PAPER MATA KULIAH

ILMU PENYAKIT DALAM VET I

“Phemphigus Foliceae”

Oleh :
Kelas : A
Nama NIM
1. Ni Ketut Suastini 1609511003
2. Yohana Cendyka Kartika Dewi Guru 1609511009
3. Fiorencia Zefanya 1609511011
4. Rr. Allamanda Ardia Wardana 1609511012
5. Ni Putu Dyah Giana Paramitha 1609511014
6. Putu Angga Prasetyawan 1609511052

FAKULTAS KEDOKTERAN HEWAN

UNIVERSITAS UDAYANA
TAHUN AJARAN
2018
 Phemphigus Foliceae

1. Definisi
Pemphigus foliaceus (PF) adalah penyakit kulit anjing dan kucing yang
biasanya dimulai pada usia pertengahan awal. Pemphigus foliaceus, kondisi kulit
autoimun yang paling umum pada anjing dan kucing, ditandai oleh pustula, erosi,
dan krusta. Penyakit ini menyebabkan pustula dan pengerasan kulit di permukaan
kulit. Penyakit ini dapat mulai di wajah dan telinga, tetapi sering menyebar untuk
mempengaruhi kulit di bagian lain dari tubuh. Itu juga bisa menyebabkan
penebalan dan keretakan pada footpads. Untungnya, organ lain tidak terkena
penyakit. Pada kucing, bagian yang sering terkena yaitu di bagian telapak dekat
kuku atau cakarnya.

A B

Gambar 1
A) Anjing yang mengalami Pemphigus foliaceus,
B) Kucing yang mengalami Pemphigus foliaceus
Sumber : https://veteriankey.com/pemphigus-foliaceus-in-a-cat/
2. Patofisiologi
Pemphigus foliaceus mempengaruhi epidermis. Untuk membantu epidermis
dalam melindungi tubuh dari lingkungan, epidermis sebagian besar terdiri dari
keratinosit yang saling melekat erat. Terdapat dua tipe struktur adhesi yang
mempertajankan keratinosit, yaitu desmosom yang bertanggung jawab untuk
adesi dari sel ke sel dan hemidesmosom yang mengikat keratinosit ke membran
basalis. (Tater dan Thierry. 2010)
Terdapat dua mekanisme toleransi sistem imun. Mekanisme pertama yaitu
seleksi secara positif oleh timus, dimana yang dipilih hanya sel T yang dapat
mengenali peptida pada molekul Histocompatability Complex (MHC) .
Mekanisme kedua yaitu seleksi negatif, dimana sel T yang mengenali antigen-
sendiri dengan afinitas yang terlalu tinggi dihapus melalui proses apoptosis dan
tidak diizinkan untuk memasuki sirkulasi tubuh. Mekanisme yang menginduksi
sistem autoimun pada kulit berkaitan dengan MHC dan gen apoptosis. Autoimun
secara langsung ditujukan terhadap komponen desmosom, yang merupakan
jembatan intraseluler utama antara keratinosit dan bertanggung jawab untuk
kohesi antar sel. Hal ini menyebabkan hilangnya kohesi antara
keratinosit,akantolisis dan terjadi pembentukan pustula.
Mekanisme terjadinya pemphigus foliaceus terjadi karena adanya keberadaan
antibodi terhadap desmoglein (Dsg). Autoantibodi anti-Dsg menyebabkan
gangguan terhadap adesi intraseluler pada keratinosit. Pada pemphigus foliaceus,
target dari autoantibodi yang paling umum adalah Dsg 1, yaitu glikoprotein yang
terdapat pada desmosom. Respon dari antibodi biasanya melibatkan IgG
(subkelas IgG4).
Pembentukan BLISTER pada pemphigus foliaceus disebabkan karena
pengikatan antibodi IgG terhadap Dsg 1 menyebabkan hilangnya hubungan
interseluler antara keratinosit dan membentuk BLISTER pada epidermis.
3. Etiologi
Pemphigus foliaceus (PF) adalah bentuk pemfigus yang paling dikenal di
semua hewan domestik, dan telah dijelaskan pada anjing, kucing, kuda dan
kambing. Penyakit autoimun ini dicirikan oleh produksi autoantibodi yang
diarahkan terhadap antigen desmosomal yang terletak di ruang interseluler
epidermis suprabasal. Pada manusia dan anjing, antigen PF utama adalah
desmoglein 1, glikoprotein 150 kDa dari kelompok cadherin molekul adhesi.
Meskipun patogenesis masih belum dipahami dengan baik, produksi autoantibodi
terhadap antigen ini tampaknya bertanggung jawab atas penghancuran jembatan
interseluler epidermis suprabasal dan untuk detasemen sel-sel (acantholysis). Sel-
sel acantholytic biasanya diamati dalam spesimen sitologi dan histologis dari
pustula utuh. Pemphigus foliaceus tampaknya tidak memiliki predileksi usia pada
spesies lain, dan kejadian pada usia muda dalam kasus yang dilaporkan
sebelumnya mungkin suatu kebetulan. Selain itu, penyebab pemphigus foliaceus
adalah:
a. Autoantibodi: tubuh menciptakan antibodi yang bereaksi terhadap jaringan
dan sel yang sehat seolah-olah mereka patogen (berpenyakit).
b. Paparan sinar matahari yang berlebihan.
c. Breed-breed tertentu tampaknya memiliki predisposisi keturunan.

4. Gejala Klinis
PF adalah versi yang lebih dangkal dari kompleks pemphigus yang lain, yang
mempengaruhi terutama pada stratum korneum. Lesi primernya adalah pustula
dangkal dan sementara. Hal ini masih sulit ditemukan, karena sangat rapuh dan
tersembunyi di bawah mantel. Pustula pecah dengan mudah, meninggalkan
eritema, remah-remah kekuningan, erosi, alopecia dan perangkat kecil perifer. Lesi
paling sering mulai muncul pada bagian dorsal moncong, secara bilateral simetris
dan menyebar secara bertahap. PF menunjukkan distribusi khas (Gambar 1) dan
paling sering mempengaruhi pinnae, daerah perioral dan periokular, planum
hidung, jembatan hidung dan bantalan kaki. Lesi footpad terdapat pada 1/3 anjing
dan ditandai oleh hiperkeratosis, retakan, pembengkakan eritematosa, dan
perubahan warna keputihan. Lesi itu bisa menjadi satu-satunya gejala. Nailbed
dapat melibatkan kuku tetapi biasanya tidak terpengaruh. Lesi di rongga mulut dan
sambungan mukokutan jarang terjadi. Pada beberapa pasien PF dapat menjadi
umum, dengan perkembangan gejala berikut pada kasus yang berat: anoreksia,
demam, depresi, limfadenomegali, dan edema ekstremitas. Pruritus hanya dapat
ditemukan pada kurang dari setengah anjing dengan 17 hingga 36% menunjukkan
gatal sedang hingga berat (Marleen,2015).
Beberapa gejala klinis yang umum muncul pada penderita Phempigud
Foliceae diantaranya :
 Pustula
 Krusta
 Erosi
 Alopecia kepala, wajah, planum hidung, pinnae, dan footpads.
 Lesi kulit menyebar ke seluruh bagian tubuh,
 Demam,
 Lesu
 Depresi
 Edema ekstremitas (Shinpei et al. 2015)

Gambar 2
Kondisi wajah anjing yang nampak disertai lesi khas dari Canine Phempigud Foliceae yaitu
scaling, alopecia dan pustula
Sumber : Marleen, 2015
 Gambar 3
Salah satu gejala dari Phempigud Foliceae yaitu ditemukannya pustula, krusta, erosi, dan
alopecia, pada wajah (A), telinga (B), dan batang tubuh (C) anjing.
Sumber : Shinpei et al. 2015

5. Diagnosa
Biopsi kulit dan pemeriksaan histopatologi kulit tetap menjadi standar emas
(gold standar) untuk diagnosis pemphigus folieaceus. Namun, diagnosis definitif
tidak dapat dicapai dalam beberapa kasus karena stadium penyakit dan kualitas
biopsi. Diagnosis pemphigus foliaceus dapat dibuat dari temuan pada spesimen
biopsi kulit yang diperoleh dari lesi kulit yang khas. Banyak sampel biopsi harus
diambil untuk meningkatkan akurasi diagnosis. Kehadiran keratinosit acantholytic
dalam vesikula adalah ciri lesi untuk diagnosis pemphigus folieaceus. Karena
acantholysis dapat dilihat pada kondisi dermatologi lainnya, spesimen biopsi
harus dievaluasi oleh ahli patologi hewan dengan keahlian dalam dermatopathies.
Diagnosis pasti pemphigus foliaceus didasarkan pada diagnostic histopatologi
fitur-fitur yang paling penting adalah pustula atau vesikula acantholysis ditandai
keratinocytes.15-17 Neutrofil atau eosinofil biasanya hadir dalam jumlah besar di
dalam pustula Direkomendasikan untuk mengirim riwayat klinis lengkap yang
mencakup breed, usia, distribusi lesi, perjalanan klinis dan riwayat pengobatan
dan kondisi kulit yang mendasari. Diagnosa banding untuk pemphigus foliaceus
berdasarkan temuan klinis termasuk folikulitis superfisial, dermatofitosis,
demodicosis, diskoid lupus erythematosus, dan pemphigus erythematosus.
 A B

Gambar 3
a) Kulit menunjukkan pustula subcorneal dengan krusta
b) Panah yang menunjukkan keratinosit acantholytic diterima dengan neutrofil
Sumber : Ganta, Chanran, 2017

6. Pengobatan
 Glukokortikoid
Glukokortikoid adalah hormon steroid yang dapat dengan mudah menyebar
melalui membran sel ketika dalam bentuk bebas mereka. Dalam sitoplasma mereka
mengikat, dengan afinitas tinggi, reseptor glukokortikoid sitoplasma. Kompleks
ligan-reseptor yang terbentuk bermigrasi ke dalam nukleus di mana ia dapat
memodifikasi transkripsi gen spesifik yang menyandikan protein yang bertanggung
jawab untuk aksi glukokortikoid. Dalam sel-T normal, ketika antigen berikatan
dengan reseptor sel-T, gugus fosfat ditambahkan ke asam amino tirosin dari beberapa
protein intraseluler. Akibatnya protein kinase C (PKC) diaktifkan dan juga
konsentrasi kalsium intraseluler meningkat. Kedua peristiwa ini (PKC dan
peningkatan kalsium) diperlukan untuk faktor transkripsi untuk mengikat promotor
gen IL-2 dan memulai transkripsi. RNA pembawa pesan (mRNA) kemudian
mentranslokasi ke sitoplasma tempat ia diterjemahkan oleh ribosom menjadi protein
IL-2 atau terdegradasi oleh RNAase. Glukokortikoid mampu melakukan intervensi
dengan beberapa langkah dari siklus sel-T ini. Mereka menghambat fosforilasi
tirosin; mereka menghambat calmodulin kinase II, enzim dari jalur kalsium dan
mereka menghambat pengikatan faktor transkripsi ke transkrip dan kemudian
menerjemahkan IL-2 mRNA ke protein yang efektif. Selanjutnya terjadi peningkatan
degradasi mRNA.
Glukokortikoid topikal dapat digunakan sebagai monoterapi untuk kasus-
kasus ringan foliaceus pemfigus, terutama pada anjing dengan lesi wajah lokal.
Mereka juga dapat digunakan dalam kombinasi dengan obat-obat sistemik lainnya
dalam kasus-kasus refrakter lebih. Berbagai glukokortikoid lebih kuat dari
hidrokortison telah digunakan secara topikal untuk foliaceus pemfigus seperti
betametason atau triamsinolon, yang keduanya tersedia dalam berbagai konsentrasi.
Karena glukokortikoid dapat menyebabkan atrofi kulit, melindungi wilayah
penerapan glukokortikoid dari trauma. Atrofi kulit ringan dapat dikelola dengan
beralih ke yang lebih rendah-potensi glukokortikoid topikal. Atrofi kulit lebih parah
harus dikelola dengan menghentikan semua glukokortikoid topikal.
Imunosupresi sistemik dengan glukokortikoid memberikan respon klinis
paling cepat pada anjing dan kucing dengan foliaceus pemfigus. Prednisone awalnya
dimulai pada 2 hari mg / kg / oral pada anjing, dan prednisolon awalnya dimulai pada
2 sampai 4 mg / kg / hari secara oral pada kucing. Dosis prednison atau prednisolon
kemudian dapat ditingkatkan jika tidak ada perbaikan dalam gejala klinis jelas dalam
satu atau dua minggu. Kucing dapat merespon lebih baik untuk glukokortikoid selain
prednison karena bioavailabilitas rendah prednison dibandingkan dengan
glukokortikoid. Jika glukokortikoid dari potensi yang berbeda digunakan, dosis setara
harus dihitung. Pada kucing, triamcinolone dapat awalnya dosis pada 2 sampai 4 mg /
kg / hari secara oral, dan deksametason dapat awalnya dosis pada 0,3-0,6 mg / kg /
hari secara oral. Dosis glukokortikoid harus dipilih berdasarkan tanda-tanda klinis.
Anjing dan kucing dengan ringan lesi foliaceus pemfigus dapat merespon
glukokortikoid dosis rendah. Dosis tinggi administrasi glukokortikoid digunakan
terutama dalam kasus-kasus yang parah foliaceus pemfigus di mana remisi cepat
tanda-tanda yang diperlukan.
Efek samping awal glukokortikoid termasuk poliuria, polidipsia, dan
polifagia. Dengan jangka panjang penggunaan, segudang efek samping lainnya dapat
mengembangkan seperti tukak lambung, hepatopathy (anjing), diabetes mellitus,
calcinosis Cutis, atrofi kulit, dan infeksi sekunder.
 Chlorambucil
Chlorambucil adalah agen alkilasi yang mengganggu sintesis DNA dan
dengan demikian sitotoksik. Ia bertindak terutama pada sel B dan dianggap sebagai
obat imunosupresif kerja lambat yang memerlukan dua (hingga enam) 11 minggu
sebelum efek terapeutik dapat dilihat. Efek samping yang dilaporkan termasuk
supresi sumsum tulang, hepatoksisitas dan gangguan gastrointestinal yang
menunjukkan melalui muntah, diare dan anoreksia. Chlorambucil digunakan sebagai
alternatif untuk azathioprine, hanya ketika terapi lain tidak ditoleransi atau dalam
kombinasi dengan kortikosteroid dan azathioprine dalam kasus yang sulit.
chlorambucil digunakan pada kucing dengan foliaceus pemfigus yang gagal untuk
merespon glukokortikoid karena azathioprine bukanlah pilihan pengobatan untuk
kucing.
 Siklosporin
Siklosporin adalah inhibitor kalsineurin yang menghalangi transkripsi gen
sitokin di diaktifkan cells. Siklosporin T adalah pengobatan disetujui untuk dermatitis
atopik anjing dan digunakan extralabel untuk berbagai lain kekebalan-dimediasi
kondisi pada anjing dan kucing. Siklosporin ini efek pada anjing dan kucing termasuk
muntah, diare, hiperplasia gingiva, hirsutisme, papillomas, psoriasiform lichenoid
seperti dermatosis, dan disebarluaskan toxoplasmosis. Bentuk mikroemulsi
siklosporin adalah lebih mudah diserap pada anjing dan kucing dan harus digunakan
sebagai pengganti bentuk lain dari siklosporin seperti Sandimmune. Jumlah darah
lengkap dan profil kimia serum harus dilakukan secara teratur sementara pasien yang
menerima terapi siklosporin. Dalam kucing menerima siklosporin, kegiatan enzim
hati yang tinggi bisa menjadi tanda toxoplasmosis.
Siklosporin lebih efektif untuk foliaceus pemfigus bila digunakan dalam
kombinasi dengan terapi lain. Dalam tiga anjing dengan foliaceus pemfigus sudah
menerima azathioprine dan glukokortikoid, siklosporin lisan sebesar 7,5 sampai 10
mg / kg / hari digunakan dengan ketokonazol lisan pada 2,5 sampai 5 mg / kg / hari
untuk berhasil menginduksi remission.61 ketokonazol ini digunakan untuk
meningkatkan siklosporin konsentrasi. Semua anjing yang kemudian dapat memiliki
glukokortikoid dihentikan dalam waktu tiga sampai 12 minggu menambahkan
siklosporin dan ketokonazol. Tanda tidak memperburuk ketika glukokortikoid
dihentikan.
 Niacinamide dengan tetrasiklin atau doksisiklin
Tetracycline adalah antibiotik yang juga memodulasi sistem kekebalan tubuh
dengan chemotaxis neutrofil menekan dan limfosit activation.65 Tetrasiklin
digunakan dalam kombinasi dengan niacinamide untuk berbagai kekebalan-dimediasi
kondisi dermatologi.
Untuk anjing <10 kg, 250 mg setiap tetrasiklin dan niacinamide diberikan
secara oral setiap delapan jam. Untuk anjing> 10 kg, dosisnya adalah 500 mg setiap
delapan jam setiap. Tetrasiklin dan niacinamide biasanya tidak digunakan pada
kucing karena sulit untuk mengelola ini berukuran lebih besar obat oral untuk
sebagian besar kucing. Doxycycline memiliki keuntungan membutuhkan dosis
kurang sering daripada tetrasiklin. Telah diganti untuk tetrasiklin dan digunakan
dengan dosis 5 sampai 10 mg / kg secara oral pada anjing setiap 12 sampai 24 jam.
Namun, tidak ada dokumentasi manfaat doksisiklin ketika digantikan dengan
tetrasiklin untuk mengobati foliaceus pemfigus anjing.
Tetrasiklin dan niacinamide tampaknya lebih bermanfaat sebagai terapi
tunggal di kasus-kasus ringan foliaceus pemfigus, terutama kasus-kasus dengan lesi
terlokalisasi pada wajah. Hal ini juga dapat digunakan dalam kombinasi dengan
glukokortikoid atau azathioprine. Hal ini dapat memakan waktu beberapa minggu
untuk tetrasiklin dan niacinamide memiliki efek klinis. Setelah remisi terjadi dengan
tetrasiklin dan niacinamide, frekuensi pemberian dapat dikurangi untuk sekali atau
dua kali sehari.
Efek samping meliputi kelesuan, anoreksia, diare, dan peningkatan risiko
kejang. Lesu dan anoreksia secara khusus dikaitkan dengan niacinamide. Jika
niacinamide perlu dihentikan, tetrasiklin (doksisiklin atau) saja dapat terus memiliki
aktivitas imunomodulator.
  Obat lain
Berbagai terapi lain telah digunakan untuk foliaceus pemfigus, termasuk
cyclophosphamide, garam emas injeksi, imunoglobulin intravena, mycophenolate
mofetil, dan dapson.
 Daftar Pustaka

Ganta, Chanran.2017. Dermatology Focus: Canine Pemphigus Foliaceus.

https://www.ksvdl.org/resources/news/diagnostic_insights/march2017/foliac
eus.html Diakses pada 18 November 2018.

Mueller, Ralf S. et all. 2006. Pemphigus Foliaceus in 91 Dogs. May/June 2006, Vol.
42

Petermann, Marleen.2015.Pemphigus foliaceus in dogs: the immune pathogenesis

and therapies.Report Faculty of Veterinary Medicine Ghen University.

Shinpei et al. 2015. Canine Pemphigus Foliaceus with Concurrent Immune-

Mediated Thrombocytopenia. J Am Anim Hosp Assoc 2015; 51:56–63.
DOI 10.5326/JAAHA-MS-6044

Tater, K.C., Olivry, Thierry. 2010. Canine and Feline Pemphigus Foliaceus:
Improving Your Chances of A Succesful Outcome. North Olmsted 105 (1).

WOHLSEIN, P., TRAUTWEIN, G., & DEEGEN, E. (1993). Pemphigus Foliaceus

in a Horse. Veterinary Dermatology, 4(1), 27–32. doi:10.1111/j.1365-
3164.1993.tb00186.x
 Pemphigus Foliaceus in 91 Dogs

A retrospective study of 91 dogs with pemphigus foliaceus was performed. Clinical signs of
the disease included crusts (n=79), pustules (n=36), and alopecia (n=33). Lesions were most
common on the trunk (n=53), inner pinnae (n=46), face (n=37), and foot pads (n=32).
Cytological evaluation revealed acantholytic keratinocytes in 37 of 48 dogs. Results of combi-
nation treatment with prednisolone and azathioprine were comparable to results with pred-
nisolone therapy alone. More than half of the dogs achieved remission with appropriate
therapy, and another 25% significantly improved. J Am Anim Hosp Assoc 2006;42:189-196.

Ralf S. Mueller, Dr.med.vet.habil., Introduction

Diplomate ACVD, FACVSc,
Diplomate ECVD
Ingar Krebs, DVM
Helen T. Power, DVM,
Diplomate ACVD
Kathryn V. Fieseler, DVM
RS
From the Department of Clinical Sciences
(Mueller, Krebs, Fieseler),
College of Veterinary Medicine
and Biomedical Sciences,
Colorado State University,
Fort Collins, Colorado 80523
and Dermatology for Animals (Power),
106 East Campbell Avenue,
Campbell, California 95008.
Doctor Mueller’s current address is
Medizinische Tierklinik,
Ludwig-Maximilians-University,
Veterinaerstr. 13, 80539, Munich, Germany.
Pemphigus foliaceus is the most common form of the pemphigus com-
plex in small animals and possibly the most common of all immune-
mediated dermatoses in the dog.1-3 Although canine pemphigus foliaceus
has been recognized since 1977, information regarding the disease has
been mostly limited to case reports.4-7 Few studies have been performed
to further characterize the disease and to evaluate the outcome of treat-
ment.8,9 Pemphigus may occur spontaneously, may be related to drug
administration, or be associated with neoplastic skin disease.10-12 All
forms of pemphigus share the presence of autoantibodies directed against
keratinocyte desmosomal proteins, resulting in a loss of adhesions
between keratinocytes.13 In pemphigus foliaceus, this loss of adhesion
leads to intraspinosal or subcorneal pustule or vesicle formation. These
primary lesions are transient in nature and ultimately result in the forma-
tion of crusts.14
Definitive diagnosis of pemphigus foliaceus is based on histological
features—the most important of which are pustules or vesicles with
marked acantholysis of keratinocytes.15-17 Neutrophils or eosinophils are
typically present in large numbers within pustules.17 The mainstay of
pemphigus foliaceus treatment is immunosuppressive therapy.1,14 In a
recent study, a poor prognosis in dogs was reported, with only 53% of
treated cases surviving >1 year after initiation of treatment.9 The purpos-
es of this study were to evaluate the clinical features, diagnostic modali-
ties, and treatment options for pemphigus foliaceus and to determine
whether they have any predictive value on the outcome of the disease in
a large number of dogs.
Materials and Methods
Medical records of dogs evaluated for pemphigus foliaceus between
September 1994 and November 2002 at three referral institutions, two
specialty clinics (i.e., Animal Skin and Allergy Clinic in Melbourne,

JOURNAL of the American Animal Hospital Association 189

190 JOURNAL of the American Animal Hospital Association
Australia [n=25] and Dermatology for Animals in
Campbell, California [n=31]), and a university veterinary
teaching hospital (i.e., Colorado State University [n=35])
were reviewed. Dogs were included in the study if a defini-
tive diagnosis of pemphigus foliaceus was made by
histopathological findings diagnostic for or highly sugges-
tive of pemphigus foliaceus in association with classical
clinical signs, supportive cytology, and response to therapy.
Breed, gender, and age of dogs, previous medications, and
the presence of pruritus at the time of disease onset were
recorded. Rate of onset of clinical signs was categorized as
rapid (i.e., clinical signs developed within a period of <1
month) or slow (i.e., clinical signs developed within a peri-
od >1 month). Lesions were categorized as pustules (super-
ficial lesions filled with pus), crusts (adherens of dried
exudates, serum, or pus to the surface of the skin), scales
(accumulation of loose fragments on the surface of the
skin), and/or alopecia (loss of hair), and the specific loca-
tions of the lesions were noted where possible. If results of
surface cytology were available, they were also recorded.
Medications used were recorded, as well as the duration of
therapy until initial improvement and complete remission.
Final treatment outcomes were recorded as remission,
euthanasia, or improvement. Improvement was defined as one
or more small areas of scaling, crusting, or erythema without
the need for changes in therapy. If a dog was euthanized, the
reason for the euthanasia was determined whenever possible.
The follow-up period was determined for each animal.
A Fisher’s exact test was used to determine if there was
a significant difference in outcomes between therapy with
prednisolone alone or in combination with azathioprine, and
any adverse effects. The times to remission with pred-
nisolone only versus prednisolone and azathioprine were
compared with a two-tailed, unpaired t-test.a A P value of
<0.05 was considered significant.
Results
Signalment and History
Ninety-one dogs were included in the study. The average age
at onset of clinical signs was 6 years (median age 6.5 years;
range 0.5 to 16 years). No gender predisposition was identi-
fied. There were 46 males (34 castrated) and 45 females (41
spayed). Affected breeds with more than one dog are listed
in Table 1. Because cases came from three different areas
and institutions, breed predispositions were not identified. In
29 dogs, the course of the disease was determined to be
rapid. The course was considered slow in 22 dogs and could
not be determined from medical records for 40 dogs. The
average time from onset of disease to presentation to a spe-
cialist was 17.2 weeks (range 3 days to 2 years).
Forty-four animals received medications before referral.
In 18 of these dogs, antibiotics and/or glucocorticoids at
various dosages were given with partial or no response.
Prior to onset of clinical signs, 26 dogs had also received
drugs for other reasons. Thirteen animals received antibi-
otics, and four were given L-thyroxine supplementation.
Heartworm preventions with ivermectin or milbemycin
May/June 2006, Vol. 42
Table 1
Most Common Dog Breeds Diagnosed
With Pemphigus Foliaceus
Breed No. of Dogs
Mixed-breed dog 40
Akita 10
Labrador retriever 9
Cocker spaniel 5
German shepherd dog 3
Chinese shar pei 3
Chow chow 2
Boston terrier 2
Shih tzu 2
Australian cattle dog 2
oxime were administered in six dogs, and one animal each
received fipronil, lufenuron, and mexiletine. In three ani-
mals, the disease onset was acute and occurred shortly after
administration of fipronil, cephalexin, and amoxicillin, indi-
cating a possible drug reaction. However, rechallenge of the
animal to verify a drug reaction was not performed.
Clinical Findings
In 62 dogs, it was possible to verify from the records the
degree of pruritus at disease onset. Forty-eight dogs showed
no pruritus; in four (6%) dogs, there was severe pruritus; in
seven (11%) animals, the pruritus was moderate; and in
three (5%) it was mild. Crusts were the most common
lesions noted (n=79). Pustules were described in 36 dogs;
alopecia occurred in 33 dogs; and scaling affected 27 dogs.
The locations of lesions are listed in Table 2. Lesions limit-
ed to the face were noted in 15 dogs, and exclusive foot pad
involvement occurred in three animals. Focal disease affect-
ing only the face and/or feet occurred in 31 dogs. In 60
dogs, pemphigus foliaceus was generalized. Of the dogs
with nonoral mucous membrane lesions (n=2), one dog had
erosions of the prepuce, and one had erosions of the vulva.
Of the 48 dogs in which results of skin surface or aspira-
tion cytology were recorded, individual acantholytic cells
were present in 37 dogs, high numbers of neutrophils were
seen in 35 dogs, and numerous eosinophils were noted in
eight cases. In the cytological preparations from two dogs,
clusters of acantholytic cells were present. Intracellular bac-
teria were seen cytologically in nine animals. Extracellular
cocci were noted in an additional 23 dogs. Histopathology
was either strongly suggestive of or diagnostic for pemphi-
gus foliaceus in all dogs, with acantholytic cells present in
intraepithelial pustules and/or serocellular crusting.
May/June 2006, Vol. 42
Table 2
Locations of Dermatological Lesions in
Dogs With Pemphigus Foliaceus
Site No. of Dogs (%)
Trunk 53 (58%)
Inner pinnae 46 (51%)
Dorsal muzzle 37 (41%)
Foot pads 32 (35%)
Periocular area 26 (29%)
Outer pinnae 23 (25%)
Planum nasale 23 (25%)
Interdigital area 10 (11%)
Lips 9 (10%)
Perianal area 5 (5%)
Mucous membranes 2 (2%)
Treatments and Outcomes
Of the 88 dogs treated for the disease, 46 went into remis-
sion with treatment, 31 improved greatly with treatment but
still had mild focal lesions, and 11 were euthanized. Four
dogs were euthanized because they had severe disease that
did not respond to treatment. Two dogs were euthanized
because of adverse effects from medications, and four were
euthanized for other diseases or unrelated causes. In one
dog, the reason for euthanasia was unknown.
The initial treatment for most dogs was glucocorticoids
alone or a combination of glucocorticoids and azathioprine
[Table 3]. Owners of three dogs refused treatment and were
lost to follow-up. For the dogs achieving complete resolu-
tion with exclusively glucocorticoids, the average time to
remission was 7 months (range 1.5 to 12 months). Sixteen
of the 39 dogs initially treated with glucocorticoids received
prednisolone long term (15 went into complete remission
with this treatment). In 13 dogs, other drugs were added or
replaced prednisolone. Adverse effects from prednisolone
therapy included signs of iatrogenic hyperadrenocorticism,
such as lethargy and polyuria/polydipsia, in 10 of 39 ani-
mals. One dog developed anorexia in addition to the
polyuria and polydipsia, one dog lost weight, and one dog
developed anorexia and pain. Of the 39 dogs treated with
glucocorticoids only [Figure 1], five were euthanized 1
month to 4.25 years after therapy was initiated. Euthanasia
for three dogs was related to other medical problems (i.e.,
an undiagnosed kidney mass, hematochezia, and collapse).
One dog was euthanized because of its advanced age, and
the reason for euthanasia in one dog was unknown.
The average time to remission for dogs that received a
combination of prednisolone and azathioprine (n=33) was
Pemphigus Foliaceus 191
Figure 1—Treatment outcomes for 39 dogs with pemphi-
gus foliaceus that were treated exclusively with glucocorti-
coid therapy.
11.7 months (range 2 to 29 months). There was no statisti-
cally significant difference between time to remission for
dogs treated with glucocorticoids alone and those treated
with azathioprine and prednisolone (P=0.089). Five dogs
treated with azathioprine and prednisolone had received
prednisolone from the referring veterinarian with unsatis-
factory results, and two of these dogs went into remission
with combination therapy. Over the long term, 17 of the 33
dogs continued to receive a combination of glucocorticoids
and azathioprine, two received only azathioprine, two
received only prednisolone, and two received a combination
of prednisolone and aurothioglucose. For four dogs, other
drugs replaced or were added to prednisolone and azathio-
prine. Adverse effects seen with the combination therapy of
prednisolone and azathioprine included iatrogenic hyper-
adrenocorticism (n=13), hepatotoxicity (n=3), anemia
(n=1), demodicosis (n=1), and gastrointestinal bleeding
(n=1). Thus, adverse effects occurred more often with this
combination than with prednisolone alone (P=0.05). Of the
33 dogs treated initially with a combination of prednisolone
and azathioprine [Figure 2], three were euthanized 3 months
to 2 years after diagnosis because of the pemphigus foli-
aceus. One dog suddenly died of unknown cause.
Eight dogs were initially treated with tetracycline and
niacinamide. Pemphigus foliaceus was controlled in three of
these dogs. In two dogs, prednisolone was added to the
Figure 2—Treatment outcomes for 33 dogs with pemphi-
gus foliaceus that were treated with a combination of aza-
thioprine and prednisolone.
192 JOURNAL of the American Animal Hospital Association May/June 2006, Vol. 42

Table 3

Drugs Used Initially for the Treatment of Pemphigus Foliaceus in 88 Dogs

Drugs No. of Dogs

Prednisone/prednisolone (1.5 to 5 mg/kg/d PO*) 38

Methylprednisolone (1.3 mg/kg/d PO) 1

Prednisone/prednisolone (1 to 4.5 mg/kg/d PO) and azathioprine (1.5 to 2.6 mg/kg/d PO) 28

Triamcinolone (0.1 mg/kg/d PO) and azathioprine (1 to 1.8 mg/kg/d PO) 2

Dexamethasone (0.2 mg/kg/d PO) and azathioprine (1.8 mg/kg/d PO) 1

Methylprednisolone (0.7 to 1.6 mg/kg/d PO) and azathioprine (0.7 to 2.6 mg/kg/d PO) 2

Tetracycline and niacinamide† 6

Prednisolone (0.5 to 1 mg/kg/d PO), tetracycline, and niacinamide† 2

Azathioprine (2 mg/kg/d PO) 1

Prednisolone (0.7 mg/kg/d PO), doxycycline (5 mg/kg/d PO), and niacinamide† 1

Essential fatty acids‡ 1

Prednisolone (3 mg/kg/d PO), azathioprine (1.5 mg/kg/d PO), and doxycycline (5 mg/kg/d PO) 1

Prednisolone, azathioprine, and fatty acids 1

Pimecrolimus topically 1

Prednisolone (0.7 mg/kg/d PO), doxycycline (5.2 mg/kg/d PO) 1

Triamcinolone (0.1 mg/kg/d PO), tetracycline† 1

* PO=per os
† Tetracycline and niacinamide were each given at 250 mg PO q 8 h in dogs <15 kg and at 500 mg PO q 8 h in dogs >15 kg.
‡ Essential fatty acid supplementation was administered according to the manufacturers’ recommendations.

therapy, and in one dog this regimen resulted in complete
remission. One dog was euthanized for lymphoma. Two
dogs did not achieve remission. Adverse effects were not
observed with this therapy.
Twenty-nine dogs had a rapid onset of disease and were
referred within 1 month. Treatment outcomes for these dogs
are illustrated in Figure 3. Two of the dogs were euthanized for
lack of response to treatment. Onset of disease was gradual in
22 dogs, and the outcomes are depicted in Figure 4. Four of
these dogs were euthanized—one from a lack of response, two
from collapse, and one from an unrelated cause. Two dogs
died suddenly of unknown causes. Control of the pemphigus
foliaceus did not differ between the two groups (P=0.16).
Treatment outcomes for 31 dogs with localized disease
are shown in Figure 5. In this group, three dogs were euth-
anized—one from severe pemphigus, one because of the
adverse effects of therapy, and one from collapse. One dog
died from unknown cause. The outcomes of 59 dogs with
generalized disease are presented in Figure 6. In this group,
three dogs were euthanized because of the severity of the
pemphigus foliaceus. Four other dogs were euthanized for
reasons other than pemphigus (i.e., lymphoma, old age,
hematochezia, unknown cause). There were no significant
differences in remission rates or death rates between dogs
with localized disease and dogs with generalized disease
(P=0.66 and P=1.0, respectively).
May/June 2006, Vol. 42
Figure 3—Treatment outcomes for 29 dogs with pemphi-
gus foliaceus that had a rapid onset (i.e., <1 month) of clin-
ical signs.
Figure 4—Treatment outcomes for 22 dogs with pemphi-
gus foliaceus that had a gradual onset (i.e., >1 month) of
clinical signs.
Figure 5—Treatment outcomes for 31 dogs with pemphi-
gus foliaceus localized to the face.
A variety of drugs were used for long-term control of the
pemphigus foliaceus [Table 4]. Two of the dogs were com-
pletely tapered off medications and had no recurrence of
clinical signs. Interestingly, one of these dogs had received
amoxicillin immediately prior to onset of the disease. Six
dogs were lost to follow-up.
For the 91 dogs evaluated in this study, the average fol-
low-up time was 1.5 years (median 1.1 years; range 1 month
to 7.9 years). Average time to improvement was 6 weeks.
Pemphigus Foliaceus 193
Figure 6—Treatment outcomes for 59 dogs with general-
ized pemphigus foliaceus.
Figure 7—Treatment outcomes for 44 dogs with pemphi-
gus foliaceus that received antibiotics either before or with
immunosuppressive therapy.
Figure 8—Treatment outcomes for 43 dogs with pemphi-
gus foliaceus that did not receive antibacterial drugs.
When achieved, average time to remission was 9.3 months
(median 6.5 months, range 1 to 36 months). Forty-three
dogs were followed for <12 months, and 12 of these dogs
were euthanized (eight for other diseases and four for lack
of response or for adverse effects of treatment for the pem-
phigus foliaceus). In 54 dogs, the follow-up time was >12
months. Four of these dogs were euthanized—one for unre-
lated cause, one for neoplastic disease, and two because of
the pemphigus foliaceus. Twenty-two dogs never achieved
194 JOURNAL of the American Animal Hospital Association May/June 2006, Vol. 42

Table 4

Drugs That Achieved Final Control of Pemphigus Foliaceus in 80 Dogs

Drugs No. of Dogs

Prednisolone (0.1 to 4 mg/kg/d PO*) and azathioprine (0.4 to 3 mg/kg/d PO) 24

Dexamethasone (0.09 to 0.1 mg/kg/d PO) and azathioprine (0.8 to 2 mg/kg/d PO) 2

Methylprednisolone (0.1 to 0.4 mg/kg/d PO) and azathioprine (1 to 2.2 mg/kg/d PO) 4

Triamcinolone (0.08 mg/kg/d PO) and azathioprine (1 mg/kg/d PO) 1

Prednisolone (0.1 to 4 mg/kg/d PO) 22

Methylprednisolone (0.1 mg/kg/d PO) 1

Triamcinolone (0.07 mg/kg/d PO) 1

Azathioprine (0.4 to 3 mg/kg/d PO) 9

Tetracycline† or doxycycline (3 to 18 mg/kg/d PO) and niacinamide† 8

Prednisolone (0.3 to 1.25 mg/kg/d PO) and aurothioglucose (0.07 to 0.15 mg/kg)‡ 2

Essential fatty acids§ PO 2

Prednisolone (1 mg/kg/d) and cyclosporine (3 mg/kg/d PO) 1

Aurothioglucose‡ (0.15 mg/kg) 1

Methylprednisolone (0.2 mg/kg/d PO), tetracycline, and niacinamide† PO 1

Tacrolimus (0.1% topically) 1

* PO=per os
† Tetracycline and niacinamide were each given at 250 mg PO q 8 h in dogs <15 kg and at 500 mg PO q 8 h in dogs >15 kg.
‡ Aurothioglucose was given weekly or every other week as an intramuscular injection.
§ Essential fatty acid supplementation was administered according to the manufacturers’ recommendations.

complete remission, and 12 of these cases were followed for
<12 months.
Treatment method (i.e., prednisolone only versus pred-
nisolone and azathioprine) had no influence on the number
of dogs euthanized (12% and 13%, respectively; P=1.0).
Similarly, the number of dogs that achieved clinical remis-
sion with only prednisolone initially (38%) was not signifi-
cantly different from the number of dogs treated with
azathioprine and prednisolone together (42%; P=0.81).
Forty-four dogs [Figure 7] were initially treated with
antibiotics, and 43 dogs [Figure 8] received immunosup-
pressive therapy without antibiotics. There were no signif-
icant differences between dogs treated with or without
antibiotics with regard to remission (55% and 49%,
respectively; P=0.83) or euthanasia rates (18% and 12%,
respectively; P=0.55).
Discussion
This retrospective study of 91 dogs with pemphigus foliaceus
was similar to previous reports, especially with respect to clin-
ical signs, diagnosis, and treatments.4-9 Based on data from
dogs in this study, prednisolone compared to prednisolone and
azathioprine treatment had approximately the same impact on
the rates of response and euthanasia. Additionally, the form of
disease (i.e., localized versus generalized) and concurrent
treatment with antibiotics had no predictive value in outcome.
May/June 2006, Vol. 42
Alopecia, scaling, crusting, and epidermal collarettes are
the commonly reported lesions of canine pemphigus foli-
aceus.1 In the study reported here, crusts were the most
common clinical feature and were seen in 87% of the ani-
mals. Pustules were noted in more than one-third of the
dogs. Pemphigus foliaceus can be localized or generalized,
and lesions have usually been noted first on the head, par-
ticularly the dorsal aspect of the nose.8 In the current study,
however, most dogs had a generalized distribution of
lesions at presentation. Foot pad involvement has been com-
mon in pemphigus foliaceus and has been associated with
lameness.19 Occasionally, the feet may be the only affected
site.18,19 In the current study, more than one-third of the
dogs had foot pad lesions, although exclusive pad involve-
ment was rare. Involvement of mucous membranes and
mucocutaneous junctions has been rare in pemphigus foli-
aceus.1 In this study, nine dogs had lesions on the lips, and
two showed changes of the nonoral mucous membranes.
Pruritus has been reported as variable in pemphigus foli-
aceus, and some dogs in this study were pruritic.1 However,
many dogs had received glucocorticoids before presentation
to the specialist, and in 29 dogs it was not possible to deter-
mine pruritus from the records.
Differential diagnoses for pemphigus foliaceus based on
clinical findings include superficial folliculitis, dermato-
phytosis, demodicosis, discoid lupus erythematosus, and
pemphigus erythematosus.1,20,21 Direct cytology can be
strongly suggestive of pemphigus foliaceus, especially the
cytological feature of acantholytic cells, either singly or in
rafts. In the study reported here, 37 (77%) of 48 cytological
specimens had acantholytic keratinocytes; thus, the pres-
ence of these cells in cytological preparations from crusted
lesions should raise the index of suspicion for pemphigus.
Microscopic evaluation of Tzank preparations or impres-
sion smears is part of a routine diagnostic workup for pus-
tular and crusty skin diseases; thus, the number of
cytological samples recorded seemed very low. However,
because of the retrospective nature of this study, it was
unknown how many cytological samples were obtained but
not recorded. Prospective studies are warranted to confirm
the above findings. It must be remembered that superficial
folliculitis (e.g., bacterial or caused by Trichophyton spp.)
can occasionally result in acantholysis—most likely sec-
ondary to enzyme release from neutrophils or the organisms
themselves. Therefore, acantholytic cells are not pathogno-
monic for pemphigus foliaceus.
Histopathologically, increased density of acantholytic
cells, the presence of acantholytic rafts, and increased pus-
tule size (with pustules spanning multiple hair follicles) are
specific for pemphigus foliaceus.16 Direct immunofluores-
cence or immunohistochemical evaluation may support the
diagnosis, but these tests are less sensitive than direct
histopathological evaluation and are often prohibitive
because of technical or cost factors.1,2 Indirect immunoflu-
orescence staining of either circulating antibodies or circu-
lating immune complexes is of little value in diagnosing
canine pemphigus foliaceus, but it might be a valuable tool
Pemphigus Foliaceus 195
in the future as sensitivity of these tests increases.2,22-24
None of the dogs in the current study underwent immune
testing.
Secondary pyoderma is common in pemphigus foliaceus
and may complicate the diagnosis.1 Initial treatment with
antibiotics prior to or concurrent with the onset of immuno-
suppressive treatment may be associated with better out-
comes.9 However, antibiotic treatment did not influence
treatment outcomes in the current study, and future prospec-
tive studies are needed to address this apparent discrepancy.
In recent studies, the initial recommended treatment for
pemphigus foliaceus was oral glucocorticoids at immuno-
suppressive doses, which were tapered based on clinical
improvement.1,8,14 The success rate with exclusively gluco-
corticoid therapy has been reported to be <50%, with a
favorable response within the first 10 days of treatment
being a good prognostic sign.1,8 The results of the study
reported here concurred with these reports. Fourteen dogs
responded to prednisolone therapy within the first 2 weeks,
and 10 of these dogs ultimately remained on prednisolone
alone to control their clinical signs. Thus, rapid response
during early treatment may be a good prognostic sign.
Concurrent use of other immunosuppressive drugs, par-
ticularly azathioprine, has been advocated as the treatment
of choice for pemphigus foliaceus.14 In the present study, 21
(64%) of 33 dogs initially treated with prednisolone and
azathioprine showed satisfactory results. The number of
dogs responding to combination therapy was not signifi-
cantly higher than the number of dogs responding to gluco-
corticoids only. However, five of these dogs received
glucocorticoid therapy prior to referral with unsatisfactory
results. It is possible that owners chose combination thera-
py more often when pemphigus foliaceus was severe or pre-
vious drug therapy had failed, masking a better success rate
with the drug combination. Prospective, blinded studies
with random allocation of dogs to different treatment proto-
cols are needed to clarify this further. The average time to
remission was longer for dogs treated with combination
therapy than for dogs exclusively treated with prednisolone
(11 versus 7 months). This longer time to remission may
have arisen because combination therapy was used in the
more severely affected dogs.
Adverse effects of combination therapy were largely
attributed to iatrogenic hyperadrenocorticism and were seen
more often when prednisolone was used alone. A reason for
this finding was not identified. In addition, hepatotoxicity
was seen in three dogs. All liver enzymes were dramatical-
ly elevated shortly after initiation of therapy and were
thought to be secondary to azathioprine administration.
Anemia was observed in one dog and was also attributed to
bone marrow suppression from azathioprine. These adverse
effects emphasize the need for appropriate regular monitor-
ing with complete blood counts and biochemical panels in
all dogs receiving azathioprine.14
Cyclophosphamide, aurothioglucose, and cylosporine,
niacinamide, and tetracycline as adjunctive or single thera-
pies have all been reported as treatments for canine pemphi-
196 JOURNAL of the American Animal Hospital Association
gus foliaceus.14 Cyclosporine used as a single therapeutic
agent had limited efficacy in one study; however, the num-
ber of dogs was small, and different formulations of
cyclosporine exist today.25 More studies are needed to eval-
uate this drug for the treatment of canine pemphigus foli-
aceus. Chrysotherapy with aurothioglucose alone or in
conjunction with corticosteroid therapy may be effective in
controlling some cases of canine pemphigus foliaceus.8 In
one study, the following treatments were used to maintain
remission of pemphigus foliaceus: chlorambucil; aurothio-
glucose; glucocorticoids with aurothioglucose; tetracycline
or doxycycline with niacinamide; prednisolone; fatty acid
supplementation; and tacrolimus.8 Animal numbers in these
treatment groups were too small to perform statistical analy-
sis.8 None of the dogs treated with these therapies showed
any adverse effects.8 Further studies are needed to evaluate
particular treatments (i.e., tetracycline in combination with
niacinamide) with promising results and rare adverse
effects.
Treatment-induced remission of canine pemphigus foli-
aceus after cessation of therapy has been recently reported.26
In the current study, two dogs remained in remission after all
drugs had been discontinued for approximately 1 year. One
of these dogs had suddenly developed clinical signs of pem-
phigus while receiving amoxicillin for another problem, and
drug-induced pemphigus may have been possible. Although
a greater percentage of dogs with a rapid onset of disease
responded better to therapy than those with gradual onset,
this difference was not statistically significant. Similarly,
control of clinical signs was achieved in dogs with general-
ized disease more than in dogs with localized disease, but the
difference was not statistically significant.
Conclusion
Based on this study, canine pemphigus foliaceus was char-
acterized by crusts and pustules and had either a generalized
pattern or was limited to the face and feet. Cytological eval-
uation, a rapid and highly informative test, showed acan-
tholytic cells in many specimens. Prednisolone or a
combination of prednisolone and azathioprine was the treat-
ment most commonly used. Side effects were more fre-
quently seen with the combination therapy. More than half
of the affected dogs achieved remission with appropriate
therapy, and another 25% were improved.
a GraphPad InStat; GraphPad Software, San Diego, CA 92121
References
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Immunopathol 1983;5:47-64.
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13. Carlotti DN. Autoimmune mediated skin diseases. J Small Anim
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of the Cat and Dog. London: Wolfe Publishing, 1994.
16. Kuhl KA, Shofer FS, Goldschmidt MH. Comparative histopathology
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17. Gross TL, Ihrke PJ, Walder EJ. Veterinary Dermatopathology: A
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Disease. St. Louis: Mosby, 1992.
18. High M. An interesting case of pemphigus foliaceus in a dog. Can
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19. Ihrke PJ, Stannard AA, Ardans AA, et al. Pemphigus foliaceus of the
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 GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2014-2015

Pemphigus foliaceus in dogs: the immune pathogenesis and therapies.

Why are some dogs not responsive to the treatment?

By

Marleen PETERMANN

Promoter: dr. Elisa Maina Literature review as part of the

Co-Promoter: Prof. dr. Eric Cox Master`s Dissertation

© 2015 Marleen Petermann

Disclaimer

Universiteit Gent, its employees and/or students, give no warranty that the information provided in this
thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any
infringement of third-party rights.

Universiteit Gent, its employees and/or students do not accept any liability or responsibility for any use
which may be made of the content or information given in the thesis, nor for any reliance which may
be placed on any advice or information provided in this thesis.
 GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2014-2015

Pemphigus foliaceus in dogs: the immune pathogenesis and therapies.

Why are some dogs not responsive to the treatment?

By

Marleen PETERMANN

Promoter: dr. Elisa Maina Literature review as part of the

Co-Promotor: Prof. dr. Eric Cox Master`s Dissertation

© 2015 Marleen Petermann

Preface

This report is the first part of my master thesis, which I could conduct in the fields of immunology and
dermatology. Both have always been of major interest for me so that I felt privileged that I could
deepen my knowledge as part of my study.

The realization of my literature study was enabled by professional assistance and familial support.

In the first place I wish to thank my promoter Dr. Elisa Maina for her time, interest, advice and critical
proof-reading.

I also want to thank my parents for their advice, support and for making my dream, becoming a
veterinarian, possible.
Table of contents:

Abstract .............................................................................................................................................. 1
Samenvatting ...................................................................................................................................... 2
1. Introduction ..................................................................................................................................... 3
1.1.The different forms of the pemphigus complex .................................................................. 3
1.1.1 Pemphigus vulgaris ........................................................................................... 4
1.1.2. Pemphigus vegetans ........................................................................................ 4
1.1.3.Pemphigus erythematosus ................................................................................. 4
1.1.4. Panepidermal pustular pemphigus .................................................................... 5
1.1.5. Paraneoplastic pemphigus ................................................................................ 5
2. Pemphigus foliaceus ....................................................................................................................... 6
2.1.Epidemiology .................................................................................................................... 6
2.2. Clinical signs .................................................................................................................... 6
2.3. Pathogenesis ................................................................................................................... 7
2.3.1. Involved components ........................................................................................ 7
2.3.1.1. Desmosomes: Function, ultrastructure and composition ..................... 7
2.3.1.2. Desmosal cadherins .......................................................................... 8
2.3.1.2.1. Distribution of certain desmosomal and non-desmosomal
adhesion molecules .......................................................................... 8
2.3.1.2.2. Desmoglein as a minor target auto-antigen ......................... 9
2.3.1.2.3. Desmocollin as a major target auto-antigen ..................... 10
2.3.1.2.4. Other auto-antigens ......................................................... 11

2.3.2. The role of neutrophils ................................................................................... 11

2.3.3. Antibodies....................................................................................................... 12
2.3.3.1. Antibody profile................................................................................ 12
2.3.3.2. IgG subclasses ................................................................................ 13
2.3.3.3. Titer and severity ............................................................................. 13
2.3.3.4. Specificity of the antibodies.............................................................. 13
2.3.4. Pathophysiology ............................................................................................. 14

2.3.4.1. Introduction ..................................................................................... 14

2.3.4.2. Role of the auto-antibodies. ............................................................. 14

2.3.4.3. Proposed mechanisms ................................................................... 14

2.4. Drug-related pemphigus ................................................................................................ 17
2.4.1 Introduction...................................................................................................... 17
2.4.2. Trimethoprim-sulfonamide .............................................................................. 17
®
2.4.3. Promeris Duo ............................................................................................... 17

2.4.4. Certifect :® Fipronil-Amitraz- S-methoprene .................................................... 18

 2.4.5. Vectra 3D ....................................................................................................... 18
2.4.6. Others ............................................................................................................ 19
2.5. Therapies....................................................................................................................... 19
2.5.1. Introduction..................................................................................................... 19

2.5.2. Most commonly used drugs in the treatment of cPF ........................................ 19

2.5.2.1. Corticosteroids ................................................................................ 19
2.5.2.2. Azathioprine .................................................................................... 22
2.5.2.3. Chlorambucil ................................................................................... 22
2.5.2.4. Chrysotherapy ................................................................................. 22
2.5.2.5 Tetracycline and niacinamide............................................................ 23

2.5.2.6. Mycophenolate mofetil ..................................................................... 23

2.5.2.7. Cyclosporine and tacrolimus ............................................................ 24
2.5.2.8. Cyclophosphamide .......................................................................... 25
2.5.2.9. Dapsone and sulfasalazine .............................................................. 25
3. Why are some dogs not responsive to the treatment ..................................................................... 26
3.1. Treatment outcome and long-term prognosis ................................................................. 26

3.2. Possible reasons why some dogs are not responding..................................................... 27

3.2.1. Incorrect diagnosis.......................................................................................... 28
3.2.2. Naturally or drug-related pemphigus ............................................................... 28
3.2.3. Drug regime ................................................................................................... 28

3.2.4. Glucocorticoid resistance ............................................................................... 29

3.2.5. Influence of 25-hydroxyvitamin D concentrations ............................................ 29

3.2.6. Tachyphylaxis to topical glucocorticoids, is it proven? ..................................... 29

4. Discussion .................................................................................................................................... 30
5. References ................................................................................................................................... 31
Abstract

This literature study reviews up-to-date knowledge on the immunopathogenesis, therapies and
prognosis of pemphigus foliaceus (PF) in dogs. PF is an auto-immune blistering skin disease
described in dogs, humans and other species. The formation of auto-antibodies, primarily IgG4,
against keratinocyte desmosomal adhesion proteins (cadherins) results in acantholysis and
intraepidermal blister formation. Desmocollin-1 was identified as the major auto-antigen and
desmoglein-1 as a minor auto-antigen. PF however appears to be a complex and multifactorial
disease where different auto-antigens, auto-antibodies and pathogenic components are involved. The
exact pathomechanism is not yet fully elucidated but different theories have been suggested. Those
include among others steric hinder by IgG-binding and thereby disabling desmosomal bond-formation;
the initiation of multistep mechanisms of intracellular events triggered by IgG-binding and also
neutrophils seem to play a pathogenic role. It is proposed that predisposing and inducing factors such
as environmental or endogenous factors, e.g. drugs, are necessary for the outbreak of the disease.
Drugs that seem to have a potential to provoke acantholysis similar to PF include trimethoprim-
sulfonamides, Certifect®, Promeris® and Vectra 3D®. In most dogs remission of the lesions is achieved
with an adequate drug regime. The first-line drugs are glucocorticoids. Due to adverse drug reactions
or an insufficient response second-line medication such as azathioprine, cyclosporine or chlorambucil
are used on a regular basis. Some dogs however do not respond to the treatment. The reason
therefore is not yet known.

Key words: Autoimmunity - Blistering - Canine pemphigus foliaceus - Desmosomes -

Glucocorticoids

1
Samenvatting

In deze literatuurstudie wordt gepoogd om de huidige kennis omtrent de immunopathogenese en de

verschillende therapeutische benaderingen bij canine pemphigus foliaceus samen te vatten. In het
bijzonder worden mogelijke hypothesen besproken waarom sommige honden niet op de therapie
reageren.

Pemphigus foliaceus is een auto-immune huidaandoening die onder andere bij de mens en de hond is
beschreven. Het lichaam vormt auto-antistoffen, voornamelijk IgG4, die zich tegen adhesie proteïnes
van de keratinocyten in de epidermis richten. Daardoor ontstaat acantholyse en intra-epidermale
blaarvorming. Een aantal jaar geleden kon desmocollin-1 worden aangetoond als het hoofd auto-
antigen en desmoglein-1 als een bijkomend auto-antigen. Pemphigus foliaceus bij honden blijkt een
multifactoriele aandoeningen te zijn met verschillende auto-antigenen, auto-antistoffen en
pathologische componenten. Een reeks aan pathologische mechanismen werden door onderzoekers
voorgesteld. Tot op heden is de exacte immunopathogenese echter nog niet volledig opgehelderd.
Voorgestelde mechanismen verantwoordelijk voor de acantholyse zijn onder andere sterische
hindering door auto-antistoffen die ter hoogte van de desmosomen vasthechten en de intercellulaire
verbinding tussen naburige cellen inhiberen. Ook werd de initiatie van een cascade aan intracellulaire
mechanismen, uitgelokt door IgG-binding, voorgedragen waarbij een reeks aan componenten
betrokken zou zijn. In canine pemphigus foliaceus werd bovendien de betrokkenheid van neutrofielen
aangetoond. In de klassieke humane vorm van pemphigus foliaceus is dit niet het geval.

Onderzoekers vonden daarboven indicaties dat de uitbraak van PF door endogene of exogene
factoren uitgelokt kan worden. Het best beschreven voorbeeld is een uitbraak na toediening van een
beperkt aantal geneesmiddelen. Bij de hond werden uitbraken beschreven na de toediening van
trimethoprim-sulfonamides, Certifect®, Promeris® en Vectra 3D®. Men vermoed dat genetische
factoren eveneens een rol spelen in de pathogenese, omdat sommige rassen gepredisponeerd zijn.

De therapie berust voornamelijk op immunosuppressie. Hierbij zijn glucocorticoide zoals prednisolone

de meest gebruikte farmaceutica. Door ernstige bijwerkingen of een onvoldoende respons kunnen
tweedelijns geneesmiddelen (onder andere azathioprine, cyclosporine of chlorambucil) als vervanger
of in combinatie met glucocorticoiden worden gebruikt. In een beperkt aantal gevallen treedt volledige
remissie op, in de meeste gevallen is levenslange behandeling echter noodzakelijk. Een ander klein
deel van de patiënten reageert niet op de behandeling. De exacte oorzaak hiervoor is niet bekend.
Een aantal hypothesen zijn door de auteur voorgesteld. Hierbij behoren het ontwikkelen van
resistentie tegen glucocortcoide, een foutieve diagnose, het gebruik van een ongeschikt geneesmiddel
protocol of een geïnduceerde vorm van pemphigus in plaats van de spontaan ontstane aandoening.

Sleutelwoorden: Autoimmuniteit – Blaarvorming - Canine pemphigus foliaceus – Desmosoom –

Glucocorticoide

2
 Pemphigus foliaceus in dogs: the immune pathogenesis and therapies.

Why are some dogs not responsive to the treatment?

1. INTRODUCTION

This literature study reviews up-to-date knowledge regarding the immunopathogenesis and available
therapies for the canine form of pemphigus foliaceus (PF). PF is a complex disease with many
components involved. The interactions of these components as well as the exact pathologic
mechanisms are not yet identified. It is also not known why some dogs do not respond to a
(glucocorticoid) treatment. This literature study strives to give a better insight in already conducted
research and proposed hypotheses.

1 2-4 2, 3
Pemphigus foliaceus (PF) is an auto-immune skin disease described in humans, cats, dogs,
horses,3, 5 goats 6 and a Barbary sheep (single case)7. The word pemphigus originates from the Greek
word for blister 8, which is one of the major characteristics describing this medical condition.
PF is part of the pemphigus complex, which comprises pemphigus vulgaris, pemphigus vegetans,
pemphigus erythematosus, pemphigus foliaceus, panepidermal pustular pemphigus, paraneoplastic
pemphigus, and drug-related pemphigus.9 The pemphigus diseases have in common that the body
produces auto-antibodies against the adhesion molecules of keratinocytes, resulting in the separation
of the keratinocytes, called acantholysis.10,
11
This becomes clinically visible as pustules, which are
very fragile, transient, and leave crusts and erosions11.

1.1. THE DIFFERENT FORMS OF THE PEMPHIGUS COMPLEX

Diseases included in the group of pemphigus can be differentiated based on the specific auto
antigens. The various forms of pemphigus recognize different auto antigens, located in different layers
12
of the skin. Thus, depending on where the acantholysis takes place, the clinical signs such as
distribution, severity and location of the lesion changes.13

Figure 1: The figure shows the location of the different pemphigus diseases within the epidermis.

3
1.1.1. Pemphigus vulgaris

Pemphigus vulgaris (PV) is the most severe and rarest form of the pemphigus complex.10, 12The auto-
antibodies bind to antigens in the deepest layer of the epidermis, close to the dermal-epidermal
junction. Acantholysis therefore occurs right above the basal cell layer, forming suprabasilar
11, 12
vesicles.
PV is divided in both, humans and dogs, in a mucosal-dominant type and a mucocutaneous type. In
the mucosal-dominant type oral lesions dominate with little or no skin involvement, whereas in the
14
mucocutaneous form oral as well as skin lesions can be seen.
The clinical signs are primarily bullae, erosions and ulcers of the skin and can be found, depending on
the type, on the mucosae, at the mucocutaneous junctions and on the trunk, especially in areas of
skin-to-skin-contact such as in the groin and axillae. Oral lesions often cause increased salivation,
halithosis and difficulties to eat.9, 10, 11, 12
PV shows varying degrees of pain and pruritus.9 Systemic
10
symptoms, such as fever, depression or anorexia are frequently seen.
Desmoglein-1 was identified as the major-autoantigen in the cutaneous form, thus causing skin-
lesions. Desmoglein-3 on the other side is the target-antigen in the mucosal form, resulting in mucosal
lesions.14 Logically, both auto-antigens (anti-Desmoglein-1 IgG and anti-Desmoglein-3 IgG) are
present in the mucocutaneous form.15
The diagnosis is made by histopathological examination and by immunofluorescence. Histology is
12
characterized by suprabasilar clefts and vesicles filled with acantholytic cells. Acantholytic cells are
round, the cytoplasm is dense and the nucleus hyperchromatic. Depending on the stadium (acute or
chronic), neutrophils and eosinophils can also be found in the vesicles.16 Another diagnostic tool is
immunofluorescence. In affected patients antibody-deposition in the intercellular spaces can be found
on the biopsy after staining with anti-IgG fluorescein.12

1.1.2. Pemphigus vegetans

9, 17
Pemphigus vegetans is extremely rare in domestic animals. It is seen as a localized and benign
18 11
variant of pemphigus vulgaris. On histology acantholysis is visible in the middle of the epidermis ,
causing pustules and erosions.17 The lesions are generalized rather than mucocutaneous.9
In humans two types of pemphigus vegetans are described: The Neumann type and the Hallopeau
type. Both are sought to be a localized form of Pemphigus vulgaris. The Neumann type is more
common. Lesions include large bullae and erosions, which heal by forming granulation tissue. The
Hallopeau type is less aggressive and on clinical examination pustules instead of bullae can be seen.
When they heal, verrucous hyperkeratotic vegetation develops.18

1.1.3. Pemphigus erythematosus

11
Pemphigus erythematosus (PE) and pemphigus foliaceus are more superficial. They affect especially
the stratum corneum, creating intracorneal or subcorneal pustules that rapidly burst, leaving superficial
4
erosions bordered by epidermal collarettes17, erythema, alopecia, scales and honey-colored to brown
crusts.10,11 Nasal depigmentation is frequently seen.9, 10, 11, 17, 19
The main difference between PE and PF is that pemphigus erythematosus is limited to the face and
10, 12
ears. It is primarily found on the bridge of the nose, around the eyes and on the ear pinnae. The
9,10,12
disease is most common in Collies, German Shepherds and Shetland sheep dogs.
Pemphigus erythematous shows clinical, histological and immunological overlap with characteristics of
pemphigus foliaceus as well as characteristics of discoid lupus erythematosus. 9, 19
It is thus still
questionable if PE is an own entity, a benign variant of PF restricted to the face or a crossover of PF
9,10,19
and discoid lupus erythematosus.

Pemphigus foliaceus and drug-related pemphigus will be discussed more in detail further on.

1.1.4. Panepidermal pustular pemphigus

Panepidermal pustular pemphigus (PPP) was introduced in 1994 by a research team for a group of
cases that were previously diagnosed as pemphigus vegetans or pemphigus erythematosus.20 An
important criteria for diagnosing PPP is that the pustules can be found throughout all levels of the
epidermis and follicular epithelium.9 This stands in contrast to PF where pustules stay limited to the
granular and upper spinous layers.19 The pustules rupture easily, leaving a thick crust on the skin.9 On
histology the pustules were found to contain neutrophils, eosinophils and acantholytic cells. 20
Desmoglein-1 could be identified as the major auto-antigen,21 just as in PF. Also the distribution of the
lesions is similar to the distribution of PF. It is apparent that features of PPP overlap with features of
PF. It is thus debatable if PPP is an own entity or a variant of PF. 19

1.1.5. Paraneoplastic pemphigus

At last there is paraneoplastic pemphigus (PNP). This form is very rare and shows blistering skin
22, 23 24, 25
lesions in association with underlying neoplasms. Envoplakin, periplakin, desmoplakin-I and –
22 23
II and desmoglein-3 were identified as target-antigens in canines.
In humans pemphigus has been associated with a range of neoplasia, such as a lymphoma, chronic
26
lymphocytic leukemia, spindle cell sarcomas, squamous cell carcinoma of the lung or thymomas. In
dogs only a limited number of cases have been reported to date. A few were linked to a thymic
lymphoma26, a sertoli cell tumor27 and another to a splenic sarcoma.22 On clinical examination severe
stomatitis and polymorphous ulcerative lesions were seen in the oral cavity, nose, vulva,
15,22,24,26
mucocutaneous junctions and haired skin. On histopathology suprabasal epithelial
acantholysis typical of PV was found as well as apoptotic keratinocytes with satellitosis that showed
similarities with erythema multiforme.22,26

5
2. PEMPHIGUS FOLIACEUS

2.1. EPIDEMIOLOGY

Pemphigus foliaceus was first described in dogs in 1977 by Halliwell and Goldschmidt 28, since then
the disease was mentioned in different species1-6 and further researches were carried out.
However, limited information about the epidemiology in dogs can be found. 19Of the pemphigus
10,11,12,19,29
complex, PF is the one most frequently seen. A study from 1987 reported an estimated
prevalence of 0,3%. More specific, three out of 1000 presented canine patients with skin diseases in
30
an animal hospital in New York were diagnosed with pemphigus foliaceus. Another study showed
that canine pemphigus foliaceus (cPF) is probably the most common auto-immune skin disease in
19 29
dogs , accounting for almost one-third of the cases. Contrary, in humans pemphigus vulgaris is the
31
most frequent variant of the pemphigus group. Authors of a retrospective study carried out in Brazil
diagnosed 102 dogs with PF over a 25-year period. That is 4.1 cases per year.32All breeds can be
10
affected , but some breeds have a higher risk. Different studies describe a higher prevalence in the
Bearded Collie, Akita, Newfoundland, Schipperke, Dachshund, Doberman pinscher, English Springer
Spaniel, English bulldog, Finish Spitz, Labrador retriever, English Cocker Spaniels, Chow Chows,
Shar-Peis and Collies19,33 There appears to be no age or sex predisposition. 10, 33,34
However, most
11, 30
dogs are middle-aged between four and six years with 4.2years as the mean age of onset.33

2.2. CLINICAL SIGNS

As mentioned before are PF and PE the more superficial versions of the pemphigus complex,
affecting primarily the stratum corneum.11, 19
The primary lesions are superficial and transient
10,17
pustules. These are yet hard to find, since being very fragile and hidden under the coat. 10The
pustules rupture easily, leaving erythema, yellowish crusts, erosions, alopecia and peripheral
collarettes.9,11,17,33The lesions most commonly start on the dorsal part of the muzzle, are bilaterally
symmetric and spread gradually.33 PF shows a typical distribution (Fig. 2,3) and most commonly
affects the pinnae, perioral and periocular region, the nasal planum, bridge of the nose and the
9,10,11,17,33
footpads. The footpad lesions are present in 1/3 of the dogs and are characterized by
hyperkeratosis, cracks, possible erythematous swelling and whitish discoloration.33 Those lesions can
10 23
be the only symptom. The nailbed can be involved the nails however are usually unaffected.
9, 17, 30
Lesions in the oral cavity and the mucocutaneous junction are rare. In some patients PF can
become generalized, with development of the following symptoms in severe cases: anorexia, fever,
10, 11, 17
depression, lymphadenomegaly and limb edema. Pruritus can only be found in less than half of
33
the dogs with 17 to 36% showing moderate to severe itching. The disease can be waxing and
10, 11, 17
waning.

Figure 2, 3: Typical facial distribution of cPF

lesions with scaling, alopecia and pustules.

6
2.3. PATHOGENESIS

2.3.1. Involved components

2.3.1.1. Desmosomes: Function, ultrastructure and composition

Desmosomes or macula adherens are adhesion molecules found in the lateral cell membrane. They
are complex structures that are particularly important in tissues that are subject to mechanical stress,
such as the epidermis or the cardiac muscle. Their main function is cell-cell-adhesion and to assure
tissue integrity. Besides giving strength to epithelia by linkage of the desmosomes to the keratin
intermediate filament of the cytoskeleton, more and more evidence is emerging that desmosomes also
14,35,36
play an important role in intracellular signaling pathways.

Desmosomes are classified as glycoproteins and contain an

intra- and an extracellular part that form a complex. They are
14
spot-like distributed across the lateral cell membrane.

Two adjacent cells each possess an outer and an inner dense

plaque. Those plaques are made of proteins of the plakin
(desmoplakin) and armadillo-family (plakoglobin and
14
plakophilin). The inner dense plaque (IDP) consists of
desmoplakin and is linked to intermediate filaments of the
cytoskeleton, to stabilize the cell and keep the desmosomes
on their place. The outer dense plaque (ODP) serves as an Figure 4: Schematic ultrastructure of a
desmosome.
anchor for the cytoplasmic domain of the cadherins. Cadherins
are a third involved protein-family, comprising a cytoplasmic and an extracellular component. Their
name derives from the circumscription “Ca2+ -dependent adhesion” and comprises the glycoproteins
desmocollin (Dsc) and desmoglein (Dsg). Those two are intracellular connected to plakophilin and
plakoglobin and form extracellular heterophilic and homophilic bonds with the adjacent cells.37

Despite their important role in cell-cell adhesion and cell integrity, the desmosomes are not static but
dynamic structures that can change their molecular composition and adhesive properties. 38 Recently
2+
two different adhesion states have been described. One is Ca independent, stable and
2+
hyperadhesive, the other is dynamic, weaker and Ca -dependent. Both are reversible through cell
38
signaling mechanisms which involve protein kinase C and epidermal growth factor receptors.

It is evident that calcium-ions play an essential role in the induction of desmosome formation. Several
studies revealed that cultured keratinocytes do not form desmosomes in low Ca2+ concentrations
(below 0.1mM). 39-41 When the Ca2+ concentration is however increased desmosomes formed within
two hours.42,43 An increase in extracellular Ca2+ is registered by a Ca2+ sensing receptor (CaR).39-42
44
CaR activates phospholipase C and triggers the production of inositol 1,4,5-triphosphate (IP3). The
2+ 45
increase in IP3 results in an elevated intracellular Ca concentration in keratinocytes. Similar
findings were made in cPF research. Seven out of seven human PF sera bound to canine footpad
epithelium under high calcium conditions, but they failed to bind when calcium was chelated (with
EDTA).46 Desmosomes are associated with even more proteins, among others accessory proteins
7
such as Perp, corneodesmosin or the armadillo protein p0071.14 The discussion of these goes beyond
the scope of this literature study. Considering the complexity and numerous components of
desmosomes it is intelligible that several of them are plausible candidate autoantigens.47

2.3.1.2. Desmosomal cadherins

2.3.1.2.1. Distribution of desmosomal and non-desmosomal adhesion molecules

In humans there are three isoforms of desmocollin (Dsc 1-3) and four isoforms of desmoglein (Dsg 1-
4). Each cell expresses several isoforms and each desmosome contains more than one type of
14
desmocollin and desmoglein.
The keratinocytes of the epidermis have different degrees of differentiation which are organized in
layers and express a variety of molecular compositions.15 Desmosomal cadherins for example are
expressed in a pattern that is typical for a specific tissue. Except for Dsc-2 and Dsg-2, the expression
of the cadherins is restricted to stratified epithelial tissues. That means that only Dsc-2 and Dsg-2 are
found, for example in the human cardiac muscle, but all seven (Dsc1-3, Dsg1-4) can be found in the
epidermis.14

Bizikova et al. carried out a research in 2011 with the goal to identify possible antigens of PF.
Therefore the research-team immunomapped the major desmosomal (desmoglein-1, desmoglein-3,
desmocollin-1, desmocollin-3, desmoplakin-1/2, plakoglobin and plakophilin-1) and non-desmosomal
adhesion proteins (E-cadherin, claudin-1, zona occludens-1 and occludin). The desmosomal
immunostaining patterns were then compared to the patterns of IF staining with canine PF sera.35
Figure 5 compares the staining patterns of desmoglein 1 and 3 in canine footpad and buccal mucosa.

Figure 5: Desmoglein-1 and -3 fluorescence patterns

in canine footpad and buccal mucosa. Intercellular
staining indicates the binding of autoantibodies to
extracellular components. The antibody deposition shows a
typical honey-comb pattern.

Of the tested cPF sera 88% showed immunofluorescence-staining laterally of the keratinocytes in the
stratum spinosum and stratum granulosum, 11% showed additional intercellular staining in the stratum
basale, one serum (2%) only bound to the stratum granulosum and 18% also showed intercellular
fluorescence of the buccal mucosae. 80% of the sera thus exhibited a restricted staining pattern to the

8
suprabasal footpad epithelium and very low staining of the buccal mucosa. Several different staining
patterns of the various cPF sera were detected, which implies an immunological heterogeneity of cPF
IgG auto-antibodies.35,48The figure below (figure 7) shows the distribution of certain desmosomal
components in the canine epidermis. The indirect immunofluorescence (IIF) staining patterns and thus
the distribution of the most relevant molecules for PF are described more in detail below.

Figure 6: The diagrams represent the staining patterns and immunofluorescence intensity of desmosomal cadherins
in the different layers of canine footpad, haired skin and buccal mucosa . The column width is in accordance with the
staining intensity.
DSG1: desmoglein-1; DSG3: desmoglein-3; DSC1: desmocollin-1; DSC3: desmocollin-3

Desmoglein-1 and -3

In the canine footpad the fluorescence intensity was highest in the stratum spinosum, with a moderate
decrease towards the stratum granulosum and the stratum basale. Dsg1 is more evenly distributed in
the haired skin with slight decreases towards the basal layer and high fluorescence intensity in the
entire stratum spinosum. Dsg1 is furthermore absent in the stratum distendum of the buccal mucosa.
Dsg1 and 3 show a reciprocal staining pattern in all examined epithelia. Dsg1 is most present in the
superficial layers whereas Dsg3 is highest in the basal layers of the footpad, haired skin and buccal
35
mucosa.

Desmocollin

Desmocollin 1 was exclusively found on the margins of suprabasal keratinocytes in the footpads as
well as in the interfollicular epidermis. Interestingly, Dsc1 was not detectable in canine buccal mucosa,
with neither of the anti-Dsc1 antibodies. Dsc3 can well be found in all three tissues, is evenly
distributed in footpad and haired skin and more present in the deeper layers of the buccal mucosa.35
The staining patterns of the human and canine adhesion-molecules showed a more or less identical
35
distribution. In general it can be said that Dsc 2-3 and Dsg 2-3 are primarily expressed in the lower
layers and that Dsc1, Dsg1and Dsg4 are more present in the upper layers of the human epidermis.14

2. 3. 1. 2. 2. Desmoglein as a minor target auto-antigen

The desmosomal cadherin desmoglein-1 is identified as the major auto-antigen in the human form of
49
pemphigus foliaceus. Different tests and studies were carried out to determine the homologous
9
canine target molecule. A study carried out by Iwasaki and his team in 1997 used
immunofluorescence and western blotting to detect candidate antigens extracted from canine
keratinocytes. By western blotting sera of canine PF patients recognized a 160kDa protein (50% of the
sera), a 85kDA protein (25%) and a 120kDa protein (31, 25%). There were indications that the 160kda
protein corresponds with the human desmoglein-1. The same study also showed that binding between
antibody and antigen could only be seen at the sites of the cell-cell adhesions and not on the entire
surface of the cells.50
Further researches were conducted about the hypothesis of desmoglein-1 being the major auto-
antigen using a novel screening strategy to detect conformational epitopes. In this study from 2006
only 6% of the sera from dogs with pemphigus foliaceus contained antibodies that recognized canine
desmoglein-1.15 Another study by Yabuzoe et al. from 2008 showed similar results. All sera (n=3)
bound to the extracellular part of the desmosomes where adjacent cells made contact. However, only
a limited number of cPF sera bound to Dsg1 specifically. These findings suggest that the target auto-
51
antigen is a desmosomal protein. They furthermore denunciate desmoglein-1 as a minor autoantigen
in cPF.46 What is more, it illustrates that pemphigus foliaceus is a heterogenous disease, with more
than one antigen being involved.

In a study that aimed at detecting the main autoantigen in cPF researchers found that calcium
depletion and glycosylation have an influence on the recognition of dsg1 in epithelial cells. The role of
calcium in expressing epitopes is described more in detail in section 2.3.1.1. To test their hypothesis,
the researchers incubated canine footpad epithelium as a substrate in 1mM calcium buffer or 5mM
ethylenediamine tetracetic acid (EDTA) for thirty minutes. Ensuing they tested the binding of human
PF sera to the substrate with calcium being respectively present or absent. All seven human PF sera
bound to canine footpad epithelium under high calcium conditions, but the IgG in the sera did not bind
when calcium was chelated. Based on those results they concluded that membrane-bound canine
dsg1 was expressed in a calcium-dependent conformation. Another interesting finding is, that the
binding of the few canine and human PF sera that contained Dsg1-antibodies, depended on
46
glycosylation. This was investigated by inhibiting protein glycosylation with tunicamycin.

2.3.1.2.3 Desmocollin as a major target auto-antigen

Desmocollin is an important molecule of the cell-cell adhesion of adjacent keratinocytes. The

importance of Dsc1 in cell-to-cell adhesion was demonstrated with the aid of genetically modified
mice. Those mice were lacking Dsc1 and showed epidermal fragility and spontaneous blister
52
formation in superficial epidermal layers as aresult.
After localizing the major desmosomal adhesion molecules those profiles were compared to the
staining profiles of cPF serum IgG. 80% of the tested cPF sera showed a similar staining profile to that
of desmocollin-1 (Dsc1). This suggests Dsc1 being a relevant candidate antigen. Desmocollin-1 is
additionally not detectable in canine buccal mucosae,35 which matches the clinical signs of PF, since
the pustules typically form in the superficial layers and the oral cavity rarely being involved.9, 17, 30

10
Further researches demonstrated that the sera of most PF affected dogs contain specific IgG
antibodies against Dsc1 that are not present in normal dogs. Those findings define Dsc1 (variant “b”)
as a major auto-antigen in canine pemphigus foliaceus. The Dsc-variants “a” and “b” differ in the
splicing pattern of the mRNA transcript that codes for the Dsc1-protein. In variant “a” an exon
containing a stop-codon is removed. The resulting coding sequence is thus translated into the longer
14
Dsc1a protein.
Similarly to human PF, serum of dogs may contain antibodies against more than one auto-antigen.
47
Sera were found in dogs that contained antibodies against Dsg1 and Dsc1 at the same time.
Interestingly, although Dsg1 is the major auto-antigen in human PF, desmocollins (Dsc1-3) are
identified as auto-antigens in some human PF patients with aberrant forms of superficial pemphigus,
especially in those with neutrophil-rich skin lesions.35,54

2.3.1.2.4. Other auto-antigens

So far desmoglein-1 and desmocollin-1 have been specified as auto-antigens in cPF. In humans they
also identified the non-desmosomal adhesion protein E-cadherin as a target. The relevance of anti E-
cadherin antibodies is not yet clear.36
In a study that involved double-sided immunogold labeling of canine PF sera the researchers detected
that more antibodies were bound to the intracellular part of the desmosomes than to the extracellular
part. The location of the detected antibodies was similar to that of desmoplakin, an intracellular
component of desmosomes. By immunoblot analysis the IgG recognized a 250kDa epidermal protein,
which is also conform with desmoplakin.51 In canine pemphigus vulgaris envoplakin and periplakin
(besides Dsg1 and -3) are well known as target antigens.55
Another study found two separate auto-antibody staining patterns in PF-dogs as well as in normal
dogs. They detected with indirect immunofluorescence one group of antibodies that bound to the
superficial layer of the epidermis (predominantly in the stratum granulosum) to peripheral cellular
antigens and another group that bound intracellular in the deep layers of the epidermis, especially the
stratum basale. Both patterns often co-existed. Most normal and PF dogs contained the superficial
antibodies, while only few sera were detected that contained the antibodies that bound to the stratum
25
basale. It is suspected that the cytoplasmic target antigen, just like in humans , is a plaque protein of
the plakin family. The author thinks that the antibodies that bind to the deeper skin layers do not play a
pathogenic role, since PF is a superficial skin disease.48 The (possible) pathogenic role of the
desmoplakin antibodies is not yet clear. The authors suggest that anti-desmoplakin antibodies can
intrude the cell after cell-membrane damage by other factors and that it might act as an accelerator of
51
the disease. They describe this phenomenon as epitope spreading.

2.3.2. The role of neutrophils

In contrast to the human form of PF, the formed pustules of the canine PF patients contain intense
neutrophilic infiltrations.15,56,57 Electron microscopy showed neutrophils next to acantholytic

11
keratinocytes, as well as in the epidermis next to the pustules. They could be found in the
intraepidermal pustules as well as in the superficial ones. 58 Neutrophil chemotaxis might be the result
of chemokine secretion by keratinocytes after IgG-binding48 which can be seen in some human PF-
59
patients that also show neutrophil-rich lesions. Eosinophils were present without pathologic findings.
Interestingly, all half-desmosomes were seen at the contact points between the granulocytes and
keratinocytes. None were observed on cell surfaces where no neutrophils made contact. The
neutrophils featured invaginations that enclosed half-desmosomes of the acantholytic keratinocytes.
Remarkably, the half-desmosomes in the invaginations had intact attachment plaques and attached
tonofilaments that did not show any retraction. (Retraction of tonofilaments and internalization of
58
desmosomes was described in humans with PV and PF).
In the early stages of acantholysis, neutrophils developed pseudopodia that were brought in between
the half-desmosomes of two adjacent keratinocytes. In a later phase, the same study showed that the
granules of the neutrophils were secreted to the surface of the acantholytic keratinocytes, resulting in
disassembly of the half-desmosomes. Those findings suggest a supplementary pathogenic role of the
neutrophils regarding the degeneration of cell- adhesion and separation of adjacent keratinocytes.56

2.3.3. Antibodies

2.3.3.1. Antibody profile

In different studies IgG was found as the main immunoglobulin deposited in a net-like manner around
the keratinocytes of canine PF patients.35,48,56 Also, in the immunomapping study from Bizikova et al,
all cPF sera (n=66) contained anti-keratinocyte IgG in the serum and IgG-depositions primarily
suprabasal and membrane-associated.35 Yabuzoe et al found IgG deposition on the keratinocytes
perilesionally and in the serum, but not that of IgA, IgM or C3.56
Bizikova and her team conducted a study in 2014 to investigate the serum antibody profiles (IgG, IgA
and IgM) of anti-keratinocyte, anti-Dsc1 and anti-Dsg1 immunoglobulines in cPF patients. They
detected the auto-antibodies by indirect immunofluorescence (IIF). More sensitive methods might
however result in higher positive numbers. Conversely, they found that 18% (n=6) of the sera
contained auto-reactive IgA, around 3% (n=1) contained (anti-desmocollin1) IgE and none of the sera
contained IgM. An IgA pemphigus without IgG-antibodies, similar to certain human PF forms, was not
discovered. Those findings suggest that canine PF is a predominantly IgG-mediated disease and that
IgE and IgA can only be detected in rare cases.60
In humans two forms of pemphigus are described that feature anti-Dsc1 IgA in a higher extent than in
the classical form.61,62 These are called atypic pemphigus and IgA pemphigus.63 In the latter IgA is the
dominant antibody isotype.63 Remarkably both of them, contrarily to the classic human PF form and
similarly to canine PF, also feature neutrophilic pustules.19, 63

12
2.3.3.2. IgG subclasses

Four IgG subclasses (IgG1-4) are described in the dog. IgG are composed of four peptide chains; two
identical light chains and two identical heavy chains. The subclasses are defined primarily by the
amino acid composition and the structure of the hinge region. Furthermore, they differ in abundance in
64
the canine serum. The predominant subclasses of antikeratinocyte antibodies present in canine PF
sera are IgG1 and IgG4. In normal healthy dogs those two autoantibodies are present in relatively
lower titers, with IgG1 being the most prominent one of the IgG subclasses. In normal and cPF dogs
no significant differences in the titers and seroprevalence of IgG1, IgG2 and IgG3 could be detected.
IgG4 however was almost exclusively found in dogs with PF (80%) and in only 7% of normal dogs.
The same author described furthermore decreasing or stable IgG4 titers when the skin lesions
resolved, whereas such development could not be seen in IgG1. IgG4 might thus be used as a marker
for disease activity since it is higher in severe cases and decreases when the skin lesions diminish.
Based on those findings the author suggests that IgG4 antibodies is most likely the pathogenic isotype
48
in cPF.

2.3.3.3. Titer and severity

Different studies suggest that the serum titers are positively correlated with the severity of the clinical
signs.46,
47, 48, 131
When skin lesions improved antikeratinocyte IgG4 titers stayed constant or
decreased. The maximum reduction was seen when the disease improved or reached remission. The
48
titer of IgG1 was not significantly affected by the degree of severity. Bizikova and colleagues
conducted a study in 2012 to investigate the relationship between anti Dsc1 IgG titers and disease
severity. They selected ten dogs with PF, applied a scoring system to score the severity of the disease
and took serum samples before and during treatment. In all dogs a noticeable clinical improvement
could be seen. The antibody titre decreased in 70% of the dogs (n=7) together with the decrease in
48
severity.

2.3.3.4. Specificity of the antibodies

As mentioned earlier, more than one auto-antigen and antibody are involved in the pathogenesis of
PF. So far anti- Dsg1 and anti-Dsc1 antibodies have been identified. The detection of auto-antibodies
is, however, apparently strongly depending on the substrate. In normal bovine esophagus antibodies
65
were detected in 65% of the cPF-patients , indirect IF on cultured canine keratinocytes was positive
66
for all tested cPF sera , when however other substrates were used the frequency of the antibody
detection decreased significantly.65

13
2.3.4. Pathophysiology

2.3.4.1 Introduction

The underlying mechanisms and factors that induce the production of pathogenic antibodies are not
fully understood yet. Nevertheless several theories and involved components are proposed. Besides a
genetic predisposition, different exogenous factors are proposed to play an important role in the
67-69
pathogenesis. Drugs are the best-recognized triggers. Other hypotheses include a suppressor T-
cell dysfunction or bypass; a modification of self-antigen; a cross-reaction with exogenous antigens;
abnormalities of the major histocompatibility complex II and access of T-cells to previously hidden self-
antigens.11

2.3.4.2 Role of the auto-antibodies

Intradermal injections of PF IgG in neonatal mice resulted in the formation of pustules that were
histologically similar to those of human and canine PF patients. The location of the lesions in the skin
were similar to those of PF as well. Passive transfer of the serum of normal dogs to the mice did not
lead to blister formation. This proves the pathogenic role of the antikeratinocyte antibodies. 48 An
important difference between the induced PF-lesions in the mice and natural ones is however that the
induced pustules lack eosinophil and neutrophil granulocytes that predominate in natural cPF.48,56

2.3.4.3. Proposed mechanisms

The mechanism between the auto-antibodies and the induction of acantholysis is not satisfyingly
explained yet. A logic and simple explanation would be, that the auto-antibodies interfere with the
extracellular adhesion by steric hinder. By this means the bond between the extracellular cadherins
14
would be disabled, resulting in the separation of the keratinocytes. A research conducted by
Waschke et al in 2005, however, has revealed that pemphigus foliaceus IgG from patients was not
70
able to break a homophilic Dsg1-Dsg1 bond.

Conversely, evidence is increasing that PF is a more complex disease with several pathogenic
components involved. Several papers suggest that serum IgG can trigger a multistep mechanism of
intracellular events.31 Research in humans proposes numerous mechanisms of acantholysis after the
antibody bound to desmosomal antigens. These include the internalization of the antibody-antigen
complex and fusion with intracellular lysosomes; an increased amount of urokinase-type plasminogen
activator in the affected epithelium, resulting in high concentrations of extracellular plasmin and thus
destruction of the adhesion molecules.71-73 Another possible (supplementary) pathologic mechanism
includes the activation and fixation of complement.74
It is proven in humans that the binding of IgG-antibodies induces the following changes in the
keratinocytes. IgG binding activates the Fas/Fas ligand cell death pathway and the mitogen activated
protein (MAP)-kinase pathway. It also induces phospholipase-C activation, inositol 1, 4, 5-triphosphate
14
generation, increases intracellular calcium concentration and a redistribution of protein kinase C (PKC)
inside the cell. Additionally, it interferes with RhoA signaling.75-78 Rhoa stands for RAS homolog family
member A and plays a role in the regulation of cytoskeletal dynamics, transcription, cell cycle
79
progression and cell transformation.
Another study regarding human PF suggests as well that IgG binding to desmoglein or other surface-
molecules activates a variety of mechanisms resulting in the disassembly of the desmosomes,
tonofilament retraction and internalization of desmosomal structures in cultured keratinocytes.14,58 A
part of this mechanism could be due to the phosphorylation of desmoglein and thus its dissociation
36
from plakoglobin. Additionally, desmoglein could disappear from the surface by
58,65
endocytosis. However, those findings are not totally in accordance with the findings in cPF. In cPF
a study examined more than twenty half-desmosomes of keratinocytes from canine PF patients. All of
them had intact intracytoplasmic dense plaques and no remarkable tonofilament retraction. That might
suggest that in cPF IgG does not trigger an alteration of the intercellular pathways and interference
56
with the cell-cell adhesion.
As described previously, it is also likely that neutrophils play a pathogenic role by forming
pseudopodia and release enzymes to break adhesion bonds.54, 56

A different article describes that the onset and course of PV is based on predisposing and inducing
factors. The inducing factors can be environmental or endogenous factors. That also implies that a
predisposed genetic background alone is not sufficient for the diseases to break out but also requires
a trigger.31 Endogenous factors might be hormone disorders, emotional stress, immune suppression
etc. Environmental factors starting the disease could be drug intake (see “Drug related pemphigus”
below), viral infections, diet, contact allergens etc. The same principle might apply to cPF. A genetic
background that predisposes certain breeds to cPF has already been mentioned. 19 The disease was
furthermore found in two Shetland sheepdog littermates.80
In humans there is an endemic form of PF described in Brazil, called “fogo selvagem”. 81 Different
authors suggest a genetic predisposition together with an environmental trigger to develop clinical
15, 31
signs. Additionally, in specific pathogen free (SPF) dogs lower frequencies and titres of
antikeratinocyte antibodies were detected, compared to normal outbred client-owned dogs. It is not
known if those findings are relevant, but it might indicate that environmental factors might play a role in
the pathogenesis.48 Pilot studies were conducted in dogs with PF. Flea infestation was one of the
exogenous factors associated with the disease82, whereas UV-rays might induce new skin lesions.83

All the mechanisms mentioned above do not exclude each other. It might be possible that several
mechanisms occur simultaneously or even trigger or enhance each other. Waschke et al describe in
their article that the binding of IgG to the extracellular part of the desmosomes causes steric
hindrance, impaired signal transduction, keratin retraction, cytoskeleton collapse and of course
37
acantholysis. The study was based on human research and not on dogs.

Kitajima et al proposes in his review article from 2013 the term “Desmosome–remodeling impairment
disease”, which shows similarities with some of the mechanisms proposed above. This theory includes

15
a mechanism of Dsg3 non-assembly and depletion from desmosomes when in the “weak adhesion
state”. In human PV the binding of autoantibodies to Dsg3 results in the activation of intracellular
events, more specific in the activation of protein kinase C (PKC). PKC induces subsequently a switch
2+-
from the Ca independent hyperadhesive state to the weak-adhesion state. This seems to be the
38,42
more susceptible state, creating keratinocytes that are more sensitive to blistering processes.
38
Then endocytosis of Dsg3 and thus a depletion of Dsg3 from the cell surface takes place.

An article by Ruocco et al from 2013 states that there must be additional auto-antigens in human
pemphigus vulgaris (hPV), besides Dsg1 and Dsg3. Otherwise, when both, anti-Dsg1 and anti-Dsg3
antibodies, would be present the epidermis would completely disintegrate. They suggest the
keratinocyte acetylcholine receptors as a non-desmoglein pemphigus antigen. The receptors play an
important role in the mediation of intercellular adhesion and in particular the expression of desmoglein.
Again, similar to the findings of the varying immunostaining-patterns of affected the dogs, it is likely
that different subsets of auto-antibodies are involved, which would explain the variety of clinical and
31
biological symptoms.

As described earlier pemphigus vulgaris in humans has a mucosal-dominant type, caused by anti-
Dsg3 autoantibodies and a mucocutaneous form with anti- Dsg1 and -3 autoantibodies. The mucosal-
dominant type can evolve into the mucocutaneous form when autoantibodies against Dsg1 develop
aswell.14 Based on those findings the “desmoglein compensation theory” emerged. This theory states
that one isoform of desmoglein can compensate the loss of another. 84 This theory, or hypothesis,
would explain why there is no blister formation in the epidermis in the mucosal-dominant PV form. The
auto-antibodies attack Dsg3, but Dsg1 expression in the basal layers can compensate for this loss of
function and keeps the epidermis intact. Blistering would, according to this theory, only occur when
both, Dsg1 and Dsg3 get attacked.14 Thus there are only superficial lesions in PF, since adhesion in
the basal epidermal layer, is maintained by Dsg3. 38 This hypothesis is however not completely
conclusive. In November 2014 a case was reported of a human patient with suprabasilar acantholysis,
85
but was at the same time found positive for Dsg-3 and negative for Dsg-1 antibodies. According to
this hypothesis also Dsg4 should be able to compensate for the loss of the other isoforms, which is
apparently not the case. 14

It is evident that the responsible immunopathogenesis is not yet clearly elucidated. Many
investigations offer however valuable clues. The current research shows a huge variety of cell biologic
processes that might be involved. Some of them are primarily pathogenic; others only trigger a
cascade of mechanisms leading finally to acantholysis. Some components are essential, others add
up to the clinical signs.38

16
2.4 DRUG-RELATED PEMPHIGUS

2.4.1. Introduction

There seems to be a condition of cPF which occurs as a response to drug therapy. Drug related
pemphigus may be further divided in drug-induced and drug-triggered PF.9,86 The first one is a
transient form of pemphigus in which clinical signs disappear after discontinuing the drug. Conversely,
19,86
the latter is permanent. Several drugs have been associated with a PF outbreak in dogs so far.
The majority of the reported patients developed localized lesions, whereas only a small percentage
showed generalized lesions. More than half of the dogs also developed systemic signs (lethargy,
fever, anorexia, pain and lameness). Interestingly, most of the dogs were of large breeds.87 Drug-
related PF is clinically, histologically and immunologically similar to the naturally occurring form of
canine pemphigus foliaceus. The history helps to differentiate naturally occurring PF from drug-related
PF.67 In the drug-related form the dog must have received the inducing drug, there is an unusually fast
onset of clinical signs, an unusually early onset and unusual clinical characteristics (e.g. oral
lesions).86
Most of the drugs related to cPF outbreaks were insecticides. In humans different pathomechanisms
are suggested on how pesticides (including insecticides) induce PF. The proposed pathomechanisms
include an alteration of the keratinocyte membrane biochemistry and/or an interaction with the
immune-system.67 Many of the described drugs contain a thiol-group, which might be an indication.31
One theory describes the blocking of keratinocyte nicotinic acetylcholine receptors and thus inhibiting
signaling which is important for cell-cell adhesion in the epidermis.69 Another possibility could be a
modification of the skin by the drug and thus creating neoantigens and secondary auto-antigens.88,89
Other theories include an increased cytokine production or an alteration in enzyme activity that are
involved in cell-adhesion. 90

2.4.2. Trimethoprim-sulfonamide

Trimethoprim together with sulfamethoxazole, sulfadiazine or sulfamethoxypyridazine may induce

9, 27, 87, 91, 92
pemphigus foliaceus as well as other skin diseases, such as erythema multiforme or
93
perforating folliculitis. Once the administration stopped the lesions resolved within a few weeks.27

2.4.4. Promeris Duo®

® 94
Promeris Duo (Zoetis Animal Health) is an insecticide, containing metaflumizone and amitraz. It is
132 86-88
associated with currently 22 cases of drug-triggered as well as drug-induced PF. Promeris is no
®
longer on the market. The lesions strongly resemble those triggered by Certifect . Systemic signs
were reported in both entities, however, it was more frequently described in Promeris-triggered PF
(PTPF; 64%) than in Certifect-triggered PF (CTPF; 43%). 91% of the dogs were large breeds and
bitches were affected more frequently (68%). Also here the majority of the patients (86%) reached

17
complete remission. Just as in natural PF ( 82%), most sera of CTPF (71%) as well as PTPF ( 75%)
dogs contained anti- Dsc1 IgG`s.67
No distinct differences between the histopathology of the skin lesions of CTPF, PTPF and natural PF
were identified. The exact trigger or component of the drug (amitraz, metaflumizone, fipronil, the
vehicles or a combination) causing the eruptions is not yet known but the clinical, histological and
67
immunological similarities imply a common pathogenesis with natural PF.
67
Promeris and Certifect both comprise amitraz , but amitraz has been widely used against other
ectoparasites such as Demodex or Sarcoptes mites without inducing PF.87 Given the large number of
amitraz treated dogs and the relatively low number of PF-outbreaks, it is likely that also other factors
influence the disease outbreak, such as the genetic background, environment, immunological or
hormonal status.67 Another interesting finding is that lesions developed in 67% of the affected dogs
within two weeks. The production of auto-antibodies however takes a minimum of three to four weeks
after primary sensitization.95 Moreover, recent articles describe contact-triggered PF also after the
application of another antiparasitic drug that does not contain amitraz but even other components
(Vectra 3D®).86,87

2.4.3. Certifect:® Fipronil-Amitraz- S-methoprene

Certifect® (Merial) was released in 2011 as an ectoparasiticide which contains fipronil, amitraz and S-
67
methoprene. It has been found to provoke acantholytic pustular dermatitis in some patients. 21
87
cases were reported to date. It shows a close clinically, histologically and immunologically similarity
to the naturally occurring form of canine pemphigus foliaceus. 67 Most of the affected dogs were larger
breeds and middle-aged or older.67 There were more females affected (71%) than male dogs.
Systemic signs were exhibited in 43% (n=9) of the dogs. In 29% (n=9) the lesions were limited to the
application site, while the rest of the affected canines also showed distant lesions. In 33% of the dogs
one application of the drug was sufficient for clinical symptoms and 29% developed symptoms after
the second application. IgG autoantibodies were found in the sera with Desmocollin-1 being the main
target antigen (79% of the cases, n=11). Canine desmoglein-1 was found not to be targeted at all.
Those findings suggest that this new ectoparasiticide is capable of triggering PF. Complete remission
was reported in most of the dogs (81%). Due to its complex composition it is not possible to identify
67
the actual pathogenic agent.
In humans pesticides are also suggested as PF-triggering factors.68

2.4.5. Vectra 3D

Vectra 3D is used as a flea preventative and contains dinotefuran, pyriproxyfen and permethrin. 67,86, 96
The insecticide has been reported to trigger PF-like lesions in three dogs to date.87, 96 Cytology and
86, 96
skin biopsy revealed the same histopathological lesions as in naturally occurring PF.

18
2.4.6. Others

Other drugs that are associated with a drug-related PF outbreak include cephalexin, oxacillin92 and
97
polymixin B. Additionally, a puppy with juvenile cellulitis was treated with amoxicillin-clavulanic acid
and topical oxytetracycline and developed ensuing signs of PF. Those symptoms resolved after drug
98
withdrawal.

2.5 THERAPIES

2.5.1. Introduction

Pemphigus foliaceus is based on auto-reactive processes. Consequently most treatments contain a

component that suppresses or modulates the immune system. The most popular and first-line choice
27
of drugs are glucocorticoids. Considering the side-effects of corticosteroids, a general consideration
should always be to keep the patient on an as low as possible dose, even though that could mean a
few remaining lesions.19 Besides glucocorticoids, azathioprine, cyclosporine, chlorambucil, tetracycline
and niacinamide are most commonly used for the treatment, according to Rosenkrantz. 27 There are
further drugs that can be used additionally to glucocorticoids or as a substitute. Possible reasons to
use second or third-line drugs are unacceptable side- effects, sparing of glucocorticoids or an
insufficient response with glucocortcicoids.27

2.5.2. Most commonly used drugs in the treatment of cPF

2.5.2.1. Corticosteroids

Historically the standard treatment for cPF was an immunosuppressive therapy with oral
-1 34, 33, 27, 99
glucocorticoids at daily dosages from 2 to 6.6mg kg . When the lesions diminish the dose
27, 33
and/or administration frequency is reduced, preferably to an alternate day intake. In some patients
33
however, immunosuppression alone did not lead to the desired remission. In those cases
corticosteroids are combined with cytotoxic or other alternative drugs. Frequently used cytotoxic drugs
are azathioprine, cyclophosphamide or chlorambucil. 27, 33, 34

Topical glucocorticoids can be used in localized lesions and if necessary in combination with systemic
therapies. Glucocorticoids with different potencies are available. It is advised to initially use an
immunosuppressive dosage, for example 2.2-4.4mg kg-1 of prednisone or prednisolone daily. If
adequate response is achieved after two weeks it is possible to switch to a less potent glucocorticoid
and/or gradually reduce this dosage over a period of 30 to 40days. If no significant improvement or
unacceptable adverse reactions appear after ten to 14 days it is possible to add another
immunosuppressive drug to the treatment regime.11 The final goal is to reach a dosage of 1mg kg-1 on
an alternate day basis or less.

19
A systemic glucocorticoid therapy is however the most used approach for cPF.27 Frequently used
glucocorticoids are prednisone, methylprednisolone or prednisolone.19 Rosenkrantz describes in his
article that he prefers to use methylprednisolone due to less mineralocorticoid effects and thus less
27
polyuria and polydipsia. Furthermore, some patients respond better to the latter. Much more potent
glucocorticoids (six to ten times stronger) are triamcinolone and dexamethasone. Both are
administered orally. Those can be used in more persevering cases, in dogs with extensive polyuria
and polydipsie or changed behavioral patterns. The starting immunosuppressive dosage for
-1 -
triamcinolone is 0.2-0.6mg kg daily and 0.2-0.4 mg kg 1 for dexamethasone. For maintenance it is
sufficient to give the drugs every third day, since they suppress the hypothalamic-pituitary-adrenal axis
-1
for 24 to 48hours. The maintenance dosage for triamcinolone lies between 0.1-0.2 mg kg every
-1
second or third day, whereas for dexamethasone only 0.05-0.1mg kg is advised every second or
third day as well.27

Glucocorticoids affect many cells and tissues of the body, thus initiate a wide range of changes that
involve different cell types simultaneously. Their main effect is anti-inflammatory as well as
immunosuppressive. Long-term administration can influence the blood count. More specifically it
results in neutrophilic leukocytosis together with eosinopenia, monocytopenia, and lymphocytopenia.
One of the main anti-inflammatory mechanisms is the hindrance of the neutrophils and monocytes to
migrate to the inflammation site. Granulocyte function is relatively less impeded in comparison to the
monocyte-macrophage function which seems to be particularly sensitive to corticosteroids. 100 Another
article also describes the inhibition of inflammatory mediators and suppression of autoantibody
levels.101

Another observed side-effect is a transient lymphocytopenia of all detectable lymphocyte

subpopulations.102 That is explained by a redistribution of the circulating lymphocytes.100 In some
species (mouse, rat, rabbit) the lymphocytopenia was also caused by induced cell-death. In humans
that phenomenon was only seen in immature or activated T-cells, but not in resting T-cells.103,104
Glucocorticoids are steroid hormones that can easily diffuse through the cell membranes when in their
free form. In the cytoplasm they bind, with high affinity, to cytoplasmic glucocorticoid receptors. The
formed ligand-receptor complexes migrate into the nucleus where it can modify the transcription of
specific genes that encode proteins responsible for the action of the glucocorticoids. In normal T-cells,
when antigens bind to the T-cell receptor, a phosphate group is added to the tyrosine amino acid of
several intracellular proteins. As a result protein kinase C (PKC) is activated and also the intracellular
calcium concentration rises. These two events (PKC and calcium increase) are necessary for the
transcription factors to bind to the IL-2 gene promotors and initiate the transcription. The messenger
RNA (mRNA) then translocates to the cytoplasm where it is either translated by ribosomes to the IL-2
protein or degraded by RNAases. Glucocorticoids are capable of intervening with several of these
steps of the T-cell cycle. They inhibit the tyrosine phosphorylation; they inhibit calmodulin kinase II, an
enzyme of the calcium pathway and they inhibit the binding of transcription factors to transcript and
later translate IL-2 mRNA to effective proteins. Furthermore there is an increase in mRNA
degradation.100
20
Cytokines are essential for the proper functioning of T-cells. IL-2 in particular promotes T-cell
proliferation and generation of effector, suppressor, and cytotoxic functions. Consequently, the
generation, proliferation, and function of helper and suppressor T cells are depressed by these
105
drugs. Cytotoxic T-cell responses are also impeded by moderate-to-high doses of glucocorticoids
because of the blockade of cytokine expression and to a lesser extent because of the lysis of reactive
106
T-cell clones.

Generally speaking the cellular immunity is more susceptible than the humoral immunity, which means
that the B-cells are relatively resistant to the immunosuppressive effects of glucocorticoids. These
drugs inhibit the proliferation of B-cells but have a minimal effect on the transformation of B-cells into
active immunoglobulin-secreting plasma cells. Their major effect on B-cells is on the antibody titre.
Low doses of glucocorticoids have no significant effect on the titre, however, daily high-doses result in
decreasing immunoglobulin levels with the greatest suppression after two to four weeks. 107 The
suppression is the result of an initial elevated catabolism followed by reduced production. Another
reason is the impeded production of cytokines and the decreased activity of helper T-cells that play an
essential role in the activation of B-cells.100

Side-effects are commonly seen in long-term treatments with corticosteroids (daily for >14days) and
more frequently with the more potent derivatives. Described side-effects of topical treatment include
atrophy, alopecia and localized pyoderma. By licking or dermal absorption systemic effects are also
possible. In systemic therapies with strong glucocorticoids iatrogenic hyperadrenocorticism is
27
described with cushingoid and diabetic effects. Due to those hormonal alterations, polyphagia,
muscle atrophy, a poor hair coat, weight gain, calcinosis cutis, hepatomegaly, and panting can be
102
seen regularly. The glucocorticoids have varying mineralocorticoid effects resulting in polyuria and
polydipsia. Since being an immunosuppressive drug, patients are also more prone to infections, such
as secondary bacterial skin infections, cystitis or demodicosis and experience delayed healing.102
Reported are also gastrointestinal side-effects, such as ulcerations, pancreatitis, vomiting and/or
27
diarrhea. Besides the physical side-effects, also behavioral changes are observed. Some dogs are
108
described to be lethargic, others become more aggressive or restless.
Due to the side-effects especially on the blood count, monitoring including complete blood counts,
chemistry profiles, urinalysis and urine cultures should be performed every six months.27

The prognosis after a glucocorticoid therapy alone is moderate. Several studies have been conducted
to analyze the treatment outcome and gained similar results. Ihrke et al registered a positive outcome
33,
with corticosteroids alone in 39% of the examined PF patients Rosenkrantz and colleagues
documented 35% of the PF cases to be adequately controlled with only glucocorticoid therapy27 and
Mueller and his team reported complete remission with only glucocorticoids in 38% of the patients
99
within 1.5-12months after treatment-begin (average 7months).

21
2.5.2.2. Azathioprine

Azathioprine is an immunosuppressive drug and can be administered solely, in combination with

27
glucocorticoids or with other immunosuppressive drugs. It is the first choice to be added to the
11
glucocorticoid treatment regimen as a glucocorticoid-sparing agent. Azathioprine is a prodrug that
inhibits DNA and RNA-synthesis by blocking purine-biosynthesis. Especially fast growing cells such as
B- and T-cells are affected because of their lack of a salvage pathway which is necessary for purine
biosynthesis. As a result the drug decreases lymphocyte proliferation, lymphocyte numbers and T-cell
109 110
dependent antibody synthesis. The main side effects are myelosuppression and gastrointestinal
111
symptoms such as vomiting, diarrhea and hepatotoxicity. Bone marrow suppression occurs after
one to two weeks after therapy onset and is reversible. Rare side-effects comprise hepatic necrosis
110
and pancreatitis. The advised doses are 1.5-2.5mg kg-1 every 24 to 48hours. It shows a delayed
onset and takes normally one or two months until clinical effects become visible. 27 It is thus important
11
to not stop or reduce the therapy too fast. Complete blood counts are recommended every two to
three weeks for the first three months after therapy onset, subsequent monitoring should be carried
out semi-annually.27
The success rate of the treatment with prednisolone and azathioprine together was reported in 55% of
the cPF patients99. Five dogs in this study did not react on prednisolone alone but underwent complete
remission when both drugs were administered. Nevertheless, more adverse drug effects were
99
described with this combination. The benefit is thus controversial.

2.5.2.3. Chlorambucil

Chlorambucil is an alkylating agent that interferes with the DNA-synthesis and is thus cytotoxic. It acts
primarily in on B-cells and is considered a slow-acting immunosuppressive drug that takes two (to
six)11 weeks before therapeutic effects can be seen.109 Reported side-effects include bone marrow
suppression, hepatoxicity and gastrointestinal distress which shows through vomiting, diarrhea and
anorexia. Myelosuppression is nevertheless rather mild and occurs one or two weeks after the
109
therapy onset. Chlorambucil is used as an alternative to azathioprine, solely when other therapies
are not tolerated or in combination with corticosteroids and azathioprine in difficult cases. 27 It is the
first choice for cats together with glucocorticoids.11

2.5.2.4. Chrysotherapy

To prevent extensive side-effects by the long-time administration of corticosteroids an alternative

27,33,34
approach is described by administering goldsalts, such as aurothiomalate and aurothioglucose.
Aurothioglucose is however no longer commercially available. Rosenkrantz found it effective for feline
27
pemphigus, but not for canine pemphigus. The exact mechanism of action is not fully elucidated, but
10, 27
it works immune-modulating and anti-inflammatory A disadvantage is a long lag phase of ten to 16
27
weeks. It can be used solely or adjunctive with glucocorticoids, and orally as well as in form of an
22
injection. Adverse reactions to the therapy include bone marrow suppression, oral ulceration and
glomerulonephropathy. Furthermore are the injections very painful and deep. One case is reported
where two dogs died from a toxic epidermal necrolysis after a sudden change from azathioprine to
112
aurothioglucose. Regular monitoring is advised. The monitoring should contain a complete blood
count every two to three weeks, and biochemistry and urinalysis every four to six weeks for the first
10
few months after start of the therapy and then repeated every three to six months.

2.5.2.5. Tetracycline and niacinamide

A few cases are also described where PF patients responded to tetracycline and niacinamide
99, 113
treatment. Tetracycline is a broad-spectrum antibiotic with anti-inflammatory properties.
Niacinamide is part of the vitamin B-group, inhibits mast-cell degranulation and also
phosphodiesterase. A common side-effect is an upset gastrointestinal tract that might result in
diarrhea, increased liver enzymes, vomiting and anorexia. In those cases decreasing or discontinuing
niacinamide can relief the symptoms. The drugs have a delayed effect. Clinical benefits are thus not
visible before one or two months after therapy onset. The dosage or the frequency of administration
may then be reduced. The advised dosage for both are 250mg three times a day for dogs under 10kg
and 500mg every 8hours for dogs above 10kg.27

2.5.2.6. Mycophenolate mofetil

Mycophenolate mofetil is the prodrug of mycophenolic acid and interferes with the synthesis of de
novo purine.27 T- and B- lymphocytes are especially sensitive since they cannot use the salvage
synthesis pathway. This feature enables the drug to selectively inhibit lymphocyte proliferation and
the production of antibodies.27,109 Further mechanisms include a suppression of dendritic cell
maturation and decreased monocyte recruitment to the inflammation site. 109 Adverse drug reactions
27, 109
include bone marrow suppression, being more prone to infections and gastrointestinal problems.
Most studies on drugs are executed retrospectively and the drugs were originally developed for
19
humans. A prospective clinical trial was, however carried out in a 16-week pilot study with
mycophenolate mofetil (20-40mg kg-1, 3 times daily). Of the eight examined dogs only three improved.
-1
Dosages between 22-39mg kg per day were used spread over three applications per day. Adverse
drug reactions were minimal. Most commonly were pyoderma, Malassezia infestations, diarrhea and
leukocytosis.27Due to high costs and a lack of proven efficiency, this drug is not recommended as a
treatment by the researchers.114

23
2.5.2.7. Cyclosporine and Tacrolimus

Cyclosporine and tacrolimus are immunosuppressive drugs115 that are extensively used in human
27
medicine, especially in patients that received organ transplants. Both drugs have the same proposed
116
mechanism of action and immunomodulating properties. Tacrolimus is however more potent and
27
more toxic for dogs.
Both drugs bind to intracellular receptors called immunophilins. More specifically, cyclosporine binds
to cyclophilin, while tacrolimus binds to FK506-binding proteins. Thereby, they inhibit calcium-
dependent pathways including that of calcineurin. Calcineurin is a calmodulin-dependent protein
phosphatase. This enzyme activates nuclear factor of activated T-cells (NF-AT) by dephosphorylation.
NF-AT is a transcription factor of T-cells that is responsible for the initiation of interleukin synthesis.
The activated NF-AT is translocated into the nucleus and upregulates interleukin-2 synthesis.
IL-2 is important for the proliferation and differentiation of T-cells.100As a consequence cyclosporine
117, 118
causes a decrease in canine lymphocytes and immunosuppression. It also affects different
cytokines, such as IL-2, IL-3, IL-4 and tissue necrosis factor alpha (TNF). IL-2 is also responsible for
lymphocyte proliferation.10,
115
It furthermore modifies the function of granulocytes, macrophages,
natural killer cells, eosinophils and mast cells.109 In a study from 2004 good results were achieved in
-1
two dogs within four to six weeks with 25mg kg per day. Another patient received 15mg kg -1
whereupon the clinical signs subsided. When the severity however got worse the patient did not
27, 119
respond to higher doses.

A pilot study on cyclosporine carried out by Olivry investigated whether cyclosporine A (CsA)
monotherapy (microemulsified CsA) would be effective for the induction of treatment in cPF. Of the
five tested dogs only one showed mild side-effects (intermittent diarrhea). The used induction doses
-1
was 5-10mg kg administered daily. None of the dogs showed complete remission of the lesions.
Four of the five dogs were even withdrawn due to worsening of the score based on the Pemphigus
foliaceus extent and severity index (PEFESI). Based on those results cyclosporine is not
recommended as a sole therapy in the proposed doses. It might nevertheless be effective in higher
115
doses or in combination with oral glucocorticoids.

Cyclosporine can be administered orally in combination with ketoconazole or oral

glucocorticoids.Ketoconazole inhibits the hepatic microsomal isoenzyme P450 system, resulting in
increased blood cyclosporine levels, allowing lower dosages and thus reduced costs. 27

The reported side-effects of cyclosporine include anorexia, emesis and diarrhea, especially during the
27, 116
first days of treatment. More severe side-effects are rare and appear to be dose-dependent.
Weight loss, nephrotoxicity, gingival hyperplasia, papillomatosis, hirsutism, hepatotoxicity,
opportunistic infections, lymphoproliferative disorders and involuntary shaking are reported.119, 116, 120
Tacrolimus shows more extensive side effects than CsA when administered orally alone or in
combination. Those adverse reactions include anorexia, vomiting, diarrhea, weight loss, impaired
glucose metabolism, marked hepatotoxicity and infections. 121 Nevertheless no side-effects and good

24
results have been achieved in discoid lupus erythematosus and pemphigus erythematosus when
tacrolimus was administered topically (0.1%). 27,122

2.5.2.8. Cyclophosphamide

Cyclophosphamide is also an alkylating agent. It interferes with DNA replication by adding an alkyl-
group to DNA. It is used, among others, as a cancer therapy agent and against auto-immune
disorders. It can be used solely or in combination with other drugs. The drug is very potent and shows
thus severe side-effects such as hemorrhagic cystitis,27 anorexia, vomiting, diarrhea and weight
123
loss. Because of those undesired reactions and sufficient alternative therapies it is not used by
-1 27
default. The recommended dosage is 1.5mg kg every alternate day.

2.5.2.9. Dapsone and sulfasalazine

Dapsone is an antibiotic with anti-inflammatory features. It inhibits neutrophil chemotaxis, reduces

complement activation, antibody production and lysosomal enzyme synthesis. As described earlier are
the lesions in dogs, contrary to the human form, infiltrated with neutrophils and is dapsone thus more
-1
effective in dogs. Dapsone should be given 1mg kg every eight hours. The drug shows a delayed
effect after one to two months. The following adverse reactions are described in dogs: anemia,
neutropenia, thrombocytopenia, hepatotoxicity, gastrointestinal signs, neuropathies and cutaneous
drug eruptions. Therefore close and regular monitoring of the thrombocytes, chemistry profiles and
urinalyses are recommended.27
-1
Sulfasalazine metabolites have anti-inflammatory features as well. It is dosed at 10-40mg kg spread
over the day, thus one dose every eight hours. A big disadvantage is the risk on developing
keratoconjunctivitis sicca. Therefore it is recommended to monitor tear production as well as blood
counts and chemistry profiles regularly. Tear production is best monitored every two to four weeks,
blood counts and chemistry profile initially (first 1.5 months) every two weeks and then reduced to
every two to four months.27

25
3. WHY ARE SOME DOGS NOT RESPONSIVE TO THE TREATMENT
3.1 TREATMENT OUTCOME AND LONG-TERM PROGNOSIS

Even after a verified diagnosis of cPF there seems to be quite some variation regarding the treatment
27
outcome and the long-term prognosis. Different studies reported different outcomes. It is assumed
that this might be due to different treatment approaches as well as due to a variation of clinical signs
and disease severity.19

In a study from 1985, 37 dogs with PF were examined and followed-up for one to seven years. The
dogs were treated with different drug regimens with the following outcome: 39% responded to
corticosteroids solely, 50% responded to prednisone and cytotoxic drugs and 55% reacted to
prednisone in combination with aurothioglucose. 47% of the dogs died in less than one year.33

A more recent study that was carried out between 1994 and 2000 with 43 cPF patients aimed at
identifying the factors that can influence the outcome of cPF. The researchers recorded a case fatality
rate of 60.5%. The majority (60%) of the dogs that passed away died within the first year of treatment.
Interestingly, the survival rate increased when antibiotics were administered in the beginning of the
immunosuppressive therapy and also lowered the number of patients with adverse drug effects. That
could be explained by the patients being less susceptible to secondary bacterial infections. What is
more, the researchers detected a positive correlation between the duration of the treatment and the
survival rate. 92% of the dogs from the non-survival group had died within one year after treatment
initiation, but only two died after twelve months. That proposes a better prognosis for survival after a
certain period of time. The median of death in the non-survival group was ten months. Also the
number of complications shows a significant association with the survival rate.
The following factors were not significantly associated with prolonged survival: the sex, age at
diagnosis, previous or additional atopic or thyroidal diseases, extent of the skin involvement, time
period between clinical signs and PF-diagnosis or the administration of gastric protectants when
glucocorticoids were used. There was also no link between the survival time and whether a veterinary
dermatologist or primary care veterinarian managed the long-term treatment.
Of the dogs that died 69% (n=18) were euthanized, four dogs for causes unrelated to PF. Most of the
euthanized patients underwent two to four changes in treatment protocol, due to unsatisfying results or
unacceptable adverse drug effects, before deciding to euthanize the patient.
Remarkably, four of the treated dogs stayed in prolonged remission with no further clinical signs of PF
after discontinuation of the treatment. Also, there was no significant link between initial treatment
protocol and survival time or rate. The used initial treatment protocols comprised prednisone (n=16),
prednisone together with azathioprine (n=23), mycophenolate mofetil (n=2), tetracycline and
niacinamide (n=1) and prednisone in combination with chlorambucil (n=1). However, due to the high-
incidence of corticosteroid-related side-effects and the great amount of deaths within the first year the
(124)
authors advice to choose a combination treatment to minimalize the required corticosteroid
124
maintenance doses. However, Mueller et al described contradictory that the combination of

26
glucocorticoids and azathioprine causes more side-effects in cPF patients.99 The benefit of a
combination therapy is thus controversial.

27 99
Three other studies, one by Rosenkrantz and colleagues, one by Mueller and colleagues and one
11
by Kummel reported a more positive outcome. Rosenkrantz reported 71% of the patients still being
27
alive after one year of treatment. Mueller and colleagues examined 91 dogs, whereof only 13%
99
(n=11) were euthanized. And Kummel documented a 75% survival rate after treatment with no or few
125 (124)
side-effects. Rosenkrantz stated, similar to the findings by Gomez, that there was no significant
difference in rate or degree of remission between dogs that were treated with prednisolone alone or
together with azathioprine. However, contrary to Gomez Rosenkrantz also reported that there is no
association between the survival rate and the use of antibiotics in combination with
99
immunosuppressive drugs.

Prolonged remission is described in a small number of cases after discontinuation of the

immunosuppressive therapy. In a study by Olivry et al 51 were either treated with immunosuppressive
doses of oral glucocorticoids or with glucocorticoids together with azathioprine. Clinical remission
occurred after 1.5 to 5months after initiation of the therapy. The doses was progressively reduced and
finally withdrawn. The total period of administering immunosuppressive therapy ranged between three
and 22 months. In six dogs clinical symptoms did not reappear for the duration of the trial (1.5 to
103
6years).
This study suggests that a relapse of the lesions does not necessarily occur after withdrawal of the
drug and that in some dogs immunosuppressive therapy can lead to a long-term remission of the
lesions. 51 dogs met the criteria for the research and six of those went into complete and long-term
remission (12%). In a study investigating long-term outcome of treatment in humans with PV it was
found that patients with mild to moderate PV were twice as likely to undergo complete remission
compared to those with severe lesions.126 A similar research by Mueller and colleagues found that the
distribution of the lesions, localized versus generalized, had no effect on resolving of the lesions or
death and that 7-22% of the affected dogs stayed in remission after ending the immunosuppressive
99
therapy. Another finding by Olivry et al was that patients with a rapid response to the
immunosuppressive therapy were more likely to benefit from prolonged remission.127 In contrast, in
dogs the severity of the disease appears to play no major role in long term remission. The average
time to undergo complete remission after initiating the therapy was two months.126

3.2 POSSIBLE REASONS WHY SOME DOGS ARE NOT RESPONDING

To the best of our knowledge, there are no studies investigating why a conspicuous number of dogs
are unresponsive to the treatment. Nevertheless, research is giving some hints why some cPF
patients might not be responsive.

27
3.2.1. Incorrect diagnosis

A possible cause could be an incorrect diagnosis. As described earlier, pemphigus foliaceus is an

heterogenous disease with a wide variety of clinical features. It is thus important to rule out other
126
differential diagnostics for pustular dermatitis , such as pustular dermatophytosis.

3.2.2. Naturally or drug-related pemphigus

In those cases where the history is unknown, it is impossible to determine with certainty if PF is
naturally occurring or if it is induced or triggered by drugs.To verify the diagnosis a drug provocation
test should be done. This provocative test is carried out rarely due to the potential risk for the patient.
The diagnosis of drug related PF is thus based on history, clinical signs, histopathology and the
response to drug withdrawal.126 Rosenkrantz states in a report that many cases that are actually drug
induced, have been misleadingly accredited to chronic diseases and have often been treated with
long-term drugs.128

3.2.3. Drug regime

In the study from Olivry et al investigating the long-term remission, it becomes obvious that there is no
static therapy regime and dosage. Each of the patients received a different drug therapy and dosage
depending on the individual response. In the six studied cases patient one did not react to initial
prednisone therapy, therefore azathioprine was added and the lesions resolved. Case number two
showed marked improvement on prednisone alone. The third and fourth case required prednisone as
well as azathioprine before clinical remission occurred. Case five and six were initially treated with
only prednisolone and due to unacceptable adverse side-effects prednisolone dosage was reduced
and azathioprine added with a positive outcome. Also, each dog received a different dosage of
glucorticoids (and azathioprine) which was adapted if necessary. For a satisfying outcome it is thus
important to find the right dosage, frequency of administration and the right combination of drugs
(antibiotics, corticosteroids, chrysotherapy, azathioprine etc or combination of those?) for each patient
126
individually.
Especially for the chrysotherapy, dapsone, azathioprine and, tetracycline and niacinamide a lag-phase
is described. Depending on the used agent it is important to not stop the therapy after a lack of
response too early.27, 109

3.2.4. Glucocorticoid resistance

Glucocorticoids are the first-line therapy for the treatment of pemphigus diseases yet some patients do
not respond. That might be due to the development of resistance. To investigate glucocorticoid
resistance in PF patients a study evaluated the glucocorticoid sensitivity. The sensitivity was analyzed
on the basis of the number of binding sites, the affinity of glucocorticoid receptors to dexamethasone
and the cytokine pattern. The number of binding sites, as well as the affinity of the receptors were
28
found to be significantly higher in pemphigus patients compared to a control group. Furthermore the
study showed a higher production of pro-inflammatory cytokines in human PF patients, especially of
IL-6 and TNFα. In summary, the study showed a pro-inflammatory cytokine profile and altered
glucocorticoid sensitivity in PF patients. That might be an indication that pro-inflammatory cytokines
129
play a role in developing glucocorticoid resistance in human pemphigus patients.
Another proposed explanation for the insufficient response could be an increased expression and/or
function of P- glycoprotein in lymphoid cells. The protein is located in the cell membrane and by being
able to pump foreign substances out of cells it can influence the bioavailability of the drugs. These
proteins might thus be capable of reducing the intracellular concentration of glucocorticoids, thereby
weakening its therapeutic effects. Research however found neither increased p-glycoprotein levels in
pemphigus patients that responded poor to steroids, nor differences in p-glycoprotein activity.
Consequently, p-glycoprotein does not seem to be involved in the poor response to steroid treatment
130
seen in some pemphigus patients.

3.2.5 Influence of 25-hydroxyvitamin D concentrations

The active form of vitamin D is well known for its properties of regulating calcium levels in the blood
and assuring bone health. As described earlier, calcium is also essential for the functioning of
glucocorticoids and has an impact on the expression of cell surface molecules. Recent research
revealed further effects of vitamin D on the immune system,132 especially on T-cells. It has been
proven in animal models that 1,25-dihydroxyvitamin D3 can prevent or suppress certain autoimmune
diseases. A proposed mechanism therefore is an influence on the cytokine profile which might
consequently suppress inflammatory T-cell activity.133 Another study investigated the relationship
between 25hydroxy-vitamin-D serum concentrations and the response to a subsequent
glucocorticoid-therapy in dogs with atopic dermatitis. Interestingly, dogs that showed a a good
response to the glucocorticoid treatment also had significantly higher pretreatment serum 25(OH)D
concentrations compared to dogs with a poor response. That might be an indication that vitamin D has
a synergistic therapeutic effect with glucocorticoids and that low vitamin D-serum concentrations might
134
attenuate the response. Further research to confirm or refute this hypothesis is necessary.

3.2.6. Tachyphylaxis to topical glucocorticoids, is it proven?

Tachyphylaxis( the fast onset of tolerance) to topical glucocorticoids has been reported in literature
and is an widely accepted belief. A study was conducted reviewing relevant published articles on the
internet, to look for evidence to support this concept. Although they found 52 relevant articles they
could not find proof that glucocorticoid treatments are susceptible to tachyphylaxis. Although some of
the described effects indeed resembled tolerance effects, the clinical significance however is not
known nor proven. The authors assume that the described phenomenon of tolerance is due to other
factors such as poor adherence of the therapy over time or that the drugs have a maximum effect in
the first weeks resulting in remission but only to a certain degree.135
29
4. Discussion

In the last decades much research regarding canine pemphigus foliaceus has been conducted and
much knowledge was gained. Nevertheless, compared with the human form of PF little is known about
the pathogenesis and even less is proven. Many pathogenic factors have been associated with the
disease and many possible pathological mechanisms proposed. However the exact pathogenic role
and importance of most of them are still unclear or questionable. Some of the studies mentioned
above even described contradictory results.

Not all the findings from human research can furthermore be extrapolated to dogs. For example cPF
involves a strong neutrophilic infiltration of the pustules, which can only be found in seldom cases of
human PF. The same applies to the therapies. Veterinary immunosuppressive therapeutics were
initially adopted from human medication and research, and thus knowledge of their therapeutic
efficacy and potential for adverse effects in dogs remains limited. Most of the studies are also
retrospective, a blinded prospective study especially regarding treatment protocols could bring more
clarity.

The majority of the canine patients reacts well to the treatment protocols and receives remission. A
few of the patients however, initially or after a relapse, do not respond to glucocorticoids (anymore).
Different reasons have been proposed but little research was conducted to investigate this issue and
no satisfactory explanation can be given yet. Thus also here, more research is needed.

In summary, this literature study shows that cPF is a very interesting and complex disease. It has
been getting an increasing degree of awareness among scientists and veterinarians in the last
decades and much progress has been made. There are however, still many open questions that
require further attention. Answers to the remaining questions about especially the
immunopathogenesis could help to develop a more specific treatment with minimal side-effects.

30
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Figures

o Figure 1: Campbell C.L. (2004) 1st edition. Small animal dermatology secrets: 32. Immune
mediated skin diseases. Hanley& Belfus. p.231-243
o Figure 2,3. : Dr. Elisa Maina. Private archive
o Figure 4: Waschke J. (2008) The desmosome and pemphigus. Histochemistry and cell biology
130:21–54
o Figure 5,6,: Bizikova et al (2010) Immunomapping of desmosomal and nondesmosomal
adhesion molecules in healthy canine footpad, haired skin and buccal mucosal epithelia:
comparison with canine pemphigus foliaceus serum immunoglobulin G staining patterns.
Veterinary Dermatology. 22(2):132-142
CASE REPORTS

Canine Pemphigus Foliaceus with Concurrent

Immune-Mediated Thrombocytopenia
Shinpei Kawarai, PhD, DVM, Masaharu Hisasue, PhD, DVM, Shinobu Matsuura, PhD, DVM, Tetsuro Ito, DVM,
Yukari Inoue, DVM, Sakurako Neo, PhD, DVM, DACVP, Yoko Fujii, PhD, DVM, DACVIM (Cardiology),
Hiroo Madarame, PhD, DVM, DJCVP, Kinji Shirota, PhD, DVM, DJCVP, Ryo Tsuchiya, PhD, DVM

ABSTRACT
A 3 yr old wirehaired fox terrier was presented to his primary care veterinarian with fever, thrombocytopenia, and
generalized crusting dermatitis. The skin lesion had progressed for at least 18 days, and thrombocytopenia had
developed 3 days before presentation. Histopathology and direct immunofluorescence studies of the skin were
consistent with pemphigus foliaceus (PF). Immunofluorescence revealed immunoglobulin G deposition around the
keratinocytes in the stratum spinosum. A diagnosis of immune-mediated thrombocytopenia (IMT) was confirmed by the
presence of platelet surface-associated immunoglobulin using flow cytometry. Systemic immunosuppressive therapy
with cyclosporine and azathioprine was effective, and the dog survived for .2 years from the initial presentation. IMT is
rarely associated with PF. This appears to be the first detailed report of a definitive diagnosis of concurrent PF and IMT
in a dog. The authors’ findings indicate that canine PF could be complicated by hematologic immune-mediated
diseases such as IMT. (J Am Anim Hosp Assoc 2015; 51:56–63. DOI 10.5326/JAAHA-MS-6044)

Introduction different epidermal staining patterns were identified by indirect IF

Canine pemphigus foliaceus (PF) is the most common autoimmune
pustular skin disease in dogs characterized by pustules, crusting,
erosions, and alopecia of the head, face, nasal planum, pinnae, and
footpads.1–3 When generalized, skin lesions spread over the entire
body, and symptoms include fever, lethargy, and limb edema.
Histopathologically, acantholytic keratinocytes are found within
intraepidermal-to-subcorneal neutrophilic and eosinophilic pus-
tules. Direct immunofluorescence (IF) has shown that antikerati-
nocyte autoantibodies are deposited in vivo in the subcorneal layers,
especially the stratum spinosum and granulosum.1–9 Indirect IF
using canine PF sera revealed the presence of circulating
antikeratinocyte immunoglobulin (Ig) G in vitro.10 Recently, several
using healthy canine footpad epithelium as a substrate.11 Compar-
isons of IF staining patterns of desmosomal and nondesmosomal
adhesion molecules of keratinocytes with those of canine PF sera
showed that desmocolin-1 was the major autoantigen of canine
PF.11,12 Although circulating antikeratinocyte autoantibodies are
considered to cause acantholysis only on the epidermis, canine PF
reportedly occurs either concurrently with or following hypothy-
roidism, leishmaniasis, and systemic lupus erythematosus
(SLE).3,13–15 Dysregulated antibody production based on polyclonal
B cell activation, cross-reacting antigens, and the innocent
bystander reaction due to adsorption of circulating immune
complexes onto keratinocytes are thought to cause these autoim-
mune disorders.3,13–15

From Laboratory of Small Animal Clinics, Veterinary Teaching Hospital
(S.K., T.I., H.M.) and Laboratory of Veterinary Internal Medicine II
(M.H., Y.I., S.N., R.T.), Laboratory of Veterinary Surgery I (Y.F.),
and Laboratory of Veterinary Pathology (K.S.), Department of
Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku,
Sagamihara, Kanagawa, Japan; and Center for Advanced Biomed-
ical Research, Boston University School of Medicine, Boston, MA
(S.M.).
CRP, C-reactive protein; IF, immunofluorescence; Ig, immunoglob-
ulin; IMT, immune-mediated thrombocytopenia; IV, intravenous
injection; PF, pemphigus foliaceus; PO, per os; PSA, platelet
surface-associated; RF, rheumatoid factor; SLE, systemic lupus
erythematosus

Correspondence: hisasue@azabu-u.ac.jp (M.H.)

56 JAAHA | 51:1 Jan/Feb 2015 Q 2015 by American Animal Hospital Association


FIGURE 1 Appearance of the wirehaired fox terrier at the time of presentation. A, B: Note the generalized dermatitis with pustules, crusts,
erosions, and alopecia, on the face (A), ear (B), and trunk (C) at the primary care hospital. The dog’s hair was clipped by the primary care
veterinarian.
Thrombocytopenia in dogs is often immune mediated.16 The
pathogenesis of canine immune-mediated thrombocytopenia
(IMT) involves platelet surface-associated (PSA) Ig binding, which
leads to the increased removal of antibody-coated platelets by
macrophages through phagocytosis in the reticuloendothelial
system.16 Canine IMT is difficult to diagnose because it occurs
not only primarily without obvious underlying causes, but also
secondarily to various systemic diseases including infectious,
neoplasias, adverse reactions to drugs (e.g., sulfonamide, cephalo-
sporin, cyclophosphamide, etc.), and systemic autoimmune
diseases such as SLE.16–20 Typically, IMT is diagnosed by excluding
other causes of thrombocytopenia, such as disseminated intravas-
cular coagulation and deficient platelet production by bone
16–20
marrow. Kristensen et al. (1994) established a direct assay
for measuring PSA-Ig using flow cytometry, which can be used to
16,17,19
diagnose canine IMT. Tsuchiya et al. (2010) recently
improved the accuracy of the assay using established artificial
21
positive-control platelets. The authors of this study routinely use
this assay when canine IMT is suspected in their hospital.
The purpose of this article is to describe a rare association of
PF with concurrent IMT. Diagnosis was made based on
histopathological findings, immunohistochemical detection of
IgG deposition around keratinocytes, and flow cytometric
detection of PSA-Ig.
Case Report
A 3 yr, 10 mo old male wirehaired fox terrier was presented to his
primary care veterinarian with pruritus and generalized dermatitis.
The owner noticed that the dog’s skin lesions had worsen 6 days
Concurrent Pemphigus Foliaceus and IMT in a Dog
before presentation. At the time of examination, the dog was
pyrexic (39.78C) and the skin lesions were bilateral, symmetrical,
and manifested as pustules, crusting, and erosions. The pustules
initially appeared on the ventral trunk, in the ear canal, and on the
pinnae. Multifocal crusting and erosion then progressed over the
entire body to cover the nasal planum, top of the head, muzzle,
dorsal and ventral trunk, feet, and paw pads over the next 18 days
(Figures 1A, B, and C). Prior to referral, commercially-available
rapid drug sensitivity testing (Monoris Inc., Tokyo, Japan) was
performed without bacterial identification from swabs collected
from underneath a crust on the ventral trunk. Although the
crusting dermatitis did not respond to a systemic antibiotic
(cephalexina, 25 mg/kg per os [PO] q 12 h) selected by the drug
sensitivity test, the fever was reduced with a single dose of
diclofenac sodiumb (1.25 mg PO). Because leukocytosis, anemia,
and thrombocytopenia were diagnosed after the administration of
cephalexin and diclofenac sodium, the dog was referred to the
study authors’ hospital for further examination, diagnosis, and
treatment.
Twelve days after the initial presentation at the primary care
hospital (day 1), the dog had lost 10% of its body weight (new body
weight was 9 kg). Body temperature, pulse, and respiratory rates
were 37.98C, 120 beats/min, and 25 breaths/min, respectively. A
physical examination revealed lameness; swelling of all limbs in the
synovial joints of the carpus, stifle, and hock; conjunctivitis; and
peripheral lymphadenopathies on the superficial cervical and
popliteal lymph nodes. A grade 2/6 systolic heart murmur, loudest
over the left thorax, was audible during cardiac auscultation. The
skin showed generalized erythema, pustules, crusting, and erosions.
JAAHA.ORG 57
Those lesions were distributed symmetrically on the dog’s face
(including the muzzle, nasal planum from the top of the muzzle to TABLE 1
the front of the glabella, the periocular region, ear canal and Complete Blood Cell Count and Serum Biochemical Profile
pinnae), ventral and dorsal trunk, feet, and paw pads. The oral Results at the Time of Initial Presentation

mucosa and mucocutaneous junctions, such as the eyelids and Variable Value Reference range
anus, were free of lesions. Impression smears were obtained from 9
WBC (3 10 /L) 1.76 (0.6–1.7)
the erosive skin lesions under the crusting. Cytology findings 12
Red blood cell count (3 10 /L) 4.3 (5.5–8.5)
showed eosinophilic, pyogranulomatous inflammation comprising Hemoglobin (g/L) 96 (120–180)
numerous degenerate and nondegenerate neutrophils, many Packed cell volume 0.29 (0.37–0.55)
eosinophils and macrophages, and a few small lymphocytes. A Mean corpuscular volume (fL) 67 (66–77)
few oval and intensely basophilic acantholytic keratinocytes with a Mean corpuscular hemoglobin (pg/cell) 22.3 (19.9–24.5)
centrally placed nucleus were individually surrounded by abundant Mean corpuscular hemoglobin concentration (g/L) 333 (320–360)
neutrophils. Infectious organisms such as bacteria and Malassezia 9
Platelet count (3 10 /L) 70 (200–500)
spp. were unremarkable in smears. Swabs collected from pustules Total protein (g/L) 47 (51–77)
in the axillary area were examined for bacterial and fungal cultures Albumin (g/L) 18 (25–40)
at Hoken Kagaku Laboratory (Kanagawa, Japan) using sheep blood Blood urea nitrogen (mmol/L) 7.43 (3.28–10.42)
agar and Sabouraud agar, respectively. Sarcoptic mange and Creatinine (lmol/L) 35.36 (44.2–132.6)
Demodex canis were not detected in skin scrapings or plucked hairs. Phosphorus (mmol/L) 1.39 (0.71–1.78)
Considering the diagnosis of hematologic abnormalities by the Calcium, total (mmol/L) 2.2 (2.15–2.85)
primary care veterinarian and based on the physical exam findings Alanine aminotransferase (lkat/L) 0.25 (0.3–1.19)
conducted at the authors’ hospital, immunologic tests, diagnostic Alkaline phosphatase (lkat/L) 68.17 (0.6–4.33)
imaging, arthrocentesis, fine-needle aspiration of lymph nodes, Total cholesterol (mmol/L) 5.26 (2.77–8.13)
skin biopsy, and a histopathological examination were performed. Triglycerides (mmol/L) 1.34 (0.19–1.15)
Table 1 shows the hematologic and serum biochemical Total bilirubin (lmol/L) 16.93 (0.0–5.13)
findings. Complete blood cell counts showed a slightly elevated Glucose (mmol/L) 4.11 (3.83–6.77)
white blood cell count (1.76 3 109/L; reference range, 0.6–1.7 109/ Sodium (mmol/L) 152.4 (142–152)
L), a low red blood cell count (4.3 3 1012/L; reference range, 5.5– Potassium (mmol/L) 4.48 (3.7–5.3)
8.5 1012/L), a packed cell volume of 0.29 (reference range, 0.37– Chloride (mmol/L) 118.6 (105–117)
0.55), and a remarkably low platelet count (PLT; 70 3 109/L;
reference range, 200–500 109/L). The ratio and cell counts of
reticulocytes were 0.0032 proportion of red blood cells and 13.76 3 phosphatase (68.17 lkat/L; reference range, 0.6–4.33 lkat/L), and
9 bilirubin (16.93 lmol/L; reference range, ,5.13 lmol/L). C-
10 /L, respectively (the range of reticulocyte counts in regenerative
response to mild anemia (a packed cell volume of 0.26–0.38) are reactive protein (CRP) was measured using a laser nephelometric
100–150 3 109/L). A slightly low serum iron concentration (16.11 immunoassayc to evaluate the degree of systemic inflammation.
lmol/L; reference range, 16.83–21.84 lmol/L) and low unsaturated Examination of globulin protein fraction by serum protein
iron binding capacity (6.80 lmol/L, reference range; 22.73–60.86 electrophoresis was performed because hypoalbuminemia was
lmol/L) indicated that chronic inflammation caused the mild observed. CRP was obviously elevated at 114.29 nmol/L (reference,
nonregenerative anemia. Blood coagulation tests showed an ,9.52 nmol/L) and serum protein electrophoresis revealed elevated
elevated plasma fibrinogen (13.35 lmol/L; reference range, 2.59– a2-globulin level.22
9.88 lmol/L), a normal prothrombin time (8.1 s; reference range, To determine the cause of the hypoalbuminemia, diagnostic
6.8–8.6 s), a slightly prolonged activated partial prothrombin time imaging was performed, focusing on possible renal and hepatic
(28.1 s; reference range, 13.1–26.9 s), and a normal fibrinogen- abnormalities. Thoracic and abdominal radiography revealed no
fibrin degradation product (,2.5 mg/L; reference range, 0–2.5 mg/ apparent abnormalities. An abdominal ultrasound showed hepatic
L). The blood coagulation test results excluded disseminated venous congestion and mild hyperechogenicity of the liver
intravascular coagulation. The serum biochemical analysis showed parenchyma and renal cortex. The dog underwent a cardiac
moderately low total protein (47 g/L; reference range, 51–77 g/L) work-up to determine the cause of its heart murmur and hepatic
and albumin (18 g/L; reference range, 25–40 g/L) with high alkaline venous congestion. Echocardiography revealed mild pulmonary

58 JAAHA | 51:1 Jan/Feb 2015


FIGURE 2 Flow cytometric analysis of platelet surface-associated (PSA) immunoglobulin (Ig) M, -IgG, and complement 3 (-C3). Platelets
from sick (filled curve) and healthy control (open curve) dogs stained with anti-canine IgM, IgG, and C3 antibodies conjugated with fluorescein
isothiocyanate. Numbers in the graphs indicate binding percentage (%) of PSA-IgM, -IgG, and -C3 on platelets obtained from sick dog. Reference
lines for PSA-IgM, -IgG, and -C3 positive platelets from healthy control are set at 5%.
hypertension on the basis of increased tricuspid regurgitation
velocity (3.41 m/s, reference range ,3.0 m/s; estimated pressure
gradient across the tricuspid valve, 46 mm Hg). The dog’s systolic
blood pressure was 120 mmHg, which was considered normal for
dogs. Urinalysis revealed a specific gravity of 1.045, a protein
content of 1.8 g/L, and a protein/creatinine ratio of 1.5, indicated
mild proteinurea (reference ranges, 1.020–1.050, ,0.5 g/L, and
,0.5, respectively). The findings of proteinurea and a hyperechoic
renal cortex indicated protein-losing nephropathy. The leakage of
antithrombin III from the kidneys is considered a possible cause of
pulmonary thromboembolism, which leads to pulmonary hyper-
tension; however, further work-up was not performed.
To exclude lymphoma, a fine-needle aspirate of the lymph
nodes was obtained, which showed mild-to-moderate reactive
lymphoid hyperplasia with mild neutrophilic infiltration. the
authors performed arthrocentesis because idiopathic polyarthritis
was suspected on the basis of the history of fever, physical findings
of lameness, and increased serum CRP level. A gross examination
of joint fluid showed turbidity and decreased viscosity. A cytologic
examination identified low-to-moderate cellularity and mild
inflammation consisting of large mononuclear cells (94%), small
mononuclear cells (5%), and neutrophils (1%). Those findings
were consistent with polyarthropathy associated with chronic
inflammation.
At that point, a mild anemia, polyarthropathy (possibly due to
chronic inflammation), thrombocytopenia, hypoalbuminemia
(probably due to protein-losing nephropathy), and pulmonary
hypertension were identified. Based on those findings, the authors’
differential diagnoses included PF, pemphigus erythematous,
panepidermal pustular pemphigus, cutaneous lymphoma, erythe-
Concurrent Pemphigus Foliaceus and IMT in a Dog
ma multiforme, cutaneous vasculitis, IMT, and SLE. An additional
skin biopsy and immunologic tests were conducted to reach a
definitive diagnosis.
A commercial laboratory (Monoris Inc, Tokyo, Japan)
performed a direct Coombs’ test and measured serum antinuclear
antibodies and rheumatoid factor (RF) to determine the presence
of SLE. The direct Coombs’ test (378C and 48C) and antinuclear
antibodies were negative, but RF was positive (titer was 320;
reference value; 0). Nine days after the initial presentation, flow
cytometry for PSA-IgM, -IgG, and -C3 was performed as described
by Tsuchiya et al (2010) for the diagnosis of canine IMT.21 The
gates for positive events were set based on healthy control samples
at 0–5%. Values .10% were judged positive. The results showed
apparently positive anti-canine IgM (89.3%) and anti-canine IgG
(47.1%) antibodies, but negative anti-canine C3 (7%) antibody
compared with the healthy control (Figure 2).
Skin biopsies obtained using a 4 mm biopsy punch from the
crusting skin lesions on the dog’s head, neck, and dorsal trunk were
examined by routine histopathology. Those findings revealed severe
crusting of the superficial epidermis and large intraepidermal to
subcorneal pustules (Figure 3A) that mainly comprised neutrophils
and eosinophils, and some included acantholytic keratinocytes
(Figure 3B). The pustules involved the hair follicles and extended
into the follicular infundibula. Neutrophilic and eosinophilic
inflammation was seen in the pustules. Primarily neutrophils and
then mononuclear cells infiltrated the superficial-to-middle dermis.
Direct IF using anti-canine IgG antibodiesd (1:800) identified IgG
deposition throughout the superficial-to-middle epidermis layer,
particularly in the stratum spinosum (Figure 3C). Canine PF and
JAAHA.ORG 59
FIGURE 3 A: Histopathological features of the superficial pustules. B: High-power magnification of the area circled in A. The pustules are
comprised of neutrophils with some acantholytic keratinocytes (arrows). C: Direct immunofluorescence shows immunoglobulin G deposition from
the superficial to middle epidermal layers, especially the stratum spinosum. Hematoxylin and eosin stainining (A and B) and Immunofluorescence
staining with anti-dog IgG (green) and antinuclear stain, DAPI (blue) (C). Bar ¼ 400 lm (A), 100 lm (B), and 100 lm (C).

IMT were diagnosed based on those histopathological and
immunologic findings.
The dog was hospitalized for 7 days and received fluid therapy
with acetate Ringer’s solution (3 mL/kg/hr) supplemented with low
e
molecular weight heparin (dalteparin sodium , 75 U/kg/day) and
monoammonium glycyrrhizinatef (1 mg/kg intravenous injection
[IV] q 12 hr). Cefazolin sodium hydrateg (25 mg/kg IV q 8 hr) and
enrofloxacinh (5 mg/kg BW q 24 hr) were administered to prevent
bacterial growth, and ursodeoxycholic acidi (10 mg/kg PO q 12 hr)
was administered as liver function support. Hand-mixed topical
j
corticosteroid (0.12% betamethasone valerate and 0.3% heparin-
oidk) and 0.5% vitamin A oill was applied to reduce the inflam-
mation and as a moisturizer. After cutaneous lymphoma had been
excluded by frozen-section histopathology by day 3 from the first
m
presentation at the authors’ institution, cyclosporine (5.5 mg/kg
PO q 24 hr) was started. A bacterial culture and sensitivity tests were
performed on day 7. Escherichia coli and Staphylococcus spp. with
methicillin resistance (i.e., penicillin, oxacillin, cephalexin, cefazo-
lin, imipenem, lincomycin, enrofloxacin, minocycline, gentamicin,
and sulfamethoxazole-trimethoprim) were isolated on the basis of
23
the Clinical Laboratory Standard Institute guidelines. Based on the
culture, the previously prescribed antibiotics were substituted with
n
fosfomycin calcium hydrate (25 mg/kg PO q 12 hr). The results of
fungal cultures were negative on day 17.
Follow-up examination on day 22 after initial therapy revealed
a 50% improvement in the skin lesions. The physical findings, PLT
(359 3 109/L), and CRP (46.67 nmol/L) were also improved.
Because of persistent erythema and pruritus on the skin lesion and
hyperthermia (39.88C), azathioprineo (2 mg/kg PO q 24 hr) was
coadministered with cyclosporine. On day 36, the red blood cell
count and packed cell volume had recovered (5.14 3 1012/L and
0.37, respectively) and CRP had decreased to 11.43 nmol/L.
Cyclosporine was discontinued immediately. On day 64, 90% of
the skin lesions were improved and were completely resolved by
day 120 (Figure 4). The dog’s physical condition and complete
blood cell count had also improved. The azathioprine dosage was
gradually tapered to 0.8 mg/kg PO q 48 hr. On day 309 from the
time of referral, PSA-IgM, -IgG, and -C3 were re-evaluated by flow
cytometry. The results were negative for IgM (7%), IgG (5.1%),
and C3 (3.6%). The skin symptoms were controlled by azathio-
prine monotherapy until day 785.
Discussion
To the author’s knowledge, this is the first report to describe a dog
definitively diagnosed with concurrent PF and IMT. Some forms of
immune-mediated skin disease have been considered as possible
causes of IMT and an epidemiological survey by Grindem et al.
(1991) found that IMT in pemphigus complex is very rare.16,18 The

60 JAAHA | 51:1 Jan/Feb 2015


FIGURE 4 Appearance of the wirehaired terrier following
treatment with azathioprine 120 days after initially being presented
to the authors’ hospital.
following forms of histological types of pemphigus are recognized
in dogs and cats: PF, pemphigus erythematosus, panepidermal
pustular pemphigus, pemphigus vulgaris, and paraneoplastic
pemphigus. 1–9
The most common form of pemphigus complex is
PF, of which symptoms are commonly less severe than in other
forms, such as pemphigus vulgaris. 1–4
Although Grindem et al.
(1991) did not specify the forms of pemphigus, recent retrospective
studies have not reported those diseases to occur simultaneous-
ly.7,8,19,20 Therefore, the presence of PF and IMT seems to be rare.
The present findings of PSA-Ig analysis reflect immunological
signs of canine PF in addition to known physiological and
hematological abnormalities. In canine PF, symptoms are generally
localized to the skin. Although systemic symptoms, including
anorexia, depression, fever, and weight loss are rare, they become
obvious when erosive skin lesions progress over the entire body.3
Various hematologic abnormalities, such as moderate to severe
leukocytosis and neutrophilia, mild nonregenerative anemia,
thrombocytopenia, mildly to moderately elevated a2-, b2-, and c-
globulins, and mild hypoalbuminemia have also been report-
ed.7,8,14,15 Those features are usually associated with the severity of
skin symptoms; however, the results of routine blood tests,
diagnostic imaging, and urinalysis have received relatively little
focus because they are not considered to support a specific
diagnosis.3 Focusing on thrombocytopenia among systemic
symptoms, IMT was diagnosed on the basis of PSA-Ig levels
measured using flow cytometry. Therefore, if dogs with PF have
obvious systemic signs, systemic work-ups and immunological tests
should be conducted to identify complicating factors and
associated diseases.
Concurrent Pemphigus Foliaceus and IMT in a Dog
A diagnosis of canine IMT is difficult because it is usually only
concluded by excluding other potential causes of thrombocytope-
nia.16–20 Dogs with IMT develop an increased risk of spontaneous
hemorrhage when PLT fall below 10–30 3 109/L, whereas O’Marra
et al. (2011) reported that signs of bleeding were absent in 19% of
73 dogs showing platelet counts ,50 3 109/L.16–20,24 Those asymp-
tomatic dogs with moderate thrombocytopenia are not thoroughly
evaluated in routine clinical practice because an invasive bone
marrow examination is needed to exclude nonimmune-mediated
thrombocytopenia. The authors of the current study diagnosed
IMT in a dog by measuring PSA-Ig using flow cytometry at the
time of the initial presentation at their hospital. Furthermore, the
positive PSA-IgM and -IgG findings became negative when the
thrombocytopenia was improved by immunosuppressive therapy
with azathioprine. The study authors thus considered that the PSA-
Ig level correlated with disease status and was useful for the
diagnosis and evaluation of treatment in dogs with IMT.
Vaughan et al. (2010) reported that complicated cases of canine
PF concurrent with allergic skin disease and other systemic diseases
are significantly associated with the presence of eosinophil
infiltration in intraepidermal pustules.8 In the present case,
eosinophil infiltration was detected in the pustules, and the dog
presented with pruritus at onset and a history of recurrent pyoderma.
Concurrent IMT was also diagnosed. Those finding are consistent
with those of Vaughan et al. (2010).8 One case, positive for
eosinophil infiltration, was provisionally diagnosed with drug
hypersensitivity against ampicillin, amoxicillin, enrofloxacin, and
metronidazole.8 Because PF and IMT, as observed in the present case,
are clinical symptoms in drug hypersensitivity, it was possible that
those symptoms persisted at the initial presentation to the authors’
hospital.2,6,8,16 If canine PF is accompanied with eosinophilic
infiltration, it may be necessary to examine underlying diseases.
Skin lesions had been evident in the case described herein
since the first examination at the primary care veterinary hospital.
However, the PLT (194 3 109/L; reference range, 200–500 3 109/L)
at that time were essentially normal and gradually decreased
thereafter. The possible causes of secondary IMT include infectious
diseases, neoplasia, adverse reactions to drugs, and systemic
autoimmune diseases such as SLE. In the current case, PF and
adverse drug reaction were considered as the possible etiologies.
IMT may also arise in association with circulating antikeratinocyte
autoantibodies.3 Secondary IMT develops following SLE in some
dogs, suggesting that immune complexes bound to platelets by
complement-mediated immune adherence or nonspecific interac-
tions cause IMT concurrent with SLE.16 The current case had IgG
antibodies on the epidermis, had IgM and IgG antibodies on the
platelet-cell surface, and was RF positive. Those findings indicate
JAAHA.ORG 61

that in this case, PF may have been the cause of hematologic
immune-mediated diseases such as IMT. The other possible cause
of IMT was an adverse drug reaction. Prior to referral, IMT
occurred after administration of cephalexin, which is a known
cause of secondary IMT. In the present case, cefazolin sodium
hydrate, which belongs to the same drug class as cephalexin, had
been administered IV for 3 days before administration of
cyclosporine. Because the symptoms did not worsen after cefazolin
administration without any immunosuppressant drug, the possi-
bility of an adverse drug reaction was considered low. Epidemi-
ological studies are warranted to determine the prevalence and
association of IMT with PF in dogs.
Conclusion
Canine PF is autoimmune pustular disease with symptoms that are
generally localized to the skin. Although a low incidence of
thrombocytopenia associated with pemphigus complex is reported,
the study authors confirmed the diagnosis of concurrent PF and
IMT based on histology findings, direct IF, and the presence of
PSA-Ig. The dermatologic and hematologic signs were treated with
immunosuppressive therapy using cyclosporine and azathioprine,
and the dog has survived for .2 yr since the day of first
presentation at the authors’ animal hospital. Positive findings for
PSA-IgM and IgG became negative when thrombocytopenia was
improved after immunosuppressive therapy. Those findings
indicate that IMT can develop with canine PF; thus, systemic
evaluation should include precise hematologic assessment.
The authors would like to thank Dr. Takao Otsuki (Heiwa Animal
Hospital, Japan) for referring patients, Dr. Yoko Kakinuma for the
technical assistance with the histological sections, Dr. Ryoji
Takahashi and Dr. Shinsuke Fujimoto for supporting patient care
at the clinic, and Jonathan Bloch for proofreading the manuscript.
The authors declare that there are no conflicts of interest associated
with the present report. This study was supported by the Grant-in-
Aid for Matching Fund Subsidy for Private University and by the
Science Research Promotion Fund of The Mutual Aid Corporation
for Private Schools of Japan.
FOOTNOTES
a
Larixin; Toyama chemical, Tokyo, Japan
b association with systemic lupus erythematosus, and subsequent
Voltaren; Novartis Pharma, Tokyo, Japan
c
Canine CRP measurement system CRP-2; Arrows Co. Ltd, Osaka, Japan
d
Fluorescein-conjugated goat affinity purified antibody to dog IgG
(whole molecule); MP bio Japan K.K., Tokyo, Japan
e 16. Scott MA, Jutkowitz LA. Immune-Mediated Thrombocytopenia. In:
Hepagumin a; Sawai Pharma, Osaka, Japan
f
Stronger neo-minophagen C, Eisai, Tokyo, Japan
62 JAAHA | 51:1 Jan/Feb 2015
g
Cefamezin a; Astellas, Tokyo, Japan
h
Baytril; Bayer Health Care, Tokyo, Japan
i
URSO; Mitsubishi Tanabe Pharma Co., Osaka, Japan
j
Rinderon-V Ointment; Shionogi Co. Ltd., Osaka, Japan
k
Hirudoid Soft Ointment; Maruho, Osaka, Japan
l
Sahne Oint; Eisai, Tokyo, Japan
m
Atopica; Novartis Animal Health, Tokyo, Japan
n
FOSMICIN; Meiji Seika Pharma Co. Ltd., Tokyo, Japan
o
Imuran, GlaxoSmithKline, Tokyo, Japan
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