行政院國家科學委員會專題研究計畫 期中進度報告
抗登革病毒非結構性蛋白 1 抗體的自體免疫分子 機制(1/3)
計畫類別： 個別型計畫
計畫編號： NSC94-3112-B-006-007-
執行期間： 94 年 05 月 01 日至 95 年 04 月 30 日 執行單位： 國立成功大學微生物免疫學研究所
計畫主持人： 林以行
共同主持人： 劉校生，莊偉哲
計畫參與人員： 鄭獻仁、萬書彣；、陳美君
報告類型： 完整報告
報告附件： 出席國際會議研究心得報告及發表論文 處理方式： 本計畫可公開查詢
中 華 民 國 95 年 5 月 18 日
行政院國家科學委員會補助專題研究計畫
抗登革病毒非結構性蛋白 1 抗體的自體免疫分子機制(1/3) Molecular Mechanisms of Autoimmunity by Antibodies against Dengue
Virus Nonstructural Protein 1 (1/3) 計畫類別：■ 個別型計畫 □ 整合型計畫
計畫編號：NSC 94－3112－B006－007
執行期間：九十四年五月一日至九十五年四月三十日
計畫主持人：林以行 教授 共同主持人：
計畫參與人員：莊偉哲、劉校生 教授
博士班研究生：鄭獻仁、萬書彣(成大醫學院基礎醫學研究所) 碩士班研究生：陳美君(成大醫學院微生物及免疫學研究所)
成果報告類型(依經費核定清單規定繳交)：□精簡報告 ■完整報告
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
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■出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、
列管計畫及下列情形者外，得立即公開查詢
□涉及專利或其他智慧財產權，□一年□二年後可公開查詢 執行單位：國立成功大學醫學院微生物及免疫學研究所
中 華 民 國 九 十 五 年 四 月 三 十 日
□ 成果報告
■ 期中進度報告
行政院國家科學委員會補助專題研究計畫成果報告
抗登革病毒非結構性蛋白 1 抗體的自體免疫分子機制(1/3) Molecular Mechanisms of Autoimmunity by Antibodies against Dengue
Virus Nonstructural Protein 1 (1/3) 計畫編號：NSC 94-3112-B006-007
執行期限：94 年 5 月 1 日至 95 年 4 月 30 日
主持人：林以行 國立成功大學微生物及免疫學研究所
一、摘要
血小板減少症、血管壁滲漏病變以及 肝臟腫大症狀是嚴重登革疾病的主要病 徵。即使證據顯示肝炎性的傷害參與登革 致病機轉，然而導致病理發生的真正機制 仍未清楚。本實驗室先前研究結果顯示了 一種以分子模擬機轉進而導致登革自體抗 體的生成。我們認為抗登革病毒(DV)非結 構性蛋白 1 (NS1)抗體(anti-DV NS1)會導致 內皮細胞誘發炎性反應及引起細胞凋亡。
在本研究中，我們遂以小鼠動物模式探討 抗體的病理效應，發現不管是主動免疫小 鼠病毒 NS1 蛋白亦或是被動給予 anti-DV NS1 抗體都會造成小鼠體內 AST 及 ALT 的表現增加。然而血清中 BUN 的表現量則 無異常。肝組織的切片病理觀察顯示部分 區域有細胞壞死、巨噬細胞浸潤以及細胞 凋亡的發生。根據本計畫的研究結果，主 動免疫小鼠 DV NS1 會產生內皮細胞自體 抗體的產生並會造成內皮細胞的傷害以及 進一步引發免疫媒介的肝炎性病理現象。
這樣的效應可以在以 anti-DV NS1 被動給 予小鼠的模式中得到驗證 anti-DV NS1 所 可能引起的肝炎性致病機制。
關鍵詞：自體免疫、登革、內皮細胞、肝 臟、小鼠
Abstract
Clinical manifestations of severe dengue diseases include thrombocytopenia, vascular leakage, and hepatomegaly. Evidence showed the involvement of hepatic injury in disease pathogenesis; however, the mechanisms remain unresolved. Our previous study suggested a mechanism of molecular mimicry in which antibodies (Abs) directed against dengue virus (DV) nonstructural protein 1 (NS1) cross-reacted with
endothelial cells and induced inflammatory activation and apoptosis. In this study, the pathogenic effects of anti-DV NS1 Abs were further examined using a murine model. The binding of anti-DV NS1 to naïve mouse vessel endothelium or the deposition of Abs to vessel endothelium of DV NS1-immunized mice was demonstrated in the liver sections. The presence of apoptotic cells was observed in the liver vessel of DV NS1-immunized mice. The levels of AST and ALT in mouse sera increased after either active immunization with DV NS1 or passive administration with anti-DV NS1 Abs.
Changes of BUN levels could not be detected.
Gross and histological examinations revealed tissue damage, macrophage infiltration, and cell apoptosis in mouse livers, but all histological examination of kidneys appeared normal without evidence of pathology. In conclusion, active immunization with NS1 caused endothelium injury and immune-mediated hepatitis-like pathology in mice. Study using passive administration further supported a role played by anti-DV NS1 Abs which lead to liver damage.
Keywords: Autoimmunity, dengue, endothelial cell, liver, mice
二、前言
Dengue virus (DV) infection causes a spectrum of illness from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).1,2 According to the World Health Organization (WHO) report, the global prevalence of dengue has grown dramatically in recent decades. The disease is now endemic in more than 100 countries; WHO estimates there may be 50 million cases of dengue infection
and several hundred thousand cases of DHF per year.3 Thus far, the pathogenesis of DHF is not fully defined. Several mechanisms have been considered such as antibody-dependent enhancement (ADE) of infection,4 viral load and serotype variation,5-7 and abnormal immune activation.8-12 There is no effective vaccine available, although evaluations of several candidate vaccines are under progress.13,14 For the safety of dengue vaccine, however, its potential pathogenic effects should be avoided.
The clinical features of DHF include plasma leakage, bleeding tendency, and liver involvement.8 The hemorrhagic syndrome is a major hallmark of DHF, yet the pathologic mechanism remains unclear. Moreover, hepatic injury is a common condition as demonstrated by hepatomegaly and elevated serum transaminase levels.15-17 In addition, defective of hemostatic agent factor VII in liver disease was involved in thrombocytopenia-associated bleeding.18,19 The liver failure leading to coagulopathy might be related to the progression of hemorrhage in DHF.9
Liver dysfunction has been observed in dengue patients.8,9,15-17 The serum levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were elevated in DHF patients. There are several mechanisms proposed for dengue hepatitis. In vitro study indicated that the severity of cytopathic effects and the increase in AST levels correlated with the virus replication rate in DV-infected liver cell lines.20Further studies showed cell activation and induction of apoptotic death of infected liver cell lines.21-23 In addition to a direct viral cytotoxic effect, the indirect route by immunopathogenesis including cytokines, chemokines, and infiltrated immune cells may also be involved.9-11 Actually, hepatic injury resulted from vessel endothelium disruption in liver has also been reported.24,25 Both endothelial cell dysfunction and endothelial cell-derived inflammatory events may cause stress leading to liver injury. It is speculated that the cause of dengue hepatic injury is multiple.
The generation of endothelial cell cross-reactive antibodies (Abs) was demonstrated in dengue patients.26 Anti-DV nonstructural protein 1 (NS1) is responsible
for the binding effects of endothelial cell autoAbs due to a mechanism of molecular mimicry. Cell apoptosis and inflammatory activation are the two major responses causing endothelial cell damage in vitro by anti-DV NS1 Abs.27-29 To elucidate the effect of anti-DV NS1 in vivo, a murine model by active immunization with recombinant DV NS1 or passive administration with anti-DV NS1 was used. Results showed liver damage in both active and passive model, thereby supporting a pathologic mechanism of liver injury mediated by anti-DV NS1 Abs. The involvement of infiltrated immune cells was also investigated.
三、研究目的
Vascular leakage and hemorrhagic syndrome are the major clinical features associated with severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in dengue virus (DV) infection, yet the mechanisms are not well understood.
Results from our previous studies showed a mechanism of molecular mimicry in which antibodies (Abs) directed against DV nonstructural protein 1 (NS1) cross-react with endothelial cells and induce these cells to undergo apoptosis and immune activation.
Several candidate self-proteins recognized by patient sera and anti-NS1 Abs have been identified. The objectives of this study are to further examine the pathogenic effects of anti-DV NS1 Abs using the murine model.
四、材料與方法
Cells and Animals. Human microvascular endothelial cell line-1 (HMEC-1) was passed in culture plates containing endothelial cell growth medium (EGM; Clonetics, Walkersville, MD) composed of 2% FBS, 1
g/ml hydrocortisone, 10 ng/ml epidermal growth factor, and antibiotics.26 C3H/HeN breeder mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained on standard laboratory food and water ad libitum in our medical college laboratory animal center. Their male 8-wk-old progeny were used for the experiments.
Antibody Preparation. Anti-DV or Japanese encephalitis virus (JEV) nonstructural protein 1 (NS1) IgG was obtained from sera of C3H/HeN mice i.p.
immunized with purified recombinant DV-2
(New Guinea C strain) or JEV (NT109 strain) NS1 proteins following IgG purification as described previously.27 The control IgG was purified from normal mouse sera.
Murine Model. For the active immunization model, C3H/HeN mice were immunized with 20 g of recombinant DV NS1, JEV NS1, or BSA proteins in complete Freund’s adjuvant and then three injections weekly of 20 g proteins in incomplete Freund’s adjuvant. For the passive immunization model, mice were administrated with 500 g of anti-DV NS1, anti-JEV NS1, or control IgG.
Endothelial Cell Binding Activity Assay.
To determine the endothelial cell binding ability of Abs, HMEC-1 cells were seeded in monolayers on sterile glass slides coated with 1% gelatin and liver specimens were obtained from C3H/HeN mice. After perfused with OCT compound to prepare tissue blocks, the liver specimens were sliced into 3-m thick sections for experiments.
Cells or liver sections were washed briefly in PBS, fixed with 1% formaldehyde in PBS at room temperature for 10 min, and washed again with PBS. Mouse anti-DV NS1, anti-JEV NS1, or control IgG were then added and incubated at 4°C for 1 h. After washing three times with PBS, samples were incubated with 1 l of 1 mg/ml HRP- or PE-conjugated goat anti-mouse IgG at 4°C for 1 h. For the detection of cell binding ability of Abs in vivo, mouse liver tissues obtained from DV NS1- or JEV NS1-immunized mice as described above were sliced. After being washed with PBS, liver sections were incubated with 1 l of 1 mg/ml HRP- or PE-conjugated goat anti-mouse IgG at 4°C for 1 h. The binding ability was observed according to the manufacturer’s instructions for the AEC substrate kit (Zymed, San Francisco, CA), counterstained with hematoxylin and then using light microscopy or fluorescence microscopy. Mice immunized with PBS plus adjuvant were used for control.
TUNEL Assay. Apoptosis was detected by TUNEL assay using the In Situ Cell Death Detection Kit (Integrin, Indianapolis, IN).
Formalin-fixed, forzen sections of liver or kidney obtained from DV NS1- or JEV NS1-immunized C3H/HeN mice as described above were assessed by TUNEL reaction according to themanufacturer’sinstructions.
The apoptotic cells were detected by colorimetric developement using AEC substrate kit (Zymed) and viewed by light microscopy.
Serum AST, ALT and BUN Detection.
Mouse sera were collected and the concentrations of aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) were determined using EKTACHEM DT60II (for BUN) and DTSCII (for AST and ALT) analyzers (Eastman Kodak, Rochester, NY).
Histopathological and Immunohistochemical Staining. To investigate the pathological changes of tissue/organ in DV NS1-immunized or anti-DV NS1-treated C3H/HeN mice, liver and kidney were prepared to tissue blocks and sliced for experiments. For histopathology, tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin wax, and 5 µm sections were stained with hematoxylin-eosin (Sigma).
For immunohistochemical analysis, 3 µm frozen sections were fixed with 1%
formaldehyde in PBS. After being washed with PBS, 1 l of 0.1 mg/ml monoclonal anti-mouse CD3 or CD56 Abs (PharMingen, San Diego, CA) or polyclonal rat anti-mouse F4/80 (Serotec, Oxford, UK) was added to sections and incubated for 1 h at 4°C. After being washed three times with PBS, tissue sections were incubated with 1lof 1 mg/ml FITC- or PE-conjugated or goat anti-rat or anti-mouse IgG (Jackson Laboratory) for 1 h at 4°C. The presence of CD3-, CD56-, or F4/80-positive cells was detected using fluorescence microscopy.
五、結果與討論
Antibodies against DV NS1 cross-react with endothelial cells in mouse liver. Our previous studies have demonstrated the cross-reactivity of anti-DV NS1 to endothelial cells.26,27 In this study, we investigated the pathogenic effect of anti-DV NS1 in vivo using the murine model. The generated Abs against DV or JEV NS1 were first confirmed for their cross-reactivity with endothelial cell line. Anti-DV NS1 cross-reacted with HMEC-1 cells as shown by immunocytochemical staining compared with anti-JEV NS1 or control IgG (Fig. 1A).
To further investigate the cross-reactivity of anti-DV NS1 to normal mouse tissues, C3H/HeN mouse liver and kidney were
frozen sectioned and tested by immunocytochemistry. Interestingly, anti-DV NS1 could bind to vessel endothelium in liver (Fig. 1B, arrowhead) but not in kidney (data not shown). The control IgG and anti-JEV NS1 Abs did not show binding activity to vessel endothelium. To characterize the binding ability of anti-DV NS1 in vivo, C3H/HeN mice were immunized with DV or JEV NS1 or PBS.
The frozen sections of liver and kidney were examined for Ab binding to vessel endothelium. Results showed that only DV NS1- but not JEV NS1- or PBS-immunized mice had Ab deposition on vessel endothelium in liver (Fig. 1C, arrowhead).
The Ab binding to vessel endothelium was not observed in kidney (data not shown).
Vasculopathy and liver injury occur in DV NS1-immunized and anti-DV NS1-treated mice. Antibodies against DV NS1 might cause endothelial cell apoptosis and inflammatory activation.27-29 The pathogenic role of anti-DV NS1 was herein investigated in the mouse model. CH3/HeN mice immunized with DV or JEV NS1 were examined for cell damage in the liver. Results showed the presence of apoptotic cells surrounding the liver vessel (Fig. 2A, arrowhead) but not kidney (Fig. 2B) after immunization with DV NS1 four times to generate anti-DV NS1 Abs. In JEV NS1- or PBS-immunized mice, there were no dead cells detectable in liver and kidney. In addition to the apoptotic cell death, the pathogenic effects of anti-DV NS1 in liver and kidney were further determined by AST, ALT and BUN concentrations in mouse sera.
In DV but not JEV NS1- or PBS-immunized mice, the serum levels of liver enzymes including AST and ALT were increased (Fig.
3). However, there was no change in the serum levels of BUN. To further confirm the liver injury caused by anti-DV NS1, we passively administrated purified IgG against DV NS1 to mice. After 2 days, there was an increase in serum levels of AST and ALT but not BUN after injection with anti-DV NS1 but not with anti-JEV NS1 or control IgG (Table 1). Again, there was no change in the serum levels of BUN. Mice treated with galactosamine plus LPS were used for the positive control of liver damage. The results indicated that hepatitis-like pathologic effect can be caused by anti-DV NS1 Abs.
To further examine liver injury mediated
by anti-DV NS1, histopathologic changes were determined. In DV NS1-immunized mice, gross and histological examinations revealed typical pathological changes including hepatic fibrosis (Fig. 4, d), fatty liver (e), cell infiltration (f), necrotic body (g), and vesicle formation (h) in mouse liver.
These pathologic changes were not observed in the liver of mice after JEV NS1 (c) or PBS (b) immunization as compared to normal mice (a). In kidney, there was no marked histopathology observed in mice of all the groups (Fig. 4B). We also observed the histopathologic changes in anti-DV NS1-treated mice. Similarly, livers showed cell infiltration in mice given anti-DV NS1, while livers in mice given anti-JEV NS1 remained intact (Fig. 4C). Mice treated with galactosamine plus LPS were used for positive control. The histology of kidney in all mice remained normal (data not shown).
Cell infiltration and apoptosis can be observed in the livers of DV NS1-immunized and anti-DV NS1-treated mice. Hepatic inflammation caused by infiltrated immune cells has been proposed.30To characterize the liver injury induced by anti-DV NS1, we identified the types of infiltrated cells by immunostaining using specific Abs against macrophage, T cell, and NK cell. First, liver tissue sections from C3H/HeN mice passively administrated with anti-DV NS1 showed Ab deposition in vessel endothelium (Fig. 5A). Staining with anti-F4/80, anti-CD3, or anti-CD56 of liver sections showed an increase of infiltrated macrophages in anti-DV NS1-treated mice, while T and NK cells were not detectable (Fig. 5B). The infiltrated macrophages could not be detected in livers of anti-JEV NS1-treated mice. By TUNEL assay, apoptotic cells in livers of mice given anti-DV NS1 were observed around the vessel (Fig. 5C). Acute hepatic injury caused by galactosamine plus LPS in mice was used for positive control. The immune cell infiltration and cell apoptosis were observed (Fig. 5B and C). These results indicated that anti-DV NS1 caused monocyte/macrophage infiltration and apoptotic cell death in mouse liver.
Concluding remarks. In studying the pathogenesis of DV infection, we have demonstrated the generation of autoAbs in dengue patients and the anti-DV NS1 Abs were responsible for the cross-reactivity to endothelial cells and inducing damage.26-29,31
These previous studies were done in vitro. In this study, we examined the effects of anti-DV NS1 in the animal model. Either active immunization of recombinant DV NS1 or passive administration with anti-DV NS1 Abs caused increased concentrations of AST and ALT, but not BUN, in mouse sera, suggesting a pathogenic effect of anti-DV NS1 on liver injury. Gross and histological examinations revealed tissue damage and macrophage infiltration in mouse livers, while kidneys were not affected. A chemotactic effect on macrophage infiltration by monocyte chemotactic protein-1 (MCP-1) derived from anti-DV NS1-activated endothelial cells is likely to contribute to tissue damage in liver.29 In the absence of DV infection, our study explored the pathogenic anti-DV NS1 on liver damage via endothelial dysfunction. Based on these results, anti-DV NS1 caused liver injury but not kidney was similar to clinical manifestations in DHF patients.
Liver impairment occurs commonly among patients with DHF.15-17,32-34
However, the causes of hepatic injury contributing to dengue hemorrhage remain unclear.
Abnormal hemostasis in DHF includes vasculopathy, platelet dysfunction, and coagulopathy. In coagulopathy, a number of studies have demonstrated the amount changes of coagulator factors, including prothrombin, fibrinogen, 2-antiplasmin, plasminogen, and activated factor VII in DHF patients. Further study explained the deficiency of prothrombin complex and factor VII due to liver damage.17,35 In physiological function, liver plays important role on constitutive expression of clotting factors. The involvement of liver injury for the progression of DHF is thus speculated.
Patients with DV infection showed acute hepatic inflammation that might lead to DHF progression. High levels of serum liver enzymes are the risk factors associated with liver injury in DHF.15-17,20,36
In addition to the viral cytotoxicity, host immunopathogenic effects including hepatocyte/immune cell interaction and inflammatory responses may also contribute to liver damage.9 In the present study, we provide evidence to correlate the relationship between anti-DV NS1 and liver injury using the murine model.
In addition to the increased AST and ALT levels, histopathologic changes were also observed. How did liver injury occur by
anti-DV NS1? Anti-DV NS1 could not bind to liver cell lines including Chang liver, Hep3B, and Huh7 as examined previously.26 The direct cytotoxic effects of Abs on hepatocytes should therefore be excluded.
Our previous studies have demonstrated the pathogenic role of anti-DV NS1 causing endothelial cell apoptosis and inflammatory activation in vitro.26-29 Endothelial cell damage was induced by anti-DV NS1 directly.
Similarly, endothelial cell apoptosis was observed in mouse liver mediated by anti-DV NS1. Also, macrophage infiltration in liver could be detected. The mechanisms involved in cell infiltration and hepatic injury need further investigation. Actually, inflammatory endothelial cell activation was induced by anti-DV NS1 Abs. The increased cytokine (i.e., IL-6), chemokines (i.e., IL-8 and MCP-1) and adhesion molecule (i.e., ICAM-1) expression, as well as the peripheral blood mononuclear cell adherence to endothelial cells29 might account for liver injury caused by infiltrated immune cells and inflammatory activation.
The causes of liver damage in DHF are multiple. In this study, we showed that autoAb-induced endothelial dysfunction may contribute to hepatic inflammation. Several autoimmune diseases associated with hepatic injury have been related to the generation of autoAbs.37,38 Liver damage in autoimmune diseases involved both cytotoxicity and inflammation facilitated by autoAbs via direct or indirect route. Our previous study showed anti-DV NS1-induced endothelial cell apoptosis via a nitric oxide (NO)-regulated pathway.27,28 The involvement of NO in hepatotoxicity has been reported.24,39-41 NO is involved in hepatocyte injury primarily through the induction of apoptosis.41,42 Whether anti-DV NS1-mediated liver damage in mice is via NO-mediated endothelial cell dysfunction needs further investigation.
Anti-DV NS1 has been reported to cross-react with host factors, including blood clotting, integrin/adhesin proteins, and endothelial cells.43 Some mice injected with NS1 hybridoma cells showed hemorrhagic symptoms around the peritoneal tissues. It is believed that the changes on vasopermeability in mice may, at least in part, due to the generation of anti-DV NS1 Abs.
Our unpublished data demonstrated that anti-DV NS1 caused an increase in
endothelial cell permeability in vitro and vascular permeability in mice. Taken together, the pathogenic role of anti-DV NS1 was demonstrated in mice showing the hepatitis-like manifestation, similar to that in the clinical symptoms. It provides important information for NS1 peptide-based vaccine development to avoid the generation of autoAbs.
六、計畫成果自評
直至今日，DHF/DSS 的真正機轉尚未 清楚。本計畫初始的目標為發展以登革病 毒非結構性蛋白 NS1 做為基礎抗原蛋白，
研發中和性登革疫苗抗體。先前的研究發 現，NS1 在登革病毒感染時參與了自體免 疫抗體生成的機制。而根據 DV 感染引起 種種的病理反應都顯示除了病毒的直接傷 害外，愈來愈多的證據顯示宿主因子可能 參與 DHF/DSS 的病程發展與機制。我們的 研究顯示在登革病患血清中測得抗血小板 與抗內皮細胞的自體抗體存在。實驗證實 DV NS1 抗體就是一種自體免疫抗體，對 血小板及血管內皮細胞均有結合作用並造 成對細胞功能上的影響。我們認為來自病 毒的直接感染和抗體的合併效應，亦或是 抗體直接的影響都有可能是造成血小板減 少症或促使內皮細胞死亡進而導致血管內 皮系統失去正常機能進而參與出血性病症 的發生。
本研究計畫以小鼠模式研究 anti-DV NS1 造成血管內皮細胞傷害的進一步組織 病變。肝臟組織的炎性反應證明抗體可能 對於體內肝細胞的傷害性。免疫染色的結 果證明抗體會結合至肝臟中的血管並引發 細胞凋亡的發生，進一步誘導免疫細胞的 浸潤現象而有可能誘導免疫媒介的傷害。
這些結果有助我們對於 anti-DV NS1 的病 理角色之了解。藉由本論文針對抗 DV NS1 抗體所研究證實的致病角色，這些資訊將 可作為發展疫苗時的參考，以利用重組蛋 白 的 技 術 對 病 毒 蛋 白 具 有 致 病 性 的 epitopes 以 truncation 或 mutation 的方式予 以去除，希望能誘導具中和病毒的保護性 抗體運用在疫苗動物模式。
七、參考文獻
1. Henchal EA, Putnak JB. The dengue viruses. Clin Microbiol Rev 1990;3:376.
2. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998;11:480.
3. World Health Organization (WHO).
Dengue and dengue hemorrhagic fever.
Report on the Fact sheet N.117. Geneva:
WHO, April 2002:
http://www.who.int/mediacentre/factshee ts/fs117/en/.
4. Halstead SB. Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 2003;60:421.
5. Bielefeldt-Ohmann H. Pathogenesis of dengue virus diseases: missing pieces in the jigsaw. Trends Microbiol 1997;5:409.
6. Diamond MS, Edgil D, Roberts TG, Lu B, Harris E. Infection of human cells by dengue virus is modulated by different cell types and viral strains. J Virol 2000;74:7814.
7. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000;181:2.
8. Rothman AL, Ennis FA.
Immunopathogenesis of dengue hemorrhagic fever. Virology 1999;257:1.
9. Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH, Liu CC. Immunopathogenesis of dengue virus infection. J Biomed Sci 2001;8:377.
10. Halstead SB. Dengue. Curr Opin Infect Dis 2002;15:471.
11. Rothman AL. Immunology and immunopathogenesis of dengue disease.
Adv Virus Res 2003;60:397.
12. Rothman AL. Dengue: defining protective versus pathologic immunity. J Clin Invest 2004;113:946.
13. Halstead SB, Deen J. The future of dengue vaccines. Lancet 2002;360:1243.
14. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, et al.
Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr.
Infect Dis J 2004;23:99.
15. Kuo CH, Tai DI, Chang-Chien CS, Lan CK, Chiou SS, Liaw YF. Liver biochemical tests and dengue fever. Am J Trop Med Hyg 1992;47:265.
16. Nguyen TL, Nguyen TH, Tieu NT. The impact of dengue haemorrhagic fever on liver function. Res Virol 1997;148:273.
17. Pancharoen C, Rungsarannont A, Thisyakorn U. Hepatic dysfunction in dengue patients with various severity. J Med Assoc Thai 2002;85:S298.
18. Caldwell SH, Chang C, Macik BG.
Recombinant activated factor VII 80
(rFVIIa) as a hemostatic agent in liver disease: a break from convention in need of controlled trials. Perspect Clin Hepatol 2004;39:592.
19. Chuansumrit A, Chantarojanasiri T, Isarangkura P, Teeraratkul S, Hongeng S, Hathirat P. Recombinant activated factor VII in children with acute bleeding resulting from liver failure and disseminated intravascular coagulation.
Blood Coagul Fibrinolysis 2000;11:S101.
20. Lin YL, Liu CC, Lei HY, Yeh TM, Lin YS, Chen RM, et al. Infection of five human liver cell lines by dengue-2 virus.
J Med Virol 2000;60:425.
21. Lin YL, Liu CC, Chuang JI, Lei HY, Yeh TM, Lin YS, et al. Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2-virus-infected human liver cells. Virology 2000;276:114.
22. Marianneau P, Cardona A, Edelman L, Deubel V, Despres P. Dengue virus replication in human hepatoma cells activates NF-B which in turn induces apoptotic cell death. J Virol 1997;71:3244.
23. Thongtan T, Panyim S, Smith DR.
Apoptosis in dengue virus infected liver cell lines HepG2 and Hep3B. J Med Virol 2004;72:436.
24. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ.
Mechanisms of hepatotoxicity. Toxicol Sci 2002;65:166.
25. Higuchi H, Gores GJ. Mechanisms of liver injury: an overview. Curr Mol Med 2003;3:483.
26. Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS, Yeh TM, et al. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol 2003;69:82.
27. Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM, Chen SH, et al. Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol 2002;169:657.
28. Lin YS, Lin CF, Liu CC, Liu HS, Yeh TM, Chen SH, et al. Antibody-mediated endothelial cell damage via nitric oxide.
Curr Pharm Design 2004;10:213.
29. Lin CF, Chiu SC, Hsiao YL, Wan SW, Lei HY, Shiau AL, et al. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell
activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol 2005;174:395.
30. Lalor PF, Shields P, Grant AJ, Adams DH. Recruitment of lymphocytes to the human liver. Immunol Cell Biol 2002;80:52.
31. Lin CF, Wan SW, Cheng HJ, Lei HY, Lin YS. Autoimmune pathogenesis in dengue virus infection. Viral Immnol 2006;19:127.
32. Couvelard A, Marianneau P, Bedel C, Drouet MT, Vachon F, Henin D, et al.
Report of a fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes and liver apoptosis. Hum Pathol 1999;30:1106.
33. Fabre A, Couvelard A, Degott C, Lagorce-Pages C, Bruneel F, Bouvet E, et al. Dengue virus induced hepatitis with chronic calcific changes. Gut 2001;49:864.
34. Wahid SF, Sanusi S, Zawawi MM, Ali RA. A comparison of the pattern of liver involvement in dengue hemorrhagic fever with classic dengue fever.
Southeast Asian J Trop Med Public Health 2000;31:259.
35. Wills BA, Oragui EE, Stephens AC, Daramola OA, Dung NM, Loan HT, et al.
Coagulation abnormalities in dengue hemorrhagic fever: serial investigations in 167 Vietnamese children with dengue shock syndrome. Clin Infect Dis 2002;35:277.
36. Hung NT, Lei HY, Lan NT, Lin YS, Huang KJ, Lien le B, et al. Dengue hemorrhagic Fever in infants: a study of clinical and cytokine profiles. J Infect Dis 2004;189:221.
37. Manns MP. Recent developments in autoimmune liver diseases. J Gastroenterol Hepatol 1997;12:S256.
38. Manns MP, Obermayer-Straub P. Viral induction of autoimmunity: mechanisms and examples in hepatology. J Viral Hepatol 1997;4:42.
39. Hon WM, Lee KH, Khoo HE. Nitric oxide in liver diseases: friend, foe, or just passerby? Ann NY Acad Sci 2002;962:275.
40. McNaughton L, Puttagunta L, Martinez-Cuesta MA, Kneteman N, Mayers I, Moqbel R, et al. Distribution of nitric oxide synthase in normal and cirrhotic human liver. Proc Natl Acad Sci USA 2002;99:17161.
41. Wang JH, Redmond HP, Wu QD,
Bouchier-Hayes D. Nitric oxide mediates hepatocyte injury. Am J Physiol 1998;275:G1117.
42. D’Ambrosio SM, Gibson-D’Ambrosio RE, Brady T, Oberyszyn AS, Robertson FM. Mechanisms of nitric oxide-induced cytotoxicity in normal human hepatocytes. Environ Mol Mutagen 2001;37:46.
43. Falconar AKI. The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis. Arch Virol 1997;142:897.
Figure legend
Fig. 1. Anti-DV NS1 Abs cross-react with vessel endothelium of liver. (A) Endothelial cell binding ability of Abs, including 5 g of control IgG, anti-JEV NS1 IgG, and anti-DV
NS1 IgG, were detected by
immunocytochemical staining, AEC substrate development, counterstained with hematoxylin, and viewed using light microscopy. Magnification: 400X. (B) Cross-reaction of anti-DV NS1 Abs with vessel endothelium of mouse liver. The liver sections were obtained from normal mice.
Specimens were fixed with 1% formaldehyde in PBS and then incubated with 5 g of control IgG (a), anti-JEV NS1 IgG (c), or anti-DV NS1 IgG (b and d) at 4°C for 60 min.
After washing with PBS, specimens were incubated with HRP (a and b) or PE (c and d) conjugated anti-mouse IgG at 4°C for 30 min.
(C) Deposition of anti-DV NS1 Abs on vessel endothelium of liver in DV NS1-immunized mice. The liver sections were obtained from PBS-, JEV NS1-, or DV NS1-immunized mice. Specimens were fixed with 1% formaldehyde in PBS and then incubated with HRP (a and b) or PE (c and d) conjugated anti-mouse IgG at 4°C for 30 min.
After washing with PBS to remove unbound Abs, specimens were developed by AEC kit and viewed using light microscopy or fluorescence microscopy. The positive signals are marked by arrowheads.
Magnification: 200X.
Fig. 2. Vessel endothelium apoptosis in liver but not in kidney after NS1 hyperimmunization. Frozen liver (A) and kidney (B) sections were prepared from PBS-,
JEV NS1-, or DV NS1-immunized mice. Cell apoptosis was detected by TUNEL assay. The positive signals are marked by arrowheads.
Magnification: 400X.
Fig. 3. Serum AST and ALT, but not BUN, increase in NS1-hyperimmunized and anti-DV NS1-treated mice. Sera were collected from C3H/HeN mice which were immunized with PBS (n = 6), JEV NS1 (n = 5), or DV NS1 (n = 13) in adjuvant, and the concentrations of AST, ALT, and BUN were measured. Untreated mice were used for normal control (n = 9). The averages of each group are shown.
Fig. 4. Histopathologic changes in the liver, but not kidney, of mice after active immunization with DV NS1 or passive administration with anti-DV NS1 Abs.
C3H/HeN mice were immunized with PBS, JEV NS1, or DV NS1 in adjuvant. The histopathologic changes of liver (A) and kidney (B) were detected using H&E staining.
In liver sections, normal (a), PBS-immunized (b), JEV NS1-immunized (c), and DV NS1-immunized (d-h) mice are shown. In kidney sections, normal, PBS-immunized,
JEV NS1-immunized, and DV
NS1-immunized mice are shown. The concentrations of serum AST, ALT, and BUN in each of the mice are also shown. In passive model (C), C3H/HeN mice were i.v. injected with 500 g of anti-JEV NS1 IgG, anti-DV NS1 IgG, or 250 g of galactosamine plus 2
g of LPS for 48 h. The abnormal pathological changes and localization of infiltrated cells are marked. Galactosamine plus LPS was used for positive control.
Magnification: 400X.
Fig. 5. Cell infiltration and apoptosis in the liver of mice after passive administration with anti-DV NS1 Abs. C3H/HeN mice were i.v. injectedwithg of anti-JEV NS1 IgG, anti-DV NS1 IgG, or 250 g of galactosamine plus 2g of LPS for 48 h. (A) The cell binding activity of Abs was shown by staining with FITC-conjugated anti-mouse IgG. (B) Immune cell infiltration including macrophages, T lymphocytes, and NK cells were detected using FITC-conjugated F4/80 or anti-CD3 and PE-conjugated anti-CD56 Abs, respectively. (C) By TUNEL assay, cell apoptosis of livers in anti-JEV NS1-, anti-DV NS1-, and galactosamine plus LPS-treated mice were detected. Galactosamine plus LPS was used for positive control. Magnification:
400X.
Table 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
