SERUM CYTOKINE EXPRESSION ACCORDING TO LAMIVUDINE THERAPY RESPONSE OF CHRONIC HEPATITIS B VIRUS INFECTION Jungyong Park 1, Younhee Park 1, Young Lan Kim 1, Kwang IL Park 1,Hyon-Suk Kim 1 Immunoserology, Laboratory Medicine Yonsei University Medical Center, Seoul, Korea 1

B ACKGROUND Cytokines A group of proteinaceous signaling compounds like hormones & neurotransmitters, used extensively for inter-cell communication Interleukin (IL) Interferon (IFN) Chemokine Colony Stimulating Factor (CSF) Epidermal Growth Factor (EGF) Vascular Endothelial Growth Factor (VEGF) PDCF etc.

Common Features Function in networks (complexity) Bind to high affinity receptors Redundancy Pleiotropic effects Cascade induction & Transient production Synergism & antagonism Normal ranges difficult to determine Profiles differ from one biologic site to another B ACKGROUND

Viral Infection & Cytokine Immune Response Modulation to Viral Challenge Anti viral function e.g) IFN viral responses against host’s immune system e.g) HIV -host cytokine receptors B ACKGROUND

Lamivudine Therapy Highly active against HBV in vitro Approved for use in chronic hepatitis B as one year therapy course Continuous, long-term use is common (should be considered experimental) Lamivudine Resistance Emergence B ACKGROUND …Which related to B viral hepatitis & recurrence after therapy? … Used in diagnosis & resistance prediction? …Which related to B viral hepatitis & recurrence after therapy? … Used in diagnosis & resistance prediction? …Used as a novel approach?

Approach Protein Biochip Assay (Randox EVIDENCE ® ) -simultaneous release of 12 cytokines B ACKGROUND Biochip surface Y Y EE

ATERIALS & METHODS Study Population Patients: 60 patients (49 males/ 11 females) Chronic HBV patients Outpatient Clinic, Yonsei University Medical Center Apr. 2003~Feb Controls: 14 healthy individuals (12 males/ 2 females) Health Promotion Center visitors. (routine medical check-up) Jan. 2006~ May 2006 Biomarkers AST, ALT, HBeAg, HBeAb, HBV-DNA M & M ATERIALS ETHODS

Cytokine Evaluation IL-1A IL-1B IL-2 IL-4 IL-6 IL-8 IL-10 TNF-a VEGF IFN-r MCP-1 EGF Statistical Analysis Kruskal-Wallis test M & M ATERIALS ETHODS

4 Categories in this study Pretreatment group Viral hepatitis(HBV) patients No Lamivudine treatment history Remission group After Lamivudine treatment AST <34, ALT<46 IU/L HBV-DNA: <0.5 pg/mL Recurrence group After Lamivudine treatment elevated AST, ALT, HBV-DNA Control group Individuals for routine physical exam M & M ATERIALS ETHODS

* : Mean±S.D. PretreatmentRemissionRecurrenceControlTotal (11)(28)(21)(14)(74) SEX (M/F) 11/021/717/412/261/13 AGE (yrs) 36.0±5.9 * 35.6± ± ± ±9.5 AST (IU/L) 123.6± ± ± ± ±65.9 ALT (IU/L) 205.4± ± ± ± ±123.1 HBV- DNA (pg/mL) ± ± ± ± ±518.2 Table 1. General characteristics of 4 groups R ESULT

R PretreatmentRemissionRecurrenceControlTotal TNF-a (pg/mL) 3.5± ± ± ± ±49.6 IL-1A (pg/mL) 0.34± ± ± ± ±1.5 IL-1B (pg/mL) 1.83± ± ± ± ±43.5 IL-2 (pg/mL) 1.1±1.30.7±1.03.4± ±2.51.7±5.8 IL-4 (pg/mL) 1.9±2.40.6±1.03.5±9.42.0±4.91.9±5.5 IL-6 (pg/mL) 13.5± ± ± ± ±165.1 IL-8 (pg/mL) 10.7± ± ± ± ±619.9 IL-10 (pg/mL) 0.7±0.80.6±2.71.6±3.60.3±0.50.8±2.6 VEGF (pg/mL) 11.1± ± ± ± ±137.6 IFN-r (pg/mL) 0.4±0.91.4±4.42.8± ±1.71.5±6.1 MCP-1 (pg/mL) 215.8± ± ± ± ±124.4 EGF (pg/mL) 27.3± ± ± ± ±88.0 Cont.

Total Pretreatment Remission Recurrence Control ANOVA (P-value 0.006) Fig.1. TNF-a Distribution R ESULT * P < 0.05 *

Fig.2. IL-6 Distribution R ESULT ANOVA (P-value 0.023) * * P < 0.05 Total Pretreatment Remission Recurrence Control

Fig.3. IL-8 Distribution R ESULT ANOVA (P-value ) * * * * P < 0.05 Total Pretreatment Remission Recurrence Control

Fig.4. IL-10 Distribution R ESULT * P < 0.05 ANOVA (P-value <0.0001) *** Total Pretreatment Remission Recurrence Control

Table 2. Comparison between HBe Ag +, HBeAb + groups HBeAg + (n=31) HBeAb + (n=19) p-value AGE (yrs) 39.3 ±10.8 *35.6 ± AST (IU/L) 113.8± ±38.1<.0001 ALT (IU/L) 189.3± ± HBV-DNA (pg/mL) 558.2± ±265.2<.0001 TNF-a (pg/mL) 33.5± ± IL-6 (pg/mL) 94.4± ± IL-8 (pg/mL) 277.5± ± IL-10 (pg/mL) 1.4±3.00.9± EGF (pg/mL) 47.9± ± VEGF (pg/mL) 35.6± ± * : Mean±S.D. R ESULT

Fig. 7. Correlation between each biomarker & cytokine ALT HBeAb TNF-a IL-8 VEGF HBeAg HBV-DNA IL-6 IL-10 EGF ρ R ESULT

Inflammation related Cytokines Recurrence group vs. Remission group TNF-a IL-6 IL-8 IL-10 HBeAg + group vs. HBeAb + group IL-10 D ISCUSSION

Cytokines in Serum Reflect recurrence HBV inflammation Cytokine profiles disease status Understand pathology, mechanisms Predictable markers of drug resistance D ISCUSSION

Limitation No. of study participants too small Pleiotropic effects HBV mutations Further study Cytokine expression in carriers or other status Understand cytokine network by simultaneous detection D ISCUSSION