Heterogeneity and coexistence of oncogenic mechanisms involved in HCV-associated B-cell lymphomas.

The association of HCV-infection with B-lymphomas is supported by the regression of most indolent/low-grade lymphomas following anti-viral therapy. Studies on direct and indirect oncogenic mechanisms have elucidated the pathogenesis of HCV-associated B-lymphoma subtypes. These include B-lymphocyte proliferation and sustained clonal expansion by HCV-envelope protein stimulation of B-cell receptors, and prolonged HCV-infected B-cell growth by overexpression of an anti-apoptotic BCL-2 oncogene caused by the increased frequency of t(14;18) chromosomal translocations in follicular lymphomas. HCV has been implicated in lymphomagenesis by a "hit-and-run" mechanism, inducing enhanced mutation rate in immunoglobulins and anti-oncogenes favoring immune escape, due to permanent genetic damage by double-strand DNA-breaks. More direct oncogenic mechanisms have been identified in cytokines and chemokines in relation to NS3 and Core expression, particularly in diffuse large B-cell lymphoma. By reviewing genetic alterations and disrupted signaling pathways, we intend to highlight how mutually non-contrasting mechanisms cooperate with environmental factors toward progression of HCV-lymphoma.

Steps and Routes of HCV Infection: The Great Promise of New Anti-Viral Targets.

A major breakthrough in understanding the steps and signalling that drives the HCV to reach a full life-cycle has been achieved by in vitro models that have facilitated elevated virus production, resulting in the discovery of pathways and factors involved in virus entry, translation and replication. The HCV enters host cells through binding of its envelope glycoproteins to cell receptors, followed by clathrin-mediated endocytosis and fusion with cell membranes, leading to virus uncoating and cell entry. This chain of events is mediated by sequential involvement of different co-receptors, for example, SR-B1, CD81 and the tight-junction proteins- claudin and occludin. HCV RNA replication and translation are coupled processes, requiring cooperation of replicase, helicase and other viral proteins with cell-regulatory factors. Virion packaging and release are highly targetable steps, although they require greater in-depth investigation. The HCV-immune response appears to be fairly ineffective, and neutralizing antibodies that inhibit E2-CD81 binding are unable to resolve infection. HCV-transmission through cell-to-cell contact has been implicated in the evolution of chronic infection. In particular, CD81-dependency and the role of other co-factors involved in entry in cell-to-cell infection, as well as virus escape from host-neutralization still require confirmation. To highlight viral and cell mechanisms implicated in HCV-infection, we review here some of the major discoveries that have been made, from virus entry to its release from infected cells, in understanding the HCV host-cell interplay, which may help in defining new molecular targets to provide therapeutic antiviral strategies.

Multifunctional anti-cancer nano-platforms are moving to clinical trials.

Cancer management needs rapid, non-invasive diagnosis and tumour-specific therapeutics which is unfortunately lacking for most cancer types. Novel approaches for cancer management aim at providing customized therapy according to individual diagnoses, determined by gene expression profiling, in particular, targeting highly selective monoclonal antibodies (mAbs) to single cancer cell antigens, in combination with highly cytotoxic drugs, thereby avoiding the unwanted side effects of conventional chemotherapy. Multifunctional nano-vectors that combine new and more powerful drugs and/or probes for diagnostic imaging with tumour surface-specific ligands/antibodies have been developed. These nano-vectors have displayed superior selective anti-tumour activity compared to antibodies or standard anti-cancer drugs/agents alone both in-vitro, and in preclinical and clinical models. Anti-cancer nano-platforms can significantly improve early cancer detection and ameliorate therapeutic strategies. In the immediate future nano-technology may enable the simultaneous early detection and selective inactivation of cancer cells before they develop into full blown tumours.

Cord blood CD133 cells define an OV6-positive population that can be differentiated in vitro into engraftable bipotent hepatic progenitors.

Cell therapy represents the most promising alternative strategy for end-stage liver diseases and hepatic progenitors are the best candidates. We have identified a reservoir of immature hepatic precursors within human cord blood, which can derive engraftable bipotent progenitors. We isolated a stem cell subset CD133+/CD34+/OV6(low) expressing a surface-marker profile consistent with that of fetal liver cells. Upon induction of hepatic commitment by a medium containing cytokines and factors involved in vivo oval-cell activation, a heterogeneous cell population displaying characteristics of functional oval-cell-like bipotent hepatic progenitors was obtained. The cells expressed markers of hepatocytes and cholangiocytes and were highly enriched in OV6, c-Met, c-Kit, and Thy-1. They also displayed liver functional activity as glycogen storage, urea production, albumin secretion, and inducible CyP2B6 activity. When injected into liver-damaged severe-combined immunodeficient mice, induced bipotent hepatic progenitors appropriately engrafted livers of recipient animals, where they formed clusters of human-derived cells expressing human leucocyte antigen-class I, Hep-Par1, and OV6 antigens. Human-specific albumin, alpha-fetoprotein, and cytokeratin 19 were also expressed. In transplanted animals, AST serum levels showed a significative reduction with regard to controls. This human model for in vitro progenitor-cell activation may provide a powerful tool for elucidating the pathways and synergies that regulate this complex process and can represent a valuable source, exploitable for liver cell-based therapies and regenerative medicine.

Liver stem cells and possible clinical applications.

The discovery of several sources of hepatic progenitors in extra-hepatic organs and tissues, both in animal models and in humans, supports opportunities to isolate, grow and expand them in vitro. Microenvironment factors involved in regulating proliferation and commitment of liver cell precursors have been identified and better characterization of liver stem cell pathobiology would greatly improve the understanding of liver differentiation/ regeneration processes, especially those leading to hepatocarcinogenesis. The goal of these researches has been to discover the most available, suitable and easy-to-use pluripotent stem cells (PSC) sources for cell-based therapies in genetic and acquired liver diseases, therapies which to date have required liver transplantation. This report reviews the efforts, so far, to either investigate the presence of PSC in hepatic and extra-hepatic districts or evaluate their capacity to differentiate in vitro and to restore in vivo liver function.

Molecular and cellular determinants of cell-to-cell transmission of HCV in vitro.

It was reported previously that HCV can be transmitted from persistently infected human bone-marrow-derived B-lymphoblastoid cells (TO.FE(HCV)) to human hepatoma cells by cell-to-cell contact. The present study confirms and characterize further such type of HCV infection in vitro. TO.FE(HCV) cells were co-cultured with 2.2.15 hepatoma cells, that are not susceptible to cell-free infection by sera containing HCV of 1b genotype. By this co-cultivation system it was demonstrated that HCV transmission to recipient cells requires de novo virus RNA replication. Several factors may favor HCV-transmission, evidence is provided that TO.FE(HCV) cells were able to select HCV-quasispecies. 5'-UTR and core sequence analysis revealed differences in the HCV-quasispecies composition in serum inoculum and in infected TO.FE B-cells at 4 months post-inoculation. It is considered that the latter may be more successful in replicating HCV in vitro and used to express surface molecules which may be involved in cell-to-cell contact. In TO.FE(HCV) cells replicate distinct, or few close related, HCV-variants correlated with those of serum inoculum. Comparative analysis of tetra-spans and integrins expression undertaken by cytofluorimetry displayed higher level of expression for TO.FE cells in comparison to other human bone-marrow-derived B-cell lines. Overall, the observed persistent in vitro HCV replication is mediated by a continuous cell-to-cell reinfection that may be favored by selection of viral variants and expression of molecules involved in cell adhesion. These observations may provide an explanation for the establishment of HCV infection, the occurrence of chronic infection and HCV-related lymphoproliferative diseases.

Stem cells: an alternative to organ transplantation in chronic, degenerative and infectious diseases?

Even in the absence of damage or illness mature animals need billions of new cells every single day of their lives in order to survive and renew circulating blood cells and intestinal and skin lining. This task is accomplished by undifferentiated cells residing in most adult organs. These cells are designated adult stem cells (ASC) since they represent the adult counterpart, present in almost every organ, of the embryonal stem cells (ES) from which the entire human body develops. Scientists first hypothesized the existence of stem cells over a century ago, and haematopoietic stem cells (HSC) have been exploited for the therapy of human diseases for two decades. Other types of stem cells also circulating in the bloodstream have been described. We briefly describe the potential uses of each of these types of cells, including autologous circulating stem cells, for disease therapy and in particular for the possible reversal of liver failure due to chronic hepatitis and/or cirrhosis.

Transmission in vitro of hepatitis C virus from persistently infected human B-cells to hepatoma cells by cell-to-cell contact.

Virus cell-to-cell spread has been reported for many different viruses and may contribute to pathogenesis of viral disease. The role played by cell-to-cell contact in hepatitis C virus (HCV) transmission was studied in vitro by cell co-cultivation experiments. A human lymphoblastoid B-cell line, infected persistently with HCV in vitro (TO.FE(HCV)), was used as HCV donor [Serafino et al., 2003]; recipient cells were the human hepatoma HepG2 cell line. Both cell types were co-cultured for 48 hr to allow the cell-to-cell contacts. The hepatoma HepG2 cells are not permissive to free-virus infection, but they were infected successfully using TO.FE(HCV) cells as source of virus. The kinetics of viral RNA synthesis and the percentage of infected cells were compared in cell-mediated-and cell-free-viral infection. After co-cultivation, a consistent proportion of hepatoma cells replicated HCV and stably expressed viral antigens. Virus produced was infectious as demonstrated by the ability to reinfect fresh B-cells. This cell model shows that permissiveness to HCV infection can be achieved in vitro in non-permissive hepatoma cells by direct cell-to-cell contacts with infected human B-cells. This mechanism of virus spread may also play a pathogenic role in vivo.

Suggested role of the Golgi apparatus and endoplasmic reticulum for crucial sites of hepatitis C virus replication in human lymphoblastoid cells infected in vitro.

Iacovacci et al. [(1997a) Research in Virology 148:147-151] described that the euploid diploid cells, of the normal human bone marrow-derived lymphoblastoid B-cell line TO.FE., are susceptible to hepatitis C virus (HCV) infection and support long term virus production. Transmission electron microscopy described some steps of HCV replication cycle in this in vitro infected cellular system [Serafino et al. (1997) Research in Virology 148:153-159]. In the present study, in order to identify the intracellular sites involved in HCV replication, the ultrastructural changes associated with infection in TO.FE. cells were correlated with the subcellular localisation of structural and nonstructural viral proteins. Transmission electron microscopy and confocal microscopy data indicate that these viral proteins appeared located in the Golgi apparatus and endoplasmic reticulum, suggesting an active involvement of these compartments in viral assembly and morphogenesis. Furthermore, transmission and scanning electron microscopic observations on cultures infected chronically support the hypothesis that these cellular compartments may serve as starting sites of the morphological changes associated to viral infection and replication, leading to cell-cell fusion, syncytia formation, and finally lysis of infected cells and virus release.

Low cell dosage of lymphoblastoid human cell lines EBV(+) is associated to chronic hepatitis in a minority of inoculated Nu/Nu mice.

It has been suggested that an atypical course of primary infection by EBV and the reactivation of EBV infection in transplanted patients may induce hepatitis. We explored the possibility to dissect the infectious activity from the ability to promote B lymphocyte proliferation in vivo by injecting in nu/nu mice a low number (2 x 10(6)-0.05 x 10(6)) of cells from CE a normal human bone marrow-derived B cell line. This line carries an endogenous EBV in episomal and linear forms. Twenty nu/nu mice were inoculated subcutaneously with the B cell line CE and a matched group with the cell line RAG obtained by EBV in vitro infection of normal human peripheral blood. The mice injected with the CE line did not develop a lymphoproliferative disease, but 5 of them displayed typical histopathological lesions of chronic hepatitis without involvement of other organs. Similar results were obtained in 2 out of 20 animals in the RAG group. A close association between liver lesions and a previous EBV infection, by putative circulating B lymphoblastoid cells releasing their EBV, was established by PCR and by in situ hybridization with BamHI "W" DNA probe. This latter probe detected the presence of about 15% of positive cells only in affected livers. In addition, the rare detection in some hepatocytes of "A" type Cowdry bodies would suggest the occurrence of continuous EBV replication although at a very low level. These data show that we succeeded in dissecting the infectious from the proliferative activity of the endogenous EBV carrier CE cell line. This provides in addition a promising model for chronic EBV-associated hepatitis.