Hepatology Research 33 (2005) 292–297

High prevalence of alpha 1 antitrypsin phenotypes in viralhepatitis B infected patients in Iran

Mohammad Hashemia, Seyed Moayed Alavianb, Saeid Ghavamia,∗, Frederick J. de Serresc,Masoud Salehid, Taher Doroudie, Amir Hossain Mahagheghi Fardf, Hamid Mehrabifarg,

Behzad Milanih, Seyed Javad Saeidi Shahrib

a Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iranb Department of Internal Medicine, School of Medicine, Baghyat Allah Medical University, Tehran, Iran

c Center for the Evaluation of Risks to Human Reproduction, Environmental Toxicology Program,National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC, USA

d Department of Infectious Disease, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Irane Tehran Hepatitis Center, Vesal Avenue, Tehran, Iran

f Department of Virology, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Irang Department of Internal Medicine, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran

h Department of Laboratory Medicine, Gargan Azad University, Gorgan, Iran

wideevelopvolved

dy, the role

wereperformeds: carrier

ic

hronic

gyp-by

Received 13 May 2005; received in revised form 17 August 2005; accepted 14 September 2005Available online 2 November 2005

Abstract

Objective: Hepatitis B virus (HBV) infection is a major global public health problem. Approximately 2 billion people are infected worldand more than 350 million of these individuals are chronic carriers of HBV. Approximately 15–40% of infected patients will dcirrhosis, liver failure, or hepatocellular carcinoma (HCC). Alpha 1 antitrypsin (AAT) deficiency is one of many factors that may be inin abnormalities such as liver and lung disease, inflammatory joint diseases, and inflammatory eye diseases. In the present stuplayed by AAT in HBV infected individuals is analyzed.Methods: AAT phenotyping and trypsin inhibitory capacity (TIC) experiments were performed on 281 HBV infected patients whoreferred to Tehran and Zahedan Hepatitis Center for a period of 3 years from June 2001 to September 2003. The same tests wereon 257 individuals who did not suffer from any systemic diseases (control group). The case group was subdivided into three group(36.7%), chronic (50.5%), and cirrhotic (12.8%).Results: The results showed that AAT phenotypes, MS, MZ, M1Z, and M1S, were significantly higher in the HBV group (p < 0.01). Inaddition, there was a significant difference in AAT phenotypes (MS, MZ, and M1Z) among inactive carriers and individuals in the chronand cirrhotic group (p < 0.001).Conclusions: There is a high prevalence of moderate AAT (MS, M1S, and MV) and severely deficient (MZ and M1Z) phenotypes in IranianHBV individuals. In addition, AAT deficiency might be a risk factor for infected HBV individuals progressing from the carrier stage to cand cirrhotic stages.© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Alpha 1 antitrypsin; Viral hepatitis B; Chronic active hepatitis; Cirrhosis

∗ Corresponding author. Present address: Manitoba Institute of Cell Biol-ogy, 675 McDermot Ave., Rm. 0N6008, Winnipeg, MB, Canada R3E 0V9.Tel.: +1 204 787 4108; fax: +1 204 787 2190.

E-mail address: ghavami@cc.umanitoba.ca (S. Ghavami).

1. Introduction

Hepatitis is a clinical condition of miscellaneous etiolowhose final effect is hepatic inflammation or injury with heatic cell necrosis. Clinically, the syndrome is characterized

1386-6346/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.hepres.2005.09.035

M. Hashemi et al. / Hepatology Research 33 (2005) 292–297 293

elevated levels of aminotransferases, ALT, AST, and by vari-able elevation of alkaline phosphatase or bilirubin. In general,the degree of ALT and/or AST elevation is more reflective ofsudden liver damage than of true severity. Hepatitis may havean acute or chronic manifestation. Chronic hepatitis requiresongoing liver damage for at least 6 months, however, clinicalexaminations often do not reveal this damage and it is onlydiscovered after performing a liver biopsy[1,2].

The causes of hepatitis are diverse and include: viralinfections, bacterial or fungal infections, immune disorders,hepatic perfusion and oxygenation problems, metabolic dis-eases, and toxic injury either from medication, environmentalor industrial toxins, or with increasing frequency from theuse of chemicals and herbs as complementary and alterna-tive medicine (CAM) therapy[3]. HBV infection accountsfor 0.5–1.2 million deaths each year and is the 10th leadingcause of death worldwide[1].

The prevalence of HBV infection varies markedly in dif-ferent geographic areas of the world, as well as in differentpopulation subgroups[2,4]. Overall, approximately 45% ofthe global population lives in areas of high chronic HBVprevalence[5]. In sub-Saharan Africa, the Pacific, and par-ticularly Asia, HBV infection is highly endemic with themajority of individuals becoming infected during childhood[2]. Outside of the endemic areas, regions with high rates ofchronic HBV infection include the Southern parts of Easterna , andt peanc area

nep inci-p sesiT ereda creasd clas-s eb ula-tw lum( top loss-op tis-s

ha 1a ainsu encyw con-t irali so-c ,t psinp thiss phe-

notype in hepatitis B infected individuals who were referredto Tehran Hepatitis Center and Zahedan Hepatitis Center.

2. Materials and methods

2.1. Reagents

The following reagents were obtained from PharmaciaDiagnostics, Uppsala, Sweden: acrylamide (N,N′-methyleneacryl amide), isoelectric focusing calibration kit, ampho-line 4.0–6.5, ampholine 3.5–5.0, repel silane ES, andacryl amide gel bound bed. Dithiothreitol (DTT) andiodoacetamide were purchased from Merck (Darmstadt,Germany). The following were purchased from Sigma(St. Louis, MO, USA): methylenebisacrylamide (N,N′,N′-tetramethylenediamine),N,N,N′,N′ tetramethylethylenedi-amine (TEMED), ammonium persulfate, sodium hydrox-ide, glycine, acetic acid, Coomassie Brilliant Blue R-250,methanol, trichloroacetic acid, sulfosalicylic acid, bovineserum albumin (BSA), dimethyl sulfoxide (DMSO), Tris basebuffer, a-N benzoyl-d-larginine-p-nitroaniline (BAPNA).Alpha 1 antitrypsin standard serum was a gift from ProfessorM.K. Fagerhol (Ullevaal University Hospital, Oslo, Norway).

2.2. Case and control cohort criteria

byt dan( mena 2–78y sAg)p fromt ep-a e ofa cir-r anyh

hys-i odw andH byu sor-b IIm estsw isitss har-a ults.I tive,l LTl velso ori verb lin-i sults( nd

nd Central Europe, the Amazon basin, the Middle Easthe Indian subcontinent. In Western and Northern Euroountries and North America, HBV infection is relatively rnd acquired primarily in adulthood[6].

Alpha 1 antitrypsin (�1AT), the archetype of the serirotease inhibitor (SERPIN) supergene family, is the pral blood-borne inhibitor of destructive neutrophil protea

ncluding: elastase, cathepsin G, and proteinase 3[7–10].his glycoprotein is secreted by liver cells and is considn acute-phase reactant because its plasma levels inuring host response to inflammation/tissue injury. Theical form of�1AT deficiency, which affects 1 in 1800 livirths in Northern European and North American pop

ions, is associated with a mutant molecule termed�1ATZhich is retained as a polymer in the endoplasmic reticu

ER) of liver cells[11,12]. Homozygotes are predisposedremature development of pulmonary emphysema by af-function mechanism in which lack of�1AT in the lungermits uninhibited proteolytic damage to the connectiveue matrix[13–15].

Despite the recent biochemical characterization of alpntitrypsin, its involvement in chronic liver disease remnclear and the association of alpha 1 antitrypsin deficiith the pathogenesis of liver disease continues to be

roversial. In previous studies, a high prevalence of vnfection in patients with alpha 1 antitrypsin deficiency asiated chronic liver disease has been found[16,17]. Howeverhere is no study in the prevalence of alpha 1 antitryhenotype in hepatitis B infected individuals. Therefore,tudy was performed to examine the alpha 1 antitrypsin

e

From 2001 to 2003, 281 patients were followedhe Hepatitis Center in Tehran (Central Iran) and ZaheSouth-East Iran). The patients studied consisted of 214nd 67 women with a mean age of 40.8 years (range, 1ears). Patients were all hepatitis B surface antigen (HBositive for at least 6 months. Patients were excluded

he study if they had any of the following conditions: htocellular carcinoma, concomitant hepatitis C, evidencutoimmune hepatitis, Wilson disease, primary biliaryhosis, history of heavy alcohol intake, or intake ofepatotoxic drugs.

Patients had to complete a history enquiry and a pcal examination during the first visit to the clinic. Bloas taken for liver biochemistry, alpha fetoprotein,BV serological markers including HBsAg and HBeAgsing commercially available enzyme-linked immunoent assay (ELISA) kits (Hepanostika HBsAg Uni-Formicroelisa system, Organon Teknika, Holland). Blood tere obtained during the first visit and in subsequent vcheduled for 3–6 month intervals. The patients were ccterized into three groups by clinical and laboratory res

nactive carrier state is characterized by HBeAg negaow, or undetectable serum of HBV DNA and normal Aevels. Chronic hepatitis B is characterized by high lef HBV DNA (>100,000) (by Amplicor 2), persistent

ntermittent elevation in ALT levels and a compatible liiopsy. The third group, cirrhotic, was characterized by c

cal evidence (splenomegaly, ascites) and paraclinical relow platelet count, prolongation of protrombin time, a

294 M. Hashemi et al. / Hepatology Research 33 (2005) 292–297

esophageal varices in upper GI endoscopy). Two hundredand fifty-seven individuals without any systemic or inflam-matory disease were matched based on age, sex, and ethnicityand enrolled into the study as a control group. All cases inboth groups were assessed by two gastroenterologists throughdetailed clinical and paraclinical exams including, ANA, RF,ESR, CRP, PTT, CBC, AST, ALT, TG, and Cholesterol.In addition, the patient (case) and control group membersenrolled in this study were familiarly unrelated (cousins upto the third grade were not enrolled into the study).

2.3. Laboratory tests

Ten-millilitre blood samples were collected from eachpatient enrolled in the study for a series of laboratorytests. After separating serum, samples were divided into twoaliquots. One aliquot was used for ANA, RF, ESR, CRP, PTT,CBC, AST, ALT, TG, and Cholesterol test. The second aliquotwas subdivided into 50�L aliquots and kept at−70◦C untilphenotyping and serum trypsin inhibitory capacity (S-TIC)tests were performed. AAT phenotyping was carried out on allserum samples from subjects by the widely accepted isoelec-tric focusing technique on polyacrylamide gels at pH 3.5–6.5[8,18,19]. Isoelectric focusing (IEF) was carried out on flatbed polyacrylamide gels (pH 3.5–6.5). Pre-focusing was car-ried out at a maximum voltage of 3000 V, 75 mA, 5 W forh onp singg ut ata onc fos-a ith as inings

lec-t ples[ baseb hd ixedw d at3 as ac ad bya .

2

d byt ds

3

s Bi werec

Table 1Comparison of alpha 1 antitrypsin phenotypes between case and controlgroups

AAT phenotypes Groups

Control Case

Normal 95.72% (246/257) 77.93% (219/281)MS* 1.95% (5/257) 3.20% (9/281)MZ* 0.00% (0/257) 4.98% (14/281)MV* 0.00% (0/257) 1.78% (5/281)M1Z* 1.95% (5/257) 7.83% (22/281)M1S* 0.38% (1/257) 3.56% (10/281)ZZ 0.00% (0/257) 0.36% (1/281)SZ 0.00% (0/257) 0.36% (1/281)

There was a significant difference in alpha 1 antitrypsin phenotypes betweenthe case and control groups (p < 0.05). The M1S, MS, MZ, M1Z, and MVphenotypes are significantly more frequent in case group than the controlgroup (p < 0.05). Asterisk (*) shows the statistically difference between twogroups.

The patients were categorized into three groups based onthe stage of the disease (inactive carrier, chronic hepatitis B,and cirrhotic) as defined by our clinical criteria. In the inac-tive carrier group, chronic and cirrhotic group there were 103individuals (70 men and 33 women), 142 (113 men and 29women), and 36 individuals (31 men and 5 women), respec-tively.

3.1. AAT deficiency tests

AAT phenotypes were compared between case and con-trol groups as shown inTable 1. The MS, MZ, M1S, M1Z,and MV phenotypes are significantly more frequent in thecase group than in the control group (p < 0.05). The distri-bution of different AAT phenotypes in different hepatitis Bgroups is shown inTable 2. The MZ and M1Z phenotypes aresignificantly more common in chronic and cirrhotic groups(p < 0.01). The S-TIC of all individuals in each case and con-trol group was calculated in order to find the serum alpha1 antitrypsin activity (data were not shown). According toS-TIC, MV, MS, and M1S can be considered as moderatelydeficient phenotypes, M1Z, MZ, SZ, and ZZ as severely defi-

Table 2Distribution of different alpha 1 antitrypsin phenotypes in different stagesof hepatitis in case group

A

N /36)M 6)M 6)M )M 6)M 6)Z )S 6)

T ronica ceb

alf an hour. Serum (5�L) was applied to sample applicatiieces (LKB production) on the surface of the pre-focuel 15 mm from the cathode strip. Focusing was carried omaximum voltage of 3000 V, 75 mA, 15 W for 2.5 h. Up

ompletion of the run, gels were fixed in a solution of sullicylic acid and trichloroacetic acid. Gels were stained wolution of Coomassie Blue R-250 and rinsed in a destaolution.

The AAT phenotypes were determined by their erophoretic pattern. S-TIC was also measured in all sam8]. Each serum sample was diluted 100 times with Trisuffer (pH 8.2, 100�M, and at 37◦C) and then mixed witiluted trypsin solution. These solutions were then mith trypsin synthetic substrate (BAPNA) and incubate7◦C for 5 min (bovine serum albumin was consideredontrol). The absorbance value for each sample was respectrophotometer at 400 nm and TIC was calculated

.4. Statistical analysis

The statistical analyses of the results were evaluatehe Chi-square and pairedt-test andp < 0.05 was considereignificant.

. Results

In the 3-year period (2001–2003), 281 viral hepatitinfected patients entered the study and these patientsompared to 257 control subjects.

AT phenotypes Hepatitis stage

Inactive carrier Chronic Cirrhosis

ormal 93.20% (96/103) 70.42% (100/142) 63.89% (23S 1.94% (2/103) 4.23% (6/142) 2.78% (1/3Z* 0.00% (0/103) 7.04% (10/142) 11.12% (4/3V 0.00% (0/103) 2.12% (3/142) 5.55% (2/36

1Z* 1.94% (2/103) 11.97% (17/142) 8.33% (3/3

1S 2.92% (3/103) 3.52% (5/142) 5.55% (2/3Z 0.00% (0/103) 0.00% (0/142) 2.78% (1/36Z 0.00% (0/103) 0.70% (1/142) 0.00% (0/3

he MZ and M1Z phenotypes are significantly more frequent in chnd cirrhotic group (p < 0.01). Asterisk (*) shows the statistically differenetween two groups.

M. Hashemi et al. / Hepatology Research 33 (2005) 292–297 295

Table 3Categorization of alpha 1 antitrypsin phenotypes

AAT phenotypes Groups

Control Case

Normal 95.72% (246/257) 77.93% (219/281)Moderately deficient* 2.33% (6/257) 8.54% (24/281)Severely deficient* 1.96% (5/257) 13.52% (38/281)

MV, MS, and M1S can be considered as moderately deficient phenotypes;MZ, M1Z, SZ, and ZZ as severely deficient; and M1M2, M1M1, and M2M2

phenotypes as normal. A significant difference could be observed among theseverely deficient, and moderately deficient phenotypes between the caseand control groups (p < 0.01). Asterisk (*) shows the statistically differencebetween two groups.

cient, and M1M2, M1M1, and M2M2 phenotypes as normal(S-TIC values for healthy adults are 2.1–3.8�mol/(min mL),M1Z heterozygous are about 55–65% healthy adults M1S,M2S heterozygous are about 65–75% healthy adults, andMV heterozygous is about 75–85% healthy individuals inliver disease patients)[20–22,8,23–25]. On the basis of thesecriteria, a significant difference could be observed amongthe severely deficient and moderately deficient phenotypesbetween the case and control groups (Table 3) (p < 0.01).

Taking the AAT phenotype in various groups of hepati-tis patients into account, major population of deficient andmoderately deficient phenotypes were observed in chronichepatitis B and cirrhotic patients (p < 0.01) (Table 4). In addi-tion, the frequencies of deficient and moderately deficientphenotypes in chronic and cirrhotic groups are significantlyhigher than the control group.

3.2. Biopsy results

Liver biopsies were preformed on 65 chronic hepatitispatients using the Tru Cut needle. Biopsy specimens werestudied extensively by a pathologist and patients were cate-gorized according to their biopsy stage (1–8). In stages higherthan 5 no moderate deficient alpha 1 antitrypsin phenotypescould be observed (Table 5).

4

4

t theh dis-

Table 5Distribution of alpha 1 antitrypsin phenotypes in different staging of liverbiopsy

Liver biopsystage

AAT phenotypes

Normal Moderatelydeficient

Severely deficient

1 66.67% (10/15) 33.33% (5/15) 00.00% (0/15)2 80.00% (8/10) 10.00% (1/10) 10.00% (1/10)3 70.00% (7/10) 00.00% (0/10) 30.00% (3/10)4 81.82% (9/11) 9.09% (1/11) 9.09% (1/11)5 50.00% (5/10) 00.00% (0/10) 50.00% (5/10)6 66.67% (2/3) 33.33% (1/3) 00.00% (0/3)7 66.67% (2/3) 00.00% (0/3) 33.33% (1/3)8 00.00% (0/1) 00.00% (0/1) 100.00% (1/1)

The severely deficient phenotypes are more frequent in stage greater than 4(p < 0.05).

poses individuals to liver disease[26,27]. On the other hand,AATD does not solely cause liver disease. In a prospec-tive study involving liver biopsies and AAT phenotyping,an increased prevalence of the MZ phenotype was found inpatients with cryptogenic cirrhosis and chronic active hep-atitis [28]. However, in another study involving 469 patientswith acute or chronic liver disease and 98 control subjectswithout known liver disease, the heterozygous AATD stateoccurred with approximately the same frequency in patientswith (4.7%) and without (6.1%) chronic liver disease[29].Thus, the relation of AATD to hepatic disease appeared to berandomly distributed, and no causal relation could be shown.Moreover, it has been shown that patients with the PiZZ phe-notype and without the clinical manifestations of chronic liverdisease may exhibit the characteristic periodic acid-Schiffpositive globules in the liver even when histological assess-ment may be otherwise normal[30]. These findings suggestthat the globules themselves are not responsible for hepaticdamage and that another factor might underlie chronic liverdisease[31].

Propst et al. have performed AAT phenotyping on 1865individuals[16]. One hundred and sixty-four individuals withhomozygous or heterozygous AATD were included in theirstudy. The AATD patients were categorized into four groupsbased on the stage of liver disease as defined by clinical andhistological criteria, cirrhotic (32%), chronic active (7%),s fre-q asf

TD atitis

A

active

N 93.20% 3/36)M 4.85% 6)S 1.95% 36)

M served llyd

. Discussion

.1. Previous studies

It has been suggested for more than 20 years thauman alpha 1 antitrypsin deficiency (AATD) state pre

able 4istribution of alpha 1 antitrypsin phenotypes in different stage of hep

AT phenotypes Groups

Control In

ormal 95.72% (246/257)oderately deficient* 2.33% (6/257)everely deficient* 1.96% (5/257)

ost severely deficient and moderately deficient phenotypes were obifference between two groups.

teatosis (48%), and with no liver disease (21%). Theuencies of the AAT phenotype in their AATD groups are

ollows:

carrier Chronic Cirrhosis

(96/103) 70.42% (100/142) 63.89% (2(5/103) 9.86% (14/142) 22.22% (8/3(2/103) 19.72% (28/142) 13.89% (5/

in the chronic and cirrhotic groups (p < 0.01). Asterisk (*) shows the statistica

296 M. Hashemi et al. / Hepatology Research 33 (2005) 292–297

Cirrhotic: MZ (83%), SZ (6%), FZ (9%), and ZZ (4%);chronic active: MZ (75%), SZ (8.3%), FZ (8.3%), and ZZ(8.3%); steatosis: MZ (79%), SZ (8%), FZ (8%), and ZZ(5%); and no liver disease: MZ (67%), SZ (19%), FZ (5%),and ZZ (9%).

In the cirrhotic group, 62% of patients were anti-HCVpositive and 33% of patients had evidence of HBV infec-tion; three patients were HBsAg positive. Eighty percent ofpatients in the chronic active hepatitis group were anti-HCVpositive. Thirty percent of patients had evidence of HBVinfection and two patients were HBsAg carriers. Eighteenpercent of patients in the steatosis group showed evidenceof past HBV infection. Five percent of these patients wereanti-HCV positive. In the last group no patient had evidenceof HCV or HBV infection.

In another study, Propst et al. analyzed 240 consecutivepatients with cirrhosis of different etiologies (AATD, HCVinfection, HBV infection, HCV and HBV infection, cryp-togenic cirrhosis, and alcoholic cirrhosis)[17]. In AATDdeficient group, 61 patients were found to have AATD-associated chronic liver disease. Of these, 98% showedevidence of heterozygosity and 2% were homozygous forAATD (83% PiMZ, 8% PiSZ, 7% FZ, and 2% PiZZ).Among these patients with AATD, 78% were found tohave additional positive viral markers; the HCV test waspositive in 51% of subjects and 45% showed signs ofH orb

4

ingf rrym cientp thant tm s. Ino c-t nica sig-n -au cia-t AATp encyo an 4(

arem mei oticcd ongt cir-r lieso cellso ich

are key in priming and directing the virus-specific T-cellresponse; and the T cells, which are the main antiviraleffectors. After infection of the hepatocyte various cellu-lar and humoral responses have been postulated that areaimed at elimination of the virus. The earliest responsesare non-specific and include the interferon system, natu-ral killer cells, and non-specific activation of Kupffer cells.The precise role of several of these unspecific mechanismsis not well understood in HBV infection although it wasrecently shown that natural killer T-cell activation inhibitsHBV replication in vivo [32,33]. After these non-specificresponses, immune responses directed specifically againstviral proteins become important. The two major arms ofthe immune system are the humoral arm, which consistsof B-lymphocytes that produce antibody, and the cellulararm, which is composed of various cell types, includingmacrophages and T-lymphocytes. On the other hand, indi-viduals with AATD more frequently develop chronic inflam-matory diseases such as rheumatoid arthritis, systemic lupuserythematosus, bronchitis, glomerulonephritis, and uveitisthan the healthy controls[8,31]. In addition, the associationbetween AATD and several immune-mediated diseases haspreviously been described. This suggests that AATD is notonly important as an anti-inflammatory protein, but also asan immune regulator with the ability to modulate lymphocyteproliferation, cytotoxicity, monocyte and neutrophil function,pd m,A iono erp

withH onst ep-a

5

ATDi ingm tiono htb nica

A

edanM er-h ine,U itho stan-d

BV infection (25% patients were HBsAg positive)oth.

.2. Current study

Of the 281 HBV patients who underwent phenotypor AAT in our study, 62 (22.1%) were found to caoderate or severe, heterozygous or homozygous defihenotypes. This prevalence is significantly higher

he control of this study (Tables 2–4) (p < 0.01), and iay reflect the differences between studied populationur study, 9.7% of AATD individuals were in the ina

ive carrier group with 69.4% and 20.9% in the chrond cirrhotic groups, respectively. Therefore, there is aificant difference in the distribution of AATD individuls among the patient group (p < 0.01). In patients whomnderwent liver biopsies, we did not find any asso

ion between the stages of the liver disease andhenotypes, except the significantly increased frequf severely deficient phenotypes in stages higher thTable 5).

The question arises whether patients with AATDore prone to develop HBV infections and if they beca

nfected, do they progress toward chronic active and cirrhonditions? As shown inTables 3 and 4AAT phenotypesiffer between patients and the control group and am

he patient groups (inactive carrier, chronic active, andhotic group) (p < 0.01). The host response to viruses ren a complex interaction of several cell systems; thef the innate immune system, the dendritic cells, wh

hagocyte motility, and chemokinesis[34–38,8,39]. Thus,ue to the regulatory role of AATD in the immune systeATD deficiency might be a risk factor for the progressf HBV infection toward chronic active and cirrhotic livathology.

In our study, males were diagnosed more frequentlyBV. This finding is consistent with previous observati

hat men have a higher risk for developing chronic viral htitis and cirrhosis[40,29].

. Conclusion

In summary, our data suggest a high prevalence of An patients with HBV associated liver disease. This find

ay explain the heterogeneity in the clinical manifestaf the inflammatory liver disease. In addition, AATD mige a risk factor for HBV individuals developing the chrond cirrhotic stages.

cknowledgments

This work was supported by a research grant from Zahedical University. We are grateful to Professor M.K. Fagol, Department of Immunology and Transfusion Medicllevaal University Hospital Oslo, Norway, for his help wur study and contributing the alpha 1 antitrypsin serumards.

M. Hashemi et al. / Hepatology Research 33 (2005) 292–297 297

References

[1] Alavian SM, Nematizadeh F. Occult HBV infection in patients withserological markers of past HBV infection. Am J Gastroenterol2003;98:937–8.

[2] Lavanchy D. Hepatitis B virus epidemiology, disease burden, treat-ment, and current and emerging prevention and control measures. JViral Hepat 2004;11:97–107.

[3] Marsano LS. Hepatitis. Prim Care 2003;30:81–107.[4] Yu MC, Yuan JM, Govindarajan S, et al. Epidemiology of hepato-

cellular carcinoma. Can J Gastroenterol 2000;14:703–9.[5] Mahoney FJ. Update on diagnosis, management, and prevention of

hepatitis B virus infection. Clin Microbiol Rev 1999;12:351–66.[6] Lok AS, Heathcote EJ, Hoofnagle JH. Management of hep-

atitis B: 2000—summary of a workshop. Gastroenterology2001;120:1828–53.

[7] de Serres FJ. Alpha-1 antitrypsin deficiency is not a rare diseasebut a disease that is rarely diagnosed. Environ Health Perspect2003;111:1851–4.

[8] Ghavami S, Hashemi M, Shahriari HA, et al. Alpha-1-antitrypsinphenotypes and HLA-B27 typing in uveitis patients in southeastIran. Clin Biochem 2005;38:425–32.

[9] Huber R, Carrell RW. Implications of the three-dimensional structureof alpha 1-antitrypsin for structure and function of serpins. Biochem-istry 1989;28:8951–66.

[10] Perlmutter DH. Liver injury in alpha1-antitrypsin deficiency: anaggregated protein induces mitochondrial injury. J Clin Invest2002;110:1579–83.

[11] Perlmutter D. In: Arias A, editor. The liver: biology and pathobiol-ogy. New York: Raven Press; 2001. p. 699–719.

[ liver6.

[ liverInvest

[ ologyItaly,

[ nt ofDis

[ tiondefi-

1–5.[ arci-

–11.[ a 1-

[ lpha2–6.

[ fibri-–2.

[ a 1Clin

[22] Fagerhol M, Cox D. The Pi polymorphism: genetic, biochemi-cal, and clinical aspects of human alpha 1-antitrypsin. Adv Genet1981;11:1–62.

[23] Kueppers F, Dickson ER, Summerskill WH. Alpha1-antitrypsin phe-notypes in chronic active liver disease and primary biliary cirrhosis.Mayo Clin Proc 1976;51:286–8.

[24] Miszczuk-Jamska B, Pajdak W, Barylak H, et al. Serum trypsininhibitory activity and alpha-1-antitrypsin level in liver diseases.Folia Histochem Cytobiol 1986;24:173–7.

[25] Theodoropoulos G, Archimandritis A, Tsomi A, et al. Serum trypsininhibitory capacity and alpha 1-antitrypsin levels in liver cirrhosisand hepatoma. Acta Hepatogastroenterol (Stuttg) 1979;26:195–7.

[26] Perlmutter DH. Liver disease associated with alpha 1-antitrypsin defi-ciency. Prog Liver Dis 1993;11:139–65.

[27] Qu D, Teckman JH, Perlmutter DH. Review: alpha 1-antitrypsindeficiency associated liver disease. J Gastroenterol Hepatol1997;12:404–16.

[28] Hodges J, Millward-Sadler G, Barbatis C, et al. Heterozygous MZalpha 1 antitrypsin deficiency in adults with chronic active hepatitisand cryptogenic cirrhosis. N Engl J Med 1981;304:557–60.

[29] Fisher RL, Taylor L, Sherlock S. Alpha-1-antitrypsin deficiencyin liver disease: the extent of the problem. Gastroenterology1976;71:646–51.

[30] Sharp HL, Mathis R, Krivit W, et al. The liver in noncirrhotic-1-antitrypsin deficiency. J Lab Clin Med 1971;78:1012–3.

[31] Sharp HL. Alpha-1-antitrypsin: an ignored protein in understandingliver disease. Semin Liver Dis 1982;2:314–28.

[32] Jung MC, Pape GR. Immunology of hepatitis B infection. LancetInfect Dis 2002;2:43–50.

[33] Kakimi K, Guidotti LG, Koezuka Y, et al. Natural killer T cellactivation inhibits hepatitis B virus replication in vivo. J Exp Med

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[ n ontes. J

[ lphaory

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[ ro-icityd has133:

[ aryed

12] Teckman J, Qu D, Perlmutter D. Molecular pathogenesis ofdisease in�1-antitrypsin deficiency. Hepatology 1996;24:1504–1

13] Crystal R. Alpha1-antitrypsin deficiency, emphysema anddisease: genetic basis and strategies for therapy. J Clin1990;95:1343–52.

14] de Serres FJ, Blanco I, Fernandez-Bustillo E. Genetic epidemiof alpha-1 antitrypsin deficiency in southern Europe: France,Portugal and Spain. Clin Genet 2003;63:490–509.

15] Janoff A. Elastases and emphysema: current assessmethe protease–antiprotease hypothesis. Am Rev Respir1985;132:417–33.

16] Propst T, Propst A, Dietz O, et al. High prevalence of viral infecin adults with homozygous and heterozygous alpha 1 antitrypsinciency and chronic liver disease. Ann Intern Med 1992;117:64

17] Propst T, Propst A, Dietz O, et al. Prevalence of hepatocellular cnoma in alpha 1 antitrypsin deficiency. J Hepatol 1994;21:1006

18] Jeppsson JO, Franzen B. Typing of genetic variants of alphantitrypsin by electrofocusing. Clin Chem 1982;28:219–25.

19] Kishimoto Y, Yamada S, Hirayama C. An association between a1 antitrypsin and chronic liver disease. Hum Genet 1990;84:13

20] Aschauer R, Holzknecht F, Platzer S. Endogenous inhibitors ofnolytic activity in liver cirrhosis. Klin Wochenschr 1973;51:1170

21] Dietz AA, Rubinstein HM, Hodges LK. Measurement of alphantitrypsin in serum, by immunodiffusion and enzymatic assay.Chem 1974;20:396–9.

2000;192:921–30.34] Ades EW, Hinson A, Chapuis-Cellier C, et al. Modulation

the immune response by plasma protease inhibitors. I. Alphmacroglobulin and alpha 1-antitrypsin inhibit natural killing aantibody-dependent cell-mediated cytotoxicity. Scand J Imm1982;15:109–13.

35] Breit SN, Luckhurst E, Penny R. The effect of alpha 1 antitrypsithe proliferative response of human peripheral blood lymphocyImmunol 1983;130:681–6.

36] Breit SN, Penny R. The role of alpha 1 protease inhibitor (a1 antitrypsin) in the regulation of immunologic and inflammatreactions. Aust N Z J Med 1980;10:449–53.

37] Breit SN, Robinson JP, Luckhurst E, et al. Immunoregulationalpha 1 antitrypsin. J Clin Lab Immunol 1982;7:127–31.

38] Breit SN, Robinson JP, Penny R. The effect of alpha 1 antitryon phagocyte function. J Clin Lab Immunol 1983;10:147–9.

39] Hudig D, Redelman D, Minning LL. The requirement for pteinase activity for human lymphocyte-mediated natural cytotox(NK): evidence that the proteinase is serine dependent anaromatic amino acid specificity of cleavage. J Immunol 1984;2647–54.

40] Eriksson S, Carlson J, Velez R. Risk of cirrhosis and primliver cancer in alpha 1-antitrypsin deficiency. N Engl J M1986;314:736–9.