14.- CONCLUSIONES DEL ESTUDIO

Replication of HCV in extrahepatic sites has been under research for more than a decade and as discussed in previous section, one of the

125 compartments thought to house HCV is the PBMCs. However, due to the low level viral replication this remains a controversial issue. Monocytes and macrophages are known to house many replicating viruses. They are permissive to a wide range of viruses including other flaviviruses (Mogensen 1979). Monocytes/Macrophages are thought to harbour sanctuary sites for HCV viral persistence even after treatment with standard HCV antiviral therapy and may act as Trojan horses for ' occult' HCV infection. They might have a role in the relapse after treatment and re-infection of the graft after transplantation.

'Occult' HCV infection is described as the presence of HCV RNA in the liver tissue and PBMCs (detected in 70% of the cases with occult infection), after clearance of HCV RNA and anti-HCV antibodies from the serum of an individual in response to anti-HCV therapy. Occult infection can be identified in 87% cases after combining the results of HCV RNA detected in PBMCs and ultracentrifuge serum samples. This could be a step towards detecting occult infection without the need for liver biopsy (Bartolome, Lopez-Alcorocho et al. 2007). Infection in PBMCs has also been reported to have a link with the presence of anti-HCV antibodies and normal ALT levels years after resolution of infection (Pham, MacParland et al. 2004, Radkowski, Gallegos- Orozco et al. 2005, Radkowski, Horban et al. 2005).

Replicating forms of HCV RNA have also been reported in the monocytes/macrophages from HCV and HIV co-infected individuals (Laskus, Radkowski et al. 2000). Naïve human monocytes/macrophages are reported to be susceptible to infection with HCV in tissue culture and it has been

126 shown that the concomitant HIV infection facilitates the HCV infection in vitro (Laskus, Radkowski et al. 2004). Numerous studies have shown evidence of active HCV RNA replication in HIV and HCV co-infected patients (Laskus, Operskalski et al. 2007, Laskus, Radkowski et al. 2004, Laskus, Radkowski et al. 1998, Laskus, Radkowski et al. 2000).Human immunodeficiency virus has been shown to facilitate HCV replication in monocytes/macrophages of the host and this may help to explain why mothers co-infected with HIV and HCV are more prone to transmit HCV infection to their off springs (Granovsky, Minkoff et al. 1998, Thomas, Newell et al. 1998). Furthermore, it is thought that the viral dormancy in PBMCs, even after treatment induced clearance of HCV from serum, can lead to the reactivation of infection especially under conditions of immune suppression and this might have a role in the extrahepatic manifestations of HCV infection (Ferri, Monti et al. 1993). Various studies have confirmed the relationship between the viral relapse and the presence of replicative intermediate in the extrahepatic sites especially PBMCs (Xu, Xie et al. 2005, Majda-Stanislawska, Bednarek et al. 2006, de Felipe, Leal et al. 2009). However, testing extrahepatic cells for HCV replication is not done routinely in order to assess the treatment outcome. Moreover, it is not known whether HCV negative strands in PBMCs effects the treatment response.

HCV infection in monocytes was first reported in the monocytes/macrophages of HIV and HCV co-infected patients (Laskus, Radkowski et al. 2000). Laskus et al. found that two out of seven cultures of purified monocytes/macrophages had negative strands isolated for 21 days. Radkowski et al. isolated fresh macrophages from healthy subjects and

127 subjected them to the sera of HCV positive patients. 16 out of 25 cultures produced HCV negative strands for 14-21 days. SSCP and sequence analysis of the 5'UTR region showed that 4 cultures had different sequence variants compared to the initial sera. Moreover, the infected cultures had larger quantities of IL-8 and TNF-α compared to the uninfected cultures (Radkowski, Bednarska et al. 2004). The same group then looked at the infection of healthy monocyte/macrophages first with HIV and later with HCV. It was found that all cultures which were infected with HIV showed HCV replication where as HCV infected cultures showed infection only in 50%, concluding that HIV might facilitate HCV infection in macrophages (Laskus, Radkowski et al. 2004). Similarly, studies with immature- and mature- dendritic cells showed positive strands for 10 and 5 days respectively while negative strand was seen for 1-2 days in both types (Navas, Fuchs et al. 2002).

The JFH-1 genome from a patient with fulminant hepatitis contains a virulent strain of HCV and is able to produce infection in Huh 7.5 cells; however studies have failed to demonstrate the infection of in vitro monocytes, macrophages, DC cells, B-cells and T-cells through JFH-1 (Marukian, Jones et al. 2008). Though able to infect a permissive cell line (Huh 7.5 cells), this could be one of the setbacks of the in vitro derived HCV which may not mimic the HCV infection of PBMCs in vivo. Also Huh 7.5 cells are hepatoma derived cells with defective HFE protein (human haemochromatosis protein) (Vecchi, Montosi et al. 2010) and RIG-I (RNA helicases) (Sumpter, Loo et al. 2005). Therefore, synthetic models should be approached with caution as they

128 might not truly replicate in vivo results. Furthermore, the unusual genome (JFH-1) could behave differently to typical HCV infections.

Caussin-Schwemling et al. infected monocytes/macrophages obtained from healthy donors by subjecting them to sera of genotype 1b positive HCV patients. Low level viral replication was seen on RT PCR. Sequence analysis of HVR1 showed distinct variant tropism to monocytes/macrophages (Caussin-Schwemling, Schmitt et al. 2001).

Cultures containing HCV infection within PBMCs have been produced by many investigators, however HCV levels start dropping after 3 weeks which is most likely due to the maturation of macrophages after 2 weeks of culturing, thus releasing cytokines that inhibit other cell types (Salahuddin, Markham et al. 1982). Revie et al. developed a culture system by infecting macrophages obtained from umbilical cord blood with the plasma or serum of HCV infected individuals and its supernatant was used to infect other cell types such as B and T cells, lymphoid cells and neuronal cells (Revie, Braich et al. 2005). It has been speculated that macrophages might modify the HCV virus by up-regulating certain factors which promote its infection or select variants which could be replicated in PBMCs. As infection of Huh 7.5s has not been carried out through this system, it is not known whether the HCV variants selected through macrophages would infect hepatocytes. In another series from the same group it was reported that the cord blood macrophages were not susceptible to infection by genotype 3 strain of HCV and it was presumed that these macrophages might preferentially select genotype 1 HCV (Revie, Alberti et al. 2010). It is not clear how HCV infects monocytes

129 and macrophages and whether HCV uses the Fc receptor to internalise into macrophages or not. Few in vitro studies with macrophages suggest that genotype 1 HCV might preferentially proliferate in macrophages (Revie and Salahuddin 2011) and it is suggested that this may explain the resistance of this genotype to interferon based therapies.

Coquillard et al. used simultaneous ultra-sensitive subpopulation staining/hybridization in situ (SUSHI) to assess HCV infection in different subsets of monocytes acquired from HCV infected individuals, with and without HIV co-infection. It was determined that CD14++/CD16++ and CD14+/CD16++ monocytes showed HCV infection while CD14++/CD16- monocytes did not. This selective monocyte tropism was analysed further and it was found that permissive subsets had up-regulation of CD81 receptors on their surfaces. Nonetheless, this study only looked at infection from genotype 1 strain of HCV and the findings cannot be reciprocated with other genotypes (Coquillard and Patterson 2009).

In summary, the work to-date on HCV entry and replication in PBMCs is inconclusive. There is a potential for artefacts in the studies of PBMCs' compartmentalisation due to sampling error induced by low copy numbers and cloning or SSCP approaches. However the overall weight of the evidence is for some entry of HCV into PBMCs and, on balance, it seems probable that low level viral replication does take place within some of the cells. Replication may be enhanced by the presence of HIV.

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