Transcript APS-1

Journal Club
2008年３月13日 8:20-8:50
B棟８階 カンファレンス室
亀田メディカルセンター 糖尿病内分泌内科
Diabetes and Endocrine Department,
Kameda Medical Center
松田 昌文
Matsuda, Masafumi
Original Article
Autoimmune Polyendocrine Syndrome Type 1 and
NALP5, a Parathyroid Autoantigen
Mohammad Alimohammadi, M.D., Peyman Björklund, Ph.D., Åsa
Hallgren, B.Sc., Nora Pöntynen, M.Sc., Gabor Szinnai, M.D., Noriko
Shikama, Ph.D., Marcel P. Keller, Ph.D., Olov Ekwall, M.D., Ph.D., Sarah
A. Kinkel, B.Sc., Eystein S. Husebye, M.D., Ph.D., Jan Gustafsson, M.D.,
Ph.D., Fredrik Rorsman, M.D., Ph.D., Leena Peltonen, M.D., Ph.D.,
Corrado Betterle, M.D., Ph.D., Jaakko Perheentupa, M.D., Ph.D., Göran
Åkerström, M.D., Ph.D., Gunnar Westin, Ph.D., Hamish S. Scott, Ph.D.,
Georg A. Holländer, M.D., and Olle Kämpe, M.D., Ph.D.
N Engl J Med
Volume 358(10):1018-1028
March 6, 2008
Autoimmune Polyendocrine Syndrome
In 1853, Thomas Addison first described the
clinical and pathological features of
adrenocortical failure in patients who also
appeared to have pernicious anemia (PA).
In 1908, Claude and Gougerot suggested a
common pathogenesis for these conditions in
an article titled "Insufficance pluriglandulaire
endocrinnienne."
In 1926, Schmidt documented the association
between adrenocortical failure and thyroiditis.
Carpenter, in 1964, expanded the syndrome
described by Schmidt to include insulindependent diabetes mellitus.
Neufeld M, Blizzard RM. Polyglandular autoimmune disease. In: Pinchera A, Doniach D, Fenzi DF,
Baschieri L, eds. Autoimmune aspects of endocrine disorders. London, UK: Academic
Press; 1980:357-65.
Type 1 (in Finland 1/25000 no HLA antigen association)
Autoimmune polyendocrine syndrome, type 1 is also known as the candidiasis-hypoparathyroidism-Addison's disease-syndrome
after its main features:
A mild immune deficiency, leading to persistent mucosal and cutaneous infections with candida yeasts. There is also decreased
function of the spleen (asplenism).
Autoimmune dysfunction of the parathyroid gland (leading to hypocalcemia) and the adrenal gland (Addison's disease:
hypoglycemia, hypotension and severe reactions in disease).
Other disease associations are:
hypothyroidism
hypogonadism and infertility
vitiligo (depigmentation of the skin)
alopecia (baldness)
malabsorption
pernicious anemia
chronic active (autoimmune) hepatitis
As opposed to type 2, this syndrome inherits in an autosomal recessive fashion and is due to a defect in AIRE ("autoimmune
regulator"), a gene located on the 21st chromosome. Normal function of AIRE, a transcription factor, appears to be to confer
immune tolerance for antigens from endocrine organs.
A EU-funded consortium is currently doing translational research on this condition and has established a webpage at EurAPS.
Type 2
Autoimmune polyendocrine syndrome, type 2 (also known as "Schmidt's syndrome") is more heterogeneous, occurs more often
and has not been linked to one gene. Rather, patients are at a higher risk when they carry a particular HLA genotype (DQ2, DQ8
and DRB1*0404).
Features of this syndrome are:
Addison's disease
hypothyroidism
diabetes mellitus (type 1)
less common associations:
hypogonadism
vitiligo
Some researchers favour splitting this syndrome into three distinct syndromes (numbering 2, 3 and 4), but research evidence for
these distinct combinations is not convincing.
Type 3
PAS 3 can be further classified into the following 3 subcategories:
PAS 3A - Autoimmune thyroiditis with immune-mediated diabetes (IMD) mellitus (type 1
diabetes mellitus)
PAS 3B - Autoimmune thyroiditis with PA
PAS 3C - Autoimmune thyroiditis with vitiligo and/or alopecia and/or other organ-specific
autoimmune disease
PAS 3 is associated with the following diseases:
organ-specific autoimmune diseases
Celiac disease
Hypogonadism
Myasthenia gravis
Organ nonspecific or systemic autoimmune diseases
Sarcoidosis
Sjögren syndrome
Rheumatoid arthritis
Other diseases
Gastric carcinoid tumor
Malabsorption due to exocrine pancreatic deficiency
XPID
The most serious but rarest form is the X-linked polyendocrinopathy, immunodeficiency and diarrheasyndrome, also called IPEX. This is due to mutation of the FOXP3 gene on the X chromosome.
Most patients develop diabetes and diarrhea as neonates and many die due to autoimmune
activity against many organs. Boys are affected, while girls are carriers and might suffer mild
disease.
GENE
AIRE (autoimmune
regulator), which codes for
a putative transcription
factor featuring 2 zinc
motifs
Bjorses P, Halonen M,
Palvimo JJ, et al: Mutations
in the AIRE gene: effects on
subcellular location and
transactivation function of
the autoimmune
polyendocrinopathycandidiasis-ectodermal
dystrophy protein. Am J
Hum Genet. Feb 2000;66(2):
378-92
Betterle et al., JCEM 83 (4): 1049. (1998)
GENE
AIRE (autoimmune
regulator), which codes for
a putative transcription
factor featuring 2 zinc
motifs
Bjorses P, Halonen M,
Palvimo JJ, et al: Mutations
in the AIRE gene: effects on
subcellular location and
transactivation function of
the autoimmune
polyendocrinopathycandidiasis-ectodermal
dystrophy protein. Am J
Hum Genet. Feb 2000;66(2):
378-92
AIRE encodes a 54-kD protein expressed
in stromal cells of primary and secondary
lymphoid tissues, including thymic
medullary epithelial cells.
AIRE regulates the transcription of tissuerestricted antigens and has hence been
linked to both central and peripheral
tolerance.
In patients with adrenal
insufficiency,
autoantibodies are reactive to 21hydroxylase, a key enzyme in
glucocorticoid synthesis
Candidate autoantigens previously
reported to be linked to
hypoparathyroidism, including the
almost ubiquitously expressed NALP5
?
calciumsensing receptor, have not been
confirmed as relevant autoantigens
Background
• The autoimmune polyendocrine
syndrome type 1 (APS-1) is a
multiorgan autoimmune disorder
caused by mutations in AIRE, the
autoimmune-regulator gene
• To identify a parathyroid-specific
autoantigen (NALP5) is important for
improved serologic diagnosis of the
disease and would provide better
understanding of the molecular
mechanisms underlying APS-1.
Associations between Clinical Manifestations of Autoimmune Polyendocrine Syndrome Type 1 (APS1) and the Presence of NACHT Leucine-Rich-Repeat Protein 5 (NALP5) Autoantibodies
11 Swedish, 18 Norwegian, and 58 Finnish patients with APS-1 who
were members of more than 50 independent kindreds.
negative control
Panel A shows the results of the comparison
of NALP5 autoantibody titers in serum
samples from 87 patients with APS-1 (83 of
whom had the AIRE mutation and 4 of whom
did not), 100 patients with other autoimmune
disorders (5 disorders and 20 patients with
each), and 193 healthy blood donors. The
dashed line indicates the upper limit of the
normal range, defined as the mean of the
values obtained for the healthy blood donors
plus 3 SD.
Panel B shows the results of confirmation of
the specificity of autoantibodies by means of
sequential immunoprecipitation of 35Smethionine–radiolabeled human parathyroid
NALP5. For the first immunoprecipitation
step, we used no antibody or serum (positive
standard, lane 1), NALP5 antiserum (negative
standard, lane 2), serum samples from each
of three patients with reactivity to NALP5
(lanes 3, 4, and 5), serum samples from each
of three patients without reactivity to NALP5
(lanes 6, 7, and 8), and serum samples from
healthy controls (lanes 9, 10, and 11).
Panel C shows the results of
autoradiography of the blots shown in Panel
B by means of semiquantitative
measurement of pixel density with
ImageQuant software.
positive control
Presence of Autoantibodies against
NALP5 in Patients with Autoimmune
Polyendocrine Syndrome Type 1
(APS-1)
Analysis of the Expression of
Messenger RNA (mRNA) of NALP5
and Its Homologues
Panel A shows the expression of NALP5
mRNA in adult human tissues as
measured by quantitative PCR assay. Note
the noncontinuous y axis and its variable
intervals. T bars are standard deviations.
Panel B shows a phylogenetic tree
(designed with ClustalW software) on the
left-hand side indicating homologies
among mRNA in the NALP protein family
and mRNA expression in a human
multiple-tissue panel on the right-hand
side. Each of the selected proteins in the
NALP protein family was detected by
means of a 38-cycle conventional PCR
assay involving the complementary DNA
(cDNA) from the tissue panel. Note the
band corresponding to NALP5 for the
parathyroid-gland specimen. These
experiments were performed to exclude
the expression of NALP5 homologues in
the parathyroid glands. Samples of cDNA
from the adrenal cortex were not yet
available when these experiments were
performed.
GAPDH denotes glyceraldehyde-3phosphate dehydrogenase.
Immunostaining on Sections from Human and Bovine Parathyroid Glands
Results of immunohistochemical
analysis of sections from a
human parathyroid gland are
shown in Panels A through D;
immunofluorescence of
cryosections of bovine
parathyroid gland is shown in
Panels E through J. Background
stainingis shown in Panels A and
E, by omission of primary serum
samples. Staining patterns with
serum samples are also shown:
samples from a patient with
autoimmune polyendocrine
syndrome type 1 (APS-1) and
hypoparathyroidism (Panels B
and F), samples from a patient
with APS-1 without
hypoparathyroidism (Panels C
and G), samples from a healthy
control (Panel H), and samples of
rabbit anti–NACHT leucine-richrepeat protein 5 (NALP5)
antiserum (Panels D, I, and J).
Primary antibodies were
developed with the use of a
fluorescein isothiocyanate–
labeled secondary antibody
(green); a nuclear counterstain
including 4́,6-diamidino-2phenylindole (blue) was also used.
Scale bars represent 50 μm.
negative control
APS-1 and
hypoparathyroidism
APS-1 without
hypoparathyr
oidism
positive control
negative control
APS-1 and
hypoparathyroidism
APS-1 without
hypoparathyr
oidism
positive control
Results of Absorption Studies to Confirm the Specificity of Autoantibodies for NACHT Leucine-RichRepeat Protein (NALP) 5 in Patients with Autoimmune Polyendocrine Syndrome Type 1 (APS-1)
Immunofluorescence was
performed on
cryosections of bovine
parathyroid gland with
the use of serum from a
patient with APS-1 and
NALP5 autoantibodies.
The serum was diluted
(1:500) and preincubated
with either no protein
(Panel A) or an equal
amount of in vitro
transcribed and
translated NALP5 labeled
with 35S-methionine
(Panel B), NALP3 (Panel
C), or luciferase (Panel D).
Scale bars represent 50
μm.
no protein
NALP3
NALP5 labeled with 35S-methionine
luciferase
Study Summary
• The autoimmune polyendocrine syndrome type 1
(APS-1) is a multiorgan autoimmune disorder
caused by mutations in AIRE, the autoimmuneregulator gene
• The authors identified reactivity to the NACHT
leucine-rich-repeat protein 5 (NALP5) by
immunoscreening a human parathyroid
complementary DNA library, using serum
samples from patients with APS-1 and
hypoparathyroidism
• The findings suggest that NALP5 is a tissuespecific autoantigen involved in
hypoparathyroidism in patients with APS-1
Conclusion
• NALP5 appears to be a tissuespecific autoantigen involved in
hypoparathyroidism in patients with
APS-1
• Autoantibodies against NALP5
appear to be highly specific and
may be diagnostic for this prominent
component of APS-1
Alain C TissotPhD a, Patrik MaurerPhD a, Prof Juerg NussbergerMD b, Robert SabatMD c,
Thomas PfisterPhD a, Stanislav IgnatenkoMD d, Prof Hans-Dieter VolkMD e, Hans StockerPhD a,
Philipp MüllerMD a, Gary T JenningsPhD a, Frank WagnerMD f and Martin F BachmannPhD email
address a Corresponding Author Information
a. Cytos Biotechnology AG, Zurich-Schlieren, Switzerland
b. Division of Angiology and Hypertension, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
c. Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, Charité, Berlin, Germany
d. Parexel GmbH, Hennigsdorf/Berlin, Germany
e. Institute of Medical Immunology and Berlin-Brandenburg Centre for Regenerative Therapies, Charité University Medicine
Berlin, Berlin, Germany
f. Charité Research Organisation, Berlin, Germany
Corresponding Author InformationCorrespondence to: Martin F Bachmann, Cytos Biotechnology AG, Wagistrasse 25, 8952
Schlieren, Switzerland
The Lancet 2008; 371:821-827
Background
Hypertension can be controlled adequately with
existing drugs such as angiotensin-converting
enzyme inhibitors or angiotensin II receptor
blockers. Nevertheless, treatment success is
often restricted by patients not adhering to
treatment. Immunisation against angiotensin II
could solve this problem. We investigated the
safety and efficacy of CYT006-AngQb—a
vaccine based on a virus-like particle—that
targets angiotensin II to reduce ambulatory
blood pressure.
Methods
In this multicentre, double-blind, randomised,
placebo-controlled phase IIa trial, 72 patients with
mild-to-moderate hypertension were randomly
assigned with a computer-generated randomisation
list to receive subcutaneous injections of either 100
μg CYT006-AngQb (n=24), 300 μg CYT006-AngQb (24),
or placebo (24), at weeks 0, 4, and 12. 24-h
ambulatory blood pressure was measured before
treatment and at week 14. The primary outcomes were
safety and tolerability. Analyses were done by
intention to treat. This study is registered with
ClinicalTrials.gov, number NCT00500786.
Summary of Findings
Two patients in the 100 μg group, three in the 300 μg group, and none
in the placebo group discontinued study treatment. All patients were
included in safety analyses; efficacy analyses did not include the five
dropouts, for whom no data were available at week 14. Five serious
severe adverse events were reported (two in the 100 μg group, two in
the 300 μg group, and one in the placebo group); none were deemed
to be treatment related. Most side effects were mild, transient
reactions at the injection site. Mild, transient influenza-like symptoms
were seen in three patients in the 100 μg group, seven in the 300 μg
group, and none in the placebo group. In the 300 μg group, there was
a reduction from baseline in mean ambulatory daytime blood pressure
at week 14 by –9・0/–4・0 mm Hg compared with placebo (p=0・015 for
systolic and 0・064 for diastolic). The 300 μg dose reduced the early
morning blood-pressure surge compared with placebo (change at
0800 h –25/–13 mm Hg; p<0・0001 for systolic, p=0・0035
for diastolic).
Conclusion: Interpretation
Immunisation with CYT006-AngQb was
associated with no serious adverse events;
most observed adverse events were
consistent with local or systemic
responses similar to those seen with other
vaccines. The 300 μg dose reduced blood
pressure in patients with mild-to-moderate
hypertension during the daytime,
especially in the early morning.