B Cells in HSVE

Sobel, et al. and Esiri, et al. demonstrated that B cells are relatively sparse and almost entirely confined to the perivascular space in HSVE and in encephalitis cases of different aetiologies (Esiri et al. 1989). B cells mature in the perivascular spaces before migrating to surrounding parenchyma as plasma cells, and play a key role in promoting antibody production. The findings for B cells in HSVE from this study are entirely in keeping with those of Esiri et al. and Sobel et al. (Sobel et al.

1986, Esiri et al. 1989).

4.4.2.4 Inflammatory Cells in HIVE

Although only mild inflammatory infiltrates in HIVE patients were observed in this study, there was a consistent pattern of an inflammatory cell response in areas linked to myelin loss. This pattern was characterised by large infiltrations of macrophages and microglia within the parenchyma of white matter. Small infiltrates of T cells in the perivascular space of the blood vessels and no discernible CD4+ cells and B cells. T cell response is mild in HIVE due to phagocytosis of HIV infected lymphocytes by macrophages/microglia. Interestingly, the giant cells positively stained with CD163 marker.

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These findings correlate well with the pathogenesis of HIVE, in which the HIV productively infects microglia and macrophages. Following the entry to CNS, the main target cells for the virus infection are macrophages and microglia. This explains the presence of numerous microglia and macrophages within the main affected areas for HIVE, particularly in the white matter. Furthermore, the multinucleated giant cells that represented a fusion of mononuclear phagocytes have also been actively infected with HIV. HIVE demonstrates similar observation to HSVE in that both infiltrating macrophages and activated microglia were stained with CD163. Once again, I was unable to discriminate between the cells. Overall, the inflammatory infiltrate, reflecting the pathological damage, was much less severe in HIVE compared to HSVE.

4.5 Conclusion

In this chapter I characterised the inflammatory cell infiltrates in HSVE, comparing with HIVE patients and RTA controls. This was important because host inflammatory cells may contribute to brain cell damage during encephalitis. Given HIV is not known to infect neurones or glial cells. The findings from the HIVE patients suggest inflammatory cells drive myelin loss. In HSVE more inflammatory cells were present in areas of severe tissue damage. I cannot confirm, from these observations, whether inflammatory cells drive neuronal loss or respond to brain cell damage. Nevertheless, given their abundance, inflammatory cells could certainly contribute to the damage. In the current study, M1 and M2 pathway was not considered but in the future it would be interesting to examine macrophage

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activation products that determine M1 and M2 pathway in the pathogenesis of HSVE.

In the next chapter I begin to examine the host response to HSV infection using a genome-wide perspective gene-expression microarrays.

111 CHAPTER 5

COMPARISON OF HOST TRANSCRIPTIONAL RESPONSES IN BRAIN TISSUE FROM HSV ENCEPHALITIS PATIENTS AND CONTROLS

5.1 Introduction

Formalin fixed paraffin embedded (FFPE) post-mortem brain tissue is widely available and routinely archived in many hospitals. In this chapter the FFPE brain tissue from HSVE and non-encephalitic RTA cases were subjected to RNA extraction following their histopathological examination. I aimed to establish the optimum RNA extraction method for FFPE tissue. Then using this method I recovered RNA from FFPE post-mortem human brain tissue from HSVE and RTA patients. Subsequently, I compared genome-wide gene-expression between the HSVE and RTA cases using a microarray approach.

5.1.1 Formalin Fixed Paraffin Embedded (FFPE) Tissue

Fresh frozen tissue is one of the best sources of tissues for nucleic acid and protein recovery (Klopfleisch et al. 2011). Fresh tissue contains good quality RNA for gene expression studies, however fresh frozen samples are not readily available. Following post-mortem, tissue is fixed and archived as FFPE. One of the most commonly used fixatives is formalin, also known as formaldehyde. Formalin is

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cheap, easy to handle, and preserve tissues efficiently by fixation of structural and cytoskeletal proteins. Formalin also prevents tissue autolysis and act as anti- microbicidal by changing highly infectious tissue into non-infectious material (Klopfleisch et al. 2011). FFPE tissue has the following advantages; such tissue is routinely archived, it is widely available from most hospitals, it preserves the morphology and integrity of the tissue for histological examination and for long term storage.

Nevertheless, there are challenges to using FFPE for molecular studies. The amount and quality of nucleic acid recovered from FFPE is less optimal compared to fresh tissue because of extensive cross linking between proteins and nucleic acid following formaldehyde fixation (Park et al. 1996, Farragher et al. 2008). Fixation also causes fragmentation of DNA and RNA in the affected tissue (van Deerlin et al. 2002).

Fragmented RNA is the main barrier to use of FFPE tissue in gene expression studies. The extent of fragmentation varies depending on tissue type and fixative used, but the typical length of fragments for RNA in FFPE is around 200 base pairs (bp) (Lehmann and Kreipe 2001). It is therefore suggested that the amplification of fragments longer than 200 bp should not be attempted due to failure of amplification process in molecular experimental studies (Lehmann and Kreipe 2001, Farragher et al. 2008).

Beside the fixative, there are other parameters that influence the quality and quantity of RNA or DNA retrieved from FFPE tissue. These parameters includes length of time from necropsy or surgical excision to fixation (pre-fixation time),

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fixation conditions, prolonged tissue hypoxia and temperature (Lehmann and Kreipe 2001). Prolonged tissue hypoxia reduces pH in tissue leading to further degradation of nucleic acids (Srinivasan et al. 2002).