high rates of local side effects were found. 8,9 Our current data raise the possibility that virus-specific IgE could result in adverse responses to future vaccinations or exposures. Of note, in the Atopic Dermatitis Vaccinia Network Registry, ADEH1 subjects reported more adverse effects from primary VZV infection or vaccination than nonatopic subjects (unpublished data, Zaccaro, Atopic Dermatitis Vaccinia Network, May 2010). Subjects with AD have an increased susceptibility to viral skin infections, and we anticipated that cell-mediated immunity to VZV vaccine would be reduced in subjects with moderate to severe AD. However, we found that the control subjects and subjects with AD had similar cellular responses. Future studies of varicella may yield larger differences by enrolling more ADEH1 patients. We were surprised to see the wide variation in cellular responses in both groups and the effect of the timing of the blood draw after VZV vaccination. In summary, controls and subjects with AD had similar cell- mediated responses to the VZV vaccine. However, ADEH1 subjects demonstrated higher VZV-specific IgE, a possible risk factor for adverse effects to booster doses of vaccine or to wild- type VZV exposure. We thank the supervising study coordinator, Irene Borras-Coughlin, and administrative assistant, Jeanne Testa, at Children’s Hospital Boston and the pediatric offices that referred subjects including Longwood Pediatrics and the Children’s Hospital Primary Care Center in Boston, Mass. Lynda Schneider, MD a Adriana Weinberg, MD b Mark Boguniewicz, MD b,c Patricia Taylor, NP c Hans Oettgen, MD, PhD a Lisa Heughan, BS a Daniel Zaccaro, MS d Brian Armstrong, MPH d Aaron Holliday, BS d Donald Y. M. Leung, MD, PhD b,c From a Children’s Hospital Boston, Mass; b the University of Colorado, Denver, Colo; c National Jewish Health, Denver, Colo; and d Rho Inc, Chapel Hill, NC. E-mail: [email protected]. Supported by federal funds from the National Institute of Allergy and Infectious Dis- eases, National Institutes of Health, Department of Health and Human Services, under contract nos. N01 AI40029 and N01 AI40033, as well as the Clinical Translational Scientific Award UL1 RR025780 (National Jewish) and UL1 RR025758-01 from the National Center for Research Resources, National Institutes of Health, to the Har- vard Catalyst Clinical and Translational Science Center (Harvard Catalyst). Disclosure of potential conflict of interest: L. Schneider has received research support from the NIH/NIAID Atopic Dermatitis Vacinnia Network. A. Weinberg has received grants and consulting fees from Merck & Co. H. Oettgen is a consultant and scientific advisor for Schering Plough, is a consultant for Genentech, and has received research support from Novartis. The rest of the authors have declared that they have no conflict of interest. REFERENCES 1. Boguniewicz M, Leung DYM. Recent insights into atopic dermatitis and implications for management of infectious complications. J Allergy Clin Immunol 2010;125:4-13. 2. Beck LA, Boguniewicz M, Hata T, Schneider LC, Hanifin J, Gallo R, et al. Pheno- type of atopic dermatitis subjects with a history of eczema herpeticum. J Allergy Clin Immunol 2009;124:260-9. 3. Gao PS, Rafaels NM, Hand T, Murray T, Boguniewicz M, Hata T, et al. Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpe- ticum. J Allergy Clin Immunol 2009;124:507-13. 4. Gao PS, Rafaels NM, Mu D, Hand T, Murrau T, Boguniewicz M, et al. Genetic var- iants in thymic stromal lymphopoietin are associated with atopic dermatitis and ec- zema herpeticum. J Allergy Clin Immunol 2010;125:1403-7. 5. Kreth HW, Hoeger PH. Members of the VZV-AD Study Group. Safety, reactogenic- ity and immunogenicity of the live attenuated varicella vaccine in children between 1 and 9 years of age with atopic dermatitis. Eur J Pediatr 2006;165:677-83. 6. Smith JG, Liu X, Kaufhold RM, Clair J, Caulfield MJ. Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin Diagn Lab Immunol 2001;8:871-9. 7. Kumar A, Grayson MH. The role of viruses in the development and exacerbation of atopic disease. Ann Allergy Asthma Immunol 2009;103:181-6. 8. Nagel JE, Whie C, Lin MS, Fireman P. IgE synthesis in man, II: comparison of tet- anus and diphtheria igE antibody in allergic and nonallergic children. J Allergy Clin Immunol 1979;63:308-14. 9. Mark A, Bjorksten B, Granstrom M. Immunoglobulin IgE responses to diphtheria and tetanus toxoids after booster with aluminium-adsorbed and fluid DT-vaccines. Vaccine 1995;13:669-73. Available online October 2, 2010. doi:10.1016/j.jaci.2010.08.010 A new case of homozygous C1-inhibitor defi- ciency suggests a role for Arg378 in the con- trol of kinin pathway activation To the Editor: Hereditary angioedema (HAE) caused by C1-inhibitor (C1-Inh) deficiency is a rare disease with an autosomal dominant pattern of FIG 2. Total IgE level (A), VZV-specific IgE (B), and VZV-specific IgG (C) in control subjects and subjects with AD without a history of EH (ADEH2) and with a history of EH (ADEH1). J ALLERGY CLIN IMMUNOL VOLUME 126, NUMBER 6 LETTERS TO THE EDITOR 1307
Transcript
FIG 2. Total IgE level (A), VZV-specific IgE (B), and VZV-specific IgG (C) in control subjects and subjects with
AD without a history of EH (ADEH2) and with a history of EH (ADEH1).
J ALLERGY CLIN IMMUNOL
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LETTERS TO THE EDITOR 1307
high rates of local side effects were found.8,9 Our current dataraise the possibility that virus-specific IgE could result in adverseresponses to future vaccinations or exposures. Of note, in theAtopic Dermatitis Vaccinia Network Registry, ADEH1 subjectsreported more adverse effects from primary VZV infection orvaccination than nonatopic subjects (unpublished data, Zaccaro,Atopic Dermatitis Vaccinia Network, May 2010).Subjects with AD have an increased susceptibility to viral skin
infections, and we anticipated that cell-mediated immunity toVZV vaccine would be reduced in subjects with moderate tosevere AD. However, we found that the control subjects andsubjects with AD had similar cellular responses. Future studies ofvaricella may yield larger differences by enrolling more ADEH1patients. We were surprised to see the wide variation in cellularresponses in both groups and the effect of the timing of the blooddraw after VZV vaccination.In summary, controls and subjects with AD had similar cell-
mediated responses to the VZV vaccine. However, ADEH1subjects demonstrated higher VZV-specific IgE, a possible riskfactor for adverse effects to booster doses of vaccine or to wild-type VZV exposure.
We thank the supervising study coordinator, Irene Borras-Coughlin, and
administrative assistant, Jeanne Testa, at Children’s Hospital Boston and the
pediatric offices that referred subjects including Longwood Pediatrics and
the Children’s Hospital Primary Care Center in Boston, Mass.
Lynda Schneider, MDa
Adriana Weinberg, MDb
Mark Boguniewicz, MDb,c
Patricia Taylor, NPc
Hans Oettgen, MD, PhDa
Lisa Heughan, BSa
Daniel Zaccaro, MSd
Brian Armstrong, MPHd
Aaron Holliday, BSd
Donald Y. M. Leung, MD, PhDb,c
From aChildren’s Hospital Boston, Mass; bthe University of Colorado, Denver, Colo;cNational Jewish Health, Denver, Colo; and dRho Inc, Chapel Hill, NC. E-mail:
FIG 1. Biochemical and functional studies on the R378C mutant: Western blot of fresh plasma samples of
the family members showing the native (105 kd) and cleaved/latent (96 kd) forms of C1-Inh. Equal plasma
volumes were loaded in each lane. Normal human plasma (NHP) and purified C1-Inh (Berinert P) are shown
as reference (A). C1-Inh’s interaction with C1s protease and kallikrein was examined by means of ELISA of
Cos-7 cell supernatants (20 ng of protein per lane) and plasma samples (B).
J ALLERGY CLIN IMMUNOL
DECEMBER 2010
1308 LETTERS TO THE EDITOR
inheritance. Clinically, HAE is characterized by episodes oflocalized swelling in the limbs, face, gut, or upper respiratorytract.1 Those affected are generally heterozygous for mutations inthe C1-inhibitor gene (C1NH), which result in either low levels ofcirculating C1-Inh (HAE type I) or normal or increased levels of anonfunctional C1-Inh (HAE type II). Acquired forms of angioe-dema (AAE) caused by accelerated C1-Inh consumptionassociated with lymphoproliferative disorders or anti–C1-Inhautoantibodies have also been described.2 HAE and AAE clinicalmanifestations are almost identical, but distinctive biochemicaltraits of patients with AAE include low amounts of C1q, C1r,and C1s proteins in serum.C1-Inh is a serum glycoprotein belonging to the serpin
superfamily of protease inhibitors, the main function of whichis to prevent the activation of the classical pathway of comple-ment, as well as the activation of kinin generation, coagulation,and fibrinolytic cascades.3
Serpins are globular proteins with a conserved structure of 9a-helices and 3 b-pleated sheets.Their reactive center is located in an exposed flexible loop
referred to as the reactive center loop (RCL). They act on theirtarget proteases by means of a suicide-substrate mechanisminvolving the cleavage of the RCL and its insertion intob-sheet A.As a result, conformational changes take place in the serpins thatultimately trap and inactivate the targeted protease. Nonphysio-logical conformations involving RCL insertion and leading tolatent states, multimeric states, or both have been attributed toseveral naturally occurring mutations.4
Although more than 200 different heterozygous mutationsleading to HAE have been described, only 2 homozygous C1-Inh–deficient subjects, 2 siblings of consanguineousorigin carryingthe I440S missense mutation, have been identified. Those patientsproduced a nonfunctional protein and exhibited an unusual diseasephenotype, with mild symptomatology and complement consump-tion. Moreover, they had a total absence of circulating C1q, thusresembling AAE.5
Herein we describe a new case of homozygous C1-Inhdeficiency in a patient with severe angioedema lacking circulatingC1q. Genetic and functional characterization of the patientshowed that he carries an R378C substitution and that this is aunique alteration resulting in partial inability of C1-Inh to controlkallikrein activation.The patient is a 33-year-old man who, for the last 10 years, has
experienced attacks of angioedema involving the face andextremities. He has had several episodes of laryngeal edemawith ventilatory compromise. Biochemical study of the comple-ment proteins (C1-Inh, C4, and C3 antigenic levels) wasperformed by means of nephelometry, C1-Inh function wasdetermined by a specific colorimetric assay (Berichrom; Siemens,Berlin, Germany), and C1q was measured by means of radialimmunodiffusion (The Binding Site, Birmingham, United King-dom). Complement profile in the propositus revealed valuesconcordant with a C1-Inh deficiency (C1-Inh, 4.38 mg/dL; C1-Inh function, 11%; C4, 1.44 mg/dL) and undetectable C1q,suggesting consumption of the classical pathway of complement.No lymphoproliferative disorder or anti–C1-Inh or anti-C1q
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autoantibodies were detected. Five additional relatives (all ofthem asymptomatic) were recruited for this study. Four of themhad values consistent with HAE type II, and only 1 relative (GZ7;III.1), a 3-year-old girl, had C1-Inh levels and function within thenormal range (see Fig E1 in this article’s Online Repository atwww.jacionline.org).Once a diagnosis ofHAEwas established, the propositus started
prophylactic treatment with the attenuated androgen stanozolol(12mg/12 hours). Clinical examinationwas performed, and bloodsamples were obtained before treatment and at 21 and 28 monthsafter treatment.After written informed consent was obtained, genetic studies of
theC1NH locuswere performed in all the familymembers, as pre-viously described.5 Sequence analysis revealed the homozygoussubstitution c.1198C>T (R378C) in exon 7 of the propositus.All of his relatives were heterozygous for the mutation (see FigE1). Data from multiplex ligation-dependent probe amplificationof the propositus ruled out a possible genomic deletion affectingthe C1NH gene and confirmed homozygosity (data not shown).Biochemical characterization of the patient’s C1-Inh was done
by means of Western blotting of fresh plasma samples. Undernonreducing conditions, native (105 kd) and inactive (cleavedor latent, 96 kd) forms of C1-Inh can be resolved. Interestingly,in the present R378C homozygote, and as reported for the other2 previously described I440S missense homozygotes,5 plasmaC1-Inh circulates almost exclusively in its cleaved or latent state.All the heterozygous relatives studied had both forms of circulat-ing C1-Inh (Fig 1).
We studied the functionality of the R378C C1-Inh mutant bymeasuring complex formation with biotinylated C1s and kalli-krein proteases by means of ELISA of plasma samples andsupernatants of stably transfected Cos-7 cells. For the plasmaassays, samples from an I440S homozygote and a patient withAAE were also analyzed. C1-Inh constructs for the Cos-7experiments were built on the previously described C1-Inhminigene.6 A mutant clone lacking exon 8 (DelEx8) was usedas a negative control for all the transfection assays.The R378C mutant protein binds C1s normally but exhibits a
strongly decreased capacity to associate with kallikrein both inplasma and Cos-7 supernatants. No complexes between C1-Inhand target proteases could be detected in the I440S, AAE, andDelEx8 samples (Fig 1).
This unexpected result prompted us to analyze the conforma-tional alterations in the R378C mutant. Cleaved and latent formsof C1-Inh can be distinguished, taking advantage of the previ-ously described, conformational-specific mAbs KII and KOK12(kindly provided by Dr Diana Wouters, Sanquin Research,Amsterdam, The Netherlands). KII specifically recognizescleaved inactivated C1-Inh, whereas KOK12 reacts with cleavedC1-Inh, complexed/latent C1-Inh, or both.7 KII signal in thehomozygous R378C patient’s plasma was higher than in normalhuman plasma and comparable with that obtained in the I440Shomozygote (an HAE type II mutation) and AAE samples. Sim-ilar detection patterns were obtained for both mutant proteinswhen screened with the KOK12 antibody (data not shown).The results of the I440S homozygote are consistent with
complete lack of functionality because of reactive-site cleavage.In the case of the R378C homozygote and considering its normalbinding to C1s, the detection of this protein by both the KII andKOK12 antibodies suggests that the R378C mutation creates alatent-like epitope recognizable by these 2 antibodies. Because
only the 96-kd band was detected in this patient by means ofWestern blotting, we hypothesize that the C1-Inh mutant mightundergo a partial insertion of the RCL into b-sheet A.
One striking similarity among both the R378C and I440Sfamilies is the absence of HAE-related symptomatology in theheterozygous subjects. However, unlike the I440S homozygotes,who only reported mild clinical manifestations, the R378Chomozygous patient described here did have recurrent and severeattacks of angioedema before treatment. Yet on prophylactictreatment with stanozolol, a decrease in the frequency and severityof symptoms was achieved in the R378C homozygote. Thisclinical amelioration coincided with a partial recovery of thepatient’s complement profile. Specifically, C1-Inh, C4, and C2concentrations were recovered to normal levels in the 2 samplesanalyzed after attenuated androgen therapy (posttreatmentsamples 1 and 2). C1q concentrations remained undetectable inposttreatment sample 1 and were only restored to normal values(10.3 mg/dL) in the second posttreatment sample (see Fig E2 inthis article’s Online Repository at www.jacionline.org).A broader discussion can be found in Appendix E1 in this article’sOnline Repository at www.jacionline.org.
Our results further characterize the complement profile associ-ated with homozygous mutations in the C1NH gene and suggestthat suspicion of homozygous deficiency cannot rely on the pres-ence of mild symptomatology. Additionally, we provide insightson the biochemical function of the R378C mutation, a substitutionthat induces a latent-like conformation of C1-Inh that specificallyinhibits kallikrein binding, suggesting that the Arginine 378 posi-tion is of strategic importance for C1-Inh control of kininformation.
We thank Dr �Angeles Rico D�ıaz (Hospital Juan Canalejo, ACoru~na, Spain)
andDrGabriel Barderas (Centro de SaludColladoVillalba,Madrid, Spain) for
referring the patients and Dr Diana Wouters (Department of Immunopathol-
ogy, Sanquin Research, Amsterdam, The Netherlands) for providing the KII
and KOK12 mAbs.
Alberto L�opez-Lera, MSa,b,c
Bertrand Favier, PhDd
Roc�ıo Mena de la Cruz, MSa,b,c
Sof�ıa Garrido, BSa,c
Christian Drouet, PhDd
Margarita L�opez-Trascasa, PhDa,b,c
From athe Immunology Unit, Hospital Universitario La Paz, Madrid, Spain; bCentro de
Investigaciones Biom�edicas en Red (CIBERER, Madrid, Spain) U-754; cHospital La
Paz Health Research Institute (IdiPAZ, Madrid, Spain); and dGREPI/TIMC-IMAG
CNRS UMR 5525, Universit�e J. Fourier and Unit�e d’Exploration de l’Angioed�eme,
Available online September 23, 2010.doi:10.1016/j.jaci.2010.07.037
Lymphoid neogenesis in the giant papillae ofpatients with chronic allergic conjunctivitis
To the Editor:Lymphoid neogenesis (tertiary lymphoid organ [TLO]) resem-
bles a secondary lymphoid organ (eg, the lymph node) and isobserved in patients with chronic inflammatory diseases.1 TLOsplay roles in immune responses against persistent antigens; how-ever, they can also induce severe tissue damage.1 It has also beenreported that the germinal centers and follicular dendritic cell(FDC) networks in TLOs relate to class-switch recombination.2
In this studywe examined the lymphoid neogenesis and lymphaticvessel formation in patients with atopic keratoconjunctivitis
TABLE I. Clinical information of the patients and summary of the res
Patient no. Age (y) Sex Total IgE (IU/mL) Spe
1 16 F 509 Pos
2 22 M 89 Pos
3 13 M 2,319 Pos
4 18 M 375 Pos
5 17 M 17,260 Pos
6 21 M 1,904 Pos
7 16 M 3,763 Pos
8 34 M 22,800 Pos
9 45 F 28 Neg
10 29 M 56 Pos
*AKC was defined as a bilateral chronic inflammation of the conjunctiva and lids associa
inflammatory condition of the conjuctiva found in subjects predisposed by their atopic back
excluded from the VKC diagnosis.
�Large lymphocyte clusters were defined as those having more than 10 lymphocytes in le
TABLE II. List of antibodies used in this study
Antigen Class
Anti–LYVE-1 Rabbit polyclonal
Anti-CD3 Murine monoclonal
Anti-CD20 Rabbit monoclonal
Anti-CD35 Murine monoclonal
Anti-PNAd Rat monoclonal (IgM)
Alexa 488–anti-mouse IgG Donkey polyclonal
Alexa 594–anti-rabbit IgG Donkey polyclonal
Alexa 488–anti-rabbit IgG Goat polyclonal
Alexa 594–anti-rat IgM Goat polyclonal
(AKC) and vernal keratoconjunctivitis (VKC),3 severe chronicforms of allergic conjunctivitis. AKC and VKC are often accom-panied by giant papillae formation in tarsal conjunctivae and/ormarked inflammatory cell infiltration in the limbal region, withprominent TH2 cytokine profiles.3 We found various degrees oflymphoid neogenesis in the giant papillae with B-cell clusters,FDCs, and a marginal T-cell zone essential for TLO formation.1
The IgE class of immunoglobulin is produced by B cells andplasma cells through class-switch recombination in the germinalcenter of lymphoid tissues.2 Preferential expression of the FceRIb-chain protein, an amplifier of allergic responses, has been ob-served in the giant papillae of patients with AKC/VKC.4 Thesefindings suggested the role of an IgE–FceRIb cascade in the path-ophysiology of severe chronic allergic conjunctivitis. The IgEmight originate from tears5 or from blood flow; however, localIgE class-switch recombination in the giant papillae itself, aswell as in the nasal polyps,6 might also be possible. RegionalIgE expression at the mast cells, dendritic cells, and Langerhanscells of the giant papillae was reported previously.7 Therefore itis important to clarify the origin of IgE production in patientswith AKC/VKC because anti-IgE antibody therapy has recentlybeen introduced for the treatment of severe atopic asthma, andit might also be effective for AKC/VKC.Ten giant papillae obtained from patients with AKC/VKC were
resected for a therapeutic purpose, as previously described.4 Theclinical information of the patients is summarized in Table I. Allthe patients had chronic allergic conjunctivitis and were treatedwith topical dexamethasone eye drops for at least 4 weeks beforesurgery. Total IgE concentrations and specific IgE titers against26 common antigens were measured by SRL, Inc (Tokyo, Japan),
ults
cific IgE Diagnosis* CD201 cell clustery FDC
itive VKC Large clusters 1itive VKC Diffuse 1itive VKC Large clusters 1itive AKC Diffuse and large clusters 1itive AKC Large clusters 1itive AKC Small clusters 1itive AKC Large clusters 1itive AKC Large clusters 1ative AKC Small clusters 1itive VKC Small clusters 1
ted with atopic dermatitis. VKC was defined as a bilateral, chronic, conjunctival
ground. Patients who had atopic dermatitis or corneal stromal neovascularization were
ngth/diameter.
Dilution Supplier
1:400 RELIATech, Wolfenb€uttel, Germany
1:100 DAKO, Glostrup, Denmark
1:200 Epitomics, South San Francisco, Calif
1:50 Dako
1:25 Becton Dickinson, Franklin Lakes, NJ
1:1,000 Invitrogen, Carlsbad, Calif
1:1,000 Invitrogen
1:1,000 Invitrogen
1:1,000 Invitrogen
APPENDIX E1
The results in the present work are in agreement with previousreports concerning attenuated androgen treatment in patients withHAE. Androgens are known to increase C1-Inh and C4 antigeniclevels in plasma of heterozygous patients with HAE.E1 Moreover,it has been shown that androgen treatment increases C1-InhmRNA expression in PBMCs.E2 Increased levels and functionof C1-Inh might restore local homeostasis between the inhibitorand its target proteases, thus controlling the activation of the clas-sical pathway of complement in the focus of edema. Nevertheless,clinical and laboratory experience regarding androgen use inpatients with HAE is restricted to heterozygous situations, inwhich the wild-type allele can be driven to increment C1-Inhproduction. To date, reports on the prophylactic intake of andro-gens in homozygous patients with HAE are lacking.Of note, the homozygous R378C substitution markedly re-
duces but does not completely suppress C1-Inh synthesis andsecretion. This basal expression of the R378C allele provides asource for the low but detectable levels of C1-Inh in the patientbefore treatment and makes the mutant allele susceptible toandrogen upregulation, explaining the patient’s good response toattenuated androgen therapy.To our knowledge, the R378C substitution described here is the
first mutation outside of the RCL reported to alter C1-Inhspecificity. The R378 residue is located in the gate region, in theturn between 2C helix and the S6A strand of b-sheet A (ProteinData Bank accession 2OAY). The importance of this region ishighlighted by studies on the model serpin a-1-antitrypsin(A1AT), in which, on interaction with proteases, the RCL insertsinto b-sheet A and is stabilized by a salt bridge formed betweenamino acids K290 and E342. The disruption of this salt bridge inthe Z variant of A1AT (carrying the E342Kmutation) reduces thestability of the molecule and induces its intracellular multi-merization by means of a loop-sheet insertion mechanism.E3
Based on structural models of C1-Inh, it is predictable thatR378 also forms a salt bridge with E429. In the R378C mutantthe positively charged arginine (homologous to K290 in A1AT)is replaced by the neutral cysteine at position 378 and probablyprevents salt bridge formation with subsequent alteration of theprotein conformation.The specific impairment in kallikrein activation control found
in the R378C mutant sheds more light on the pathogenesis ofHAE. The situation seems to be opposite to that of the familyreported byWisnieski et al,E4 in which an Ala443Val substitutionin C1-Inh prevented C1r binding but maintained full inhibitorycapacity to kallikrein and coagulation factor XII.E5,E6 Althoughthe R378C homozygous patient has had recurrent episodes ofedema, no family member with the Ala443Val mutation, all ofwhom also had complement system activation, ever had symp-toms of HAE, suggesting that classical complement activationalone does not result in angioedema.
REFERENCES
E1. Gelfand JA, Sherins RJ, Alling DW, Frank MM. Treatment of hereditary angioe-
dema with danazol. Reversal of clinical and biochemical abnormalities. N Engl
J Med 1976;295:1444-8.
E2. Pappalardo E, Zingale LC, Cicardi M. Increased expression of C1-inhibitor mRNA
in patients with hereditary angioedema treated with danazol. Immunol Lett 2003;
86:271-6.
E3. Janciauskiene S, Eriksson S, Callea F, Mallya M, Zhou A, Seyama K, et al. Dif-
ferential detection of PAS-positive inclusions formed by the Z, Siiyama, and
Mmalton variants of alpha1-antitrypsin. Hepatology 2004;40:1203-10.
