et al. Demonstration of epitope-spreading phenomena in bullous pemphigoid: Results of a prospective multicenter study. J Invest Dermatol 2011;131: 2271-80 Försti AK, Jokelainen J, Timonen M, Tasanen K. Increasing incidence of bullous pemphigoid in northern Finland: A retrospective database study in Oulu university hospital. Br J Dermatol 2014;171: 1223-6
EP
Foureur N, Mignot S, Senet P, Verpillat P, Picard-Dahan C, Crickx B et al. Correlation between the presence of type-2 anti-pemphigoid antibodies and dementia in elderly subjects with no clinical signs of pemphigoid. Ann Dermatol Venereol 2006;133: 439-43
AC C
Franzke CW, Tasanen K, Borradori L, Huotari V, Bruckner-Tuderman L. Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface. J Biol Chem 2004;279: 245219 Franzke CW, Bruckner P, Bruckner-Tuderman L. Collagenous transmembrane proteins: Recent insights into biology and pathology. J Biol Chem 2005;280: 4005-8 Herukka SK, Hallikainen M, Soininen H, Pirttila T. CSF Abeta42 and tau or phosphorylated tau and prediction of progressive mild cognitive impairment. Neurology 2005;64: 1294-7 Huilaja L, Hurskainen T, Autio-Harmainen H, Hofmann SC, Sormunen R, Rasanen J et al. Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes. Matrix Biol 2008;27: 190-200
14
ACCEPTED MANUSCRIPT Hurskainen T, Moilanen J, Sormunen R, Franzke CW, Soininen R, Loeffek S et al. Transmembrane collagen XVII is a novel component of the glomerular filtration barrier. Cell Tissue Res 2012;348: 579-88. Hurskainen T, Kokkonen N, Sormunen R, Jackow J, Löffek S, Soininen R, et al. Deletion of the major bullous pemphigoid epitope region of collagen XVII induces blistering, autoimmunization, and itching in mice. J Invest Dermatol 2015;135:1303-10
RI PT
Joly P, Baricault S, Sparsa A, Bernard P, Bedane C, Duvert-Lehembre S et al. Incidence and mortality of bullous pemphigoid in France. J Invest Dermatol 2012;132: 1998-2004 Kasperkiewicz M, Zillikens D, Schmidt E. Pemphigoid diseases: Pathogenesis, diagnosis, and treatment. Autoimmunity 2012;45: 55-70 Kunzli K, Favre B, Chofflon M, Borradori L. One gene but different proteins and diseases: The complexity of dystonin and bullous pemphigoid antigen 1. Exp Dermatol 2016;25: 10-6
SC
Langan SM, Groves RW, West J. The relationship between neurological disease and bullous pemphigoid: A population-based case-control study. J Invest Dermatol 2011;131: 631-6
M AN U
Langan SM, Smeeth L, Hubbard R, Fleming KM, Smith CJ, West J. Bullous pemphigoid and pemphigus vulgaris--incidence and mortality in the UK: Population based cohort study. BMJ 2008;337: a180 McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of alzheimer's disease: Report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on alzheimer's disease. Neurology 1984;34: 939-44
TE D
Messingham KA, Aust S, Helfenberger J, Parker KL, Schultz S, McKillip J et al. Autoantibodies to collagen XVII are present in parkinson's disease and localize to tyrosine-hydroxylase positive neurons. J Invest Dermatol 2016;136: 721-3 Nishie W. Update on the pathogenesis of bullous pemphigoid: An autoantibody-mediated blistering disease targeting collagen XVII. J Dermatol Sci 2014;73: 179-86
EP
Ong EL, Goldacre R, Taghipour K. The relationship between motor neuron disease and bullous pemphigoid: An english cohort study. J Am Acad Dermatol 2013;69: 836-7
AC C
Powell AM, Sakuma-Oyama Y, Oyama N, Black MM. Collagen XVII/BP180: A collagenous transmembrane protein and component of the dermoepidermal anchoring complex. Clin Exp Dermatol 2005;30: 682-7 Prussmann W, Prussmann J, Koga H, Recke A, Iwata H, Juhl D et al. Prevalence of pemphigus and pemphigoid autoantibodies in the general population. Orphanet J Rare Dis 2015; doi:10: 63,0150278-x Recke A, Oei A, Hubner F, Fechner K, Graf J, Hagenah J et al. Parkinson's disease and multiple sclerosis are not associated with autoantibodies against structural proteins of the dermal-epidermal junction [e-pub ahead of print]. Br J Dermatol doi:10.1111/bjd.14538 Sakuma-Oyama Y, Powell AM, Oyama N, Albert S, Bhogal BS, Black MM. Evaluation of a BP180-NC16a enzyme-linked immunosorbent assay in the initial diagnosis of bullous pemphigoid. Br J Dermatol 2004;151: 126-31 Schmidt E, Zillikens D. Pemphigoid diseases. Lancet 2013;381: 320-32
15
ACCEPTED MANUSCRIPT Schmidt T, Sitaru C, Amber K, Hertl M. BP180- and BP230-specific IgG autoantibodies in pruritic disorders of the elderly: A preclinical stage of bullous pemphigoid? Br J Dermatol 2014; 171: 212-9 Schumann H, Baetge J, Tasanen K, Wojnarowska F, Schäcke H, Zillikens D, et al. The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases. Am J Pathol 2000;156:685-95.
RI PT
Seppälä TT, Koivisto AM, Hartikainen P, Helisalmi P, Soininen H, Herukka S-K. Longitudinal changes of CSF Biomarkers in Alzheimer’s disease. J Alzheimers Dis 2011; 25:583-94. Seppänen A. Collagen XVII: A shared antigen in neurodermatological interactions? Clin Dev Immunol 2013: http://dx.doi.org/10.1155/2013/240570 Seppänen A, Autio-Harmainen H, Alafuzoff I, Särkioja T, Veijola J, Hurskainen T et al. Collagen XVII is expressed in human CNS neurons. Matrix Biol 2006;25: 185-8
SC
Seppänen A, Suuronen T, Hofmann SC, Majamaa K, Alafuzoff I. Distribution of collagen XVII in the human brain. Brain Res 2007;1158: 50-6
M AN U
Stinco G, Codutti R, Scarbolo M, Valent F, Patrone P. A retrospective epidemiological study on the association of bullous pemphigoid and neurological diseases. Acta Derm Venereol 2005;85: 136-9 Tampoia M, Giavarina D, Di Giorgio C, Bizzaro N. Diagnostic accuracy of enzyme-linked immunosorbent assays (ELISA) to detect anti-skin autoantibodies in autoimmune blistering skin diseases: A systematic review and meta-analysis. Autoimmun Rev 2012;12: 121-6 Tapiola T, Alafuzoff I, Herukka SK, Parkkinen L, Hartikainen P, Soininen H et al. Cerebrospinal fluid beta-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain. Arch Neurol 2009;66: 382-9
TE D
Van den Bergh F, Giudice GJ. BP180 (type XVII collagen) and its role in cutaneous biology and disease. Adv Dermatol 2003;19: 37-71 Winblad B, Amouyel P, Andrieu S, Ballard C, Brayne C, Brodaty H et al. Defeating Alzheimer's disease and other dementias: A priority for European science and society. Lancet Neurol 2016; 15: 455-532
AC C
EP
Yang YW, Chen YH, Xirasagar S, Lin HC. Increased risk of stroke in patients with bullous pemphigoid: A population-based follow-up study. Stroke 2011;42: 319-23
16
ACCEPTED MANUSCRIPT Table 1. Characteristics, bullous pemphigoid autoantibodies and Alzheimer’s disease biomarkers of neurologically healthy controls and patients with Alzheimer’s disease AD patients
P-value
n
40
115
Sex M/F
14/26 (35/65 %)
41/74 (36/64 %)
ns.
Age1
66.8±7.0
72.0±7.8
<0.001
MMSE2
na.
20 (8–29)
BP180 U/ml3
2.0 (0.5–12.3)
2.4 (0–47.7)
BP180 -/+3
37/1 (97/3%)
91/20 (82/18%)
BP230 U/ml
1.35 (0–24.1)
2.0 (0–35.8)
BP230 -/+
37/3 (92.5/7.5%)
101/14 (88/12%)
ns.
Aβ1-42 pg/ml
728 (263–1078)4
443 (139–1294)
<0.001
Tau pg/ml
266 (90–582)
p-Tau181 pg/ml1
51±14
RI PT
Controls
0.05
0.019
M AN U
SC
ns.
554 (48–1994)
<0.001
90±35
<0.001
Data given as median (range) unless otherwise indicated. Abbreviations: ns, not significant; MMSE, Mini-Mental State Examination; na, not available; Αβ1-42, beta-amyloid; tau, total tau; p-Tau181, hyperphosphorylated tau. Data given as mean ± standard deviation
2
Data available for 89 patients with Alzheimer’s disease
3
Two controls and four patients with Alzheimer’s disease were excluded due to lack of reactivity
TE D
1
against BP180 in immunoblotting
EP
Due to a method change, the levels are 10 % higher than in AD group.
AC C
4
17
ACCEPTED MANUSCRIPT Figure legends Figure 1. BP180 and BP230 autoantibody values of control subjects and patients with Alzheimer’s disease. Sera from controls (circles) and patients with Alzheimer’s disease (AD) (squares) were tested for IgG reactivity against BP180 and BP230 using ELISA kits. BP180 ELISA assay was performed as duplicates for all samples. (a) Increased anti-BP180 values (cut-off value
RI PT
>9U/ml, dashed line) were detected in 3/40 control samples and in 24/115 AD samples. The increased anti-BP180 values varied between 9.2 and 47.7 U/ml. Two control samples and four AD samples were excluded since they did not recognize full-length BP180 in immunoblotting. (b) Increased anti-BP230 values (cut-off value >9U/ml, dashed line) were detected in 3/40 control
SC
samples and in 14/115 AD samples. The increased anti-BP230 values varied between 9.6 and 35.8
M AN U
U/ml.
Figure 2. Autoantibodies present in the sera of patients with Alzheimer’s disease detect BP180 in immunoblotting. Twenty-four Alzheimer’s disease (AD) samples and three control samples, all of which showed increased anti-BP180 levels in ELISA assay, were further analysed by immunoblotting using human recombinant full-length BP180 expressed in COS-7 cells as a target
TE D
antigen. Anti-human IgG was used as a secondary antibody. (a) Human polyclonal anti-BP180 (NC16A) antibody and sera taken from a patient with bullous pemphigoid (BP), a control patient and an AD patient all recognized a 180-kDa protein that was not detected in collagenase-treated cell extract. (b) Four examples of immunoblotting, which verified that anti-BP180 ELISA positive AD
AC C
full-length BP180.
EP
samples recognize the full-length BP180. The ELISA positive control sample did not recognize the
18
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT