Laser Capture Microscopy and Immunostaining Point to Brain Microglia as Cells Harboring Hepatitis C

1
Laser Capture Microscopy and Immunostaining Point
to Brain Microglia as Cells Harboring Hepatitis C
Virus

• Jeffrey Wilkinson, Marek Radkowski, Debra Adair,
  Andrzej Horban, Tomasz Laskus
• Mayo Clinic Scottsdale, USA Medical University
  in Warsaw, AIDS Center and Infectious Diseases
  Hospital, Warsaw, Poland
•  

JR092203
2
HCV Tropism

• HCV is not strictly hepatotropic
• Recent findings point to the presence of
  replication in lymphocytes macrophages

3
HCV Replication Negative StrandHCV RNA

• HCV is strand RNA virus and replicates through
  negative strand intermediate
• The presence of negative strand HCV RNA could be
  regarded as evidence of replication
• In the WIHS cohort of gt1,500 HIV women 39 are
  HCV and 40 of these have evidence of
  replication in PBMC

4
Detection of HCV RNA in PBMC Subsets from 10
HIV-Infected Subjects
Positive Negative
Strand Strand PBMC
10/10 6/10 CD4
10/10 1/10 CD8 10/10
2/10 CD19 10/10
1/10 Monocytes 10/10 4/10
J Virol 2000, 74, 1014 J Inf Dis 2000, 181, 442
5
Analysis of HCV RNA Sequences from Cultured
Macrophages

J Gen Virol 2004, 85, 47-59 Blood 2004, 103,
3854-59
6

7
Can HCV Affect the CNS?

• Fatigue and depression (Foster et al. 1999,
  Singh et al. 1999)
• Mild neurocognitive dysfunction (Forton et al.
  2002, Hilsabeck et al. 2002)
• More cognitive impairment in HCV/HIV coinfection
  than in HIV infection(Ryan et al 2004 Letendre
  et al. 2005 Richardson et al, in press)

8
Analysis by SSCP of 5UTR HCV Sequences from
Brain Samples and LNs
J Virol 76600-608 2002
9
Identification of HCV-positive cells in CNS
Patients

• Autopsy brain tissue from 4 HCV pts (2 HIV)
• Frontal white matter and cerebellum tested

10
Identification of HCV-positive cells in CNS
Methods

• Laser capture microscopy (LCM)
• Staining for various phenotypes detect () and
  () HCV RNA strand
• Staining for viral proteins and determine cell
  phenotype
• Double Immunostaining

11
Staining of 4 basic brain cell types
12
Laser capture microscopy (LCM)
13
Real-time detection of HCV RNA positive and
negative strands in CD68 cells
14
Staining with anti-NS3
15
(No Transcript)
16
Real-time PCR phenotyping of brain cells
(microglia, astrocyte, neuron, oligodendrocyte)
17
Phenotyping of NS3-positive cells by real-time
RT-PCR
18
Double staining for NS3 and CD68
19
Results Summary

• HCV RNA was amplified from CD68 cells obtained
  by LCM from all 4 HCV brains (frontal white
  matter and cerebellum)
• Negative strand HCV RNA was found in CD68 cells
  from 3 patients
• Positive HCV RNA strand, but not negative strand,
  was occasionally found in other cells but at a
  markedly lower level

20
Results Summary

• NS3 cells separated by LCM and phenotyped by
  amplification of cell-specific transcripts were
  identified as being CD68
• Double staining with anti-CD68 and anti-NS3
  pointed again to CD68 cells as the site of HCV
  replication

21
Conclusions

• The brain cells harboring HCV were identified as
  being CD68 (microglia/ macrophage)
• Since the same cells are also infected by HIV,
  there is a potential for local interactions
  between both pathogens
• Our findings could help explain the biological
  basis of neurocognitive abnormalities associated
  with HCV infection

22