Gastroenterology
Volume 137, Issue 4 , Pages 1214-1218, October 2009

The Promise of Advanced Imaging Techniques for the Detection of Hepatitis C Virus Antigens in the Infected Liver

  • Benno Wölk, MD

      Affiliations

    • Institute of Virology, Medizinische Hochschule Hannover, Hannover, Germany
    • ,
  • Darius Moradpour, MD

      Affiliations

    • Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland
    • Corresponding Author InformationReprint requests Address requests for reprints to: Darius Moradpour, MD, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, BU44/07/2421, Rue du Bugnon 44, CH-1011 Lausanne, Switzerland

published online 31 August 2009.

Article Outline

 

See “Visualizing hepatitis C virus infections in human liver by two-photon microscopy,” by Liang Y, Shilagard T, Xiao S–Y, et al, on page 1448.

Hepatitis C virus (HCV) is primarily a hepatotropic virus. This may seem trivial to the readers of this journal, but how do we actually know? Historically, indirect evidence has been the first and strongest argument for the liver tropism of this virus: HCV infection leads to acute and chronic hepatitis. However, the detection of viral antigens and replicating genomes in the liver represents a major challenge in the case of HCV. Therefore, even 20 years after the identification of HCV and despite great advances in the molecular virology, diagnosis and treatment of hepatitis C, information about the type, number, and distribution of infected cells in the liver remains limited and conflicting.1 In addition, there is considerable controversy as to whether HCV infects cells other than hepatocytes, such as B cells or other lymphohematopoietic cells.2, 3 This disappointing situation was mainly attributed to the fact that the level of antigen expression is low in HCV infection.

Carefully performed immunostaining studies performed to date reported varying numbers of HCV antigen-positive hepatocytes in a subset of specimens analyzed.1, 4 In contrast, some in situ hybridization studies revealed much more widespread infection of the liver.5 A wide range in the proportion of infected cells, with widespread infection in some patients, is suggested by recent studies of host factors that are cleaved and thereby inactivated by the viral nonstructural protein 3 (NS3)-4A protease, including Cardif, an essential adaptor molecule in the RIG-I antiviral pathway and T-cell protein tyrosine phosphatase.6, 7

Reverse transcription-polymerase chain reaction (RT-PCR) allows for the reliable detection of HCV RNA in infected tissue. However, PCR-based HCV RNA amplification from crude tissue samples does not indicate the type of cell harboring replicating viral genomes. Detection of viral negative-strand RNA in individual cells would be more convincing, yet this is technically challenging. In addition, because PCR-based methods are inherently susceptible to false-positive detection in an HCV-focused working environment, they do not provide the type of experimental evidence which will convince also the last skeptic.

In this issue of gastroenterology, Liang et al8 address these challenging issues by advanced imaging techniques. By combining 2-photon excitation (2PE) microscopy with quantum dot (Qdot) technology, they achieve unprecedented sensitivity of HCV antigen detection in the liver of chronically infected patients. In this proof-of-concept study, the authors examined frozen sections of wedge resection specimens from 9 patients with HCV-induced cirrhosis who underwent liver surgery for hepatocellular carcinoma. Remarkably, they were able to stain specifically for core, NS3, and NS5A as well as double-stranded RNA (dsRNA) in all samples analyzed. dsRNA was identified by a specific monoclonal antibody and was used as a marker for cells harboring replicating HCV, because the viral replicative intermediates represent dsRNA not otherwise found in cells. The authors demonstrate the specificity of the immunostaining in 3 ways. First, only liver tissue from the patients with chronic hepatitis C but not that of 3 HCV-negative control patients was positive over a defined cutoff. Second, binding of the Qdot-coupled anti-core antibody was drastically reduced by preincubation of the tissue specimen with the unlabeled version of the same but not a different antibody. Third, cells that stained positive for core also stained positive for NS3 or dsRNA in tissue colocalization experiments. Last but not least, the authors demonstrate core antigen also in one formalin-fixed, paraffin-embedded liver biopsy specimen after antigen retrieval. At this point, however, it is unknown if the fixation and antigen retrieval procedures will permit an equally sensitive HCV antigen detection in a larger panel of liver biopsy samples.

A major finding was that HCV antigen-positive cells clustered in groups that were distributed unevenly between the different fields of view analyzed in the same sample. The authors carefully quantified their findings by counting the number of positive cells in 20 fields of view for each specimen. Between 1.7% and 21.6% of hepatocytes (7.2% and 21.6% in patients with a serum HCV RNA > 105 IU/ml) were found to be positive for core antigen. By contrast, the number of positive hepatocytes was clearly lower in the control patients. Yet, there were still up to 0.6% false-positive cells in the controls. Choosing an arbitrary cutoff of 3%, the authors show that only in patients infected with HCV between 3 and 18 out of the examined 20 fields had >3% positive cells. The presence of infected cells in clusters supports the recent notion of HCV cell-to-cell transmission in vivo.9 Interestingly, hepatocytes at the periphery of such clusters often displayed less abundant dsRNA staining when compared with viral antigen staining, which may suggest more recent infection and, thereby, reveal insights into the dynamics of HCV infection in vivo.

So, why were the authors of this study so successful in reliably detecting different HCV antigens and dsRNA in naturally infected hepatocytes? They propose that the combination of 2PE microscopy and Qdot technology greatly improved the signal-to-noise ratio by enhancing the sensitivity and at the same time reducing the autofluorescence inherent to liver tissue.

2PE microscopy was pioneered by Winfried Denk in the early 1990s.10 Whereas in conventional (one-photon) fluorescence microscopy, a single photon promotes a fluorescent molecule from the ground state (S0) to an excited state (S*), in 2PE microscopy 2 photons of low energy are absorbed to produce the same molecular transition (Figure 1).11, 12 The fluorophore then emits a single photon with a wavelength that depends on the type of fluorophore used. As a rule of thumb, 2 photons produce the same effect if they have twice the wavelength of the single photon used in conventional fluorescence microscopy. Because typical 1-photon excitation spectra are in the range of ultraviolet light (<400 nm) or visible light (400–750 nm), 2PE is performed with infrared light (>750 nm). 2PE requires that 2 photons are absorbed within 10−16–10−17 second, that is., nearly simultaneously. This is a very rare event at ordinary light intensities and typically requires a pulsed laser to produce a high density of excitatory photons. Because 2 photons need to be absorbed to excite a fluorophore, the probability for fluorescent emission increases quadratically with the excitation intensity. As a result, fluorescence is generated almost exclusively in the tiny focal volume (∼0.1 μm3) where the laser beam is tightly focused (Figure 1).11, 12 This high localization of excitation as well as deeper tissue penetration and reduced photobleaching represent the principal advantages of 2PE. Nonlinear optical microscopy techniques, such as 2PE, are also very well suited to efficiently excite Qdots.13 Qdots are semiconductor nanocrystals that have emerged as an alternative to organic dyes and fluorescent proteins, and are brighter and more stable against photobleaching than conventional fluorescent molecules. The emission spectrum of Qdots can be controlled by their size. For biological applications, Qdots are covalently linked with biomolecules, such as antibodies, to specifically label a target molecule of interest (Figure 1). These combined properties of 2PE and Qdots increase the signal-to-noise ratio between specific fluorescence and tissue autofluorescence, which is often limiting in the liver.

  • View full-size image.
  • Figure 1. 

    Quantum dot (Qdot) staining and 2PE microscopy of HCV antigens in the liver. (A) Liver specimens from HCV-infected patients were obtained during liver surgery for hepatocellular carcinoma or by percutaneous liver biopsy. (B) HCV-specific antigens and dsRNA were immunolabeled with monoclonal antibodies that were directly conjugated with Qdots (illustrated here) or, indirectly, using Qdot-coupled secondary antibodies (not illustrated). (C) Liver tissue was then analyzed by 2PE microscopy. Whereas in conventional 1-photon fluorescence microscopy a single photon (with a wavelength of 510 nm, for example) is able to promote a fluorescent molecule from a ground state (S0) to an excited state (S*), in 2PE microscopy 2 photons (1020 nm) are simultaneously absorbed whose total energy produces the same molecular transition and results in identical emission (530 nm in this example). The effect requires very high excitatory photon densities that are reached only in the focal volume (green) but not in the remaining illumination path (red), thereby eliminating out-of-focus fluorescence.

The work by Liang et al8 has important implications for the field. First, it should now be possible to address fundamental questions about HCV infection, such as the number and distribution of infected hepatocytes (and eventually other cell types) as well as their spatial relation to, among others, immune cells, activated stellate cells, and dysplastic or cancerous hepatocytes. It should also become possible to validate at the single cell level in vivo the increasing number of host factors that are potentially involved in HCV infection.14 For example, the technology described by Liang et al8 will be indispensable to further study the mechanisms of induction of and evasion from innate immune responses by HCV. Indeed, current evidence indicates that HCV induces a vast array of interferon-stimulated genes in the infected liver and yet it has developed a number of mechanisms to evade and counteract these responses.15 With 2PE microscopy and Qdot technology, it should now become possible to investigate the specific components of the interferon system that must be inactivated to enable HCV replication in the infected hepatocyte. Finally, it will be very interesting to correlate these findings with the clinical course and outcome of acute and chronic hepatitis C.

A particularly puzzling observation relates to the detection of viral antigens in the vicinity of a hepatocellular carcinoma in a patient that had previously responded to interferon-based therapy and whose serum HCV RNA was repeatedly negative (patient H9 in the study by Liang et al8) Similar observations were reported previously.16 Unfortunately, patient H9 could not be characterized further and it is, therefore, unknown whether HCV RNA was detectable in her liver. Could this represent a case of “occult HCV infection”?17 Or could it reflect the persistence of viral genetic material in an integrated cDNA form, as shown for other RNA viruses?18 The detection of dsRNA in the liver of this patient argues for a replicating infection but additional investigations will be necessary to resolve these intriguing issues.

In conclusion, the proof-of-concept study by Liang et al8 represents an important breakthrough. Future studies by the authors themselves as well as by other groups that dispose of such advanced imaging techniques should address whether the number and distribution of infected hepatocytes are similar in noncirrhotic tissue. In addition, for broader clinical applications and better preservation of the cellular morphology, it will be important to investigate in a larger panel whether 2PE microscopy and Qdot technology can be applied as successfully to fixed tissue samples obtained by needle biopsy. Finally, given the high genetic variability of HCV, it will be important to dispose of a collection of antibodies, such as the ones used by the authors, that recognize highly conserved epitopes. In the meanwhile, Liang et al8 set a new standard for follow-up studies, which will require a similarly thorough quantitative and controlled approach. Yet, the future glows bright for the detection of HCV antigens in the infected liver.

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References 

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 Conflicts of interest The authors disclose no conflicts.

 Funding Research in the authors' laboratories is supported by the Swiss National Science Foundation (3100A0-122447), the Swiss Cancer League/Oncosuisse (OCS-01762-08-2005) and the German Federal Ministry of Education and Research (01EO0802).

PII: S0016-5085(09)01493-0

doi:10.1053/j.gastro.2009.08.027

Refers to article:

  • Additional Online Content AvailableEditorial Accompanies Article Visualizing Hepatitis C Virus Infections in Human Liver by Two-Photon Microscopy , 27 July 2009

    Yuqiong Liang, Tuya Shilagard, Shu–Yuan Xiao, Ned Snyder, Daryl Lau, Luca Cicalese, Heidi Weiss, Gracie Vargas, Stanley M. Lemon
    Gastroenterology October 2009 (Vol. 137, Issue 4, Pages 1448-1458)

Gastroenterology
Volume 137, Issue 4 , Pages 1214-1218, October 2009

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