Autoantibodies to full body vascular cell junctions colocalize with

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Autoantibodies to full body vascular cell junctions colocalize with MYZAP, ARVCF, desmoplakins I and II and p0071 in endemic pemphigus in Colombia, South America Ana M. Abreu Velez1, MD, PhD, DSc Michael S. Howard1, MD

1 Georgia Dermatopathology Associates, Atlanta, GA, USA, 2Robert P. Apkarian Integrated Electron Microscopy Core, Emory University Medical Center, Atlanta, GA, USA, and 3Department of Oral € Pathology, Faculty of Odontology, Malmo €, Sweden University, Malmo

, Hong Yi2, MS, Gunnar Warfvinge3, OD, and

Abstract Background We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF). Methods Here we aimed to investigate disease autoreactivity to vessels in all body organs/systems. We compared 57 patients and 57 controls from the endemic area, matched by demographics, age, sex, and work activity. We performed immunofluorescence, immunohistochemistry, confocal microscopy, immunoblotting, indirect

Correspondence Ana M. Abreu-Velez, MD, PHD Georgia Dermatopathology Associates 1534 North Decatur Rd. NE, Suite 206 Atlanta, GA 30307-1000 USA E-mail: [email protected] Funding: Our work was performed with funding from Georgia Dermatopathology Associates(GDA); Mineros, SA, Medellin, Colombia, South America; Department of Oral Pathology, Faculty of Odontology, € University, 205 06, Malmo €, Sweden, Malmo ~ora del Carmen, El Hospital Nuestra Sen Bagre, Colombia, South America, and the El Bagre Mayoral Office.

immune electron microscopy studies, and autometallographic studies. We performed ultrasonography on large patient arteries, investigating for vascular anomalies. In addition, we reviewed autopsies on seven patients who died affected by El Bagre-EPF. We immunoadsorbed any positive vessel immunofluorescence with desmoglein (Dsg1), investigating for new autoantigens. Results Overall, 57/57 patients affected by El Bagre-EPF displayed autoantibodies to vessels in all the organs/systems of the body via all methods (P < 0.01). The autoreactivity was polyclonal, and the patient’s antibodies colocalized with commercial antibodies to desmoplakins I and II, p0071, ARVCF, and MYZAP (all from Progen Biotechnik, Germany; P < 0.01; all present at cell junctions). Immunoadsorption with Dsg1 on positive vessel immunofluorescence showed that the immune response against the vessels was directed against non-Dsg1 antigen(s). Autometallographic studies showed deposits of metals and metalloids in vessel cell junctions and in erythrocytes of 85% of patients (P < 0.01). Conclusions Immune response to these vascular antigens is likely altering endothelial cells and vessel shapes, thus disturbing hemodynamic flow. The flow alterations likely lead

Conflicts of interest: None.

to inflammation and may play a role in the atherogenesis often seen in these patients.

doi: 10.1111/ijd.13827

A new focus of EPF was described in El Bagre, Colombia,

Introduction

South America.3 Furthermore, studies in the endemic area con-

Endemic pemphigus foliaceus (EPF) is an autoimmune disease

firmed a new clinicopathologic variant of EPF (pemphigus

characterized by the presence of cutaneous vesicles and blis-

Abreu-Manu).5 The new variant shares features with Senear-

ters, and well-documented autoantibodies to desmoglein 1

Usher Syndrome. As in lupus, a localized form accounts for about 30% of the cases; a systemic form accounts for about

(Dsg1).1,2 By direct immunofluorescence (DIF) and indirect

30% of the cases, affecting multiple organs.5–12 The patients

immunofluorescence (IIF), the “hallmark” of pemphigus and EPF has been focused on the autoantibodies that create inter-

are primarily gold miners who also work in agricultural activi-

cellular staining (ICS) between epidermal keratinocytes.1–3

ties.5,6 The endemic area is in the tropics and rich in metals,

primarily

metalloids, and xenobiotics such as mercury and cyanide.5,6

documented in rural areas of South and Central America, and

We have previously reported El Bagre EPF autoantibodies to

in Tunisia, Africa.1–4 In Latin America, the disease affects

sebaceous and Meibomian glands, hair follicular units, nerves,

Indians and colonos, descendants of workers brought to these areas for gold, nickel and other metal mining activities and road

neural receptors, neurovascular bundles, melanocytes, heart, smooth and skeletal muscle, and kidney.7–12 The aim of this

construction.1–3

study was to confirm the presence of autoantibodies against

Endemic

pemphigus

foliaceus

has

ª 2017 The International Society of Dermatology

been

International Journal of Dermatology 2017

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Autoantibodies to vessel in endemic pemphigus

vessels in multiple organ/systems, utilizing differential techniques in a case–control study.

Materials and methods We tested 57 patients affected by El Bagre-EPF and 57 controls from the endemic area matched by age, gender, and work activities, clinical history, and physical examination. The patients and controls were evaluated by hematoxylin and eosin (H&E), DIF, IIF, confocal microscopy (CFM), immunoblotting

Statistical analysis We used Fisher’s exact test to compare two nominal variables (e.g., positive and negative) of the antibody response. We also compared the differences when evaluating (i) positivity of the El Bagre-EPF autoantibodies between patient cases and controls, and (ii) patient antibody results versus the commercial antibodies to MYZAP, p0071, DP I-II, and ARVCF. P < 0.05 with a 95% of confidence or more was considered statistically significant. We used the software GraphPad QuickCalcs from GraphPad Software Inc. (La Jolla, CA, USA).

(IB) using skin and vessels as antigens, indirect immune electron microscopy (IEM), and autometallography studies (see File S1 for these methods). We used cow, mouse, rat, and

Results

human tissues including musculoskeletal (including bone and

The H&E results showed perivascular inflammation (primarily by

cartilage); central and peripheral nervous; cardiovascular and

lymphohistiocytic cells) in all cases of El Bagre-EPF, compared with

lymphatic; digestive, respiratory, and urinary systems;

none in the controls (P < 0.01). In addition, most dermal vessels (especially those under the blisters) were either dilated and/or chan-

reproductive organs (male and female); and sensory organs and endocrine glands in both cases and controls.

ged in shape (e.g., tortuosity), and/or revealed a rouleaux phe-

We also tested the patients and controls using B-Mode two-

nomenon compared with none of these changes in the controls

dimensional ultrasound imaging. Autometallographic studies in

(P < 0.01). Less often, a mild vasculitis was present in some skin

skin biopsies of cases and controls were also performed

biopsies of the El Bagre-EPF patients (13/57). Our testing demon-

(Table S1). Only patients meeting diagnostic criteria for El

strated positive staining with multiple antibodies on vessels and

Bagre-EPF were included, specifically: (i) patients displayed

their cell junctions, confirmed via several immunological methods

clinical and epidemiologic features described for this disease;5–14

including immune electron microscopy. In the skin, the autoantibodies were noted on all vessels in the dermal neurovascular vascular

(ii) patients lived in the endemic area; and (iii) patient serum displayed intercellular staining (ICS) between epidermal

plexus, around all skin appendices, and in neural/mechanoreceptor

keratinocytes and to the basement membrane zone of the skin,

areas. The autoantibodies were also present in all organs/systems

via either DIF or IIF using fluorescein isothiocyanate (FITC)-

reviewed, including the heart, kidney, brain, meninges, oral, muscu-

conjugated monoclonal antibodies to human total IgG, as

loskeletal, and intervertebral disks (Figs. 1 and 2). Overall, 57/57

described elsewhere.5,6 Furthermore, (iv) each patient’s serum

patients affected by El Bagre EPF demonstrated autoantibodies to

was positive by IB for reactivity against Dsg1, as well as for

vessels, primarily in their cell junctions (including lymphatics)

plakin molecules as previously described5,6; (v) each patient’s

(P < 0.01) utilizing DIF, IIF, IHC, IB, CFM, and IEM. We found no differences in the staining in all the organs/systems examined in all

serum immunoprecipitated (IP) a Concanavalin A (Con A) affinity-purified 45 kDa fragment of Dsg1;3 and (vi) each

the animal and human tissues reviewed. The reactivity was poly-

patient’s serum yielded a positive result ELISA14 test when

clonal, with IgG (57/57), IgM (53/57), C1q (53/57), C3c (53/53),

screening for autoantibodies to pemphigus foliaceus (PF)

kappa (57/57), lambda (54/57), fibrinogen (50/57), albumin (50/57),

antigens. Written consents were obtained from all patients and

and IgE (37/57) observed. Fewer cases stained with IgA (37/57)

controls, and Institutional Review Board permission from the ~ora de El Carmen, in El Bagre, Colombia, Hospital Nuestra Sen

and IgD (28/57) (P < 0.01) (CI 98%) (Table S1). A few control

was obtained. All the cases and controls were tested via the

sels in the skin by DIF for IgG (6/57) and fibrinogen (6/57), kappa (6/57), and lambda (6/57); these control cases were all genetically

same methods and conditions.

cases from the endemic area (6/57) showed some reactivity to ves-

related to the El Bagre-EPF patients. For IIF, rat bladder epithelium

B-mode two-dimensional ultrasound imaging We tested for any possible vascular abnormalities in the clinical setting, especially for atherosclerosis. We utilized a VisualSonics ultrasound from Fujifilm, Toronto, Ontario, Canada, and the testing was performed as described.15 We classified carotid artery plaques into four categories: echolucent, predominantly echolucent, predominantly echogenic, or echogenic. Autopsies were reviewed from seven deceased patients with El Bagre-EPF. These represented El Bagre-EPF patients not included as cases in this study. International Journal of Dermatology 2017

(a substrate) also displayed positive staining with all the El BagreEPF sera because of the desmoplakins present there. In all the organs we studied, the patient autoantibodies perfectly colocalized with the commercial antibodies to desmoplakins I and II, p0071, ARVCF, and MYZAP (P < 0.01, CI 98%). The patient autoantibodies also colocalized with our vascular markers and the JAM-A gap cell junction marker. Specifically, colocalizations were noted with CD31, CD34, von Willebrand Factor/VWF and D2-40 by DIF, IIF, and IHC staining (P < 0.01, CI 98%). Furthermore, we noted positive staining on mesenchymal-endothelial cell junctions in the dermis. ª 2017 The International Society of Dermatology

Abreu Velez et al.


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Figure 1 Clinical features, histology, and detection of autoantibodies to vessels. (a) A clinical picture of one El Bagre-EPF patient with plaque lesions on his back (white arrow). (b) H&E staining demonstrates dilated upper dermal plexus vessels, surrounded by a lymphohistiocytic infiltrate (black arrows) (2009). (c) DIF of negative controls using anti-human IgG FITC conjugated showing non specific selffluorescence of the dermal vessels (white arrows). (d) DIF of the skin showing positive light green staining on a dermal vessel using FITC conjugated anti-human IgG (+++) (overlapping with orange staining from MYZAP) (white arrows) (2009). (e) IIF, showing light green positive staining on a network of bovine cardiac vessels utilizing FITC conjugated anti-human IgM (+++), colocalizing with orange staining for p0071 (white arrow) (4009). (The muscle in fuchsia) (f) IHC, demonstrating positive staining of an El Bagre-EPF patient skin biopsy with C3c on vessels in the papillary dermis (brown staining; red arrow)

Immunoadsorption of the skin cell junction autoantibodies, and testing with the vascular autoantibodies Original patient serum, affinity tryptic purified to remove desmoglein antibodies and eluted from the affinity columns was tested. We preincubated then tested this serum via DIF and IIF on El Bagre-EPF patient tissue. We observed positive reactivity to the ª 2017 The International Society of Dermatology

vessels; the vascular reactivity persisted, indicating that the vessel antigens are different than Dsg1.

CFM studies The CFM studies also demonstrated 100% colocalization of the patient autoantibodies and all the antibodies from Progen (P < 0.01), and with JAM-A (Fig. 2). International Journal of Dermatology 2017

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Figure 2 Detection of autoantibodies to vessels cell junctions, and colocalizations with MYZAP, ARVCF, DP I-II and p0071. (a) An IEM photograph of rat cardiac tissue, showing El Bagre-EPF serum labeled with 10 nm Gold-conjugated protein A antibodies and reacting with endothelial cell junctions (red arrows; black dots) (100 kV). (b) IIF, showing positive staining of splenic vessels using FITC conjugated antihuman IgG and anti-fibrinogen antibodies (light green staining), that colocalized with yellow-red-orange staining for MYZAP (white arrow) (4009). (c) A CFM data image showing perfect colocalization of the peaks of fluorescence (white arrow), indicating that the antibodies to MYZAP (red staining) colocalized with the patient antibodies (green staining). The blue peaks represent DAPI nuclear counterstaining. (d) A series of IBs show colocalization of patient autoantibodies via molecular weight with commercial antibodies. Upper left shows the colocalization of the patient antibodies (Lane 2) with the commercial antibody to p0071 (Lane 3) (at P, 105 kDa). Lane 1, a negative control. Lower left shows the colocalization of DP I-II (250 and 215 kDa, respectively) and a patient’s antibodies (Lane 1 is a representative patient serum, and Lane 2 is the commercial DP I-II antibody). The right panel shows the colocalizations of control markers (Lane 1) with patient antibodies; Lanes 2 and 3 are two patient sera, both colocalizing with ARVCF (A; 105 kDa) and MYZAP (M, 54 kDa). Full IB correlation was found between these commercial antibodies from Progen and the patient autoantibodies (P < 0.001; 98% CI)

IEM and IB studies Immune electron microscopy showed positive staining for patient autoantibodies on vascular endothelial cells and their cell junctions (Fig. 2). The IB confirmed multiple antigens detected by patient sera, that also perfectly colocalized with the Progen antibodies (P < 0.01, CI 98%) (Fig. 2).

cardiovascular valves,

some

system

showed

atheromatous

some

plaques

enlarged ranging

cardiac between

classes III and IV, several vessels with dilations and inflammation, and congestion in the pial and lung vessels. The spleens were very congested (P < 0.01). The H&E staining showed a lymphohistiocytic infiltrate around most vessels.

Patient autopsies All the autopsies showed vascular alterations. The most frequent cause of death in our study patients was cardiovascular. Patient autopsy data for the vessels and the International Journal of Dermatology 2017

In Figure 3 and in Table S1, we show a summary of our patient DIF testing (and the percentage of positive findings for immunoglobulins, complement, fibrinogen, and albumin). IB data (including 320, 260, 200, 160, 126, 116, 105, 97, 80, 67, 66, ª 2017 The International Society of Dermatology

Abreu Velez et al.

45, 48, and 31–34 kDa bands) (P < 0.01) is summarized, as well as the IP and ELISA assay data.


Report

vessels, and in erythrocytes (Fig. 3). We observed these metals/metalloids in three genetically related controls for the El Bagre-EPF patients.

B-mode two-dimensional ultrasound imaging We detected early and advanced carotid artery atherosclerotic plaques in 40/57 of the El Bagre-EPF patients (P < 0.01) and in three male controls from the endemic area suffering from psoriasis at ages 53, 57, and 63. We confirmed atherosclerotic lesions as protrusions of the intima media; we recorded the number of visualized plaques, as well as total plaque area or total plaque volume. Some plaques contained a large lipid core and appeared echolucent; other plaques with fibrosis and calcification tended to appear echogenic. We found multiple types of carotid plaques, displaying echolucent, predominantly echolucent, and predominantly echogenic features. These findings are consistent with flow alterations in the vessels. Autometallography assay Mercury and amalgamations of other metals and metalloids were present in 85% (P < 0.01) of the El Bagre-EPF patient vessel cell junctions and endothelia (including lymphatics, arteries, and veins of all sizes) (Fig. 3). We also detected the presence of these metals/metalloids in the plasma inside the

Discussion We document for the first time autoantibodies to vessels and their cell junctions (different from Dsg1) in all organs/systems, and colocalizing with MYZAP, ARVCF, p0071, and desmoplakins 1 and 2 in El Bagre EPF. These autoantibodies also colocalize with mercury/metalloids and within erythrocytes. As confirmed here, MYZAP, desmoplakins I-II, ARVCF, and p0071 are components of vascular cell junctions in blood vessels, arteries, veins, lymphatics, and capillaries. The multiple organ/system reactivity to the vessel cell junctions may account for the multi-organ clinical lesions seen in 30% of El Bagre-EPF patients. Vasculitis is one of the complications of lupus; in El Bagre-EPF, only a few cases present a mild vasculitis. Our findings may explain the enlargement, atherosclerosis, vascular shape alterations, and rouleaux we previously reported in these patients, as well in patients with other autoimmune blistering diseases.16 The autoantibodies to the vessels may also result in histologic tissue edema and inflammation.16,17 In El

Figure 3 Autometallographic studies on vessels. In (a) and (b), we show positive staining with metals and metalloids at the small dermal blood vessel and lymphatic cell junctions, respectively (dark staining; red arrows). In (c) and (d), we show the same staining in larger vessels (red arrows), as well as in plasma and erythrocytes (black arrows) ª 2017 The International Society of Dermatology


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Bagre-EPF and in other autoimmune blistering diseases including PF, bullous pemphigoid (BP), pemphigus vulgaris (PV), epi-

We found immunohistologic abnormalities in the vessels, and autoantibodies mainly directed against their cell junctions. We also

dermolysis bullosa acquisita, linear IgA disease, and dermatitis

detected atherosclerotic plaques in the El Bagre-EPF patients of

herpetiformis, we have noted patient vessels overexpressing

differential echolucencies, which were indicative of alterations in

HLA-DPDQDR antigen, ribosomal protein s6-ps240, cyclo-oxy-

vessel flow. The alterations may be because of a direct deposition

genase 2, and C5-b9/MAC. These patient biopsies also

of the antibodies in vascular cell junctions, likely changing confor-

revealed vascular rouleaux phenomenon and other vascular

mations of molecules. Alterations could then occur in vessel

inflammatory markers, including CD3, CD20, CD1a, HAM56,

shape, attracting immune cells around the vessels and inflamma-

CD68, and S100, and autoantibodies to the vessels.16,18–25 We confirmed our data by IHC, which prevents the “autofluores-

tory markers and altering flow dynamics.20 It is possible that the autoantibodies stimulate endothelial cells to express adhesion

cence” possible when using DIF/IIF. In most skin biopsies on all

molecules and various chemokines; this may lead to a recruitment

autoimmune skin blistering diseases, dilated vessels and papil-

of monocytes, leukocyte recruitment with the secretion of cytoki-

lary dermal edema are noted under the blisters, with additional

nes and other inflammatory markers. Deposition of fibrin, fibrino-

mild dermal perivascular inflammation.16,18–26

gen and activated platelets on damaged endothelium may

Of interest, the largest histopathology series on Brazilian

expresses tissue and von Willebrand factor, creating a prothrom-

EPF demonstrated that most cases have dilated capillaries in

botic milieu and leading to clinical vascular disorders.21,24 The

the dermal papillae and upper dermis; there was a variable degree of edema, with an inflammatory infiltrate that could be

polyclonality of the immune response may partially result from pollution by metals and metalloids in the endemic area, as well as

diffuse, or present a perivascular and periadnexal distribution.27

patient exposure to tropical and parasitic diseases.20,21,24,25

It was often composed of neutrophils, eosinophils, and lympho-

Other authors have reported in BP, PV, and PF patients that

cytes, which were variably represented in small or moderate

soluble E-selectin was found to be significantly increased; in

numbers.27

addition, autoreactivity to dermal vessels was noted involving

For the DIF, IIF, CFM, and immunoadsorption experiments,

fibrin and C3c.31–33

we partially solubilized the cell membranes with Triton X and

Of interest, all of the patient autoantibodies perfectly colocal-

blocked with normal goat serum. These methods helped to allow permeation of the antibodies into the cells, including their

ized with the Progen antibodies p0071, ARVCF, MYZAP, and DP I-II.34–38 These molecules are located in cell junctions in all

membranes and cell junctions. We also blocked any nonspecific

organs/systems; some are members of the armadillo protein

immune responses that can be seen when IIF or DIF are per-

family and represent specific components of dermal microvas-

formed on vascular tissues. El Bagre-EPF has autoantibodies

cular endothelial intercellular junctions.

to multiple organs as likely occurs in Senear-Usher syndrome

Our data correlates with a recent study demonstrating cardio-

that may require further studies investigating cross-reactivity or

vascular alterations in patients with inflammatory skin diseases

multiple autoimmunity.28

(including pemphigus and pemphigoid) versus controls; 2002–

The autometallographic technique detects silver sulfides/selenides, metallic silver, and gold in addition to mercuric sulfides/

2012 US national inpatient hospitalizations (n = 72 108, 077 adults) were reviewed and correlated with cardiovascular and

selenides.9,29,30 To differentiate between mercury, silver, and

cerebrovascular associated diseases.39 In our current and previ-

gold, additional steps were performed (Table S1). In our study,

ous studies, we present evidence for vascular alterations in

we clearly demonstrate the presence of mercury and other met-

most autoimmune skin diseases.16,18–25

alloids located in the cell junctions of the vessels, including the lymphatics (where the main autoantigens were found in this study) as well as in erythrocytes. Once absorbed in the body,

Conclusion

the metals/metalloids may exist in the circulation as an ionic species, or bound to serum proteins or erythrocytes. Inorganic

Patients affected by El Bagre-EPF have autoantibodies to most of the tested vascular cell junctions in most tissues. Autometal-

mercury will bind to serum proteins, and methylmercury will bind

lographic studies show deposits of metals and metalloids in

to erythrocytes. Erythrocytes and plasma are typically the main

these cell junctions. These findings may explain the clinical and

transport routes of metals and or metalloids. These metals bind

histologic edema in their tissues and observed alterations in

to protein sites by displacing original metals from their natural

vessel morphology. The chronic inflammation of the vascular

binding sites, causing malfunctioning of the molecule and ulti-

endothelial cells and observed atherosclerosis suggest that the

mately toxicity. In our cases, these metals/metalloids likely

autoantibodies are causing and/or potentiating hemodynamic

changed the conformations of proteins, possibly triggering autoimmunity. Three relatives of El Bagre-EPF patients showed

alterations in these patients. Moreover, the antibodies to the vessels and their cell junctions perfectly colocalize with multiple

metal/metalloid deposits similar to the El Bagre-EPF patients;

commercial antibodies from Progen. Further research to detect

we speculate that this finding could indicate a genetic predispo-

autoantibodies against vessels in autoimmune blistering dis-

sition toward the deposits.

eases is encouraged.



Abreu Velez et al.

Acknowledgments We thank Jonathan Jones, HT (ASCP) at GDA for excellent technical assistance.

References 1 Abreu-Velez AM, Messias-Reason LJ, Howard MS, et al. Endemic pemphigus foliaceus over a century: part 1. N Am J Med Sci 2010; 2: 51–59. 2 Castro RM, Proencßa NG. Similarities and differences between Brazilian wild fire and pemphigus foliaceus Cazenave. Hautarzt 1982; 33: 574–577. 3 Robledo MA, Prada S, Jaramillo D, et al. South American pemphigus foliaceus: study of an epidemic in El Bagre and Nechi, Colombia 1982 to 1986. Br J Dermatol 1988; 118: 737–744. 4 Morini JP, Jomaa B, Gorgi Y, et al. Pemphigus foliaceus in young women. An endemic focus in the Sousse area of Tunisia. Arch Dermatol 1993; 129: 69–73. u-Velez AM, Hashimoto T, Bollag WB, et al. A unique form 5 Abre of endemic pemphigus in Northern Colombia. J Am Acad Dermatol 2003; 49: 599–608. u-Velez AM, Beutner EH, Montoya F, et al. Analyses of 6 Abre autoantigens in a new form of endemic pemphigus foliaceus in Colombia. J Am Acad Dermatol 2003; 49: 609–614. 7 Abreu-Velez AM, Howard MS, Hashimoto K, et al. Autoantibodies to sweat glands detected by different methods in serum and in tissue from patients affected by a new variant of endemic pemphigus foliaceus. Arch Dermatol Res 2009; 301: 711–718. 8 Abreu-Velez AM, Howard MS, Smoller BR. Antibodies to pilosebaceous units along their neurovascular supply routes in a new variant of endemic pemphigus foliaceus in Colombia, South America. Eur J Dermatol 2011; 3: 371–375. u Ve lez AM, Warfvinge G, Herrera WL, et al. Detection of 9 Abre mercury and other undetermined materials in skin biopsies of endemic pemphigus foliaceus. Am J Dermatopathol 2003; 25: 384–391. 10 Abreu-Velez AM, Zhe J, Howard MS, et al. Cardiac autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America. J Clin Immunol 2011; 31: 985–987. 11 Abreu-Velez AM, Howard MS, Grossniklaus HE, et al. Neural system antigens are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in Colombia. Clin Immunol 2011; 31: 356–358. https://doi.org/10. 1007/s10875-010-9495. 12 Abreu-Velez AM, Howard MS, Hashimoto T. Palms with a polyclonal autoimmune response in patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America. Eur J Dermatol 2010; 20: 74–81. u-Ve lez AM, Patin ~o PJ, Montoya F, et al. The tryptic 13 Abre cleavage product of the mature form of the bovine desmoglein 1 ectodomain is one of the antigen moieties immunoprecipitated by all sera from symptomatic patients affected by a new variant of endemic pemphigus. Eur J Dermatol 2003; 13: 359–346. u-Ve lez AM, Yepes MM, Patin ~o PJ, et al. A cost effective, 14 Abre sensitive and specific enzyme linked immunosorbent assay useful for detecting a heterogeneous antibody population in sera from people suffering a new variant of endemic pemphigus. Arch Dermatol Res 2004; 295: 434–441. ª 2017 The International Society of Dermatology


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15 Steinl DC, Kaufmann BA. Ultrasound imaging for risk assessment in atherosclerosis. Int J Mol Sci 2015; 16: 9749–9769. 16 Abreu-Velez AM, Smoller BR, Howard MS. Rouleaux and autoagglutination of erythrocytes associated with fibrin-like material in skin biopsies form patients with autoimmune blistering diseases. Our Dermatol Online 2013; 4: 613–615. 17 Howard MS, Yepes MM, Maldonado JG, et al. Broad histopathologic patterns of non-glabrous skin and glabrous skin from patients with a new variant of endemic pemphigus foliaceus (Part 1). J Cutan Pathol 2010; 2: 222–230. 18 Abreu-Velez AM, Calle-Isaza J, Howard MS. HLA-DPDQDR is expressed in lesional skin from patients with autoimmune skin diseases. Our Dermatol Online 2014; 2: 125–128. 19 Abreu-Velez AM, Googe PB Jr, Howard MS. Ribosomal protein s6-ps240 is expressed in lesional skin from patients with autoimmune skin blistering diseases. N Am J Med Sci 2010; 5: 604–608. 20 Abreu-Velez AM, Howard MS, Hashimoto T, et al. Human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia, South America. J Am Acad Dermatol 2010; 62: 437–447. 21 Abreu-Velez AM, Calle-Isaza J, Howard MS. Cyclo-oxygenase 2 is present in the majority of lesional skin from patients with autoimmune blistering diseases. Our Dermatol Online 2013; 4: 4476–4478. 22 Abreu-Velez AM, Calle-Isaza J, Howard MS. CD1a, HAM56, CD68 and S-100 are present in lesional skin biopsies from patients affected by autoimmune blistering diseases. Our Dermatol Online 2014; 5: 113–117. 23 Abreu-Velez AM, Yepes-Naranjo MM, Avila IC, et al. Tissue inhibitor of metalloproteinase 1, Matrix metalloproteinase 9, alpha-1 antitrypsin, metallothionein and urokinase type plasminogen activator receptor in skin biopsies from patients affected by autoimmune blistering diseases. Our Dermatol Online 2013; 4: 275–280. 24 Abreu-Velez AM, Googe PB Jr, Howard MS. Immunohistochemistry versus immunofluorescence in the diagnosis of autoimmune blistering diseases. Our Dermatol Online 2013; 4: 585–595. 25 Abreu-Velez AM, Googe PB Jr, Howard MS. In situ immune response in skin biopsies from patients affected by autoimmune blistering diseases. Our Dermatol Online 2013; 4: 606–612. 26 Lever WF. Pemphigus and pemphigoid. A review of the advances made since 1964. J Am Acad Dermatol 1979; 1: 2–31. 27 Furtado TA. Histopathology of pemphigus foliaceus. AMA Arch Derm 1959; 80: 66–71. rez-Pe rez ME, Avalos-Dıaz E, Herrera-Esparza R. 28 Pe Autoantibodies in Senear-Usher syndrome: cross-reactivity or multiple autoimmunity? Autoimmune Dis 2012; 2012: 1–7. 29 Danscher G, Møller-Madsen B. Silver amplification of mercury sulphides and selenides. A histochemical method for light and electron microscopic localization of mercury in tissue. J Histochem Cytochem 1985; 31: 219–228. 30 Danscher G, Rungby J. Differentiation of histochemically visualized mercury and silver. Histochem J 1986; 18: 109–114. 31 D’Auria L, Cordiali Fei P, Pietravalle M, et al. The serum levels of sE-selectin are increased in patients with bullous pemphigoid or pemphigus vulgaris. Correlation with the number of skin lesions and recovery after corsticosteroid therapy. Br J Dermatol 1997; 137: 59–64.


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32 Ameglio F, D’Auria L, Cordiali-Fei P, et al. Bullous pemphigoid and pemphigus vulgaris: correlated behavior of serum VEGF, sE selectin and TNF-alpha levels. J Biol Regul Homeost Agents 1997; 11: 148–153. 33 Peterson JD, Worobec SM, Chan LS. An erythrodermic variant of pemphigus foliaceus with puzzling histologic and immunopathologic features. J Cutan Med Surg 2007; 11: 179–184. 34 Pieperhoff S, Rickelt S, Heid H, et al. The plaque protein myozap identified as a novel major component of adhering junctions in endothelia of the blood and the lymph vascular systems. J Cell Mol Med 2012; 16: 1709–1719. 35 Franke WW, Rickelt S, Barth M, et al. The junctions that don’t fit the scheme: special symmetrical cell-cell junctions of their own kind. Cell Tiss Res 2009; 338: 1–17. 36 Calkins CC, Hoepner BL, Law CM, et al. The Armadillo family protein p0071 is a VE-cadherin- and desmoplakin-binding protein. J Biol Chem 2003; 278: 1774–17783. 37 Schmelz M, Franke WW. Complexus adhaerentes, a new group of desmoplakins containing junctions in endothelial cells: the syndesmos connecting retothelial cells of lymph nodes. Eur J Cell Biol 1993; 61: 274–289.


38 Andreeva ER, Pugach IM, Gordon D, et al. Continuous subendothelial network formed by pericyte-like cells in human vascular bed. Tissue Cell 1998; 30: 127–135. 39 Kwa MC, Silverberg JI. Association between inflammatory skin disease and cardiovascular and cerebrovascular co-Morbidities in US adults: analysis of Nationwide Inpatient Sample Data. Am J Clin Dermatol 2017; https://doi.org/10.1007/s40257-0170293-x. [Epub ahead of print].

Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Shows a summary of the autoantibodies detected by different methods in the patients and controls. File S1. In all the cases and controls, skin biopsies were taken for H&E, PAS, DIF, IHC, CFM, EM and autometallographic studies
