1. Volume 130, Issue 4, Pages 1107-1116 (April 2006)
Productive Replication of Hepatitis C Virus in Perihepatic Lymph Nodes In Vivo: Implications of HCV Lymphotropism 
Sampa Pal, Daniel G. Sullivan, Sean Kim, K. Kay–Yin Lai, John Kae, Scott J. Cotler, Robert L. Carithers, Brent L. Wood, James D. Perkins, David R. Gretch 
Gastroenterology 
Volume 130, Issue 4, Pages (April 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Staining of positive control cell lines by in situ hybridization (ISH). Positive control HeLa cell lines expressing either positive strand (A and C) or negative strand (E and G) subgenomic HCV RNA and negative control HeLa cells (B, D, F, and H) were stained with HCV riboprobes of opposite polarity, followed by immunologic detection of bound probes and enzymatic colorometry as described in the Materials and Methods section. Positive signal is red; nuclei are counterstained green. A and E demonstrate detection of genotype 1a-positive and -negative strand RNA using genotype 1a antisense and sense riboprobes, respectively, and C and G demonstrate detection of genotype 1b-positive and -negative strand RNA with genotype 1b antisense and sense riboprobes, respectively. Negative control HeLa cell lines were stained with the same HCV sense and antisense HCV riboprobe preparation used in the experiment presented in the left adjacent panel. Original magnification, ×40.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
Immunocytochemistry (ICC) staining of HCV replicon cell lines with anti-HCV core and NS3 antibodies. (A) Huh7-replicon cells stained with anti-NS3 antibodies followed by detection with DAB (brown signal). (B) Huh7-replicon cells stained with anticore antibodies followed with vector red (VR) detection (red signals). (C) Huh7-replicon cells stained with a mixture of anticore and anti-NS3 antibodies; core and NS3 antigens were simultaneously detected using different substrate systems, as described for A and B. (D) Huh7 cells without replicon, stained with the same antibodies as in C. Original magnification, ×100.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Detection of HCV genomes in follicular regions of human lymph node biopsy specimens by strand-specific ISH. Lymph node biopsy specimens were obtained at the time of liver transplantation, fixed and frozen in OCT buffer, and frozen sections were stained by ISH using antisense HCV riboprobes as in Figure 1, as described in the Materials and Methods section. A shows a low-power image of positive-staining HCV genomes in a lymph node follicle obtained from an HCV-infected patient (original magnification, ×10), and B shows detection of abundant HCV genomes (red punctate signals) throughout the cytoplasms of infected intrafollicular lymphocytes at high power (original magnification, ×100). Cell nuclei are counterstained blue. C shows negative ISH results using the antisense riboprobe and an uninfected lymph node specimen (original magnification, ×10). D illustrates negative results when staining a lymph node biopsy specimen obtained from an HCV-positive individual with an irrelevant riboprobe synthesized from the human MxA gene coding strand (original magnification, ×100). Note that neither the nucleus-associated MxA gene nor cytoplasmic MxA transcripts were recognized by the plus strand MxA riboprobe, confirming specificity of the ISH method for RNA of the opposite strand polarity. E and F illustrate ISH staining of human liver biopsy specimens from an HCV-infected patient (E) and an HCV-negative individual (F) using the antisense HCV riboprobes (original magnification, ×40).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Detection of HCV replicative intermediate RNA in human lymph node specimens. In A–C, HCV sense riboprobes were used to detect the viral replicative intermediate RNA by strand-specific ISH. A and C are ×100 magnification; the image in B was photo enlarged. Replicative intermediate RNA were detected at relatively high levels in a lower percentage of cells than genomic RNA in most infected lymph nodes. D shows staining of a lymph node biopsy specimen obtained from an HCV-negative individual with the HCV sense riboprobes (original magnification, ×100). E illustrates abundant positive ISH staining of HCV replicative intermediate RNA in periportal inflammatory cells within a liver biopsy specimen from an HCV-infected individual, and F shows negative staining of a similar lesion in a liver biopsy specimen from an uninfected patient (original magnification, ×40).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
Identification of HCV core and NS3 antigens within lymph node follicles by immunocytochemistry (ICC). The lymph node specimens in A and B were immunostained with anticore antibodies, and the specimens in C and D were stained with anti-NS3 antibodies followed by detection with VectorRed (VR). Abundant positive signals were evident (red) at ×100 magnification in the lymph node specimen taken from an HCV-infected subject (A and C) but not the lymph node specimen from an uninfected control subject (B and D). E illustrates negative staining of an HCV-infected lymph node specimen using an isotype-matched antibody specific for the HIV nef protein. F illustrates positive identification of B cells using anti-CD20, a B-cell marker, and G illustrates dual-label ICC staining of an infected lymph node specimen with a mixture of anticore (brown signal) and anti-CD20 (red signal) antibodies. The results demonstrate HCV core localization in the CD20-positive cells. All images are ×100 magnifications, except for F (×40).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
Analysis of HCV quasispecies in serum, liver, lymph node, and PBMC compartments. (A) Autoradiograms showing the complex nature of HCV hypervariable regions amplified and cloned from serum, liver, PBMC, and lymph node tissue compartments in 1 case. Quasispecies variants were analyzed by the clonal frequency analysis technique described in the Materials and Methods section. Each unique band profile represents a unique quasispecies variant. (B) Bar graph summarizing the frequencies of the 5 predominant quasispecies variants in the 4 tissue compartments. Note that, whereas variants A and E were recovered from all 4 compartments, variant B was only recovered from lymph node (31% of clones) and serum (22% of clones) compartments, and variant C was only recovered from PBMCs (2% of clones) and serum (14% of clones). (C) Phylogenetic tree of 33 unique quasispecies variants isolated from the 4 compartments. Colored ovals indicate variants isolated from hematopoietic compartments; boxes with letters identify the 5 predominant quasispecies variants identified in the patient. See text for details.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions