BACKGROUND:
In 1991-92 a
large collaborative post-mortem study of adults and children was
undertaken in the West African city of Abidjan, Cote d'lvoire, in order to
document the impact of human immunodeficiency virus (HIV) infection on the
already high mortality from infectious disease. As part of this original study,
brain tissue preserved in paraffin wax blocks was retained from 78 HIV
positive children (76 HIV-1 and 2 HIV-2) and 77 age-matched HIV-negative
children. The baseline neuropathological findings were published and the
material was stored as an archive at the Western General Hospital, Edinburgh.
Further study of this unique collection of cases was clearly warranted.HYPOTHESES AND AIMS OF THE PRESENT STUDY:
In the Initial Study, an attempt was made to quantify white matter cell
numbers using a small sample of the whole cohort. The hypothesis was that it
would be possible to obtain reproducible numbers for total cell counts in the
white matter of HIV negative children and that the numbers were likely to be
significantly higher in HIV positive children. The Main Study focussed on the
extent of inflammatory change in HIV negative and positive cases, with the
prediction that HIV positive children would show a higher level of
inflammatory infiltrate than HIV negative children, despite the high level of
background brain pathology in the latter group, and that this would be
associated with evidence of greater white matter damage in the HIV positive
cases. Lastly in the Apolipoprotein E (APOE) Study, it was predicted that the
inflammatory response, particularly the degree of microglial activation would
be influenced by the APOE genotype, being most evident in children
possessing one or more APOE e4 alleles.MATERIALS AND METHODS:
For the Initial Study, eight age-matched HIV negative and positive cases were
selected from the African cohort, together with an additional HIV negative
case at either end of the age spectrum. Four regions of the brain were selected
from each of these 10 cases, including cerebral convexity, hippocampus, basal
ganglia and the cerebellum. Sections were stained routinely with haematoxylin
and eosin, Luxol fast blue and by immunohistochemistry. Total cell counts
were performed in an area of one mm2, and subsequently for individual cell
types identified using a combination of simple morphology and
immunostaining for glial fibrillary acidic protein (GFAP). Although there is no
immunostain that reliably identifies all of the members of a particular glial
population, whether they be astrocytes, oligodendrocytes or microglia,
separation of these cell types was attempted on the basis of GFAP positivity or
on simple nuclear morphology. The results were tabulated in Excel.In the Main Study 40 further cases from the African cohort were examined.
These included 20 HIV negative and 20 positive, chosen to represent three age
groups, lower, middle and upper, up to the age of six years. The basal ganglia
and the hippocampus were selected for examination in these cases. In about
half of these cases, significant pathology other than HIV-associated changes
was present and the spectrum of CNS disease included malaria, meningitis and
non-HIV encephalitis. Sections were stained routinely with haematoxylin and
eosin, Luxol fast blue and by immunohistochemistry for inflammatory cell
markers (CD 14, CD 16 and CD68) or lymphocyte markers (CD8 and CD20).
Sections were examined by routine microscopy, and by simple quantitation or
image analysis.In the APOE study genotyping was undertaken first on the forty cases used
in the main study. Twenty further cases were added subsequently, 10 HIV
positive and 10 negative, and within the same age range as those in the main
study. DNA was extracted from paraffin sections according to a standard
protocol and amplified using polymerase chain reaction (PCR). The degree of
inflammation, as detected by CD 16 and CD68 image analysis in these cases,
was correlated with the different APOE genotypes.
All cases used in this study were anonymised. Ethical permission was sought
and obtained for this study (LREC/2003/6/6).RESULTS:
The results of the Initial Study showed that the total cell numbers varied
from case to case in the HIV negative group, and from one brain region to
another, but that these differences were not statistically significant. The total
cell counts also varied between individual HIV positive cases and these
differences were not statistically significant either. However, comparison ofthe
means for HIV negative cases with those of the HIV positive cases revealed
significant increases in the latter group. Despite the obvious limitations of the
morphological approach to individual cell typing, subset counts from the
cerebral convexity, basal ganglia, hippocampus and cerebellum, putatively of
astrocytes, oligodendrocytes, microglia and endothelial cells, showed
significantly higher levels in the HIV positive cases.In the Main Study, changes in the white matter assessed by routine and
Luxol fast blue staining, and by immunohistochemistry for myelin basic
protein (MBP), P-amyloid precursor protein (BAPP) and GFAP, and graded
according to a simple system, showed only limited differences between HIV
positive and negative groups. A general increase in activation of
macrophage/microglial cells and of lymphocyte numbers was found in both
basal ganglia and hippocampus in the HIV positive group. This increase in the
HIV positive group was detected despite a reasonable balance of background
brain pathology between the HIV positive and negative subsets. In the case of
CD68 immunostaining, the increase was statistically significant for the grey
(p=0.002) and white (p=0.009) matter of the basal ganglia. For counts of CD8
positive perivascular cells, the increase was also significant in grey (p=0.001)
and white matter (p=0.000) of the basal ganglia, and in grey (p=0.002) and
white (p=0.000) matter of the hippocampus.In the APOE study, DNA extraction was successful in 47 cases in total. The
distribution of APOE s3 and s4 alleles in this group proved in accordance with
the known West African distributions where the s4 allele approaches a
frequency of up to 29%. In this study, though the case numbers are small, the
frequency of the e4 allele was 41% in HIV negative children and 23% in HIV
positive children (p=0.01). These rates were significantly higher than in most
Caucasian populations (p=0.001). Satisfactory immunostaining for
inflammatory markers was achieved in 44 of the genotyped cases. Correlations
with the degree of neuroinflammation in grey and white matter of the basal
ganglia were confined to APOE s3/e3 and APOE e3/e4 cases because of the
small numbers of cases with rarer APOE allelic patterns. For HIV negative
children, there was little difference for CD 16 or CD68 staining between APOE
e3/e3 and APOE e3/e4 cases, either in white or grey matter. For HIV positive
children, subjects with e3/e4 genotypes did show a trend for higher levels of
CD68 activation than those with 3/3 genotypes, particularly in the basal
ganglia white matter, but failed to reach significance (p=0.07 for white matter
and p=0.1 for grey matter).CONCLUSIONS:
1. Although the results of the initial study are limited in their possible
application to any studies other than in this cohort, the increases in total
and individual cell counts in the HIV positive group are intriguing. The
limitations of a study based on morphological assessment are
recognised.
2. In the main study the failure to demonstrate significant white matter
change in either HIV negative or positive groups may mean that there is
none, but wider sampling in each case and examination of a larger
number of cases would increase confidence in this result. It is noted
that incomplete myelination in these young subjects is a possible
confounding factor in this study. Inflammatory changes, both innate
and acquired, proved to be significantly greater in both grey and white
matter of HIV positive compared with negative cases, there being a
relative balance of co-existing disease in the two groups. This study
shows, as predicted, that HIV infection is accompanied by significant
microglial activation in these West African children, and that this
occurs even in the absence of productive viral infection in the brain.
This finding is in keeping with what is known in Western paediatric
populations infected with HIV.
3. APOE genotyping revealed a distinctive allele distribution especially in
respect of the s4 allele, which was found to be twice as common in this
African cohort as in the Scottish population. Although there was trend
for greater microglial activation in APOE c4 HIV positive children, the
differences between these and APOE e3 HIV positive children did not
reach significance
