• 대한한의학회지
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• 제19권2호
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• pp.244-270
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• 1998

This study was performed to investigate an anti-HBV activities of the aqueous extracts from 10 Korean herbal medicines in the HepG2 2.2.15 cell culture system and the results were as follows: 1. The extracts of 6 plants (Herba Artemisiae Capillaris, Radix et Rhizoma Rhei, Cortex Cinnamomi, Fructus Chebulae, Fructus Rubi and Radix Rubi) decreased, significantly and dose-dependently, the levels of extracellular HBV virion in the concentrations (10, 100, 500 and $1,000\;{\mu}g/m{\ell}$) tested. 2. However, others (Radix lsatidis, Lignum Sappan, Herba Lysimachiae and Fructus Lycii) did not show any effect either on the replication of HBV or on the levels of virion DNA in the culture media of HepG2 2.2.15 cell. 3. Among the 6 plants which showed the inhibitory potency on the production of extracellular HBV virion, Radix et Rhizoma Rhei, Cortex Cinnamomi, Fructus Chebulae, Fructus Rubi and Radix Rubi except Herba Artemisiae Capillaris also showed the inhibition of the replication of intracellular HEV DNA in the range of $100{\sim}500\;{\mu}g/m{\ell}$. Considering the above results, it is thought that 6 plants(Herba Artemisiae Capillaris, Radix et Rhizoma Rhei, Cortex Cinnamomi, Fructus Chebulae, Fructus Rubi and Radix Rubi) possess the anti-HBV activities in the HepG2 2.2.15 cell culture system. We thus suggest that these plants possess a potential as a therapeutic agent for the chronic viral hepatitis. These results might be useful as a basic data for the development of the new preventive drugs for HBV diseases.

• IMMUNE NETWORK
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• 제9권1호
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• pp.20-26
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• 2009

We previously reported that $IFN-{\gamma}$ producing T cell responses induced by the combined therapy of DNA vaccine and lamivudine for one year are important for the induction of sustained virological response (SVR). However, $IFN-{\gamma}$ production is not sufficient to predict sustained viremia control in chronic hepatitis B (CHB) carriers treated. Methods: Twelve CHB carriers were intramuscularly immunized 12 times at a 4-week interval with 8mg of HBV DNA vaccine during the standard lamivudine treatment (100mg/daily/1 year). The level of cytokines during and after the combined therapy in plasma of all 12 CHB carriers treated was determined by each ELISA kit. Six out of 12 CHB carriers revisited the clinic, and their HBV DNA levels were examined. Results: The combined therapy increased plasma IL-12 and IL-12/p40 ratio during the treatment (baseline vs. peak level: $41.8{\pm}8.3$ vs. $163.1{\pm}29.2\;pg/ml$; p<0.01 and $0.96{\pm}0.25$ vs. $3.58{\pm}0.86$; p<0.01, respectively), and the peak level of plasma IL-12 and IL-12/p40 ratio was evoked at 6 to 10 months during the combined therapy. In particular, CHB carriers with SVR had two and three-fold higher level of the peak plasma IL-12 and plasma IL-12/p40 ratio than non-virological responders (NVRs), respectively ($218.0{\pm}41.4$ vs. $108.1{\pm}28.6\;pg/ml$; p=0.09 and $5.35{\pm}1.38$ vs. $1.80{\pm}0.29$; p<0.05, respectively), while p40 level was consistent during the combined therapy. In addition, there was no significant temporal correlation between the peak IL-12/p40 ratio and the elevation of serum alanine amino-transferase (ALT) in this study, contrast to $IFN-{\alpha}$ therapy which induced peak IL-12 level following ALT flares. Conclusion: Our results indicate that the combined therapy induces the increase of plasma IL-12 and IL-12/p40 ratio, which are associated with long-term SVR in CHB carriers.

• Clinical and Experimental Pediatrics
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• 제51권11호
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• pp.1165-1171
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• 2008

목 적: 현재 우리나라에서는 B형 간염 백신접종 후 일률적으로 항체검사를 권장하지는 않으나, 아직도 우리나라의 성인 및 소아의 보유율이 외국에 비해 매우 높고, 영아기의 어린이집 등 단체생활 증가와 이로 인한 수평감염의 위험성이 높다고 생각되므로 영아기의 면역획득여부를 확인하는 것이 중요하다고 생각된다. 이에 본 연구에서는 출생 후 B형 간염 백신을 접종한 만삭아들을 대상으로 항체검사를 실시하여 산모가 보유자인 경우와 그렇지 않은 경우의 두 군으로 나누어 기본접종 후 항체 양전율을 평가하였고, 무반응자에서의 재접종 시 효과를 비교 분석하였다. 방 법: 2004년 10월부터 2007년 6월까지 문화병원에서 출생한 716명의 만삭아들을 대상으로 산모의 B형 간염 보유여부에 따라 두 군으로 나누어 현재 추천되고 있는 일정으로 기본접종 후 생후 7-12개월(산모가 보유자인 경우는 생후 9-15개월)에 항체가를 측정하여 기본접종의 효과를 비교하였다. 또한 각 군의 무반응자에게 3회 재접종을 실시하고 1-3개월 후 항체가를 측정하여 재접종의 효과를 비교하였다. 또한 보유자인 산모로부터 출생한 영아에서 산모의 HBeAg 양성 여부가 주산기 예방조치의 실패와 관련이 있는지 알아보았다. 결 과: HBsAg이 음성인 산모에서 태어난 총 662명의 건강한 만삭아에서 B형 간염 기본접종 후 623명(94.1%)에서 항체 양전되었고, HBsAg이 양성인 산모에서 태어난 만삭아중 감염된 4명의 영아를 제외한 50명에서는 39명(78%)이 기본접종 후 항체 양전되어 산모가 보유자가 아닌 경우가 더 높은 항체 양전율을 나타냈다(P<0.001). 또한 건강한 만삭아의 무반응자 39명중 32명에서 재접종 후 31명(96.9%)에서 항체가 양전되었고, 산모가 보유자인 무반응자 11명 중 8명에서 재접종 후 7명(87.5%)이 항체 양전되어 두 군의 무반응자에서의 재접종은 매우 효과적이었다. 또한 두 군에서 마지막 기본접종 후 항체검사시기에 따른 항체 양전율은 거의 차이가 없었다(P>0.05). HBeAg이 음성이고 HBsAg만 양성인 산모로부터 출생한 영아 40명 모두 예방이 되었고, HBeAg과 HBsAg 모두 양성인 산모로부터 출생한 14명중에서는 4명(28.6%)이 예방조치가 실패하였다. 이와 같이 산모의 HBeAg 양성여부는 주산기 예방조치의 실패와 밀접한 관련이 있었다(P<0.001). 결 론: 보유자가 아닌 산모로부터 출생한 건강한 만삭아에서 기본접종 후 무반응자의 재접종의 효과는 매우 좋았기 때문에, 가족 내에 보유자가 있거나 보유자가 없다 하더라도 확실한 효과를 위해서 기본접종 후 항체검사 및 재접종을 고려해야 할 것으로 생각되나, 이를 위해서는 현재 시행되지 않고 있는 일률적인 항체검사의 비용효과적인 측면에 대한 연구가 뒷받침되어야 할 것으로 사료되며, 산모가 보유자인 경우에는 항체 양전율이 감소하므로 수직감염이 되지 않았다 하더라도 산모로부터 수평감염의 위험이 높으므로 현재 추천되는 방법으로 반드시 항체검사를 실시하여 항체 양전여부를 확인해야 할 것이다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권1호
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• pp.225-229
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• 2012

Background: Males have a higher prevalence of hepatocellular carcinoma (HCC) than females in general, but the reasons for the sex disparity are still obscure. DNA copy number alteration (CNA) is a major feature of solid tumors including HCC, but whether CNA plays a role in sex-related differences in HCC development has never been evaluated. Methods: High-resolution array comparative genomic hybridization (CGH) was used to examine 17 female and 46 male HCC patients with chronic hepatitis B virus (HBV) infection in Shanghai, China. Two-tailed Fisher's exact or ${\chi}^2$ tests was used to compare CNAs between females and males. Results: The overall frequencies and patterns of CNAs in female and male cases were similar. However, female HCC tumors presented more copy number gains compared to those in males on 1q21.3-q22 (76.5% vs. 37.0%, P = 0.009), 11q11 (35.3% vs. 0.0%, P = 0.0002) and 19q13.31-q13.32 (23.5% vs. 0.0%, P = 0.004), and loss on 16p11.2 (35.3% vs. 6.5%, P = 0.009). Relative to females, male cases had greater copy number loss on 11q11 (63.0% vs. 17.6%, P = 0.002). Further analyses showed that 11q11 gain correlated with 19q13.31-q13.32 gain (P = 0.042), 11q11 loss (P = 0.011) and 16p11.2 loss (P = 0.033), while 1q21.3-q22 gain correlated with 19q13.31-q13.32 gain (P = 0.046). Conclusions: These findings suggest that CNAs may play a role in sex-related differences in HBVassociated HCC development.

• 미생물학회지
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• 제28권1호
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• pp.13-18
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• 1990

adr아형 B형 간염바이러스의 preS2유전자 부위를lacZ 유전자의 5말단에 연결하여 preS2-$\beta$-galactosidase 융합단백질을 생성하는 플라스미드, pTSZ를 건설하였다. 갈본된 preS2 유전자의 3' 및 5발단을 결손시켜 얻은 재조합 플라스미드를 발현시킨 후 결손된 preS2를 포함하는 융합단백질의 항원성을 단일클론항체 H8을 사용하여 비교하며 보았다. 양말단에서 일정부위까지의 결손은 항원성에 영향을 미치지 않았지만 그 이상의 결손에 의하여는 항윈성이 소실됨을 볼 수 있었다. 이상의 항원성 전한부위를 DNA 염기서열 분석에 의하여 결정할 수 있었다. 그 결과 항원결정인자의 양말단은 preS2 서열 중 아미노산 전기 130-132와 140→142 사이에 각각 존재함을 알 수 있었고, 아미노산 143번의 결손은 항원성의 부분적인 감소를 초래하는 것으로 보아 항원성 결정에 보충적 역할을 한다고 생각된다. 한편 adr과 adw2아형 간의 아미노산서열의 차이가 항원결정부위 중 130, 132 및 141번 위치에 존재하며 단일를론항체 H8이 adr아형에만 특이하게 결합하는 것으로 부터, 세 잔기 중 하나 혹은 그 이상이 아형특이성에 관여한다고 추정된다.

• BMB Reports
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• 제37권6호
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• pp.741-748
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• 2004

The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1~447) and its four different derivatives, denoted as NS5A-A (aa 1~150), -B (aa 1~300), -C (aa 300~447) and D (aa 150~447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and $centaurin{\delta}$ 2 ($CENT{\delta}2$). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, $CENT{\delta}2$ and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and $CENT{\delta}2$ were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.

• BMB Reports
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• 제31권2호
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• pp.123-129
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• 1998

Foreign proteins, $endo-{\beta}-1,4-glucanase$ of Bacillus subtilis, preS1+S2 region of hepatitis B virus large surface antigen, human ${\beta}_2-adrenergic$ receptor ($h{\beta}_{2}AR$), and bovine growth hormone (bGH) were expressed in Saccharomyces cerevisiae and secreted into the medium. These proteins were expressed using the alcohol dehydrogenase I (ADH1) promoter of Saccharomyces cerevisiae and secreted by signal sequence of the 97 K killer toxin gene of doublestranded linear DNA plasmid (pGKL1) of S. cerevisiae. All these proteins underwent severe modifications; in particular, N-glycosylation in the case of $endo-{\beta}-1,4-glucanase$, $h{\beta}_2AR$, and preS1+S2. Seventy four percent of the expressed $endo-{\beta}-1,4-glucanase$ was secreted into the culture medium. Highly modified proteins were detected in the culture medium and in the cell. Expressed $h{\beta}_2AR$, which has seven transmembrane domains, remained in the cell. The degrees of secretion and modification and the states of proteins in the culture medium and in the cell were quite different. These results indicated that the nature of the protein has a critical role in its secretion and modifications.

• Asian Pacific Journal of Cancer Prevention
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• 제17권4호
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• pp.1705-1712
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• 2016

Background: The aim of this study was to determine and evaluate the association of different viral infections, with hepatitis B and C viruses, Epstein-Barr virus, cytomegalovirus and human herpes virus-8 (HBV, HCV, EBV, CMV, HHV-8) with the risk of lymphomas (Hodgkin and non-Hodgkin) among Egyptian patients, and correlate with the histopathological staging and typing as well as the prevalence of combined infections. Materials and Methods: A total of 100 newly diagnosed lymphoma patients with 100 healthy age and sex matched normal controls were assayed for viral infection using enzyme linked immunosorbant assay (ELISA) followed by real time polymerase chain reaction (RT-PCR). Results: Our results showed a high statistical significant difference between cases and controls as regards clinical and laboratory findings (P<0.001 and=0.003). A high statistical difference was seen for the association of most viruses and lymphoma cases (p<0.001) except for positive HBs Ag, positive CMV IgG and HHV-8 (p=0.37, 0.70 and 1.0 respectively). No statistical significant difference was found between Hodgkin (HL) and non-Hodgkin (NHL) as regards viral prevalence except HCV antigen, 57.1% for HL and 26.5% for NHL (p = 0.03). Only, HBV DNA showed a high significant value among infiltrated bone marrow cases (p=0.003) and finally, a high significant association of 2 combined viral infections with infiltrated bone marrow lymphoma cases (p=0.04). Conclusions: Our results showed that infection with HBV, HCV, CMV and EBV were associated with increased risk of lymphoma among the Egyptian population. Detection of new associations between infectious agents and risk of cancer development will facilitate progress in elaboration of prophylactic measures, early diagnostic methods and, hopefully, novel therapy of malignant tumours.

• IMMUNE NETWORK
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• 제3권3호
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• 대한임상검사과학회지
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• 제38권3호
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• pp.196-202
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• 2006

최근 만성 B형간염의 치료에 B형간염 바이러스 복제를 저해하여 감염력을 약화시키는 lamivudine이 많이 사용되고 있다. 그러나 이 약제를 장기간 복용할 경우 B형간염 바이러스 DNA polymerase 유전자의 YMDD motif에 아미노산 치환을 일으켜 lamivudine 저항성 B형간염 바이러스가 나타나는 점이 문제시 되고 있다. 본 연구의 목적은 lamivudine 치료를 받은 만성 B형간염 환자에서 PCR-direct sequencing 법을 이용하여 B형간염 바이러스 DNA 중합효소 유전자의 YMDD motif(codon 552)와 codon 528에서 돌연변이 출현빈도를 구하고, HBeAg과의 관련성을 보고자 하였다. 방법은 만성 B형간염 환자의 혈청에서 DNA를 추출하고 DNA 중합효소 유전자의 codon 552와 528을 포함하는 부위에서 선택한 두 쌍의 primer를 이용하여 nested PCR을 실시하였다. 증폭된 PCR 산물은 2% agarose gel에서 전기영동을 한 후, 자동염기서열분석기를 이용하여 sequencing을 실시하였다. 총 207명 중에서 돌연변이는 124명(60%)에서 발견되었으며, 남, 녀에서 차이는 발견되지 않았고, 돌연변이군에서 비돌연변이군에 비해 평균나이가 약간 높게 나타났으나 유의성은 없었다. Codon 552에서 단일돌연변이로는 M552I(50.8%)가 가장 높게 나타났고, 다음으로 M552V(43.5%), M552I/V(5.7%)의 순서로 나타났다. Codon 528에서는 67.0%의 L528M 돌연변이가 발견되었다. Codon 552와 codon 528에서 동시에 발생한 중복돌연변이로는 M552V/L528M(43.6%)이 가장 높게 나타났고, 다음으로 M552I/L528(33.1%) 그리고 M552I/L528M(17.7%)의 순으로 나타났다. Codon 552에서 serine 돌연변이(M528S)는 발견되지 않았으며, L528M은 M552V 돌연변이와 거의 동시에 검출되었다. 본 연구에서 만성 B형간염환자에서 HBeAg의 유무와 lamivudine 돌연변이율과의 상관성은 발견되지 않았으며, PCR-direct sequencing법은 고가의 자동염기서열분석 장비와 숙련된 기술자가 필요하다는 문제점은 있으나, 검체 수가 많은 큰 임상검사실에서는 활용성이 클 것으로 판단된다. 향 후 lamivudine으로 인한 HBV 돌연변이형과 환자의 임상결과의 관련성에 대한 연구가 추가적으로 실시되면, lamivudine을 복용하는 만성 HBV 환자의 치료와 예후에 유용할 것으로 사료된다.