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Keywords:

  • ground glass hepatocyte;
  • hepatocarcinogenesis;
  • endoplasmic reticulum;
  • endoplasmic reticulum stress;
  • pre-S mutant

Abstract

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

The discovery of “ground glass” hepatocytes (GGH) that contain hepatitis B virus (HBV) surface antigens by Hadziyannis and Popper in 1973 represents a historical landmark in the pathology of chronic HBV infection. Different types of GGH have been correlated to the expression patterns of surface/core antigens and the stages of virus replication. The original two types (designated types I & II) of GGH were found to contain specific pre-S mutants with deletions over either pre-S1 or pre-S2 regions, respectively. Type II GGH consistently harbor pre-S2 deletion mutants, which can escape from immune attack and grow preferentially to form clusters. Both types of pre-S mutants can induce endoplasmic reticulum (ER) stress and oxidative DNA damage. The pre-S2 mutants, albeit inducing a weaker level of ER stress signals, could additionally initiate ER stress-independent retinoblastoma/adenovirus E2 promoter binding factor/cyclin A signaling through their interaction with c-Jun activation domain binding protein 1 to degrade p27, illustrating the growth advantage of type II GGH. The combined effects of genomic instability and the proliferation of hepatocytes harboring pre-S mutants could potentially lead to hepatocarcinogenesis over the decades of chronic HBV infection. The presence of pre-S mutants in sera was reported to carry a high risk of developing hepatocellular carcinoma (HCC). Furthermore, transgenic mice harboring pre-S2 mutant plasmids have been shown to develop a dysplastic change of hepatocytes and HCC. Therefore, in addition to being a histological marker of chronic HBV infection, GGH, particularly type II GGH, may represent the preneoplastic lesions of HBV-related HCC.


Historical background of GGH

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

In the early 1970s when the Australia antigen was found to be associated with hepatitis B virus (HBV) infection, Hadziyannis and Popper first recognized the cytoplasmic surface antigen in “ground glass” hepatocytes (GGH) of HBV carriers.1,2 The identification of GGH as a histological marker of chronic HBV infection has become a major event in liver pathology since then. Shikata later introduced the histochemical orcein stain to identify the surface antigens in GGH.3,4 Under electron and immuno-electron microscopy, GGH are characterized by an abundance of endoplasmic reticulum (ER), among which viral particles of surface antigens accumulate.3 It is the overloaded ER that make the cytoplasms of hepatocytes become “foggy” or “glassy”. After the introduction of immunohistochemistry, a series of studies demonstrated that different types of GGH correlated to the expression patterns of HBV surface/core antigens and the replicative stages of chronic HBV infection.5,6 In the original paper by Hadziyannis et al.1 two types of GGH were described, designated later by us as types I and II GGH.7 Type I GGH are usually scattered singly in the hepatic lobules with the expression of dense homogeneous or “inclusion-like” pattern of surface antigens (Fig. 1A). This type of GGH usually occurs at the early carrier stage or in patients with active diseases, frequently co-expressed with a nuclear or cytoplasmic core antigen.5,8,9 Type II GGH express a unique pattern of surface antigens at the cell margin or periphery (Fig. 1A). Most interestingly and importantly, type II GGH consistently cluster in nodules and usually occur at the advanced or low replicative stages of virus replication, and are frequently associated with cirrhosis or hepatocellular carcinoma (HCC). Conversion from type I GGH to type II GGH could be demonstrated in the serial biopsies from the same individual, frequently associated with hepatitis B e antigen (HBeAg) seroconversion.10

image

Figure 1. (a) Ground glass hepatocytes (GGH) and the expression patterns of hepatitis B surface antigens. Type I GGH are usually scattered singly (i, hematoxylin–eosin [HE] stain ×200) and express an inclusion-like pattern of the surface antigen (ii, immunohistochemical stain for the surface antigen), while type II GGH (iii) consistently cluster in nodules and express a marginal pattern of the surface antigen (iv). (b) By laser capture microdissection DNA extraction, and cloning sequencing, we found that type I GGH contain pre-S1 deletion mutants while type II GGH contain pre-S2 deletion mutants. In hepatoma tissues, a complex combination of deletions at pre-S1 and pre-S2 regions may occur. HBV, hepatitis B virus.

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Identification of pre-S deletion mutants in GGH

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

The consistent clustering distribution of type II GGH suggest to us that type II GGH may represent clonally-proliferated, adenomatous, or preneoplastic lesions of HCC.11 By dissecting the cirrhotic nodules containing type II GGH, the HBV genomes were clonally proliferated and integrated, supporting the concept of a clonal or adenomatous lesion of type II GGH. Since type II GGH express a unique pattern of “marginal” type surface antigens, it will be interesting to unravel the mystery behind the two unique biological phenomena linked to type II GGH, that is, the clustering proliferation of GGH and the marginal expression of surface antigens. The clustering distribution of type II GGH makes it available to manually isolate enough hepatocytes for the molecular cloning of the surface genes before the age of laser capture microdissection. To our surprise, we found that type II GGH consistently harbored pre-S mutants with in-frame deletions over the pre-S2 regions (predominantly, nt 2–55 or 4–57) with or without point mutations at the start codon (ATG–ATA) of the S promoter (Fig. 1b).11 The point mutation at the start codon of the S promoter could lead to the defective synthesis of the middle surface antigen. It is interesting to note that the deletion site at the pre-S2 region coincides with the epitope of the cytotoxic T-cell and neutralization responses and suggests that the pre-S2 deletion mutants may represent an immune escape mutant.12 This assumption is supported by the observation that the hepatic lobules, which contain type II GGH, usually show no inflammatory activities or lymphocyte infiltration.1,10,13 Pre-S2 deletion mutants therefore represent a selection mutant under immune pressure, which prevails at the advanced phase of chronic HBV infection. After the introduction of laser capture microdissection, we started to clone the surface genes in type I GGH, which are usually scattered singly and can only be isolated by laser capture microdissection. Unexpectedly, we found that type I GGH contain entirely different pre-S mutants with variable deletions over the pre-S1 regions. The deletion sites at the pre-S1 region may interfere with the transcriptional activities of the pre-S1 promoter and affect the regulation of HBV replication.14,15 In HCC tissues, the pre-S mutants are prevalent for up to 60%, including mutants with combinations of deletions over the pre-S1 and pre-S2 regions (Fig. 1b).

The HBV surface gene contains three gene segments (pre-S1, pre-S2, and S) that encode three surface proteins with common C-terminus. The large surface protein (LHB) encodes from all three gene fragments, the middle surface protein (MHB) contains pre-S2 and S fragments, and the small surface protein (HB) encodes for only the S region. Previous studies have shown that mutations on the S gene usually focus on the a-determinant region, which is exposed on the surface of virions and is also the immune dominant epitope of the HB protein. The a-determinant variants usually accumulate in patients with chronic hepatitis B, but no correlation with cancer incidence has ever been reported. The pre-S1 containing LHB exhibits a dual topology, and only half of the LHB translocate post-translationally into the ER lumen.16,17 The pre-S regions that remain in cytoplasmic orientation can bind to the cytosolic heat shock protein Hsc7018 and presumably interact with the core particle during virion assembly.16 The cytoplasmic orientation of the pre-S region is required for the transcriptional activator function of LHB and MHB in vitro.19,20 The luminal orientation of the pre-S domain, after virus maturation and secretion, is exposed on the surface of the virion and is involved in virus attachment and recognition.21,22 Naturally-occurring pre-S mutants are frequently affected by deletions in the pre-S1 or pre-S2 regions and by mutations that inactivate the pre-S2 start codon. The corresponding mutants therefore express shorter forms of LHB with internal deletions. Mutants with various types of in-frame deletion in pre-S1 region were found to be replication competent in vitro,23,24 and can be the predominant strain in vivo.25–27 As shown in Figure 2, the prevalence of pre-S mutants, particularly pre-S2 mutants, in serum and liver tissues increases with the natural course of chronic HBV infection.

image

Figure 2. Correlation of disease progression of chronic hepatitis B virus (HBV) infection to the prevalence of ground glass hepatocyte (GGH) expression patterns of surface and core antigens and the prevalence of pre-S mutants. In serum and liver tissues from patients with hepatocellular carcinoma (HCC), the prevalence of pre-S mutants may account for up to 60% with predominance of pre-S2 mutants. Numbers in parentheses indicate the prevalence of pre-S2 mutants.

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ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

The consistent demonstration of mutated surface genes in GGH suggests that GGH may represent hepatocytes that contain malfolded surface proteins entrapped and accumulated in the ER. By double-labeled immunofluorescence microscopy studies, the surface antigens were demonstrated to exist in the ER. The demonstration of mutated pre-S mutants in the ER confers the new biological significance in addition to its being a histological marker of chronic HBV infection. ER stress has been found to be associated with human diseases induced by host or viral proteins due to either malfolded proteins or a defect in the trafficking from ER to Golgi.28 By the in vitro expression of wild-type and mutant pre-S constructs in the Huh-7 cell line, the expression patterns of surface antigens are different and closely reflect the in vivo expression.11 Northern and Western blot analyses revealed that the pre-S1 mutant induced stronger levels of ER chaperone (Grp78 and 94) response, calcium release, cyclooxygenase-2 (COX-2) and inflammatory cytokines, and oxidative stress intermediates, which tend to result in apoptosis.7,29,30 The oxidative stress induced by pre-S mutants may lead to oxidative DNA damage and genomic instability. Preliminary data from our laboratory also revealed that the pre-S mutants could upregulate the expression of vascular endothelial growth factor (VEGF) through ER stress with the activation of Akt/mammalian target of rapamycin (mTOR) signaling in GGH (ZC Yang and IJ Su, pers. comm., 2007). The pre-S2 mutants, albeit inducing a weaker level of ER stress signal, exhibited higher levels of mutation frequency and transforming capabilities using primary hepatocyte cell line H4.7 Under ER stress conditions, hepatocytes expressing the pre-S2 mutant protein could sustain cell cycle progression and provide cell survival in the presence of strong ER stress inducer brefeldin A, while Huh-7 cells expressing either wild-type or pre-S1 mutants were arrested in the G1 phase and underwent apoptosis.31 A complementary DNA microarray was employed to dissect the genes differentially expressed and regulated by HBV pre-S mutant proteins (HC Wang and IJ Su, pers. comm.., 2008). The pre-S2 mutant protein exhibited stronger transactivator activities associated with cell growth, such as G-protein-coupled receptors and Ras-associated signals. In addition, the pre-S2 mutant can specifically downregulate apoptosis-related genes, such as tumor necrosis factor-related death ligand-1á, an activator of apoptosis human gene harakiri and candidate tumor suppressor gene deleted in breast cancer-2. Together, these data suggest that pre-S2 mutant proteins may provide survival or growth signals to protect hepatocytes from ER stress-induced cell cycle arrest or apoptosis, consistent with the clustering pattern of type II GGH.

Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

As described earlier, the pre-S1 and pre-S2 mutants are distinct in ER stress-independent responses. Such a distinction is considered significant for the difference in their carcinogenic preferences. The pre-S2 mutant, but not the wild-type or pre-S1 mutant LHB, was found to directly interact with c-Jun activation domain binding protein 1 (JAB1), which enhances activator protein-1 transcriptional activity and cell proliferation. Through its binding to JAB1, the pre-S2 mutant large hepatitis B surface antigen induces JAB1 nuclear translocation. The nuclear JAB1 targets the cyclin-dependent kinase (cdk) inhibitor p27Kip1 to cytosolic 26S proteasome to be degraded. Through this pathway, the downstream molecule cdk2 was activated, resulting in retinoblastoma (Rb) hyperphosphorylation and cell cycle progression.32 These findings provide a molecular mechanism for the growth advantage induced by the pre-S2 mutant. The p27Kip1 degradation, cdk2 activation, and Rb hyperphosphorylation were also found in the pre-S2 mutant transgenic mice as in the in vitro cell cultures. JAB1 is known to regulate multiple protein stabilities via transporting them to 26S proteasome. The targets of JAB1 include p53, Smads 4 and 7, and estrogen receptors, suggesting that JAB1 may regulate carcinogenesis via multiple pathways.33 JAB1 is also the constitutive photomorphogenic-9 (COP9) signalosome subunit-5) subunit of COP9 signalosome, a multisubunit complex regulating proliferation and differentiation.34 It is unclear whether the JAB1-associated COP9 signalosome affects hepatocyte differentiation through pre-S2 mutant LHBS. In a separate study, the pre-S2 mutant was found to induce cyclin A overexpression.31 The cyclin A expression was aberrantly located in the cytoplasm in type II GGH and in the livers of transgenic mice harboring pre-S2 mutants. The significance of the aberrant expression of cyclin A in GGH remains to be clarified.

From these findings and our own, we conclude that both types of pre-S mutants accumulate in the ER and can induce ER stress signaling, while pre-S2 mutants can additionally induce an ER stress-independent signal pathway to initiate the growth of type II GGH, explaining the clustering distribution and growth advantage of type II GGH.

Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

The emergence of pre-S1 deletion mutants was associated with a poor outcome of patients with chronic HBV infection.35 In many patients at the advanced stages of diseases, the pre-S2 variants represent the only detectable or the predominant virus, indicating that these variants can replicate autonomously.27,36,37 Based on a horizontal cross-section studies on patients with chronic HBV infection (Fig. 2), the pre-S mutants emerged at a frequency of 3% by the age of 30, 15% by the age of 40, and 30% by the age of 50. Distinct from pre-S1 variants, the pre-S2 deletion mutants are infrequently detected in HBeAg-positive patients, but are predominantly found in anti-HBeAg-positive patients and in patients with HCC.11,27,38,39 In patients with HCC, the prevalence of pre-S mutants reached a level of 60%.40–42 The emergence of the pre-S2 mutant is found to be the most important risk factor for the development of HCC.42 Besides the pre-S mutants, a C-terminal-truncated pre-S2 protein was identified as a potential transactivator to initiate a Ras/Raf/extracellular signal-regulated kinase signaling and act as tumor promoters in the development of HCC.19,43,44

Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

HBV transgenic mice have been produced both with complete HBV genomes that can support viral replication and with selected viral genes, including three surface genes, HBx, core, and precore genes.45 The expression of large surface antigens in transgenic mice has been shown to be cytopathic and could lead to liver injuries, regenerative hyperplasia, chronic inflammation, oxidative DNA damage, and eventually progress to HCC.46–49[Correction made after online publication on 7 July 2008: reference citation ‘46–48’ has been corrected to ‘46–49’.] In our laboratory, the transgenic mouse model of the pre-S2 deletion mutant was created, and the dysplastic change of hepatocytes was demonstrated with HCC formation at 2 years of age.48[Corrections made after online publication on 7 July 2008: reference citation ‘49’ has been corrected to ‘48’ and reference 50 has been deleted.] In the studies of the transgenic mouse model using pre-S2 mutants, the hepatic lobules usually revealed minimal inflammatory and necrotic activities, the findings similar to the liver pathology in the non-tumorous part of human HCC. These results provide evidence for a direct role of HBV in carcinogenesis and highlight the potential role of the pre-S2 mutant protein as a viral oncoprotein.

GGH represent the precursor lesions of HBV-related HCC

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

The in vitro and in vivo evidence we provide strongly suggest that GGH, in addition to their role as histological markers of chronic HBV infection, represent preneoplastic lesions of HBV-related HCC. As summarized in Figure 3, the retention of both types of pre-S mutants in the ER plays a key event in initiating the ER stress-dependent malfolded protein response signals, leading to oxidative stress DNA damage and the genomic instability of GGH. The pre-S mutant-induced oxidative stress may also activate two signal pathways, one involving VEGF/Akt/mTOR and the other nuclear factor-êB/COX-2 to protect cells from apoptosis. For type II GGH and pre-S2 mutants, an additional ER stress-independent JAB1/adenovirus E2 promoter binding factor/Rb/cyclin A growth signal was activated, leading to clustering proliferation and the growth advantage of type II GGH. The combined effects of genomic instabilities and cell proliferation will finally lead to hepatocarcinogenesis over the decades of chronic HBV infection. The oncogenic implication of GGH provides an entirely new mechanism for HBV-related hepatocarcinogenesis and also offers a good model for tumorigenesis associated with ER stress. The trans-ER membrane topology of pre-S mutants should be clarified in the future to explain the differential biological activities conferred by different types of GGH described in this review.

image

Figure 3. Schematic depiction of the potential signals induced by pre-S mutants and in ground glass hepatocyte (GGH). Both types of pre-S mutants can induce endoplasmic reticulum (ER) stress signals, which may lead to oxidative stress and DNA damage, leading to genomic instability. The pre-S mutants may also activate two signal pathways to protect the hepatocytes from apoptosis, one involving nuclear factor (NF)-êB to upregulate cyclooxygenase-2 (COX-2) and the other vascular endothelial growth factor to activate Akt/mammalian target of Rapamycin (mTOR) signaling. Pre-S2 mutant can additionally induce an ER stress-independent c-Jun activation domain binding protein 1 (JAB1)/p27/retinoblastoma (Rb)/adenovirus E2 promoter binding factor/cyclin A signal to initiate cell cycle progression. Combined effects of genomic instability and cell proliferation will potentially result in carcinogenesis. Cdk2, cyclin-dependent kinase 2; HBV, hepatitis B virus; ROI, reactive oxygen intermediate.

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References

  1. Top of page
  2. Abstract
  3. Historical background of GGH
  4. Identification of pre-S deletion mutants in GGH
  5. ER stress signals, oxidative DNA damage, and transforming capabilities induced by different pre-S mutants
  6. Pre-S2 deletion mutants could additionally induce ER stress-independent c-Jun activation domain binding protein 1/p27/retinoblastoma/cyclin signaling, explaining the clustering proliferation and growth advantage of type II GGH
  7. Pre-S mutants are prevalent in serum samples from patients with different stages of chronic HBV infection and are associated with a higher risk of developing HCC
  8. Transgenic mice of pre-S2 mutants revealed dysplastic changes and HCC formation
  9. GGH represent the precursor lesions of HBV-related HCC
  10. Acknowledgments
  11. References

[Correction made after online publication on 7 July 2008: reference 50 has been deleted.]