Research ArticleSerum Cytokine of IL-10 and IL-12 in Chronic Liver DiseaseThe Immune and Inflammatory Response

Hoda Mohamed El-Emshaty1 Wesam Ahmad Nasif23 and Ibrahim Eldsoky Mohamed4

1Gastroenterology Laboratories Gastroenterology Surgical Center Mansoura University Mansoura 35516 Egypt2Molecular Biology Department Genetic Engineering and Biotechnology Research Institute Sadat University Sadat City Egypt3Biochemistry Department Faculty of Medicine Umm Al-Qura University Makkah Saudi Arabia4Pathology Department Faculty of Medicine Mansoura University Mansoura 35516 Egypt

Correspondence should be addressed to Hoda Mohamed El-Emshaty elemshaty hyahoocom

Received 5 October 2015 Revised 12 November 2015 Accepted 23 November 2015

Academic Editor Leigh A Madden

Copyright copy 2015 Hoda Mohamed El-Emshaty et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The current study was designed to investigate the potential association of serum interleukin-10 and interleukin-12 with HCVinfection in chronic liver disease and to evaluate their possible role as new biomarkers in HCC developmentMaterial andMethodsForty-one patients suffering from chronic liver disease (33 patients harbor HCV infection and 8 are HCV-negative patients) wereenrolled in the present study and histopathologically diagnosed into 15 patients with HCC 16 patients with LC and 10 patients withliver histology compatible with precirrhotic hepatitis (PCH) Ten patients complaining of cholecystitis were included as nondiseasecontrol Serum levels of IL-10 and IL-12 were measured by enzyme linked immunosorbent assay (ELISA) Results HCV-infectedpatients showed elevated expression of IL-10 and IL-12 compared to nondisease controls (119875 lt 00001) but there is no significantdifference with respect to their expression in HCV-negative patients Serum IL-10 and IL-12 were elevated significantly with diseaseprogression (119875 lt 00001) and a positive correlation coefficient was detected between IL-10 IL-12 (119903 = 0785 119875 lt 00001) andtransaminase values suggesting their possible role in chronic inflammation progression leading to HCC Conclusion IL-10 and IL-12 might be involved in chronic inflammation progression leading to HCC and their evaluation could be used as new biomarkersto reflect the degree of inflammation in HCC development

1 Introduction

Hepatitis C virus (HCV) infection affects almost 3 ofthe worldrsquos population with the highest prevalence (15)in Egypt [1] HCV infection results in chronic hepatitis inmore than 70 of infected patients while 20ndash30 of patientsrecover spontaneously [2] The pathogenesis and outcomeof viral infections are significantly influenced by the hostimmune response The immune system is able to eliminatemany viruses in the acute phase of infection However someviruses like hepatitis C virus (HCV) and hepatitis B virus(HBV) can evade the host immune responses and establish apersistent infection [3]

The precise role of the immune response in patients withHCV infection in particular the relationship between thelevels of inflammatoryregulatory cytokines and the course

of HCV infection is still unclear Recent work has suggestedthat these cytokines can trigger distinct patterns of protectiveor immunopathological responses and that they are involvedin the clearance or establishment of chronic HCV infection[4] However the balance of proinflammatory and regulatorycytokines appears to be important in determining the courseof HCV infection [5]

Cytokines play an important role in differentiationmaturation and functional activation of immune cells [6]Cytokines are produced bymultiple cell types such asNKcellsand macrophages CD4+T cells and CD8+T cells Responsesare referred to as Th1-like and Th2-like after the originaldescription of the cytokine profiles produced by subsets ofCD4+T cells [7]Th1-like responses include IL-2 TNF-120572 andIFN-120574 secretion and are required for generation of cytotoxic Tlymphocytes and NK cell activation during the host antiviral

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 707254 7 pageshttpdxdoiorg1011552015707254

2 Disease Markers

immune response Th2-like responses produce IL-4 and IL-10 which help augment antibody production and inhibitdevelopment of the Th1 response [8]

IL-10 is a pleiotropic cytokine produced by macrophagesT-helper 2 (Th2) cells and B-lymphocytes and both canstimulate and suppress the immune response IL-10 has beenshown to inhibit various immune reactions [9] Interleukin-12 (IL-12) is one of the most important proinflammatorycytokines produced mainly by antigen presenting cells asa result of IFN-120574 stimulation [10] and presented with theinitiation of immune response so IL-12 can be consideredas one of the most clearly defined factors determining Th1andTh2 differentiation [11] Therefore the current study wasdesigned to investigate serum expression of inflammatoryand immunoregulatory cytokines (IL-10IL-12) with HCVinfection in chronic liver disease and to evaluate their possiblerole as new biomarkers in chronic inflammation progressionleading to HCC

2 Material and Methods

21 Study Population Forty-one (34 males and 7 femalesmean age 4758 plusmn 1242 yrs) patients suffering from chronicliver diseases were enrolled in the present study 33 (805)patients had positive reactivity for HCV infection withdetectable HCV RNA and with no serological evidenceof coinfection with other hepatotropic viruses or humanimmunodeficiency virus and 8 (195) patients had nega-tive reactivity for HCV infection All patients gave writteninformed consent at gastroenterology surgical center Man-soura University Egypt The studied group was subjected toclinical examination imaging radiology laboratory investi-gation and liver biopsy Liver tissue and blood samples of allindividuals were collected and sera were stored at minus70∘Cuntilbeing used

Liver tissue specimens of all cases were routinely pro-cessed for histopathological diagnosis fixed in 10 neutralbuffered formalin embedded in paraffin and cut into 3ndash5 120583mthick sections The studied group was histopathologicallydiagnosed and categorized into 15 (3658) patients (13 maleand 2 female mean age 5706 plusmn 1258 yrs) with HCC 16(390) patients (12 male and 4 female mean age 4606 plusmn79 yrs) with liver cirrhosis and 10 (244) patients (9 maleand 1 female mean age 358 plusmn 522 yrs) with liver histologycompatible with precirrhotic hepatitis (Metavir score A2F3)Tumor grading of HCC was histopathologically assessed asgrade I in 2 (133) grade II in 4 (267) and grade III in 9(60) of 15 HCC cases

Also ten (10) patients complaining of cholecystitis with-out a clinical history of hepatitis and without symptoms orsigns of liver disease were included as nondisease controlgroup (7 male and 3 female mean age 356plusmn31 yrs) (Table 1)

211 Biochemical and Virological Determination Liver bio-chemistries including albumin globulin ALT AST alkalinephosphatase and total bilirubin were measured by an auto-analyzer Serological diagnosis of HBV and HCV was per-formed by automated immunoassayHBsAgwasmeasured by

a commercial immunoassay kit (Abbott Laboratories USA)Serum HCV Ab was analyzed by third-generation ELISA kit(BIOKIT SA Barcelona Spain)

212 Detection of Genomic HCV RNA Strands by RT-PCRTotal HCV RNA was isolated from 125 120583L of the serum andpurified by using SV Total RNA isolation system (PromegaCo USA) Qualitative detection of the genomic HCV RNAwas done using a strand specific real-time RT-PCR with theuse of thermostable enzyme (Tth) for the synthesis of cDNAat a high temperature For the amplification of the genomicHCV RNA strand two sets of oligonucleotide primers (Bio-Synthesis USA) deduced from the highly conserved 51015840-noncoding region of HCV genome were induced in the firstand second stage respectively

1st Stage

Sense C196 (51015840-CCATGGCGTTAGTATGAGTG-31015840)Antisense Seq-CR (51015840-TGCTCATGGTGCACGGTC-TA-31015840)

2nd Stage

Sense Seq-3 (51015840-AGAGCCATAGTGGTCTGCGG-31015840)Antisense Seq-4 (51015840-CTTTCGCGACCCAACACT-AC-31015840)

213 Enzyme Linked Immunosorbent Assays for Il-10 andIL-12 Serum levels of IL-10 and IL-12 were measured byELISA kit (Diaclone Research Besancon France) accordingto the manufacturerrsquos instruction which can detect up to3 pgmL of human IL-10 and 5 pgmL of human IL-12 Briefly50 120583L of standards and samples were added in duplicate tothe precoated strip well plate then 50120583L of biotinylatedantibody was added to each well as a conjugated antibodyfor 2 hrs After washing three times 100120583L of streptavidinhorseradish peroxidase (HRP) was added to each well for30min followed by 100 120583L of TMB substrate solution for30min Finally 100120583L of stop solutionwas added to eachwelland the absorbance of the plate was detected on a plate readerat 450 nm All incubations and recording were done at roomtemperature

22 Statistical Analysis Results were expressed as mean plusmnSD (median) The differences in mean were assessed byKruskal Wallis (Mann-Whitney U test) and all differenceswere considered to be significant at 119875 le 005 Correlationswere calculated by Spearmanrsquos correlation coefficient

3 Results

Clinical virological and biochemical characteristics of thestudied group were listed according to the pathological diag-nosis in Table 1 Mean serum level of ALT AST total proteinand S bilirubin and prothrombin time showed elevatedexpressionwith disease progression Only ALT andASTwere

Disease Markers 3

Table 1 Clinical biochemical and virological characteristics of the studied group

Chronic liver disease (119899 = 41) NDC (119899 = 10)HCC LC PCH

No () 1541 (366) 1641 (390) 1041 (244) 1010 (100)Gender (MF) 132 124 91 73Age (years) 5706 plusmn 1258 (600) 4606 plusmn 79 (450) 358 plusmn 52 (375) 356 plusmn 31 (355)Albumin (gdL) 422 plusmn 058 (43) 415 plusmn 0467 (40) 45 plusmn 042 (46) 383 plusmn 029 (385)T protein (gdL) 795 plusmn 0603 (80) 76 plusmn 067 (78) 766 plusmn 049 (785) 722 plusmn 053 (715)T bilirubin (mgdL) 169 plusmn 257 (12) 128 plusmn 154 (10) 099 plusmn 054 (09) 057 plusmn 021 (055)AST (120583mL)lowast 928 plusmn 6567 (670) 570 plusmn 266 (525) 348 plusmn 1144 (305) 266 plusmn 55 (265)ALT (120583mL)lowastlowast 5873 plusmn 3686 (460) 490 plusmn 1735 (490) 326 plusmn 148 (255) 277 plusmn 41 (275)Proth time (Sec) 143 plusmn 17 (140) 1358 plusmn 466 (146) 132 plusmn 477 (133) 604 plusmn 0568 (61)Proth conc () 7226 plusmn 1378 (700) 70375 plusmn 1299 (690) 731 plusmn 190 (780) 2584 plusmn 1406 (2560)HCV infection

Positive 1115 (733) 1416 (875) 810 (80) 010 (00)Negative 415 (267) 216 (125) 210 (20) 1010 (100)

Tumor siteRight lobe 715 (467)Left lobe 815 (533)

HCC gradeGrade I 215 (133)Grade II 415 (267)Grade III 915 (600)

Child scoreA 315 (200)B 615 (400)C 615 (400)

Data expressed as mean plusmn SD (median)Hepatocellular carcinoma (HCC) liver cirrhosis (LC) precirrhotic hepatitis (PCH) nondisease control (NDC)lowastSignificant difference was detected between PCH and LC (119875 = 0014) and HCC (119875 = 002)lowastlowastSignificant difference was detected between PCH and LC (119875 = 0027) and HCC (119875 = 0003)

elevated significantly among disease groups The differencesin ALT were recorded between precirrhotic hepatitis (PCH)and other groups (with LC 119875 = 0027 and with HCC 119875 =0003) and also the differences in AST levels were recordedbetween PCH and other groups (with LC 119875 = 0014 andwith HCC 119875 = 002) On contrary serum albumin levelwas decreased with liver disease state but with no significantdifference

The severity of HCCwas determined through a combina-tion of clinical and laboratory evaluation within the contextof Child-Pugh scoring system Child score was recorded inHCCpatients as Child A in 20 (315) Child B in 40 (615)and Child C in 40 (615) Serum expression of IL-10 andIL-12 according to the pathological diagnosis was listed inTable 2 IL-10 and IL-12 showed significant elevation withdisease progression and the highest expression was detectedin HCC compared to LC and PCH (119875 lt 00001) but there isno significant difference between LC and PCH with respectto IL-12 However serum level of Il-10 and IL-12 showed nosignificant difference with respect to tumor site tumor gradeor Child score

Table 2 IL-10 and IL-12 in patients with different pathologies of thestudied group

IL-10 119875 value IL-12 119875 valueChronic liverDiseaseHCC (119899 = 15) 1613 plusmn 11 lt00001 6569 plusmn 1911 lt00001LC (119899 = 16) 143 plusmn 0765 5058 plusmn 208PCH (119899 = 10) 647 plusmn 0596 4877 plusmn 172NDC (119899 = 10) 604 plusmn 0568 2584 plusmn 1406

HCC gradingGrade I (119899 = 2) 166 plusmn 0565 087 6565 plusmn 34648 09Grade II (119899 = 4) 163 plusmn 072 65975 plusmn 2298Grade III (119899 = 9) 159 plusmn 133 6557 plusmn 169

Child scoreA (119899 = 3) 1613 plusmn 0305 07 6643 plusmn 1604 059B (119899 = 6) 1598 plusmn 115 6583 plusmn 236C (119899 = 6) 1628 plusmn 14 6518 plusmn 191

Data expressed as mean plusmn SD119875 value lt005 is considered significant

4 Disease Markers

Table 3 IL-10 and IL-12 serum expression in chronic liver disease with HCV infection

+ve HCV-chronic liver diseaselowastlowastlowast minusve HCV-chronic liver disease NDCPCH LC HCC Total PCH LC HCC Total

IL-10(ngmL)lowast

6437plusmn 0518

1449plusmn 0615

1639plusmn 1015

13169plusmn 402

66plusmn 113

130plusmn 014

154plusmn 116

1261plusmn 395 604 plusmn 0568

IL-12(ngmL)lowastlowast

484875plusmn 1623

50186plusmn 191

65645plusmn 171

54927plusmn 7915

4990plusmn 2263

5335plusmn 212

65825plusmn 27035

5873plusmn 795 2584 plusmn 1406

lowastIL-10 expression showed significant difference in HCV-infected (119875 lt 00001) and noninfected patients (119875 lt 0003) compared to nondisease controllowastlowastIL-12 expression showed significant difference in HCV-infected and noninfected patients (119875 lt 00001) compared to nondisease controllowastlowastlowast+ve HCV-chronic liver disease shows that there is asymptotic significance (119875 lt 00001) within groups (Kruskal-Wallis test) and the difference betweengroups was detected by Mann-Whitney showing that there is a significant (119875 lt 00001) difference between each group and the other one

1800

1600

1400

1200

1000

800

600

20000 30000 40000 50000 60000 70000

R2 linear = 0616

IL-12 (ngmL)

IL-10

(ng

mL)

Figure 1 Scatter plot of IL-10 and IL-12 in patients with differentpathological diagnosis (119903 = 0785 119875 lt 00001)

IL-10 showed a significant positive correlation with IL-12in patients with different liver pathologies (119903 = 0785 119875 lt00001) (Figure 1) and a significant correlation coefficient wasdetected between IL-10 and IL-12 with transaminase values ofpatients with chronic liver disease (Figure 2) IL-10 showedsignificant positive correlation with ALT (119903 = 0441 119875 =0001) and AST value (119903 = 0498 119875 lt 00001) Also Il-12showed a significant positive correlation with ALT (119903 = 039119875 = 0004) and AST value (119903 = 048 119875 lt 00001)

IL-10 and IL-12 were elevated significantly (119875 lt 00001)in the sera of patients with HCV infection compared tonondisease control group (Table 3) and the level was elevatedsignificantly among disease groups (119875 lt 00001) HCV-infected patients showed IL-10 at higher level than in HCV-negative patients and in opposite to IL-12 expression patternin HCV-positive and HCV-negative cases The difference incytokine expression level was low between HCV-positive andHCV-negative cases and did not differ significantly betweenthem Furthermore a significant positive correlation (119903 =0617 119875 lt 00001) was detected between IL-10 and IL-12(Figure 3) in HCV-infected patients

4 DiscussionCytokines play an important role in viral clearance infectioncontrol inflammation regeneration and fibrosis and also are

implicated in the pathological processes occurring in the liverduring viral infection [12]Therefore our study was designedto investigate serum level of IL-10 and IL-12 in chronic liverdisease and their association with HCV infection and toevaluate their possible role as new biomarkers in chronicinflammation progression leading to HCC

IL-12 is one of the most important proinflammatorycytokines presented with the initiation of immune responsedetermining Th1 and Th2 differentiation [1] Capone etal [13] compared the serum level of numerous cytokineschemokines and growth factors in HC and LC patients withrespect to those in HCC patients tested in their study Theyfound that the mean concentrations of all of these moleculeswere higher in HCC patients than in those with LC Ourresults are in consistence with these findings where serumlevels of IL-12 was more elevated in HCC patients than inthose with LC and PCH (119875 lt 00001) and the elevationwas increased with disease progression This suggests thatthe expression of these proinflammatory molecules tends toincrease in the chronic inflammation progression that leadsto LC and HCC and thus their evaluation could be used forprognostic studies [12] In spite of controversial data in theliterature several reports described that the serum level ofIL-12 was significantly higher in their patients with chronicHCV infection than in healthy donors [14 15] In currentstudy serum expression of IL-12 inHCV-chronic liver diseaseshowed significant elevation compared to nondisease indi-viduals and the level was enhanced with disease progressionsuggesting that a strong proinflammatory cytokine responsecould play an important role in the development of hepaticinjury in patients with chronic hepatitis C and thereforeapart from contributing to viral clearance this polarizedimmunological profile may contribute to the pathogenesis ofliver disease [16]

Serum IL-10 concentration has been reported to besignificantly elevated in patients with chronic HCV and IL-10 may be related to hepatocarcinogenesis with suppressionof immune surveillance [17] The measurement of IL-10 con-centrations in serum samples of patients with chronic HCVinfection by enzyme linked immunosorbent assay (ELISA)has showed contrasting results Kakumu et al [14] demon-strated greater spontaneous IL-10 production by peripheralblood mononuclear cells (PBMc) in patients with CHC andliver cirrhosis than in healthy controls and its decrease

Disease Markers 5

16000

14000

12000

10000

8000

6000

4000

2000

600 800 1000 1200 1400 1600 1800

R2 linear = 0194

600 800 1000 1200 1400 1600 1800

R2 linear = 0248

AST

(120583m

L)

00

5000

10000

15000

20000

25000

R2 linear = 0231

AST

(120583m

L)

00

5000

10000

15000

20000

25000

20000 30000 40000 50000 60000 70000

R2 linear = 0154

2000

4000

6000

8000

10000

12000

14000

16000

20000 30000 40000 50000 60000 70000

ALT

(120583m

L)A

LT (120583

mL)

IL-10 (ngmL)

IL-12 (ngmL)IL-12 (ngmL)

IL-10 (ngmL)

Figure 2 Serum levels of IL-10 and Il-12 showed significant correlation with transaminases in patients with chronic liver disease

during IFN treatment On the contrary Yamashiki et al [18]reported that the level of IL-10 in the monocytemacrophagesupernatants of patients with CHC was significantly lowerthan in healthy controls In consistence with Othman etal [19] and Hattori et al [20] IL-10 concentration waselevated in patients with HCV cirrhosis and HCC andthe concentrations are associated with disease progressionindicating that IL-10 reflects the degree of inflammation inthe liver and may be related to the development of HCCHowever increased circulating IL-10 has been reported inpatients with different types of tumors including resectableHCC [21]

These results may be explained on the basis that the highserum IL-10 levels in patients withHCC result from the secre-tion of IL-10 by tumor cells in addition to the production atthe site of inflammatory changes with activated infiltratingmononuclear cells in the liver [22] The immunosuppressiveeffects of IL-10 may play a major role in the developmentof neoplastic process by suppressing macrophage activation

and interferon-gamma production thereby crippling twopotential mediators of an antitumor response this mayhelp the tumor cells escape host immune surveillance andpotentate tumor cells to metastasize [19] Also the functionalconsequences of IL-10 binding to its receptors on tumorcells could be the prevention of programmed cell death andthe promotion of proliferation [23] Therefore it has beenproposed that IL-10 plays a key role in the oncogenetic andmetastatic ability of neoplasms [24]

Analysis of the sequential serum data reported by Wu etal [25] indicated a significant correlation between Il-10 andIL-12 in tolerance phase (correlation coefficient 042 119875 lt00001) suggesting that IL-10 may be bifunctional during thecourse of HBV infection and that its role may depend onserum levels and cooperative cytokines like IL-12 and down-stream IL-2 In current study a significant positive correlation(119903 = 0617 119875 lt 00001) was detected between IL-10 and IL-12 in HCV-chronic liver disease patients IL-10 was elevatedin patients with HCV infection compared to HCV-negative

6 Disease Markers

1800

1600

1400

1200

1000

800

600

45000 50000 55000 60000 65000 70000

R2 linear = 0381

IL-10

(ng

mL)

IL-12 (ngmL)

Figure 3 Scatter plot of IL-10 and IL-12 in patients with HCV-chronic liver disease (119903 = 0617 119875 lt 00001)

patients and in contrary to IL-12 expression pattern in HCV-positive and HCV-negative cases These results suggest thatelevation of serum IL-10might be involved in downregulationof the inflammatory response in chronic liver disease [14]Furthermore interleukin-12 production by DC appears to bedownregulated by IL-10 while with maturation the DC canproduce large amounts of IL-12 and become resistant to thesuppressive effects of antigen presenting function by IL-10[26]

In patients with chronic inflammation IL-1120572 IL-2RMIFand 120573-NGF showed significant correlation and a positive cor-relation coefficient with the transaminase values which werehigher in these patients than in healthy controls Thereforethese proteins could be considered to be an index of immuneactivation In particular these results were in agreement withliterature data reporting that IL-1 and IL-2R participate inthe progression from liver injury to fibrosis [27 28] andthat 120573-VGF is involved in liver cancer growth and metastasisand can be used as an index of chronic infection leading toLC and HCC [29 30] In agreement with these studies asignificant positive correlation was detected between IL-10and IL-12 in our study and a significant correlation coefficientwas detected with the transaminase levels which were higherin all liver pathologies than in nondisease controls theelevation was significantly recorded with disease progressionsuggesting that IL-10 and IL-12 could be used as an index toreflect the degree of inflammation in the liver with ultimatedevelopment of HCC However Liu et al [31] reported thatthe levels of TNF-alpha IL-1 Il-10 and IL-12 in patients withchronic liver failure were increased and the increase of IL-10is secondary to elevation of IL-12

5 Conclusion

Significant elevation of IL-10 and IL-12 with disease progres-sion and transaminase values might be involved in chronic

inflammation progression leading to HCC and their evalua-tion could be used as new biomarkers for HCC development

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S S Youssef A M Abd El-Aal A Saad M H Omran TEl Zanaty and S M Seif ldquoImpact of IL12B gene rs 3212227polymorphism on fibrosis liver inflammation and response totreatment in genotype 4 Egyptian hepatitis c patientsrdquo DiseaseMarkers vol 35 no 5 pp 431ndash437 2013

[2] M H Omran N E Ibrahim S S Youssef et al ldquoRelation ofinterleukin-1120573 gene to treatment response in chronic patientsinfected with HCV genotype 4rdquo Journal of Infection in Develop-ing Countries vol 7 no 11 pp 851ndash858 2013

[3] E Billerbeck T Bottler and R Thimme ldquoRegulatory T cells inviral hepatitisrdquoWorld Journal of Gastroenterology vol 13 no 36pp 4858ndash4864 2007

[4] K Hiroishi T Ito and M Imawari ldquoImmune responses inhepatitis C virus infection and mechanisms of hepatitis C viruspersistencerdquo Journal of Gastroenterology andHepatology vol 23no 10 pp 1473ndash1482 2008

[5] A Guobuzaite S Chokshi L Balciuniene et al ldquoViral clearanceor persistence after acute hepatitis C infection interim resultsfrom a prospective studyrdquoMedicina vol 44 no 7 pp 510ndash5202008

[6] L Wan Y-J Kung Y-J Lin et al ldquoTh1 and Th2 cytokinesare elevated in HCV-infected SVR(-) patients treated withinterferon-120572rdquo Biochemical and Biophysical Research Communi-cations vol 379 no 4 pp 855ndash860 2009

[7] A K Abbas andA H Lichtman ldquoCytokines and effectormech-anisms of cell-mediated immunityrdquo in Cellular and MolecularImmunology pp 243ndash317 Saunders Philadelphia Pa USA 5thedition 2003

[8] S-L Tsai Y-F Liaw M-H Chen C-Y Huang and G CKuo ldquoDetection of type 2-like T-helper cells in hepatitis Cvirus infection implications for hepatitis C virus chronicityrdquoHepatology vol 25 no 2 pp 449ndash458 1997

[9] W E Naugler and M Karin ldquoThe wolf in sheeprsquos clothing therole of interleukin-6 in immunity Inflammation and cancerrdquoTrends in Molecular Medicine vol 14 no 3 pp 109ndash119 2008

[10] M Del Vecchio E Bajetta S Canova et al ldquoInterleukin-12biological properties and clinical applicationrdquo Clinical CancerResearch vol 13 no 16 pp 4677ndash4685 2007

[11] W T Watford M Moriguchi A Morinobu and J J OrsquoShealdquoThe biology of IL-12 coordinating innate and adaptiveimmune responsesrdquo Cytokine and Growth Factor Reviews vol14 no 5 pp 361ndash368 2003

[12] S Costantini F Capone E Guerriero P Maio G Colonna andGCastello ldquoSerumcytokine levels as putative prognosticmark-ers in the progression of chronic HCV hepatitis to cirrhosisrdquoEuropean Cytokine Network vol 21 no 4 pp 251ndash256 2010

[13] F Capone S Costantini E Guerriero et al ldquoSerum cytokinelevels in patients with hepatocellular carcinomardquo EuropeanCytokine Network vol 21 no 2 pp 99ndash104 2010

[14] S Kakumu A Okumura T Ishikawa K Iwata M Yano and KYoshioka ldquoProduction of interleukins 10 and 12 by peripheral

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

immune response Th2-like responses produce IL-4 and IL-10 which help augment antibody production and inhibitdevelopment of the Th1 response [8]

IL-10 is a pleiotropic cytokine produced by macrophagesT-helper 2 (Th2) cells and B-lymphocytes and both canstimulate and suppress the immune response IL-10 has beenshown to inhibit various immune reactions [9] Interleukin-12 (IL-12) is one of the most important proinflammatorycytokines produced mainly by antigen presenting cells asa result of IFN-120574 stimulation [10] and presented with theinitiation of immune response so IL-12 can be consideredas one of the most clearly defined factors determining Th1andTh2 differentiation [11] Therefore the current study wasdesigned to investigate serum expression of inflammatoryand immunoregulatory cytokines (IL-10IL-12) with HCVinfection in chronic liver disease and to evaluate their possiblerole as new biomarkers in chronic inflammation progressionleading to HCC

2 Material and Methods

21 Study Population Forty-one (34 males and 7 femalesmean age 4758 plusmn 1242 yrs) patients suffering from chronicliver diseases were enrolled in the present study 33 (805)patients had positive reactivity for HCV infection withdetectable HCV RNA and with no serological evidenceof coinfection with other hepatotropic viruses or humanimmunodeficiency virus and 8 (195) patients had nega-tive reactivity for HCV infection All patients gave writteninformed consent at gastroenterology surgical center Man-soura University Egypt The studied group was subjected toclinical examination imaging radiology laboratory investi-gation and liver biopsy Liver tissue and blood samples of allindividuals were collected and sera were stored at minus70∘Cuntilbeing used

Liver tissue specimens of all cases were routinely pro-cessed for histopathological diagnosis fixed in 10 neutralbuffered formalin embedded in paraffin and cut into 3ndash5 120583mthick sections The studied group was histopathologicallydiagnosed and categorized into 15 (3658) patients (13 maleand 2 female mean age 5706 plusmn 1258 yrs) with HCC 16(390) patients (12 male and 4 female mean age 4606 plusmn79 yrs) with liver cirrhosis and 10 (244) patients (9 maleand 1 female mean age 358 plusmn 522 yrs) with liver histologycompatible with precirrhotic hepatitis (Metavir score A2F3)Tumor grading of HCC was histopathologically assessed asgrade I in 2 (133) grade II in 4 (267) and grade III in 9(60) of 15 HCC cases

Also ten (10) patients complaining of cholecystitis with-out a clinical history of hepatitis and without symptoms orsigns of liver disease were included as nondisease controlgroup (7 male and 3 female mean age 356plusmn31 yrs) (Table 1)

211 Biochemical and Virological Determination Liver bio-chemistries including albumin globulin ALT AST alkalinephosphatase and total bilirubin were measured by an auto-analyzer Serological diagnosis of HBV and HCV was per-formed by automated immunoassayHBsAgwasmeasured by

a commercial immunoassay kit (Abbott Laboratories USA)Serum HCV Ab was analyzed by third-generation ELISA kit(BIOKIT SA Barcelona Spain)

212 Detection of Genomic HCV RNA Strands by RT-PCRTotal HCV RNA was isolated from 125 120583L of the serum andpurified by using SV Total RNA isolation system (PromegaCo USA) Qualitative detection of the genomic HCV RNAwas done using a strand specific real-time RT-PCR with theuse of thermostable enzyme (Tth) for the synthesis of cDNAat a high temperature For the amplification of the genomicHCV RNA strand two sets of oligonucleotide primers (Bio-Synthesis USA) deduced from the highly conserved 51015840-noncoding region of HCV genome were induced in the firstand second stage respectively

1st Stage

Sense C196 (51015840-CCATGGCGTTAGTATGAGTG-31015840)Antisense Seq-CR (51015840-TGCTCATGGTGCACGGTC-TA-31015840)

2nd Stage

Sense Seq-3 (51015840-AGAGCCATAGTGGTCTGCGG-31015840)Antisense Seq-4 (51015840-CTTTCGCGACCCAACACT-AC-31015840)

213 Enzyme Linked Immunosorbent Assays for Il-10 andIL-12 Serum levels of IL-10 and IL-12 were measured byELISA kit (Diaclone Research Besancon France) accordingto the manufacturerrsquos instruction which can detect up to3 pgmL of human IL-10 and 5 pgmL of human IL-12 Briefly50 120583L of standards and samples were added in duplicate tothe precoated strip well plate then 50120583L of biotinylatedantibody was added to each well as a conjugated antibodyfor 2 hrs After washing three times 100120583L of streptavidinhorseradish peroxidase (HRP) was added to each well for30min followed by 100 120583L of TMB substrate solution for30min Finally 100120583L of stop solutionwas added to eachwelland the absorbance of the plate was detected on a plate readerat 450 nm All incubations and recording were done at roomtemperature

22 Statistical Analysis Results were expressed as mean plusmnSD (median) The differences in mean were assessed byKruskal Wallis (Mann-Whitney U test) and all differenceswere considered to be significant at 119875 le 005 Correlationswere calculated by Spearmanrsquos correlation coefficient

3 Results

Clinical virological and biochemical characteristics of thestudied group were listed according to the pathological diag-nosis in Table 1 Mean serum level of ALT AST total proteinand S bilirubin and prothrombin time showed elevatedexpressionwith disease progression Only ALT andASTwere

Disease Markers 3

Table 1 Clinical biochemical and virological characteristics of the studied group

Chronic liver disease (119899 = 41) NDC (119899 = 10)HCC LC PCH

No () 1541 (366) 1641 (390) 1041 (244) 1010 (100)Gender (MF) 132 124 91 73Age (years) 5706 plusmn 1258 (600) 4606 plusmn 79 (450) 358 plusmn 52 (375) 356 plusmn 31 (355)Albumin (gdL) 422 plusmn 058 (43) 415 plusmn 0467 (40) 45 plusmn 042 (46) 383 plusmn 029 (385)T protein (gdL) 795 plusmn 0603 (80) 76 plusmn 067 (78) 766 plusmn 049 (785) 722 plusmn 053 (715)T bilirubin (mgdL) 169 plusmn 257 (12) 128 plusmn 154 (10) 099 plusmn 054 (09) 057 plusmn 021 (055)AST (120583mL)lowast 928 plusmn 6567 (670) 570 plusmn 266 (525) 348 plusmn 1144 (305) 266 plusmn 55 (265)ALT (120583mL)lowastlowast 5873 plusmn 3686 (460) 490 plusmn 1735 (490) 326 plusmn 148 (255) 277 plusmn 41 (275)Proth time (Sec) 143 plusmn 17 (140) 1358 plusmn 466 (146) 132 plusmn 477 (133) 604 plusmn 0568 (61)Proth conc () 7226 plusmn 1378 (700) 70375 plusmn 1299 (690) 731 plusmn 190 (780) 2584 plusmn 1406 (2560)HCV infection

Positive 1115 (733) 1416 (875) 810 (80) 010 (00)Negative 415 (267) 216 (125) 210 (20) 1010 (100)

Tumor siteRight lobe 715 (467)Left lobe 815 (533)

HCC gradeGrade I 215 (133)Grade II 415 (267)Grade III 915 (600)

Child scoreA 315 (200)B 615 (400)C 615 (400)

Data expressed as mean plusmn SD (median)Hepatocellular carcinoma (HCC) liver cirrhosis (LC) precirrhotic hepatitis (PCH) nondisease control (NDC)lowastSignificant difference was detected between PCH and LC (119875 = 0014) and HCC (119875 = 002)lowastlowastSignificant difference was detected between PCH and LC (119875 = 0027) and HCC (119875 = 0003)

elevated significantly among disease groups The differencesin ALT were recorded between precirrhotic hepatitis (PCH)and other groups (with LC 119875 = 0027 and with HCC 119875 =0003) and also the differences in AST levels were recordedbetween PCH and other groups (with LC 119875 = 0014 andwith HCC 119875 = 002) On contrary serum albumin levelwas decreased with liver disease state but with no significantdifference

The severity of HCCwas determined through a combina-tion of clinical and laboratory evaluation within the contextof Child-Pugh scoring system Child score was recorded inHCCpatients as Child A in 20 (315) Child B in 40 (615)and Child C in 40 (615) Serum expression of IL-10 andIL-12 according to the pathological diagnosis was listed inTable 2 IL-10 and IL-12 showed significant elevation withdisease progression and the highest expression was detectedin HCC compared to LC and PCH (119875 lt 00001) but there isno significant difference between LC and PCH with respectto IL-12 However serum level of Il-10 and IL-12 showed nosignificant difference with respect to tumor site tumor gradeor Child score

Table 2 IL-10 and IL-12 in patients with different pathologies of thestudied group

IL-10 119875 value IL-12 119875 valueChronic liverDiseaseHCC (119899 = 15) 1613 plusmn 11 lt00001 6569 plusmn 1911 lt00001LC (119899 = 16) 143 plusmn 0765 5058 plusmn 208PCH (119899 = 10) 647 plusmn 0596 4877 plusmn 172NDC (119899 = 10) 604 plusmn 0568 2584 plusmn 1406

HCC gradingGrade I (119899 = 2) 166 plusmn 0565 087 6565 plusmn 34648 09Grade II (119899 = 4) 163 plusmn 072 65975 plusmn 2298Grade III (119899 = 9) 159 plusmn 133 6557 plusmn 169

Child scoreA (119899 = 3) 1613 plusmn 0305 07 6643 plusmn 1604 059B (119899 = 6) 1598 plusmn 115 6583 plusmn 236C (119899 = 6) 1628 plusmn 14 6518 plusmn 191

Data expressed as mean plusmn SD119875 value lt005 is considered significant

4 Disease Markers

Table 3 IL-10 and IL-12 serum expression in chronic liver disease with HCV infection

+ve HCV-chronic liver diseaselowastlowastlowast minusve HCV-chronic liver disease NDCPCH LC HCC Total PCH LC HCC Total

IL-10(ngmL)lowast

6437plusmn 0518

1449plusmn 0615

1639plusmn 1015

13169plusmn 402

66plusmn 113

130plusmn 014

154plusmn 116

1261plusmn 395 604 plusmn 0568

IL-12(ngmL)lowastlowast

484875plusmn 1623

50186plusmn 191

65645plusmn 171

54927plusmn 7915

4990plusmn 2263

5335plusmn 212

65825plusmn 27035

5873plusmn 795 2584 plusmn 1406

lowastIL-10 expression showed significant difference in HCV-infected (119875 lt 00001) and noninfected patients (119875 lt 0003) compared to nondisease controllowastlowastIL-12 expression showed significant difference in HCV-infected and noninfected patients (119875 lt 00001) compared to nondisease controllowastlowastlowast+ve HCV-chronic liver disease shows that there is asymptotic significance (119875 lt 00001) within groups (Kruskal-Wallis test) and the difference betweengroups was detected by Mann-Whitney showing that there is a significant (119875 lt 00001) difference between each group and the other one

1800

1600

1400

1200

1000

800

600

20000 30000 40000 50000 60000 70000

R2 linear = 0616

IL-12 (ngmL)

IL-10

(ng

mL)

Figure 1 Scatter plot of IL-10 and IL-12 in patients with differentpathological diagnosis (119903 = 0785 119875 lt 00001)

IL-10 showed a significant positive correlation with IL-12in patients with different liver pathologies (119903 = 0785 119875 lt00001) (Figure 1) and a significant correlation coefficient wasdetected between IL-10 and IL-12 with transaminase values ofpatients with chronic liver disease (Figure 2) IL-10 showedsignificant positive correlation with ALT (119903 = 0441 119875 =0001) and AST value (119903 = 0498 119875 lt 00001) Also Il-12showed a significant positive correlation with ALT (119903 = 039119875 = 0004) and AST value (119903 = 048 119875 lt 00001)

IL-10 and IL-12 were elevated significantly (119875 lt 00001)in the sera of patients with HCV infection compared tonondisease control group (Table 3) and the level was elevatedsignificantly among disease groups (119875 lt 00001) HCV-infected patients showed IL-10 at higher level than in HCV-negative patients and in opposite to IL-12 expression patternin HCV-positive and HCV-negative cases The difference incytokine expression level was low between HCV-positive andHCV-negative cases and did not differ significantly betweenthem Furthermore a significant positive correlation (119903 =0617 119875 lt 00001) was detected between IL-10 and IL-12(Figure 3) in HCV-infected patients

4 DiscussionCytokines play an important role in viral clearance infectioncontrol inflammation regeneration and fibrosis and also are

implicated in the pathological processes occurring in the liverduring viral infection [12]Therefore our study was designedto investigate serum level of IL-10 and IL-12 in chronic liverdisease and their association with HCV infection and toevaluate their possible role as new biomarkers in chronicinflammation progression leading to HCC

IL-12 is one of the most important proinflammatorycytokines presented with the initiation of immune responsedetermining Th1 and Th2 differentiation [1] Capone etal [13] compared the serum level of numerous cytokineschemokines and growth factors in HC and LC patients withrespect to those in HCC patients tested in their study Theyfound that the mean concentrations of all of these moleculeswere higher in HCC patients than in those with LC Ourresults are in consistence with these findings where serumlevels of IL-12 was more elevated in HCC patients than inthose with LC and PCH (119875 lt 00001) and the elevationwas increased with disease progression This suggests thatthe expression of these proinflammatory molecules tends toincrease in the chronic inflammation progression that leadsto LC and HCC and thus their evaluation could be used forprognostic studies [12] In spite of controversial data in theliterature several reports described that the serum level ofIL-12 was significantly higher in their patients with chronicHCV infection than in healthy donors [14 15] In currentstudy serum expression of IL-12 inHCV-chronic liver diseaseshowed significant elevation compared to nondisease indi-viduals and the level was enhanced with disease progressionsuggesting that a strong proinflammatory cytokine responsecould play an important role in the development of hepaticinjury in patients with chronic hepatitis C and thereforeapart from contributing to viral clearance this polarizedimmunological profile may contribute to the pathogenesis ofliver disease [16]

Serum IL-10 concentration has been reported to besignificantly elevated in patients with chronic HCV and IL-10 may be related to hepatocarcinogenesis with suppressionof immune surveillance [17] The measurement of IL-10 con-centrations in serum samples of patients with chronic HCVinfection by enzyme linked immunosorbent assay (ELISA)has showed contrasting results Kakumu et al [14] demon-strated greater spontaneous IL-10 production by peripheralblood mononuclear cells (PBMc) in patients with CHC andliver cirrhosis than in healthy controls and its decrease

Disease Markers 5

16000

14000

12000

10000

8000

6000

4000

2000

600 800 1000 1200 1400 1600 1800

R2 linear = 0194

600 800 1000 1200 1400 1600 1800

R2 linear = 0248

AST

(120583m

L)

00

5000

10000

15000

20000

25000

R2 linear = 0231

AST

(120583m

L)

00

5000

10000

15000

20000

25000

20000 30000 40000 50000 60000 70000

R2 linear = 0154

2000

4000

6000

8000

10000

12000

14000

16000

20000 30000 40000 50000 60000 70000

ALT

(120583m

L)A

LT (120583

mL)

IL-10 (ngmL)

IL-12 (ngmL)IL-12 (ngmL)

IL-10 (ngmL)

Figure 2 Serum levels of IL-10 and Il-12 showed significant correlation with transaminases in patients with chronic liver disease

during IFN treatment On the contrary Yamashiki et al [18]reported that the level of IL-10 in the monocytemacrophagesupernatants of patients with CHC was significantly lowerthan in healthy controls In consistence with Othman etal [19] and Hattori et al [20] IL-10 concentration waselevated in patients with HCV cirrhosis and HCC andthe concentrations are associated with disease progressionindicating that IL-10 reflects the degree of inflammation inthe liver and may be related to the development of HCCHowever increased circulating IL-10 has been reported inpatients with different types of tumors including resectableHCC [21]

These results may be explained on the basis that the highserum IL-10 levels in patients withHCC result from the secre-tion of IL-10 by tumor cells in addition to the production atthe site of inflammatory changes with activated infiltratingmononuclear cells in the liver [22] The immunosuppressiveeffects of IL-10 may play a major role in the developmentof neoplastic process by suppressing macrophage activation

and interferon-gamma production thereby crippling twopotential mediators of an antitumor response this mayhelp the tumor cells escape host immune surveillance andpotentate tumor cells to metastasize [19] Also the functionalconsequences of IL-10 binding to its receptors on tumorcells could be the prevention of programmed cell death andthe promotion of proliferation [23] Therefore it has beenproposed that IL-10 plays a key role in the oncogenetic andmetastatic ability of neoplasms [24]

Analysis of the sequential serum data reported by Wu etal [25] indicated a significant correlation between Il-10 andIL-12 in tolerance phase (correlation coefficient 042 119875 lt00001) suggesting that IL-10 may be bifunctional during thecourse of HBV infection and that its role may depend onserum levels and cooperative cytokines like IL-12 and down-stream IL-2 In current study a significant positive correlation(119903 = 0617 119875 lt 00001) was detected between IL-10 and IL-12 in HCV-chronic liver disease patients IL-10 was elevatedin patients with HCV infection compared to HCV-negative

6 Disease Markers

1800

1600

1400

1200

1000

800

600

45000 50000 55000 60000 65000 70000

R2 linear = 0381

IL-10

(ng

mL)

IL-12 (ngmL)

Figure 3 Scatter plot of IL-10 and IL-12 in patients with HCV-chronic liver disease (119903 = 0617 119875 lt 00001)

patients and in contrary to IL-12 expression pattern in HCV-positive and HCV-negative cases These results suggest thatelevation of serum IL-10might be involved in downregulationof the inflammatory response in chronic liver disease [14]Furthermore interleukin-12 production by DC appears to bedownregulated by IL-10 while with maturation the DC canproduce large amounts of IL-12 and become resistant to thesuppressive effects of antigen presenting function by IL-10[26]

In patients with chronic inflammation IL-1120572 IL-2RMIFand 120573-NGF showed significant correlation and a positive cor-relation coefficient with the transaminase values which werehigher in these patients than in healthy controls Thereforethese proteins could be considered to be an index of immuneactivation In particular these results were in agreement withliterature data reporting that IL-1 and IL-2R participate inthe progression from liver injury to fibrosis [27 28] andthat 120573-VGF is involved in liver cancer growth and metastasisand can be used as an index of chronic infection leading toLC and HCC [29 30] In agreement with these studies asignificant positive correlation was detected between IL-10and IL-12 in our study and a significant correlation coefficientwas detected with the transaminase levels which were higherin all liver pathologies than in nondisease controls theelevation was significantly recorded with disease progressionsuggesting that IL-10 and IL-12 could be used as an index toreflect the degree of inflammation in the liver with ultimatedevelopment of HCC However Liu et al [31] reported thatthe levels of TNF-alpha IL-1 Il-10 and IL-12 in patients withchronic liver failure were increased and the increase of IL-10is secondary to elevation of IL-12

5 Conclusion

Significant elevation of IL-10 and IL-12 with disease progres-sion and transaminase values might be involved in chronic

inflammation progression leading to HCC and their evalua-tion could be used as new biomarkers for HCC development

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S S Youssef A M Abd El-Aal A Saad M H Omran TEl Zanaty and S M Seif ldquoImpact of IL12B gene rs 3212227polymorphism on fibrosis liver inflammation and response totreatment in genotype 4 Egyptian hepatitis c patientsrdquo DiseaseMarkers vol 35 no 5 pp 431ndash437 2013

[2] M H Omran N E Ibrahim S S Youssef et al ldquoRelation ofinterleukin-1120573 gene to treatment response in chronic patientsinfected with HCV genotype 4rdquo Journal of Infection in Develop-ing Countries vol 7 no 11 pp 851ndash858 2013

[3] E Billerbeck T Bottler and R Thimme ldquoRegulatory T cells inviral hepatitisrdquoWorld Journal of Gastroenterology vol 13 no 36pp 4858ndash4864 2007

[4] K Hiroishi T Ito and M Imawari ldquoImmune responses inhepatitis C virus infection and mechanisms of hepatitis C viruspersistencerdquo Journal of Gastroenterology andHepatology vol 23no 10 pp 1473ndash1482 2008

[5] A Guobuzaite S Chokshi L Balciuniene et al ldquoViral clearanceor persistence after acute hepatitis C infection interim resultsfrom a prospective studyrdquoMedicina vol 44 no 7 pp 510ndash5202008

[6] L Wan Y-J Kung Y-J Lin et al ldquoTh1 and Th2 cytokinesare elevated in HCV-infected SVR(-) patients treated withinterferon-120572rdquo Biochemical and Biophysical Research Communi-cations vol 379 no 4 pp 855ndash860 2009

[7] A K Abbas andA H Lichtman ldquoCytokines and effectormech-anisms of cell-mediated immunityrdquo in Cellular and MolecularImmunology pp 243ndash317 Saunders Philadelphia Pa USA 5thedition 2003

[8] S-L Tsai Y-F Liaw M-H Chen C-Y Huang and G CKuo ldquoDetection of type 2-like T-helper cells in hepatitis Cvirus infection implications for hepatitis C virus chronicityrdquoHepatology vol 25 no 2 pp 449ndash458 1997

[9] W E Naugler and M Karin ldquoThe wolf in sheeprsquos clothing therole of interleukin-6 in immunity Inflammation and cancerrdquoTrends in Molecular Medicine vol 14 no 3 pp 109ndash119 2008

[10] M Del Vecchio E Bajetta S Canova et al ldquoInterleukin-12biological properties and clinical applicationrdquo Clinical CancerResearch vol 13 no 16 pp 4677ndash4685 2007

[11] W T Watford M Moriguchi A Morinobu and J J OrsquoShealdquoThe biology of IL-12 coordinating innate and adaptiveimmune responsesrdquo Cytokine and Growth Factor Reviews vol14 no 5 pp 361ndash368 2003

[12] S Costantini F Capone E Guerriero P Maio G Colonna andGCastello ldquoSerumcytokine levels as putative prognosticmark-ers in the progression of chronic HCV hepatitis to cirrhosisrdquoEuropean Cytokine Network vol 21 no 4 pp 251ndash256 2010

[13] F Capone S Costantini E Guerriero et al ldquoSerum cytokinelevels in patients with hepatocellular carcinomardquo EuropeanCytokine Network vol 21 no 2 pp 99ndash104 2010

[14] S Kakumu A Okumura T Ishikawa K Iwata M Yano and KYoshioka ldquoProduction of interleukins 10 and 12 by peripheral

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

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Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 3

Table 1 Clinical biochemical and virological characteristics of the studied group

Chronic liver disease (119899 = 41) NDC (119899 = 10)HCC LC PCH

No () 1541 (366) 1641 (390) 1041 (244) 1010 (100)Gender (MF) 132 124 91 73Age (years) 5706 plusmn 1258 (600) 4606 plusmn 79 (450) 358 plusmn 52 (375) 356 plusmn 31 (355)Albumin (gdL) 422 plusmn 058 (43) 415 plusmn 0467 (40) 45 plusmn 042 (46) 383 plusmn 029 (385)T protein (gdL) 795 plusmn 0603 (80) 76 plusmn 067 (78) 766 plusmn 049 (785) 722 plusmn 053 (715)T bilirubin (mgdL) 169 plusmn 257 (12) 128 plusmn 154 (10) 099 plusmn 054 (09) 057 plusmn 021 (055)AST (120583mL)lowast 928 plusmn 6567 (670) 570 plusmn 266 (525) 348 plusmn 1144 (305) 266 plusmn 55 (265)ALT (120583mL)lowastlowast 5873 plusmn 3686 (460) 490 plusmn 1735 (490) 326 plusmn 148 (255) 277 plusmn 41 (275)Proth time (Sec) 143 plusmn 17 (140) 1358 plusmn 466 (146) 132 plusmn 477 (133) 604 plusmn 0568 (61)Proth conc () 7226 plusmn 1378 (700) 70375 plusmn 1299 (690) 731 plusmn 190 (780) 2584 plusmn 1406 (2560)HCV infection

Positive 1115 (733) 1416 (875) 810 (80) 010 (00)Negative 415 (267) 216 (125) 210 (20) 1010 (100)

Tumor siteRight lobe 715 (467)Left lobe 815 (533)

HCC gradeGrade I 215 (133)Grade II 415 (267)Grade III 915 (600)

Child scoreA 315 (200)B 615 (400)C 615 (400)

Data expressed as mean plusmn SD (median)Hepatocellular carcinoma (HCC) liver cirrhosis (LC) precirrhotic hepatitis (PCH) nondisease control (NDC)lowastSignificant difference was detected between PCH and LC (119875 = 0014) and HCC (119875 = 002)lowastlowastSignificant difference was detected between PCH and LC (119875 = 0027) and HCC (119875 = 0003)

elevated significantly among disease groups The differencesin ALT were recorded between precirrhotic hepatitis (PCH)and other groups (with LC 119875 = 0027 and with HCC 119875 =0003) and also the differences in AST levels were recordedbetween PCH and other groups (with LC 119875 = 0014 andwith HCC 119875 = 002) On contrary serum albumin levelwas decreased with liver disease state but with no significantdifference

The severity of HCCwas determined through a combina-tion of clinical and laboratory evaluation within the contextof Child-Pugh scoring system Child score was recorded inHCCpatients as Child A in 20 (315) Child B in 40 (615)and Child C in 40 (615) Serum expression of IL-10 andIL-12 according to the pathological diagnosis was listed inTable 2 IL-10 and IL-12 showed significant elevation withdisease progression and the highest expression was detectedin HCC compared to LC and PCH (119875 lt 00001) but there isno significant difference between LC and PCH with respectto IL-12 However serum level of Il-10 and IL-12 showed nosignificant difference with respect to tumor site tumor gradeor Child score

Table 2 IL-10 and IL-12 in patients with different pathologies of thestudied group

IL-10 119875 value IL-12 119875 valueChronic liverDiseaseHCC (119899 = 15) 1613 plusmn 11 lt00001 6569 plusmn 1911 lt00001LC (119899 = 16) 143 plusmn 0765 5058 plusmn 208PCH (119899 = 10) 647 plusmn 0596 4877 plusmn 172NDC (119899 = 10) 604 plusmn 0568 2584 plusmn 1406

HCC gradingGrade I (119899 = 2) 166 plusmn 0565 087 6565 plusmn 34648 09Grade II (119899 = 4) 163 plusmn 072 65975 plusmn 2298Grade III (119899 = 9) 159 plusmn 133 6557 plusmn 169

Child scoreA (119899 = 3) 1613 plusmn 0305 07 6643 plusmn 1604 059B (119899 = 6) 1598 plusmn 115 6583 plusmn 236C (119899 = 6) 1628 plusmn 14 6518 plusmn 191

Data expressed as mean plusmn SD119875 value lt005 is considered significant

4 Disease Markers

Table 3 IL-10 and IL-12 serum expression in chronic liver disease with HCV infection

+ve HCV-chronic liver diseaselowastlowastlowast minusve HCV-chronic liver disease NDCPCH LC HCC Total PCH LC HCC Total

IL-10(ngmL)lowast

6437plusmn 0518

1449plusmn 0615

1639plusmn 1015

13169plusmn 402

66plusmn 113

130plusmn 014

154plusmn 116

1261plusmn 395 604 plusmn 0568

IL-12(ngmL)lowastlowast

484875plusmn 1623

50186plusmn 191

65645plusmn 171

54927plusmn 7915

4990plusmn 2263

5335plusmn 212

65825plusmn 27035

5873plusmn 795 2584 plusmn 1406

lowastIL-10 expression showed significant difference in HCV-infected (119875 lt 00001) and noninfected patients (119875 lt 0003) compared to nondisease controllowastlowastIL-12 expression showed significant difference in HCV-infected and noninfected patients (119875 lt 00001) compared to nondisease controllowastlowastlowast+ve HCV-chronic liver disease shows that there is asymptotic significance (119875 lt 00001) within groups (Kruskal-Wallis test) and the difference betweengroups was detected by Mann-Whitney showing that there is a significant (119875 lt 00001) difference between each group and the other one

1800

1600

1400

1200

1000

800

600

20000 30000 40000 50000 60000 70000

R2 linear = 0616

IL-12 (ngmL)

IL-10

(ng

mL)

Figure 1 Scatter plot of IL-10 and IL-12 in patients with differentpathological diagnosis (119903 = 0785 119875 lt 00001)

IL-10 showed a significant positive correlation with IL-12in patients with different liver pathologies (119903 = 0785 119875 lt00001) (Figure 1) and a significant correlation coefficient wasdetected between IL-10 and IL-12 with transaminase values ofpatients with chronic liver disease (Figure 2) IL-10 showedsignificant positive correlation with ALT (119903 = 0441 119875 =0001) and AST value (119903 = 0498 119875 lt 00001) Also Il-12showed a significant positive correlation with ALT (119903 = 039119875 = 0004) and AST value (119903 = 048 119875 lt 00001)

IL-10 and IL-12 were elevated significantly (119875 lt 00001)in the sera of patients with HCV infection compared tonondisease control group (Table 3) and the level was elevatedsignificantly among disease groups (119875 lt 00001) HCV-infected patients showed IL-10 at higher level than in HCV-negative patients and in opposite to IL-12 expression patternin HCV-positive and HCV-negative cases The difference incytokine expression level was low between HCV-positive andHCV-negative cases and did not differ significantly betweenthem Furthermore a significant positive correlation (119903 =0617 119875 lt 00001) was detected between IL-10 and IL-12(Figure 3) in HCV-infected patients

4 DiscussionCytokines play an important role in viral clearance infectioncontrol inflammation regeneration and fibrosis and also are

implicated in the pathological processes occurring in the liverduring viral infection [12]Therefore our study was designedto investigate serum level of IL-10 and IL-12 in chronic liverdisease and their association with HCV infection and toevaluate their possible role as new biomarkers in chronicinflammation progression leading to HCC

IL-12 is one of the most important proinflammatorycytokines presented with the initiation of immune responsedetermining Th1 and Th2 differentiation [1] Capone etal [13] compared the serum level of numerous cytokineschemokines and growth factors in HC and LC patients withrespect to those in HCC patients tested in their study Theyfound that the mean concentrations of all of these moleculeswere higher in HCC patients than in those with LC Ourresults are in consistence with these findings where serumlevels of IL-12 was more elevated in HCC patients than inthose with LC and PCH (119875 lt 00001) and the elevationwas increased with disease progression This suggests thatthe expression of these proinflammatory molecules tends toincrease in the chronic inflammation progression that leadsto LC and HCC and thus their evaluation could be used forprognostic studies [12] In spite of controversial data in theliterature several reports described that the serum level ofIL-12 was significantly higher in their patients with chronicHCV infection than in healthy donors [14 15] In currentstudy serum expression of IL-12 inHCV-chronic liver diseaseshowed significant elevation compared to nondisease indi-viduals and the level was enhanced with disease progressionsuggesting that a strong proinflammatory cytokine responsecould play an important role in the development of hepaticinjury in patients with chronic hepatitis C and thereforeapart from contributing to viral clearance this polarizedimmunological profile may contribute to the pathogenesis ofliver disease [16]

Serum IL-10 concentration has been reported to besignificantly elevated in patients with chronic HCV and IL-10 may be related to hepatocarcinogenesis with suppressionof immune surveillance [17] The measurement of IL-10 con-centrations in serum samples of patients with chronic HCVinfection by enzyme linked immunosorbent assay (ELISA)has showed contrasting results Kakumu et al [14] demon-strated greater spontaneous IL-10 production by peripheralblood mononuclear cells (PBMc) in patients with CHC andliver cirrhosis than in healthy controls and its decrease

Disease Markers 5

16000

14000

12000

10000

8000

6000

4000

2000

600 800 1000 1200 1400 1600 1800

R2 linear = 0194

600 800 1000 1200 1400 1600 1800

R2 linear = 0248

AST

(120583m

L)

00

5000

10000

15000

20000

25000

R2 linear = 0231

AST

(120583m

L)

00

5000

10000

15000

20000

25000

20000 30000 40000 50000 60000 70000

R2 linear = 0154

2000

4000

6000

8000

10000

12000

14000

16000

20000 30000 40000 50000 60000 70000

ALT

(120583m

L)A

LT (120583

mL)

IL-10 (ngmL)

IL-12 (ngmL)IL-12 (ngmL)

IL-10 (ngmL)

Figure 2 Serum levels of IL-10 and Il-12 showed significant correlation with transaminases in patients with chronic liver disease

during IFN treatment On the contrary Yamashiki et al [18]reported that the level of IL-10 in the monocytemacrophagesupernatants of patients with CHC was significantly lowerthan in healthy controls In consistence with Othman etal [19] and Hattori et al [20] IL-10 concentration waselevated in patients with HCV cirrhosis and HCC andthe concentrations are associated with disease progressionindicating that IL-10 reflects the degree of inflammation inthe liver and may be related to the development of HCCHowever increased circulating IL-10 has been reported inpatients with different types of tumors including resectableHCC [21]

These results may be explained on the basis that the highserum IL-10 levels in patients withHCC result from the secre-tion of IL-10 by tumor cells in addition to the production atthe site of inflammatory changes with activated infiltratingmononuclear cells in the liver [22] The immunosuppressiveeffects of IL-10 may play a major role in the developmentof neoplastic process by suppressing macrophage activation

and interferon-gamma production thereby crippling twopotential mediators of an antitumor response this mayhelp the tumor cells escape host immune surveillance andpotentate tumor cells to metastasize [19] Also the functionalconsequences of IL-10 binding to its receptors on tumorcells could be the prevention of programmed cell death andthe promotion of proliferation [23] Therefore it has beenproposed that IL-10 plays a key role in the oncogenetic andmetastatic ability of neoplasms [24]

Analysis of the sequential serum data reported by Wu etal [25] indicated a significant correlation between Il-10 andIL-12 in tolerance phase (correlation coefficient 042 119875 lt00001) suggesting that IL-10 may be bifunctional during thecourse of HBV infection and that its role may depend onserum levels and cooperative cytokines like IL-12 and down-stream IL-2 In current study a significant positive correlation(119903 = 0617 119875 lt 00001) was detected between IL-10 and IL-12 in HCV-chronic liver disease patients IL-10 was elevatedin patients with HCV infection compared to HCV-negative

6 Disease Markers

1800

1600

1400

1200

1000

800

600

45000 50000 55000 60000 65000 70000

R2 linear = 0381

IL-10

(ng

mL)

IL-12 (ngmL)

Figure 3 Scatter plot of IL-10 and IL-12 in patients with HCV-chronic liver disease (119903 = 0617 119875 lt 00001)

patients and in contrary to IL-12 expression pattern in HCV-positive and HCV-negative cases These results suggest thatelevation of serum IL-10might be involved in downregulationof the inflammatory response in chronic liver disease [14]Furthermore interleukin-12 production by DC appears to bedownregulated by IL-10 while with maturation the DC canproduce large amounts of IL-12 and become resistant to thesuppressive effects of antigen presenting function by IL-10[26]

In patients with chronic inflammation IL-1120572 IL-2RMIFand 120573-NGF showed significant correlation and a positive cor-relation coefficient with the transaminase values which werehigher in these patients than in healthy controls Thereforethese proteins could be considered to be an index of immuneactivation In particular these results were in agreement withliterature data reporting that IL-1 and IL-2R participate inthe progression from liver injury to fibrosis [27 28] andthat 120573-VGF is involved in liver cancer growth and metastasisand can be used as an index of chronic infection leading toLC and HCC [29 30] In agreement with these studies asignificant positive correlation was detected between IL-10and IL-12 in our study and a significant correlation coefficientwas detected with the transaminase levels which were higherin all liver pathologies than in nondisease controls theelevation was significantly recorded with disease progressionsuggesting that IL-10 and IL-12 could be used as an index toreflect the degree of inflammation in the liver with ultimatedevelopment of HCC However Liu et al [31] reported thatthe levels of TNF-alpha IL-1 Il-10 and IL-12 in patients withchronic liver failure were increased and the increase of IL-10is secondary to elevation of IL-12

5 Conclusion

Significant elevation of IL-10 and IL-12 with disease progres-sion and transaminase values might be involved in chronic

inflammation progression leading to HCC and their evalua-tion could be used as new biomarkers for HCC development

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S S Youssef A M Abd El-Aal A Saad M H Omran TEl Zanaty and S M Seif ldquoImpact of IL12B gene rs 3212227polymorphism on fibrosis liver inflammation and response totreatment in genotype 4 Egyptian hepatitis c patientsrdquo DiseaseMarkers vol 35 no 5 pp 431ndash437 2013

[2] M H Omran N E Ibrahim S S Youssef et al ldquoRelation ofinterleukin-1120573 gene to treatment response in chronic patientsinfected with HCV genotype 4rdquo Journal of Infection in Develop-ing Countries vol 7 no 11 pp 851ndash858 2013

[3] E Billerbeck T Bottler and R Thimme ldquoRegulatory T cells inviral hepatitisrdquoWorld Journal of Gastroenterology vol 13 no 36pp 4858ndash4864 2007

[4] K Hiroishi T Ito and M Imawari ldquoImmune responses inhepatitis C virus infection and mechanisms of hepatitis C viruspersistencerdquo Journal of Gastroenterology andHepatology vol 23no 10 pp 1473ndash1482 2008

[5] A Guobuzaite S Chokshi L Balciuniene et al ldquoViral clearanceor persistence after acute hepatitis C infection interim resultsfrom a prospective studyrdquoMedicina vol 44 no 7 pp 510ndash5202008

[6] L Wan Y-J Kung Y-J Lin et al ldquoTh1 and Th2 cytokinesare elevated in HCV-infected SVR(-) patients treated withinterferon-120572rdquo Biochemical and Biophysical Research Communi-cations vol 379 no 4 pp 855ndash860 2009

[7] A K Abbas andA H Lichtman ldquoCytokines and effectormech-anisms of cell-mediated immunityrdquo in Cellular and MolecularImmunology pp 243ndash317 Saunders Philadelphia Pa USA 5thedition 2003

[8] S-L Tsai Y-F Liaw M-H Chen C-Y Huang and G CKuo ldquoDetection of type 2-like T-helper cells in hepatitis Cvirus infection implications for hepatitis C virus chronicityrdquoHepatology vol 25 no 2 pp 449ndash458 1997

[9] W E Naugler and M Karin ldquoThe wolf in sheeprsquos clothing therole of interleukin-6 in immunity Inflammation and cancerrdquoTrends in Molecular Medicine vol 14 no 3 pp 109ndash119 2008

[10] M Del Vecchio E Bajetta S Canova et al ldquoInterleukin-12biological properties and clinical applicationrdquo Clinical CancerResearch vol 13 no 16 pp 4677ndash4685 2007

[11] W T Watford M Moriguchi A Morinobu and J J OrsquoShealdquoThe biology of IL-12 coordinating innate and adaptiveimmune responsesrdquo Cytokine and Growth Factor Reviews vol14 no 5 pp 361ndash368 2003

[12] S Costantini F Capone E Guerriero P Maio G Colonna andGCastello ldquoSerumcytokine levels as putative prognosticmark-ers in the progression of chronic HCV hepatitis to cirrhosisrdquoEuropean Cytokine Network vol 21 no 4 pp 251ndash256 2010

[13] F Capone S Costantini E Guerriero et al ldquoSerum cytokinelevels in patients with hepatocellular carcinomardquo EuropeanCytokine Network vol 21 no 2 pp 99ndash104 2010

[14] S Kakumu A Okumura T Ishikawa K Iwata M Yano and KYoshioka ldquoProduction of interleukins 10 and 12 by peripheral

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Disease Markers

Table 3 IL-10 and IL-12 serum expression in chronic liver disease with HCV infection

+ve HCV-chronic liver diseaselowastlowastlowast minusve HCV-chronic liver disease NDCPCH LC HCC Total PCH LC HCC Total

IL-10(ngmL)lowast

6437plusmn 0518

1449plusmn 0615

1639plusmn 1015

13169plusmn 402

66plusmn 113

130plusmn 014

154plusmn 116

1261plusmn 395 604 plusmn 0568

IL-12(ngmL)lowastlowast

484875plusmn 1623

50186plusmn 191

65645plusmn 171

54927plusmn 7915

4990plusmn 2263

5335plusmn 212

65825plusmn 27035

5873plusmn 795 2584 plusmn 1406

lowastIL-10 expression showed significant difference in HCV-infected (119875 lt 00001) and noninfected patients (119875 lt 0003) compared to nondisease controllowastlowastIL-12 expression showed significant difference in HCV-infected and noninfected patients (119875 lt 00001) compared to nondisease controllowastlowastlowast+ve HCV-chronic liver disease shows that there is asymptotic significance (119875 lt 00001) within groups (Kruskal-Wallis test) and the difference betweengroups was detected by Mann-Whitney showing that there is a significant (119875 lt 00001) difference between each group and the other one

1800

1600

1400

1200

1000

800

600

20000 30000 40000 50000 60000 70000

R2 linear = 0616

IL-12 (ngmL)

IL-10

(ng

mL)

Figure 1 Scatter plot of IL-10 and IL-12 in patients with differentpathological diagnosis (119903 = 0785 119875 lt 00001)

IL-10 showed a significant positive correlation with IL-12in patients with different liver pathologies (119903 = 0785 119875 lt00001) (Figure 1) and a significant correlation coefficient wasdetected between IL-10 and IL-12 with transaminase values ofpatients with chronic liver disease (Figure 2) IL-10 showedsignificant positive correlation with ALT (119903 = 0441 119875 =0001) and AST value (119903 = 0498 119875 lt 00001) Also Il-12showed a significant positive correlation with ALT (119903 = 039119875 = 0004) and AST value (119903 = 048 119875 lt 00001)

IL-10 and IL-12 were elevated significantly (119875 lt 00001)in the sera of patients with HCV infection compared tonondisease control group (Table 3) and the level was elevatedsignificantly among disease groups (119875 lt 00001) HCV-infected patients showed IL-10 at higher level than in HCV-negative patients and in opposite to IL-12 expression patternin HCV-positive and HCV-negative cases The difference incytokine expression level was low between HCV-positive andHCV-negative cases and did not differ significantly betweenthem Furthermore a significant positive correlation (119903 =0617 119875 lt 00001) was detected between IL-10 and IL-12(Figure 3) in HCV-infected patients

4 DiscussionCytokines play an important role in viral clearance infectioncontrol inflammation regeneration and fibrosis and also are

implicated in the pathological processes occurring in the liverduring viral infection [12]Therefore our study was designedto investigate serum level of IL-10 and IL-12 in chronic liverdisease and their association with HCV infection and toevaluate their possible role as new biomarkers in chronicinflammation progression leading to HCC

IL-12 is one of the most important proinflammatorycytokines presented with the initiation of immune responsedetermining Th1 and Th2 differentiation [1] Capone etal [13] compared the serum level of numerous cytokineschemokines and growth factors in HC and LC patients withrespect to those in HCC patients tested in their study Theyfound that the mean concentrations of all of these moleculeswere higher in HCC patients than in those with LC Ourresults are in consistence with these findings where serumlevels of IL-12 was more elevated in HCC patients than inthose with LC and PCH (119875 lt 00001) and the elevationwas increased with disease progression This suggests thatthe expression of these proinflammatory molecules tends toincrease in the chronic inflammation progression that leadsto LC and HCC and thus their evaluation could be used forprognostic studies [12] In spite of controversial data in theliterature several reports described that the serum level ofIL-12 was significantly higher in their patients with chronicHCV infection than in healthy donors [14 15] In currentstudy serum expression of IL-12 inHCV-chronic liver diseaseshowed significant elevation compared to nondisease indi-viduals and the level was enhanced with disease progressionsuggesting that a strong proinflammatory cytokine responsecould play an important role in the development of hepaticinjury in patients with chronic hepatitis C and thereforeapart from contributing to viral clearance this polarizedimmunological profile may contribute to the pathogenesis ofliver disease [16]

Serum IL-10 concentration has been reported to besignificantly elevated in patients with chronic HCV and IL-10 may be related to hepatocarcinogenesis with suppressionof immune surveillance [17] The measurement of IL-10 con-centrations in serum samples of patients with chronic HCVinfection by enzyme linked immunosorbent assay (ELISA)has showed contrasting results Kakumu et al [14] demon-strated greater spontaneous IL-10 production by peripheralblood mononuclear cells (PBMc) in patients with CHC andliver cirrhosis than in healthy controls and its decrease

Disease Markers 5

16000

14000

12000

10000

8000

6000

4000

2000

600 800 1000 1200 1400 1600 1800

R2 linear = 0194

600 800 1000 1200 1400 1600 1800

R2 linear = 0248

AST

(120583m

L)

00

5000

10000

15000

20000

25000

R2 linear = 0231

AST

(120583m

L)

00

5000

10000

15000

20000

25000

20000 30000 40000 50000 60000 70000

R2 linear = 0154

2000

4000

6000

8000

10000

12000

14000

16000

20000 30000 40000 50000 60000 70000

ALT

(120583m

L)A

LT (120583

mL)

IL-10 (ngmL)

IL-12 (ngmL)IL-12 (ngmL)

IL-10 (ngmL)

Figure 2 Serum levels of IL-10 and Il-12 showed significant correlation with transaminases in patients with chronic liver disease

during IFN treatment On the contrary Yamashiki et al [18]reported that the level of IL-10 in the monocytemacrophagesupernatants of patients with CHC was significantly lowerthan in healthy controls In consistence with Othman etal [19] and Hattori et al [20] IL-10 concentration waselevated in patients with HCV cirrhosis and HCC andthe concentrations are associated with disease progressionindicating that IL-10 reflects the degree of inflammation inthe liver and may be related to the development of HCCHowever increased circulating IL-10 has been reported inpatients with different types of tumors including resectableHCC [21]

These results may be explained on the basis that the highserum IL-10 levels in patients withHCC result from the secre-tion of IL-10 by tumor cells in addition to the production atthe site of inflammatory changes with activated infiltratingmononuclear cells in the liver [22] The immunosuppressiveeffects of IL-10 may play a major role in the developmentof neoplastic process by suppressing macrophage activation

and interferon-gamma production thereby crippling twopotential mediators of an antitumor response this mayhelp the tumor cells escape host immune surveillance andpotentate tumor cells to metastasize [19] Also the functionalconsequences of IL-10 binding to its receptors on tumorcells could be the prevention of programmed cell death andthe promotion of proliferation [23] Therefore it has beenproposed that IL-10 plays a key role in the oncogenetic andmetastatic ability of neoplasms [24]

Analysis of the sequential serum data reported by Wu etal [25] indicated a significant correlation between Il-10 andIL-12 in tolerance phase (correlation coefficient 042 119875 lt00001) suggesting that IL-10 may be bifunctional during thecourse of HBV infection and that its role may depend onserum levels and cooperative cytokines like IL-12 and down-stream IL-2 In current study a significant positive correlation(119903 = 0617 119875 lt 00001) was detected between IL-10 and IL-12 in HCV-chronic liver disease patients IL-10 was elevatedin patients with HCV infection compared to HCV-negative

6 Disease Markers

1800

1600

1400

1200

1000

800

600

45000 50000 55000 60000 65000 70000

R2 linear = 0381

IL-10

(ng

mL)

IL-12 (ngmL)

Figure 3 Scatter plot of IL-10 and IL-12 in patients with HCV-chronic liver disease (119903 = 0617 119875 lt 00001)

patients and in contrary to IL-12 expression pattern in HCV-positive and HCV-negative cases These results suggest thatelevation of serum IL-10might be involved in downregulationof the inflammatory response in chronic liver disease [14]Furthermore interleukin-12 production by DC appears to bedownregulated by IL-10 while with maturation the DC canproduce large amounts of IL-12 and become resistant to thesuppressive effects of antigen presenting function by IL-10[26]

In patients with chronic inflammation IL-1120572 IL-2RMIFand 120573-NGF showed significant correlation and a positive cor-relation coefficient with the transaminase values which werehigher in these patients than in healthy controls Thereforethese proteins could be considered to be an index of immuneactivation In particular these results were in agreement withliterature data reporting that IL-1 and IL-2R participate inthe progression from liver injury to fibrosis [27 28] andthat 120573-VGF is involved in liver cancer growth and metastasisand can be used as an index of chronic infection leading toLC and HCC [29 30] In agreement with these studies asignificant positive correlation was detected between IL-10and IL-12 in our study and a significant correlation coefficientwas detected with the transaminase levels which were higherin all liver pathologies than in nondisease controls theelevation was significantly recorded with disease progressionsuggesting that IL-10 and IL-12 could be used as an index toreflect the degree of inflammation in the liver with ultimatedevelopment of HCC However Liu et al [31] reported thatthe levels of TNF-alpha IL-1 Il-10 and IL-12 in patients withchronic liver failure were increased and the increase of IL-10is secondary to elevation of IL-12

5 Conclusion

Significant elevation of IL-10 and IL-12 with disease progres-sion and transaminase values might be involved in chronic

inflammation progression leading to HCC and their evalua-tion could be used as new biomarkers for HCC development

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S S Youssef A M Abd El-Aal A Saad M H Omran TEl Zanaty and S M Seif ldquoImpact of IL12B gene rs 3212227polymorphism on fibrosis liver inflammation and response totreatment in genotype 4 Egyptian hepatitis c patientsrdquo DiseaseMarkers vol 35 no 5 pp 431ndash437 2013

[2] M H Omran N E Ibrahim S S Youssef et al ldquoRelation ofinterleukin-1120573 gene to treatment response in chronic patientsinfected with HCV genotype 4rdquo Journal of Infection in Develop-ing Countries vol 7 no 11 pp 851ndash858 2013

[3] E Billerbeck T Bottler and R Thimme ldquoRegulatory T cells inviral hepatitisrdquoWorld Journal of Gastroenterology vol 13 no 36pp 4858ndash4864 2007

[4] K Hiroishi T Ito and M Imawari ldquoImmune responses inhepatitis C virus infection and mechanisms of hepatitis C viruspersistencerdquo Journal of Gastroenterology andHepatology vol 23no 10 pp 1473ndash1482 2008

[5] A Guobuzaite S Chokshi L Balciuniene et al ldquoViral clearanceor persistence after acute hepatitis C infection interim resultsfrom a prospective studyrdquoMedicina vol 44 no 7 pp 510ndash5202008

[6] L Wan Y-J Kung Y-J Lin et al ldquoTh1 and Th2 cytokinesare elevated in HCV-infected SVR(-) patients treated withinterferon-120572rdquo Biochemical and Biophysical Research Communi-cations vol 379 no 4 pp 855ndash860 2009

[7] A K Abbas andA H Lichtman ldquoCytokines and effectormech-anisms of cell-mediated immunityrdquo in Cellular and MolecularImmunology pp 243ndash317 Saunders Philadelphia Pa USA 5thedition 2003

[8] S-L Tsai Y-F Liaw M-H Chen C-Y Huang and G CKuo ldquoDetection of type 2-like T-helper cells in hepatitis Cvirus infection implications for hepatitis C virus chronicityrdquoHepatology vol 25 no 2 pp 449ndash458 1997

[9] W E Naugler and M Karin ldquoThe wolf in sheeprsquos clothing therole of interleukin-6 in immunity Inflammation and cancerrdquoTrends in Molecular Medicine vol 14 no 3 pp 109ndash119 2008

[10] M Del Vecchio E Bajetta S Canova et al ldquoInterleukin-12biological properties and clinical applicationrdquo Clinical CancerResearch vol 13 no 16 pp 4677ndash4685 2007

[11] W T Watford M Moriguchi A Morinobu and J J OrsquoShealdquoThe biology of IL-12 coordinating innate and adaptiveimmune responsesrdquo Cytokine and Growth Factor Reviews vol14 no 5 pp 361ndash368 2003

[12] S Costantini F Capone E Guerriero P Maio G Colonna andGCastello ldquoSerumcytokine levels as putative prognosticmark-ers in the progression of chronic HCV hepatitis to cirrhosisrdquoEuropean Cytokine Network vol 21 no 4 pp 251ndash256 2010

[13] F Capone S Costantini E Guerriero et al ldquoSerum cytokinelevels in patients with hepatocellular carcinomardquo EuropeanCytokine Network vol 21 no 2 pp 99ndash104 2010

[14] S Kakumu A Okumura T Ishikawa K Iwata M Yano and KYoshioka ldquoProduction of interleukins 10 and 12 by peripheral

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 5

16000

14000

12000

10000

8000

6000

4000

2000

600 800 1000 1200 1400 1600 1800

R2 linear = 0194

600 800 1000 1200 1400 1600 1800

R2 linear = 0248

AST

(120583m

L)

00

5000

10000

15000

20000

25000

R2 linear = 0231

AST

(120583m

L)

00

5000

10000

15000

20000

25000

20000 30000 40000 50000 60000 70000

R2 linear = 0154

2000

4000

6000

8000

10000

12000

14000

16000

20000 30000 40000 50000 60000 70000

ALT

(120583m

L)A

LT (120583

mL)

IL-10 (ngmL)

IL-12 (ngmL)IL-12 (ngmL)

IL-10 (ngmL)

Figure 2 Serum levels of IL-10 and Il-12 showed significant correlation with transaminases in patients with chronic liver disease

during IFN treatment On the contrary Yamashiki et al [18]reported that the level of IL-10 in the monocytemacrophagesupernatants of patients with CHC was significantly lowerthan in healthy controls In consistence with Othman etal [19] and Hattori et al [20] IL-10 concentration waselevated in patients with HCV cirrhosis and HCC andthe concentrations are associated with disease progressionindicating that IL-10 reflects the degree of inflammation inthe liver and may be related to the development of HCCHowever increased circulating IL-10 has been reported inpatients with different types of tumors including resectableHCC [21]

These results may be explained on the basis that the highserum IL-10 levels in patients withHCC result from the secre-tion of IL-10 by tumor cells in addition to the production atthe site of inflammatory changes with activated infiltratingmononuclear cells in the liver [22] The immunosuppressiveeffects of IL-10 may play a major role in the developmentof neoplastic process by suppressing macrophage activation

and interferon-gamma production thereby crippling twopotential mediators of an antitumor response this mayhelp the tumor cells escape host immune surveillance andpotentate tumor cells to metastasize [19] Also the functionalconsequences of IL-10 binding to its receptors on tumorcells could be the prevention of programmed cell death andthe promotion of proliferation [23] Therefore it has beenproposed that IL-10 plays a key role in the oncogenetic andmetastatic ability of neoplasms [24]

Analysis of the sequential serum data reported by Wu etal [25] indicated a significant correlation between Il-10 andIL-12 in tolerance phase (correlation coefficient 042 119875 lt00001) suggesting that IL-10 may be bifunctional during thecourse of HBV infection and that its role may depend onserum levels and cooperative cytokines like IL-12 and down-stream IL-2 In current study a significant positive correlation(119903 = 0617 119875 lt 00001) was detected between IL-10 and IL-12 in HCV-chronic liver disease patients IL-10 was elevatedin patients with HCV infection compared to HCV-negative

6 Disease Markers

1800

1600

1400

1200

1000

800

600

45000 50000 55000 60000 65000 70000

R2 linear = 0381

IL-10

(ng

mL)

IL-12 (ngmL)

Figure 3 Scatter plot of IL-10 and IL-12 in patients with HCV-chronic liver disease (119903 = 0617 119875 lt 00001)

patients and in contrary to IL-12 expression pattern in HCV-positive and HCV-negative cases These results suggest thatelevation of serum IL-10might be involved in downregulationof the inflammatory response in chronic liver disease [14]Furthermore interleukin-12 production by DC appears to bedownregulated by IL-10 while with maturation the DC canproduce large amounts of IL-12 and become resistant to thesuppressive effects of antigen presenting function by IL-10[26]

In patients with chronic inflammation IL-1120572 IL-2RMIFand 120573-NGF showed significant correlation and a positive cor-relation coefficient with the transaminase values which werehigher in these patients than in healthy controls Thereforethese proteins could be considered to be an index of immuneactivation In particular these results were in agreement withliterature data reporting that IL-1 and IL-2R participate inthe progression from liver injury to fibrosis [27 28] andthat 120573-VGF is involved in liver cancer growth and metastasisand can be used as an index of chronic infection leading toLC and HCC [29 30] In agreement with these studies asignificant positive correlation was detected between IL-10and IL-12 in our study and a significant correlation coefficientwas detected with the transaminase levels which were higherin all liver pathologies than in nondisease controls theelevation was significantly recorded with disease progressionsuggesting that IL-10 and IL-12 could be used as an index toreflect the degree of inflammation in the liver with ultimatedevelopment of HCC However Liu et al [31] reported thatthe levels of TNF-alpha IL-1 Il-10 and IL-12 in patients withchronic liver failure were increased and the increase of IL-10is secondary to elevation of IL-12

5 Conclusion

Significant elevation of IL-10 and IL-12 with disease progres-sion and transaminase values might be involved in chronic

inflammation progression leading to HCC and their evalua-tion could be used as new biomarkers for HCC development

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S S Youssef A M Abd El-Aal A Saad M H Omran TEl Zanaty and S M Seif ldquoImpact of IL12B gene rs 3212227polymorphism on fibrosis liver inflammation and response totreatment in genotype 4 Egyptian hepatitis c patientsrdquo DiseaseMarkers vol 35 no 5 pp 431ndash437 2013

[2] M H Omran N E Ibrahim S S Youssef et al ldquoRelation ofinterleukin-1120573 gene to treatment response in chronic patientsinfected with HCV genotype 4rdquo Journal of Infection in Develop-ing Countries vol 7 no 11 pp 851ndash858 2013

[3] E Billerbeck T Bottler and R Thimme ldquoRegulatory T cells inviral hepatitisrdquoWorld Journal of Gastroenterology vol 13 no 36pp 4858ndash4864 2007

[4] K Hiroishi T Ito and M Imawari ldquoImmune responses inhepatitis C virus infection and mechanisms of hepatitis C viruspersistencerdquo Journal of Gastroenterology andHepatology vol 23no 10 pp 1473ndash1482 2008

[5] A Guobuzaite S Chokshi L Balciuniene et al ldquoViral clearanceor persistence after acute hepatitis C infection interim resultsfrom a prospective studyrdquoMedicina vol 44 no 7 pp 510ndash5202008

[6] L Wan Y-J Kung Y-J Lin et al ldquoTh1 and Th2 cytokinesare elevated in HCV-infected SVR(-) patients treated withinterferon-120572rdquo Biochemical and Biophysical Research Communi-cations vol 379 no 4 pp 855ndash860 2009

[7] A K Abbas andA H Lichtman ldquoCytokines and effectormech-anisms of cell-mediated immunityrdquo in Cellular and MolecularImmunology pp 243ndash317 Saunders Philadelphia Pa USA 5thedition 2003

[8] S-L Tsai Y-F Liaw M-H Chen C-Y Huang and G CKuo ldquoDetection of type 2-like T-helper cells in hepatitis Cvirus infection implications for hepatitis C virus chronicityrdquoHepatology vol 25 no 2 pp 449ndash458 1997

[9] W E Naugler and M Karin ldquoThe wolf in sheeprsquos clothing therole of interleukin-6 in immunity Inflammation and cancerrdquoTrends in Molecular Medicine vol 14 no 3 pp 109ndash119 2008

[10] M Del Vecchio E Bajetta S Canova et al ldquoInterleukin-12biological properties and clinical applicationrdquo Clinical CancerResearch vol 13 no 16 pp 4677ndash4685 2007

[11] W T Watford M Moriguchi A Morinobu and J J OrsquoShealdquoThe biology of IL-12 coordinating innate and adaptiveimmune responsesrdquo Cytokine and Growth Factor Reviews vol14 no 5 pp 361ndash368 2003

[12] S Costantini F Capone E Guerriero P Maio G Colonna andGCastello ldquoSerumcytokine levels as putative prognosticmark-ers in the progression of chronic HCV hepatitis to cirrhosisrdquoEuropean Cytokine Network vol 21 no 4 pp 251ndash256 2010

[13] F Capone S Costantini E Guerriero et al ldquoSerum cytokinelevels in patients with hepatocellular carcinomardquo EuropeanCytokine Network vol 21 no 2 pp 99ndash104 2010

[14] S Kakumu A Okumura T Ishikawa K Iwata M Yano and KYoshioka ldquoProduction of interleukins 10 and 12 by peripheral

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Disease Markers

1800

1600

1400

1200

1000

800

600

45000 50000 55000 60000 65000 70000

R2 linear = 0381

IL-10

(ng

mL)

IL-12 (ngmL)

Figure 3 Scatter plot of IL-10 and IL-12 in patients with HCV-chronic liver disease (119903 = 0617 119875 lt 00001)

patients and in contrary to IL-12 expression pattern in HCV-positive and HCV-negative cases These results suggest thatelevation of serum IL-10might be involved in downregulationof the inflammatory response in chronic liver disease [14]Furthermore interleukin-12 production by DC appears to bedownregulated by IL-10 while with maturation the DC canproduce large amounts of IL-12 and become resistant to thesuppressive effects of antigen presenting function by IL-10[26]

In patients with chronic inflammation IL-1120572 IL-2RMIFand 120573-NGF showed significant correlation and a positive cor-relation coefficient with the transaminase values which werehigher in these patients than in healthy controls Thereforethese proteins could be considered to be an index of immuneactivation In particular these results were in agreement withliterature data reporting that IL-1 and IL-2R participate inthe progression from liver injury to fibrosis [27 28] andthat 120573-VGF is involved in liver cancer growth and metastasisand can be used as an index of chronic infection leading toLC and HCC [29 30] In agreement with these studies asignificant positive correlation was detected between IL-10and IL-12 in our study and a significant correlation coefficientwas detected with the transaminase levels which were higherin all liver pathologies than in nondisease controls theelevation was significantly recorded with disease progressionsuggesting that IL-10 and IL-12 could be used as an index toreflect the degree of inflammation in the liver with ultimatedevelopment of HCC However Liu et al [31] reported thatthe levels of TNF-alpha IL-1 Il-10 and IL-12 in patients withchronic liver failure were increased and the increase of IL-10is secondary to elevation of IL-12

5 Conclusion

Significant elevation of IL-10 and IL-12 with disease progres-sion and transaminase values might be involved in chronic

inflammation progression leading to HCC and their evalua-tion could be used as new biomarkers for HCC development

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S S Youssef A M Abd El-Aal A Saad M H Omran TEl Zanaty and S M Seif ldquoImpact of IL12B gene rs 3212227polymorphism on fibrosis liver inflammation and response totreatment in genotype 4 Egyptian hepatitis c patientsrdquo DiseaseMarkers vol 35 no 5 pp 431ndash437 2013

[2] M H Omran N E Ibrahim S S Youssef et al ldquoRelation ofinterleukin-1120573 gene to treatment response in chronic patientsinfected with HCV genotype 4rdquo Journal of Infection in Develop-ing Countries vol 7 no 11 pp 851ndash858 2013

[3] E Billerbeck T Bottler and R Thimme ldquoRegulatory T cells inviral hepatitisrdquoWorld Journal of Gastroenterology vol 13 no 36pp 4858ndash4864 2007

[4] K Hiroishi T Ito and M Imawari ldquoImmune responses inhepatitis C virus infection and mechanisms of hepatitis C viruspersistencerdquo Journal of Gastroenterology andHepatology vol 23no 10 pp 1473ndash1482 2008

[5] A Guobuzaite S Chokshi L Balciuniene et al ldquoViral clearanceor persistence after acute hepatitis C infection interim resultsfrom a prospective studyrdquoMedicina vol 44 no 7 pp 510ndash5202008

[6] L Wan Y-J Kung Y-J Lin et al ldquoTh1 and Th2 cytokinesare elevated in HCV-infected SVR(-) patients treated withinterferon-120572rdquo Biochemical and Biophysical Research Communi-cations vol 379 no 4 pp 855ndash860 2009

[7] A K Abbas andA H Lichtman ldquoCytokines and effectormech-anisms of cell-mediated immunityrdquo in Cellular and MolecularImmunology pp 243ndash317 Saunders Philadelphia Pa USA 5thedition 2003

[8] S-L Tsai Y-F Liaw M-H Chen C-Y Huang and G CKuo ldquoDetection of type 2-like T-helper cells in hepatitis Cvirus infection implications for hepatitis C virus chronicityrdquoHepatology vol 25 no 2 pp 449ndash458 1997

[9] W E Naugler and M Karin ldquoThe wolf in sheeprsquos clothing therole of interleukin-6 in immunity Inflammation and cancerrdquoTrends in Molecular Medicine vol 14 no 3 pp 109ndash119 2008

[10] M Del Vecchio E Bajetta S Canova et al ldquoInterleukin-12biological properties and clinical applicationrdquo Clinical CancerResearch vol 13 no 16 pp 4677ndash4685 2007

[11] W T Watford M Moriguchi A Morinobu and J J OrsquoShealdquoThe biology of IL-12 coordinating innate and adaptiveimmune responsesrdquo Cytokine and Growth Factor Reviews vol14 no 5 pp 361ndash368 2003

[12] S Costantini F Capone E Guerriero P Maio G Colonna andGCastello ldquoSerumcytokine levels as putative prognosticmark-ers in the progression of chronic HCV hepatitis to cirrhosisrdquoEuropean Cytokine Network vol 21 no 4 pp 251ndash256 2010

[13] F Capone S Costantini E Guerriero et al ldquoSerum cytokinelevels in patients with hepatocellular carcinomardquo EuropeanCytokine Network vol 21 no 2 pp 99ndash104 2010

[14] S Kakumu A Okumura T Ishikawa K Iwata M Yano and KYoshioka ldquoProduction of interleukins 10 and 12 by peripheral

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 7

blood mononuclear cells (PBMC) in chronic hepatitis C virus(HCV) infectionrdquo Clinical and Experimental Immunology vol108 no 1 pp 138ndash143 1997

[15] M Sarih N Bouchrit and A Benslimane ldquoDifferent cytokineprofiles of peripheral blood mononuclear cells from patientswith persistent and self-limited hepatitis C virus infectionrdquoImmunology Letters vol 74 no 2 pp 117ndash120 2000

[16] E Gigi M Raptopoulou-Gigi A Kalogeridis et al ldquoCytokinemRNA expression in hepatitis C virus infection TH1 pre-dominance in patients with chronic hepatitis C and TH1ndashTH2cytokine profile in subjects with self-limited diseaserdquo Journal ofViral Hepatitis vol 15 no 2 pp 145ndash154 2008

[17] A Taylor J Verhagen K Blaser M Akdis and C A AkdisldquoMechanisms of immune suppression by interleukin-10 andtransforming growth factor-120573 the role of T regulatory cellsrdquoImmunology vol 117 no 4 pp 433ndash442 2006

[18] M Yamashiki A Nishimura and H Suzuki ldquoPrognosis ofchronic hepatitis C patients correlates with circulating mono-cytemacrophage functionrdquoClinical Science vol 93 no 4 p 3811997

[19] M S Othman A M Aref A A Mohamed and W AIbrahim ldquoSerum levels of interleukin-6 and interleukin-10 asbiomarkers for Hepatocellular carcinoma in Egyptian patientsrdquoISRN Hepatology vol 2013 Article ID 412317 9 pages 2013

[20] E Hattori K Okumoto T Adachi et al ldquoPossible contributionof circulating interleukin-10 (IL-10) to anti-tumor immunityand prognosis in patients with unresectable hepatocellularcarcinomardquo Hepatology Research vol 27 no 4 pp 308ndash3132003

[21] D Y Kim JW Kim R Kuromatsu S H Ahn T Torimura andM Sherman ldquoControversies in surveillance and early diagnosisof hepatocellular carcinomardquoOncology vol 81 no 1 pp 56ndash602011

[22] A-RN ZekriM S El-dinAshour AHassanHMA El-DinAM R El-Shehaby andM A Abu-Shady ldquoCytokine profile inEgyptian hepatitis C virus genotype-4 in relation to liver diseaseprogressionrdquo World Journal of Gastroenterology vol 11 no 42pp 6624ndash6630 2005

[23] M E El-Houseini M S Mohammed W M Elshemey T DHussein O S Desouky andA A Elsayed ldquoEnhanced detectionof hepatocellular carcinomardquo Cancer Control vol 12 no 4 pp248ndash253 2005

[24] C-Y Hsia T-I Huo S-Y Chiang et al ldquoEvaluation ofinterleukin-6 interleukin-10 and human hepatocyte growthfactor as tumor markers for hepatocellular carcinomardquo Euro-pean Journal of Surgical Oncology vol 33 no 2 pp 208ndash2122007

[25] J-F Wu T-C Wu C-H Chen et al ldquoSerum levels ofinterleukins-10 and interleukin-12 predict early spontaneoushepatitis B virus e antigen seroconversionrdquo Gastroenterologyvol 138 pp 165ndash172 2010

[26] G Schuler and RM Steinman ldquoDendritic cells as adjuvants forimmune-mediated resistance to tumorsrdquo Journal of Experimen-tal Medicine vol 186 no 8 pp 1183ndash1187 1997

[27] A-R N Zekri H M A El-Din A A Bahnassy et al ldquoSerumlevels of soluble Fas soluble tumor necrosis factor-receptor IIinterleukin-2 receptor and interleukin-8 as early predictors ofhepatocellular carcinoma in Egyptian patients with hepatitis Cvirus genotype-4rdquo Comparative Hepatology vol 9 article 1 12pages 2010

[28] R G Gieling K Wallace and Y-P Han ldquoInterleukin-1 partici-pates in the progression from liver injury to fibrosisrdquo American

Journal of PhysiologymdashGastrointestinal and Liver Physiologyvol 296 no 6 pp G1324ndashG1331 2009

[29] Y Tokusashi K Asai S Tamakawa et al ldquoExpression of NGF inhepatocellular carcinoma cells with its receptors in non-tumorcell componentsrdquo International Journal of Cancer vol 114 no 1pp 39ndash45 2005

[30] G Rasi A Serafino L Bellis et al ldquoNerve growth factorinvolvement in liver cirrhosis and Hepatocellular carcinomardquoWorld Journal of Gastroenterology vol 13 no 37 pp 4986ndash49952007

[31] G Liu K Tang Q Li G Yuan W Cao and W Lu ldquoChangesof IL-1 TNF-120572 IL-12 and IL-10 levels with chronic liver failurerdquoSurgical Science vol 2 no 2 pp 69ndash72 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom