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Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver.

ABSTRACT: Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples.We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0-1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. Single-cell laser capture microdissection was used to capture more than 1000 hepatocytes in grids, to preserve geometric relationships. HCV vRNA and interferon-induced transmembrane protein 3 (IFITM3) messenger RNA (the transcript of an interferon-stimulated gene) were measured in the same hepatocytes by quantitative polymerase chain reaction and assembled in maps to identify areas of high and low HCV replication.Patients' serum levels of HCV RNA ranged from 6.87 to 7.40 log10 IU/mL; the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of vRNA ranged from 1 to 50 IU/hepatocyte. Infection was not random; we identified clustering of HCV-positive hepatocytes using infected-neighbor analysis (P < .0005) and distance to the kth nearest neighbor compared with random distributions, obtained by bootstrap simulations (P < .02). Hepatocytes that expressed IFITM3 did not appear to cluster and were largely HCV negative.We used single-cell laser capture and high-resolution analysis to show that in human liver HCV infects hepatocytes in nonrandom clusters, whereas expression of antiviral molecules is scattered among hepatocytes. These findings show that quantitative single-cell RNA measurements can be used to estimate the abundance of HCV vRNA per infected human hepatocyte and are consistent with cell-cell propagation of infection in the absence of clustered IFITM3.

SUBMITTER: Kandathil AJ

PROVIDER: S-EPMC4005338 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver.

Kandathil Abraham J AJ Graw Frederik F Quinn Jeffrey J Hwang Hyon S HS Torbenson Michael M Perelson Alan S AS Ray Stuart C SC Thomas David L DL Ribeiro Ruy M RM Balagopal Ashwin A

Gastroenterology 20130822 6

<h4>Background & aims</h4>Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples.<h4>Methods</h4>We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0-1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. ...[more]

PMID: 23973767

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Project description:The aim of this study is to elucidate the biogenetic modification of donor and recipient interleukin-28B (IL-28B) genotypes in liver graft biopsies after living donor liver transplantation (LDLT) for chronic hepatitis C virus- (HCV-) related, end-stage liver disease. Fifty liver graft biopsies were collected from recipients during LDLT treatment for HCV-related, end-stage liver disease. DNA was extracted from all 50 liver tissues, and the IL-28B single-nucleotide polymorphisms (SNPs) rs8099917 and rs12979860 were studied for allelic discrimination by real-time PCR analysis. Blood samples were obtained from donors and recipients on postoperative day 0 (POD0), POD7, and POD30. We randomly selected five liver biopsies and isolated the hepatocytes by laser capture microdissection (LCM) to evaluate genotype modifications resulting from LDLT. After LDLT, the IL-28B SNP rs8099917 was identified not only in the liver graft biopsies and donors' sera (TT = 41?:?43; GT = 9?:?5; GG = 0?:?2), but also in liver graft biopsies and recipients' sera on POD0 (TT = 41?:?44; GT = 9?:?4; GG = 0?:?2), POD7 (TT = 41?:?30; GT = 9?:?18; GG = 0?:?2), and POD30 (TT = 41?:?29; GT = 9?:?19; GG = 0?:?2). A significant difference was observed between the rs8099917 allele frequencies of liver graft biopsies and recipients' sera on POD30 (p = 0.039). In addition, a significant difference was also noted between the rs12979860 allele frequencies of liver graft biopsies and donors' sera (CT = 49?:?39; TT = 1?:?10) (p = 0.012) and of liver graft biopsies and recipients' sera on POD0 (CT = 49?:?39; TT = 1?:?11) (p = 0.002), POD7 (CT = 49?:?42; TT = 1?:?8) (p = 0.016), and POD30 (CT = 49?:?41; TT = 1?:?9) (p = 0.008). This phenomenon was confirmed by pyrosequencing of hepatocytes isolated by LCM. Following LDLT, the TT-to-GT IL-28B genotype modification predominated in rs8099917, and the CC-to-CT modification predominated in rs12979860. In conclusion, these modified phenomena suggested that the selected donor with a predictable and favourable IL-28B genotype will not confer a benefit on the recipient in the living donor liver transplantation setting.

Dataset Information

Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver.

Publications

Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver.

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