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Volumn 284, Issue 5415, 1999, Pages 825-829

Viral clearance without destruction of infected cells during acute HBV infection

Author keywords

[No Author keywords available]

Indexed keywords

VIRUS DNA;

EID: 0033617583     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5415.825     Document Type: Article
Times cited : (1099)

References (28)
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    • R. Zinkernagel and B. Bloom, Eds. (Current Biology, London)
    • L. G. Guidotti and F. V. Chisari, in Current Opinion in Immunology, R. Zinkernagel and B. Bloom, Eds. (Current Biology, London, 1996), vol 8, pp. 478-483.
    • (1996) Current Opinion in Immunology , vol.8 , pp. 478-483
    • Guidotti, L.G.1    Chisari, F.V.2
  • 7
    • 0344276197 scopus 로고    scopus 로고
    • note
    • 7 HBV genome equivalents in 0.5 ml of serum from transgenic mouse lineages 1.3.32 and 1.3.46, respectively. Before inoculation and each week thereafter, blood was obtained by venipuncture and liver tissue was obtained by needle biopsy.
  • 8
    • 0345570262 scopus 로고    scopus 로고
    • note
    • Serum HBsAg, anti-HBs, anti-HBc, HBeAg, and anti-HBe were measured by solid-phase enzyme-linked immunosorbent assay (ELISA) (Auszyme for HBsAg, Ausab for anti-HBs, Corzyme for anti-HBc, and Abbott-HBe EIA for HBeAg and anti-HBe; Abbott Laboratories, Abbott Park, IL). HBsAg and anti-HBs concentrations were quantified by reference to internal standards (Bureau of Biologics, Bethesda, MD, and World Health Organization, respectively). Serum and liver HBV DNA were analyzed and quantified by direct competitive polymerase chain reaction (PCR). A pair of highly conserved diagnostic primers located within the HBV envelope coding region that yielded a PCR product of 441 base pairs (HBV 246S, 5′-GAGTCTAGACTCGTGGTGGA and HBV 687AS, 5′-CGAACCACTGAACAAATGGC, ayw subtype, Galibert sequence) were used under conditions previously described (26). A novel Eco Rl site was introduced by creating a point mutation at nucleotide 482 (T to G) with the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, Wl). The competitor plasmid was linearized by Hind III digestion, and different concentrations of competitor plasmid (from 0.3 fg to 1 fg) were used to contaminate chimp serum DNA extracts. After PCR, the amplified fragments were subjected to Eco Rl digestion, electrophoresed through 1.8% agarose gets in the presence of of ethidium bromide (0.1 μg/ml), and examined on an ultraviolet transilluminator. The amplified competitor plasmid produced two bands of 205 and 236 nucleotides, respectively, which migrated as a single band under the conditions used. Serum HBV DNA was quantified by comparing the relative intensity of the band corresponding to the competitor plasmid with the band obtained by amplification of serial dilutions of chimp serum DNA extracts. Hepatic needle biopsies, obtained on a weekly basis from anesthetized animals, were fixed in 10% neutral-buffered formalin, paraffin-embedded, sectioned (3 μm), and stained with hematoxylin and eosin, as previously described (1). The intracellular distribution of HBcAg and PCNA was assessed exactly as described (1). Quantitation of HBcAg-positive hepatocyte nuclei (wide bars) was carried out by counting a minimum of 10,000 hepatocytes with a 20× objective. Although HBcAg was detectable in both cytoplasms and nuclei, the results reflect nuclear staining. Apoptotic hepatocytes were identified as cells containing one or more of the following features: nuclear chromatin margination, pyknotic nuclei, and cytoplasmic fragmentation. The number of apoptotic hepatocytes was also calculated on selected liver sections by TUNEL assay that was performed according to the manufacturer's instructions (Oncor, Gaithersburg, MD). Similar numbers of apoptotic hepatocytes were obtained by the two independent techniques.
  • 13
    • 0027941175 scopus 로고
    • K. Kajino et al., J. Virol. 68, 5792 (1994).
    • (1994) J. Virol. , vol.68 , pp. 5792
    • Kajino, K.1
  • 19
  • 28
    • 0344276192 scopus 로고    scopus 로고
    • note
    • We thank D. Bylund and the Scripps Immunology Reference Laboratory for performing serum HBV DNA quantitation; R. Engle, Division of Molecular Virology, Georgetown University Medical Center, for performing serological assays; R. Koch, H. McClary, and M. Pagels for technical assistance; and J. Newmann for help with manuscript preparation. Supported by grants R37 CA40489, R01 AI20001, and R01 AI40696 from the NIH. This is manuscript number 11995-MEM from The Scripps Research Institute.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.