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1 From the The Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School; and the 2 Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston.
Abstract
PURPOSE. To demonstrate the specific binding of autoantibodies present in the sera of patients with ocular cicatricial pemphigoid (OCP) to human ß4 integrin present in the normal human conjunctiva (NHC) and to study the role of OCP autoantibodies and antibody to human ß4 integrin in the pathogenesis of subepithelial lesion formation in OCP.
METHODS. Indirect immunofluorescence assay and in vitro organ culture method
using NHC were used. Sera and IgG fractions from 10 patients with OCP;
immunoaffinity-purified OCP autoantibody; antibodies to human ß4,
ß1, 
6, and 5 integrins; and sera from patients with pemphigus
vulgaris, bullous pemphigoid (BP), and chronic atopic and chronic
ocular rosacea cicatrizing conjunctivitis; and normal human serum (NHS)
were used.
RESULTS. Nine of 10 OCP sera or IgG fractions, immunoaffinity-purified OCP
autoantibody, antibodies to human ß4 and 6 integrins, and sera
from patients with BP showed homogenous, smooth linear binding along
the basement membrane zone (BMZ) of the NHC. NHS, antibodies to other
integrins, and sera from patients with chronic cicatrizing
conjunctivitis from other causes showed no such binding. When NHC was
first absorbed with OCP sera and then reacted with anti-ß4 antibodies
or vice versa, the intensity of the BMZ binding was dramatically
reduced or completely eliminated, indicating that there were
autoantibodies in OCP sera specific for the ß4 integrin. BMZ
separation developed 48 to 72 hours after addition of total OCP sera,
IgG fractions from OCP sera, immunoaffinity-purified autoantibodies
from sera of patients with OCP, or anti-ß4 antibodies to the NHC
cultures, but not after addition of normal control sera, sera from
patients with chronic cicatrizing conjunctivitis from causes other than
OCP, or sera from patients with OCP in clinical remission.
CONCLUSION. Circulating anti-ß4 integrin antibody may have an important role in the pathogenesis of OCP.
Ocular cicatricial pemphigoid (OCP) is an uncommon, chronic, subepithelial, scarring systemic autoimmune disease that mainly affects the conjunctiva and other mucous membranes derived from stratified squamous epithelium and (occasionally) the skin.1 2 3 4 5 The pathologic process of chronic conjunctivitis and the accompanying progressive subepithelial fibrosis, results in trichiasis, distichiasis, conjunctival keratinization, xerosis, and, eventually, blindness secondary to corneal damage as a consequence of these changes in the ocular environment.
OCP has some pathophysiological features in common with other bullous diseases, such as linear IgA bullous disease, epidermolysis bullosa acquisita, bullous pemphigoid (BP), and cicatricial pemphigoid (CP). The process of identifying anti-BMZ antibodies in the sera of many patients with CP is difficult and variable. It is likely that a variety of anti-BMZ autoantibodies with different specificities recognize different target molecules present in the BMZ. This may account in part for the wide spectrum of clinical manifestations and clinical courses of these bullous diseases. For example, BP sera bind to 180-kDa hemidesmosome (BPAg2) and to 230-kDa desmoplakin (BPAg1) proteins. On direct immunofluorescence examination of perilesional conjunctiva of patients with OCP, deposition of immunoglobulin and/or complement along the BMZ, although pathognomic, can often be difficult to demonstrate or inconclusive.
Circulating anti-BMZ antibodies have been observed in the sera of
patients with OCP.5
6
7
8
The autoantigen against which these
anti-BMZ antibodies are produced has not been well defined or fully
characterized. In our earlier studies we demonstrated that sera of
patients with OCP bind to a 205-kDa protein in human skin, conjunctiva,
and tumor cell lysates in an immunoblot.6
7
In our further
studies we observed that the antibody to a 205-kDa protein in OCP sera
recognizes the cytoplasmic domain of human ß4 integrin.6
These observations collectively suggest that sera of patients with OCP
specifically contain antibodies to human ß4 integrin. A subset of
patients with OCP with clinical features similar to CP has been
characterized by the presence of autoantibodies against epiligrin,
which is now identified as the 3-subunit of laminin
5,9
10
a ligand for 6ß4 integrin.11
Binding of oral pemphigoid autoantibodies to the 6 integrin subunit
has been reported.12
The purpose of this study was to investigate whether the anti-BMZ autoantibodies in sera of patients with OCP specifically bind to the human ß4 integrin present in human conjunctiva. We describe an in vitro model that may facilitate understanding of some of the events that produce vesicular lesions in conjunctival epithelium in OCP.
Materials and Methods
Serum Samples
The method section confirms adherence to the Declaration of
Helsinki. Sera used in this study were obtained from 10 patients with
OCP in the acute and active phase of the disease before the institution
of therapy. All patients were evaluated in the Immunology Service at
the Massachusetts Eye and Ear Infirmary between July 1 and December 31,
1997. The diagnosis of OCP was confirmed in each patient by direct and
indirect immunofluorescence (IIF) analysis of bulbar conjunctiva biopsy
specimens, as previously described.1
Four patients had
involvement of other mucous membranes or the skin. Oral mucosa was
involved in three patients. In one patient there was oral and skin
involvement. All the sera studied bound to a 205-kDa protein (ß4
integrin) on an immunoblot assay using human conjunctiva as
substrate.6
13
Additionally, when tested on salt-split
skin, the anti-BMZ autoantibody at 1:20 dilution bound to the epidermal
side of the split.14
Sera were obtained from 10 normal healthy people, from 10 patients with established BP (cutaneous only, without oral involvement) who had anti-BMZ antibody of 1:1280 or greater titer, when tested by IIF with monkey esophagus as substrate;15 from 3 patients with pemphigus vulgaris (PV); from 2 patients with chronic active cicatrizing conjunctivitis secondary to rosacea or atopy; and from 3 patients with OCP in prolonged clinical remission who had received no systemic therapy for at least 3 years. All sera were obtained after appropriate description of the nature of this study, which had been appropriately reviewed and approved by our Institutional Review Board. The sera were frozen and stored at –70°C until use.
Conjunctiva Collection
Normal human bulbar conjunctiva was obtained during cataract
surgery from six patients after informed consent and ethical permission
was obtained for their use in a human organ culture model. Conjunctiva
was used immediately after surgical excision or snap frozen in liquid
nitrogen, embedded in compound (Optimum Cutting Temperature;
Tissue-Tek, Miles Scientific, Elkhart, IN), and stored at –70°C
until sectioning.
Reagents
Immunoaffinity-purified OCP autoantibodies were eluted from
nitrocellulose blots, as previously described.6
16
Briefly, protein samples were transferred from gels to nitrocellulose
membrane. The band of choice was cut horizontally, incubated with sera
from patients with OCP, and washed. Autoantibodies were eluted and
recovered with retention of biologic activity and antigen specificity.
This eluted antibody had been characterized to be anti-ß4 antibody by
an immunoblot assay.6
Mouse monoclonal anti-CD104 (human
ß4 integrin) antibodies, monoclonal antibodies to human 6 integrin
(Ancell Corporation, Bayport, MN), and antibodies to human 5 and
ß1 integrin (Immunotech, Westbrook, ME) were purchased. Rabbit
polyclonal antibody to human ß4 integrin was kindly provided by
Martin Hemler (Dana Farber Cancer Institute, Boston, MA).
Indirect Immunofluorescence
Standard IIF was performed using normal human bulbar conjunctiva
as substrate to test for the presence of anti-BMZ antibodies, as
described earlier.1
2
17
Test reagents included sera from
10 patients with active OCP, IgG fraction, immunoaffinity-purified OCP
autoantibodies, sera from 3 patients with OCP in remission, monoclonal
(10 µg/ml) and polyclonal antibodies to human ß4 integrin (1:100
dilution), and monoclonal antibodies to 6, 5, and ß1 integrins
(10 µg/ml). Phosphate buffered saline (PBS) and normal human serum
(NHS) and sera from patients with chronic active cicatrizing
conjunctivitis secondary to rosacea or atopy served as negative
controls. Positive controls for this assay included sera from patients
with PV and BP. Four-micrometer sections of the conjunctiva were first
incubated at room temperature with normal goat serum 1:20 dilution for
25 minutes and then washed three times with PBS. Primary antibodies
were diluted in PBS-bovine serum albumin 1% in 1:10 to 1:320 dilutions
and incubated at room temperature for 1 hour. The sections were then
washed three times in PBS, incubated at room temperature with the
appropriate fluorescein isothiocyanate (FITC)–conjugated secondary
antibody for 30 minutes, and viewed under a fluorescence microscope.
Incubation with the primary antibody was omitted in PBS control
experiments.
Absorption and Blocking Studies
Blocking studies were performed as follows: Multiple sections of
NHC were preabsorbed for 60 minutes repeatedly with the following six
reagents: sera of patients with OCP that contained high titer (1:1000
by immunoblot) of anti-BMZ antibodies, the IgG fraction from OCP sera,
sera from patients with BP containing antibodies to BPAg1 and BPAg2,
monoclonal antibodies to human 6 integrin, monoclonal antibody to
human ß1 integrin, and normal human serum. These sections were then
washed four times with PBS and incubated with monoclonal or polyclonal
antibodies to the human ß4 integrin. The sections were then stained
with the appropriate FITC-conjugated secondary antibodies and viewed
under a fluorescence microscope to assess binding at the BMZ. In the
reverse experiment, multiple sections of NHC were repeatedly
preabsorbed with monoclonal and polyclonal antibodies to human ß4
integrin, washed four times with PBS, and reacted with the six reagents
listed earlier in the paragraph. The sections were washed and then
stained with the appropriate FITC-conjugated secondary antibodies and
viewed under the fluorescence microscope for BMZ staining.
In Vitro Culture of Normal Human Conjunctiva
The in vitro model used to evaluate the effects of specific
reagents in the pathogenesis of OCP was based on earlier models that
used skin in organ culture to study the effects of PV autoantibody on
intercellular adhesion.18
19
Pieces of NHC (2–3
mm2 ) obtained during cataract surgery were
floated in 2 ml complete RPMI-1640 medium in a 12-well tissue culture
plate immediately after surgical excision. Three different
concentrations of the test reagent were studied. Total serum (10%,
20%, and 30% by volume); purified IgG fractions; 100, 200 and 300
µg of immunoaffinity-purified autoantibodies from sera of patients
with OCP; and 10, 20, and 50 µg of anti-ß4 and anti-6 integrin
antibodies were added to the wells and incubated at 37°C with 5%
CO2 for 12 to 72 hours. These optimal volumes and
concentrations were determined by pilot experiments using the three
concentrations to titrate the ability of test reagents to produce BMZ
separation. In these pilot experiments, the cultures were terminated at
12, 24, 48, 72, and 96 hours. The conjunctival sections were processed
for routine histology using hematoxylin and eosin stain and were
examined for subepidermal blister formation. There was some degree of
variability in the BMZ separation, but the best results were seen in
cultures that were incubated for at least 48 hours. NHS and normal
human IgG and sera of three patients with PV and three patients with BP
with high titers of antibodies to BPAg1 and BPAg2, two patients with
chronic active cicatrizing conjunctivitis secondary to rosacea or atopy
were used as controls. Sera from three patients with disease in
prolonged clinical remission who had received no therapy for 3 years
were also evaluated. Sera of the patients with OCP in clinical
remission did not show the presence of anti-BMZ binding using
immunoblot assays.13
After incubation, the tissue samples
were examined by routine histology, using hematoxylin and eosin
staining procedures. The experiment was repeated five times using the
same panel of reagents.
Blocking of Blister Formation in an In Vitro Model
OCP sera, immunoaffinity-purified OCP antibody, antibody to human
ß4 integrin, and sera from patients with PV were absorbed with
lysates from ß4 integrin–expressing cell lines (UM-SC-22), as
previously described6
7.
Thereafter, they were tested in
the immunoblot assay for the presence of antibody to human ß4
integrin.6
These antibodies were then used in the in vitro
organ culture system for BMZ separation, as described earlier.
Sera from patients with active PV and with high titers of PV autoantibody were absorbed with UM-SC-22 cell lines. Absorbed and nonabsorbed PV sera were used in the in vitro NHC culture model.
Results
Indirect Immunofluorescence
NHC sections incubated with sera from patients with OCP showed
homogenous, continuous linear BMZ staining indistinguishable from that
observed during direct immunofluorescence analysis of conjunctiva from
9 of 10 patients with OCP who had active disease (Fig. 1)
. Strikingly similar observations were made when NHC was incubated with
purified IgG fractions of sera from patients with active OCP, with
immunoaffinity-purified OCP antibodies, with monoclonal and polyclonal
antibodies to human ß4 integrin, and with monoclonal antibodies to
the 6 integrin. The monoclonal antibody to ß4 integrin exhibited
more intense staining than did the polyclonal antibody to ß4 integrin
and OCP sera. Sera of patients with active PV showed binding to
intercellular spaces between the epithelial cells of the NHC (data not
shown). Sera of patients with active BP that contained antibodies to
BPAg1 and BPAg2, determined by immunoblot analysis, showed smooth
linear binding to BMZ of NHC sections (data not shown). The marked
reduction in background was caused by prior absorption of the NHC
sections with goat serum for the IIF assay and NHS for the blocking
experiments. No binding was observed in sections of NHC incubated with
normal human serum and PBS (Fig. 1)
and normal human IgG (Fig. 2
D). No staining was observed with monoclonal antibodies to the 5 or
ß1 integrins or sera of patients with chronic active cicatrizing
conjunctivitis other than OCP.
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6 integrin in the BMZ. Absorption of NHC sections with
high-titer BP sera reduced but did not eliminate the binding of
antibodies to 6 and ß4 integrins. Binding of the PV sera to the
intercellular cement substance of the NHC was unaffected by earlier
absorption of NHC sections with both OCP sera and antibodies to ß4
integrin (data not shown).
In Vitro Culture of Normal Human Conjunctiva
No morphologic changes were observed before 24 hours of culture.
NHC cultured for 48 to 72 hours showed subepithelial separation from
the BMZ in the presence of OCP sera, IgG fractions from sera of
patients with OCP, immunoaffinity-purified antibody from OCP sera, but
not in the presence of NHS (Fig. 3)
, sera from patients with chronic active cicatrizing conjunctivitis
secondary to rosacea or atopy, or sera from patients with OCP in
clinical remission. The effect of treatment with OCP sera,
immunoaffinity-purified OCP antibody, and antibody to ß4 integrin on
induction of BMZ separation is summarized in Table 1
. The data presented are the mean results of five experiments, using the
same panel of reagents. In treatment with 10% volume of OCP sera, only
5% to 10% of explants showed BMZ separation after 48 to 72 hours of
incubation. The maximum explants showing BMZ separation (60%–65%)
were observed in treatment with a 30% volume of OCP sera during 48 to
72 hours of incubation. In treatment with 100, 200, and 300 µg of
immunoaffinity-purified OCP antibody, no BMZ separation was observed
during a 12- to 24-hour incubation period. However, BMZ separation was
observed in 45% to 50% of explants when treated with 300 µg
immunoaffinity-purified OCP antibody and incubated for 48 to 72 hours.
In treatment with 10, 20, and 50 µg of the monoclonal antibody to
human ß4 integrin, no BMZ separation was observed in the explants
incubated for 12 to 24 hours. Using 20 µg, only 15% to 20% of
explants showed BMZ separation during 48 to 72 hours’ incubation.
Forty percent to 50% of explants showed BMZ separation when treated
with 50 µg of antibody to ß4 integrin and incubated for 48 to 72
hours. Prominent tissue necrosis was observed in explants incubated for
96 hours. BMZ separation was observed in sections of organ culture of
NHC incubated with antibodies to human ß4 integrin (Fig. 4)
.
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5, 6, and ß1
human integrins or antibodies to BPAg1 or BPAg2. Sections of NHC
cultures with serum from patients with acute PV showed typical
acantholysis (Fig. 4)
. This positive control indicates the validity of
the experimental design and technique. No BMZ separation was observed
in the presence of NHS, normal human IgG, and sera of patients with OCP
in prolonged clinical remission.
Blocking of Blister Formation in an In Vitro Model
OCP sera, immunoaffinity-purified OCP antibodies, and
antibody to human ß4 integrin absorbed with lysates of ß4
integrin–expressing cell lines (UM-SC-22) did not produce blister
formation in the in vitro model. These antibodies did not bind to the
205-kDa protein in an immunoblot assay using normal human conjunctiva
and skin as substrate.
Sera from patients with PV demonstrated binding to 130-kDa protein in the same substrates. When sera from patients with PV were similarly absorbed with lysate of ß4 integrin–expressing cell lines and used in the in vitro model, it resulted in production of acantholysis of conjunctival epithelial cells, identical with that produced by unabsorbed sera. No BMZ separation was seen in NHC cultures treated with absorbed or unabsorbed PV sera.
Discussion
In this study, we focused on two specific issues in the pathogenesis of OCP. First is the observation that the autoantibody in OCP was targeted against human ß4 integrin in the conjunctival BMZ. Second, an in vitro organ culture model provided evidence that OCP autoantibodies and antibodies to human ß4 integrin were capable of causing conjunctival BMZ separation that histologically resembled OCP. We realize that unequivocal demonstration of blister formation may not be possible in many patients with OCP. Nonetheless, this model provided an opportunity to study the mechanism of separation of epithelial cells from underlying subepithelial structures and in so doing to provide indirectly information on factors that contribute to the integrity of conjunctival BMZ.
Indirect immunofluorescence and blocking experiments using IIF assays
clearly indicated that OCP sera, IgG fractions from OCP sera, and
immunoaffinity-purified OCP autoantibodies from OCP sera bound to the
target antigen identically. The IIF assay also showed that conjunctival
BMZ contained 6, BPAg1, and BPAg2, along with other integrins and
adhesion molecules, and that OCP sera did not cross-react with them.
This experimental strategy was similar to that previously published in
studying the process of acantholysis in PV. In those studies, sera from
patients with PV bound to the intercellular cement substance
(desmoglein III) of the skin in a pattern identical with that seen in
vivo in patients with PV. The validity of our model is further enforced
by the observation that when NHC was incubated with serum from patients
with PV, the pemphigus autoantibody bound in a manner identical with
that seen on the normal human epidermis or in vivo in patients with PV.
Furthermore, BP sera bound to conjunctival BMZ in a smooth linear
manner identical with that of the skin. Normal human sera and sera of
patients with other cicatrizing ocular diseases did not bind to the
conjunctiva in a similar pattern. Observations in this report may
suggest that the subset of patients with OCP presented herein may be a
distinct subset, because they had a high frequency of extra ocular
involvement and universal presence of immunoreactants on conjunctival
BMZ. Therefore, it appears that the possibility of such patients having
a higher concentration of circulating autoantibodies in their sera to
different specificities was more likely to be detected in our assay
system than from other patients with OCP.
Interestingly, several investigators have demonstrated that sera of patients with OCP contain antibodies to BPAg1 and BPAg2. However, our studies clearly indicated that the antibodies to BPAg1 and BPAg2 did not block the binding of the antibodies to conjunctival BMZ by OCP sera. The binding sites for BP antibodies and CP antibodies were on the different epitopes of BPAg2.20
Studies indicate that ß4 and 6 form a heterodimeric molecule
associated with hemidesmosomes.21
Yet, our blocking
experiments demonstrated that, within the limitations of the IIF assay,
OCP sera and anti-ß4 antibodies did not block the binding of
anti-6 integrin antibody to conjunctival BMZ. This observation
further supports the hypothesis that production of anti-ß4 antibodies
in patients with OCP is a specific, early, disease-related event and is
not a consequence of damage or immune injury. Additional support for
this notion of a pathogenic role of anti-ß4 integrin antibodies in
OCP comes from the observation that such antibodies were not present in
the sera of patients with disease in prolonged clinical remission who
were receiving no systemic therapy.
In our in vitro conjunctival organ culture model, antibodies to ß4
integrin were capable of causing BMZ separation that was histologically
similar to that produced by incubation of NHC with OCP sera, IgG from
patients with OCP, and immunoaffinity-purified OCP antibodies. These
microvesicles created in vitro had histology fairly identical with or
similar to that observed in the conjunctiva of patients with OCP early
in the course of the disease. Interestingly, antibody to 6 integrin
did not produce any microvesicles, indicating that antibody to ß4
integrin was specific in its action. Further evidence for the
specificity of antibody to human ß4 integrin–producing blister came
from the blocking experiments. OCP sera, immunoaffinity-purified OCP
antibodies, and antibody to human ß4 integrin absorbed with lysate of
ß4 integrin–expressing cell lines did not produce lesions seen in
incubation with unabsorbed antibodies. These experiments, therefore,
within the limitation of the organ culture system, provide additional
potent evidence that anti-ß4 integrin antibody may play an important
role in the production of vesicles or bullae in the conjunctiva of
patients with OCP.
The concept that this in vitro model is a reasonable mechanism to study events that may mimic the in vivo phenomenon in tissue dysadhesion is valuable. Evidence for such a hypothesis comes from the experiments in which typical acantholysis, which is a histologic hallmark of PV, was observed in conjunctiva incubated with PV sera. The results observed in the conjunctiva were identical with previously published observations when normal human skin was incubated with PV sera.19 Absorption of PV sera with ß4-expressing cell lines did not affect the ability of PV sera to produce acantholysis. The earlier in vitro model culture for the study of PV eventually provided valuable insights in understanding the molecular pathogenesis of PV.
In our conjunctival organ culture, we did not observe any vesicle formation when NHC was incubated with BP sera. Using normal human skin, Gammon et al.22 have advanced our understanding of the pathogenesis of BP, showing that BP sera alone is incapable of causing skin BMZ separation but requires the addition of complement and polymorphonuclear leukocytes for lesion production. The data on the relationship between the duration of incubation and concentration of antibodies indicate that these events require time for the biologic processes to evolve. The process appeared to be dose dependent, indicating that an appropriate concentration of antibody is required to bind to available antigen sites to accomplish the separation of the epithelium from the submucosa. Similarly, the process of blister formation was time dependent, because no separation was seen in 12 to 24 hours. Our organ culture model permits the evaluation of the specific roles of polymorphonuclear leukocytes, complement, cytokines, and other biologic agents in the process of BMZ separation mediated by an autoantibody.
The pathogenesis of OCP is a complicated process that is probably
heterogeneous and may involve multiple immunologic events and biologic
agents. Our in vitro organ culture model is simply one that allowed us
to study the binding of circulating antibodies to the conjunctiva and
the phenomenon associated with the separation of epithelial cells from
the underlying submucosa and tissue matrix. There are no animal models
for OCP. At least two interesting possible explanations emerge from our
observations on BMZ separation: first, that autoantibody in patients’
sera or exogenously added anti-ß4 antibody dissolves ß4 integrin
and that vesicles are a result of physical dissolution of the binding
of ß4 integrin molecule to its ligand; and second, that binding of
antibody to ß4 integrin on the conjunctival epithelial cell surface
may trigger an intracellular signal that ultimately results in the
movement of the epithelial cells away from the basement membrane, a
consequence of multiple intracellular events. Precedence for such
observations has been reported recently. Antibodies to 6ß4
integrin heterodimer can influence the movement of tumor
cells.23
Earlier observations in the study of PV
demonstrated that the breaking of cell adhesion in the epidermis
(acantholysis) is not caused by dissolution of desmoglein III, but
rather by activation of plasminogen to plasmin.24
This
activation occurs as a direct consequence of unregulated serine
protease production in the suprabasal epidermal cells. The signal for
the upregulation of serine protease production is generated by the
binding of PV antibodies to desmoglein III on epidermal cell surfaces.
The in vitro model presented in this study allows investigators to
examine closely the consequences of the binding of anti-ß4 integrin
antibodies to ß4 integrin on conjunctival epithelial cell surfaces
and the processes that occur secondary to such binding.
Acknowledgements
The authors thank Tong-zhen Zhao in the Hilles Laboratory of the Massachusetts Eye and Ear Infirmary for valuable help.
Footnotes
RYC and KB contributed equally to this study.
Supported by Grant EY08378 from the National Institutes of Health.
Submitted for publication January 14, 1999; revised April 5, 1999; accepted May 3, 1999.
Proprietary interest category: N.
Corresponding author: A. Razzaque Ahmed, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115.
References
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6ß4 integrin is a receptor for both laminin and kalinin Exp Cell Res 211,360-367[Medline][Order article via Infotrieve]
6ß4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures J Cell Biol 139,1873-1884This article has been cited by other articles:
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 E. Letko, K. Bhol, F. Anzaar, V. L. Perez, A. R. Ahmed, and C. S. Foster Chronic Cicatrizing Conjunctivitis in a Patient With Epidermolysis Bullosa Acquisita. Arch Ophthalmol, November 1, 2006; 124(11): 1615 - 1618. [Abstract] [Full Text] [PDF]
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 C. Sitaru, A. Kromminga, T. Hashimoto, E. B. Brocker, and D. Zillikens Autoantibodies to Type VII Collagen Mediate Fc{gamma}-Dependent Neutrophil Activation and Induce Dermal-Epidermal Separation in Cryosections of Human Skin Am. J. Pathol., July 1, 2002; 161(1): 301 - 311. [Abstract] [Full Text] [PDF]
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 L. S. Chan, A. R. Ahmed, G. J. Anhalt, W. Bernauer, K. D. Cooper, M. J. Elder, J.-D. Fine, C. S. Foster, R. Ghohestani, T. Hashimoto, et al. The First International Consensus on Mucous Membrane Pemphigoid: Definition, Diagnostic Criteria, Pathogenic Factors, Medical Treatment, and Prognostic Indicators Arch Dermatol, March 1, 2002; 138(3): 370 - 379. [Abstract] [Full Text] [PDF]
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 K.C. Bhol, L. Goss, S. Kumari, J.E. Colon, and A.R. Ahmed Autoantibodies to Human {alpha}6 Integrin in Patients with Oral Pemphigoid Journal of Dental Research, August 1, 2001; 80(8): 1711 - 1715. [Abstract] [PDF]
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 S. Kumari, K. C. Bhol, R. K. Simmons, M. S. Razzaque, E. Letko, C. S. Foster, and A. R. Ahmed Identification of Ocular Cicatricial Pemphigoid Antibody Binding Site(s) in Human {beta}4 Integrin Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 379 - 385. [Abstract] [Full Text]
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 K. C. Bhol, M. J. Dans, R. K. Simmons, C. S. Foster, F. G. Giancotti, and A. R. Ahmed The Autoantibodies to {alpha}6{beta}4 Integrin of Patients Affected by Ocular Cicatricial Pemphigoid Recognize Predominantly Epitopes Within the Large Cytoplasmic Domain of Human {beta}4 J. Immunol., September 1, 2000; 165(5): 2824 - 2829. [Abstract] [Full Text] [PDF]
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