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Human immunodeficiency virus infection of dorsal root ganglion neurons detected by polymerase chain reaction in situ hybridization.

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Human Immunodeficiency
Virus Infection of Dorsal
Root Ganglion Neurons
Detected by Polymerase
Chain Reaction In Situ
Hybridization
Thomas H. Brannagan 111, MD,* Gerard J. Nuovo, MD,$
Arthur P. Hays, MD,t and Norman Latov, MD, PhD*
A predominantly sensory peripheral neuropathy is common with human immunodeficiency virus (HIV) infection, but the cause is unknown. Formalin-fixed dorsal
root ganglia (DRG), obtained at postmortem from patients with neuropathy and HIV infection and from
control subjects, were examined for the presence of
HIV DNA by using polymerase chain reaction (PCR)amplified in situ hybridization. Viral message RNA was
detected using reverse transcription in situ PCR with
gag-specific primers. HIV DNA and RNA sequences were
detected in many satellite cells, mononuclear cells, and
occasional neurons in 5 of 5 patients with HJY and neuropathy. HIV DNA was detected only in rare interstitial
and satellite cells from 3 of 4 patients with HIV infection
without neuropathy and was not detected in 6 patients
without HIV infection. HIV infection of DRG neurons
and supporting cells may contribute to the HIVassociated sensory neuropathy.
Brannagan TH 111, Nuovo GJ, Hays AP,
Latov N. Human immunodeficiency virus
infection of dorsal root ganglion neurons detected
by polymerase chain reaction in sku
hybridization. Ann Neurol 1997;42:368 -372
A predominantly sensory neuropathy is common with
human immunodeficiency virus (HIV) infection, but
the pathogenesis is unknown [I]. HIV can infect ganglion neural cells in vitro [2] and gpI2O can bind to
dorsal root ganglion (DRG) neurons [ 3 ] . However,
previous studies of DRG in patients with HIVassociared peripheral neuropathy, using immunohistochemistry and in situ hybridization, have demonstrated
HIV infection of only macrophages [4, 51. We inves-
From the Departments of *Neurology and tNeuropathology, Columbia University College of Physicians and Surgeons, New York;
and $MGN Medical Research Laboratory, Setauket, NY.
Received Jan 21, 1997. Accepted for publication Mar 3, 1997.
Address correspondence to Dr Brannagan, Department of Neurology, Allegheny University, Broad and Vine Streets, Philadelphia, PA
19102.
368
Copyrighr 0 1997 by the American Neurological Association
tigated whether the more sensitive methods of polymerase chain reaction (PCR)-amplified in situ hybridization or reverse transcription (RT) in situ hybridization would reveal the presence of HIV infection of
DRG neurons in patients with peripheral neuropathy.
Materials and Methods
Lumbosacral DRG fixed for 10 days in 10% buffered formalin and paraffin embedded were obtained. Five patients
with acquired immunodeficiency syndrome (AIDS) and peripheral neuropathy and 4 patients with AIDS without clinical neuropathy who came to autopsy were invescigaced. Autopsy tissue from 6 patients without known HIV infection
were used as controls. They were assessed blindly by PCRamplified in situ hybridization and RT in situ PCR for HIV
infection.
PCR In Situ Hybridization
Our procedure for detection of PCR-amplified HIV-1 DNA
has been previously described [6, 71. In brief, 4-pm sections
are placed on silane-coated slides. Hot-start PCR is performed using a solucion, containing PCR buffer (GeneAmp
kit, Perkin-Elmer, Nonvalk, CT), 4.5 mM MgCI,, 200 FM
dNTPs, 1.0 FM concentration of the primers, 1 mg/ml bovine serum albumin, and 2.5 U/25 ~1 Taq DNA polymerase. The primers SK38 and SK39 (nucleotides 1,541-1,578
and 1,665-1,638) and SK145 and SK431 (nucleotides
1,369-1,395 and 1,507-1,481) are used with the probes
SK19 and SK102, respectively. After an initial denaturing
step of 94°C for 3 minutes, 35 cycles are completed by using
the following protocol: annealing/extension at 55°C for 2
minutes and denaturing at 94°C for 1 minute.
After PCR, the digoxigenin-labeled probe (SKI 9 for
SK38/39 and SK102 for SK145/431) at 50 nglml in a solution of 10% forrnamide, 5% dextran sulfate, and 300 m M
NaC1) and amplified DNA are simultaneously denatured by
heating to 100°C for 5 minutes. After the hybridization and
wash steps, the slides are incubated in the anti-digoxigeninalkaline phosphatase conjugate (1:50 dilution) at 37°C for
30 minutes and the probe/target complex detected by incubation in the chromogen NBTIBCIP (nitro blue tetrazolium/
5-bromo-4-chloro-3-indoyl phosphate) (Oncor, Gaithersburg,
MD). A hybridization signal is evident as a dark blue precipitate, whereas the counterstain, nuclear fast red, stains nuclei and cytoplasm pale pink.
Reverse Transcription In Situ PCR
Procedural modifications for RT in situ PCR include, after
protease digestion, incubating tissue overnight in a KNasefree DNase solution (Boehringer Mannheim, 10 units per
section) at 37"C, which eliminates nonspecific DNA repair
and mispriming; the DNase digestion permits direct incorporation of digoxigenin dUTP (10 pM) into the PCR product. The RT and PCR steps are performed in one reaction
mixture by the use of rTth, as previously described [8]. Viral
RNA detection is performed with the gag-specific primers
listed above. Controls for viral nucleic acid detection include
pooled lymphocytes infected with HIV- 1 or sham-infected
and lymph nodes from HIV-1-seropositive and seronegative
patients (kindly provided by Dr Roy Steigbigel, Stony
Brook, NY). An additional negative control for each case was
the use of HPV (human papillomavirus)-specific primers.
Results
The results of PCR in situ hybridization for HIV
DNA and of RT in situ PCR for gag protein RNA are
presented in the Table. HIV DNA was found in DRG
neurons in 5 of 5 patients with peripheral neuropathy
(Fig A), but not in neurons in 4 patients with AIDS
and equivalent CD4 counts without clinical manifestations of neuropathy (Fig D). Another indication of the
specificity of the results is that serial sections showed
identical findings for HIV DNA, using two different
primer pairs (SK38/39 and SK145/431) and analogous
probes (SK19 and SK102). HIV DNA was also not
seen in 6 patients without known HIV infection, and
no signal was found using human papillomavirus-16
primers or omitting magnesium, HIV-I primers, or
Taq polymerase (Fig C) in any of the patients.
Serial sections for Patients I to 5 , examined by RTPCR for gag RNA, revealed RNA signal in the same
cells that contained HIV-1 DNA. These included neurons, mononuclear cells, and satellite cells.
Patient 6 had rare mononuclear cells with HIV RNA
corresponding to the rare DNA-positive cells. Patients
7 to 9 had absent HIV RNA. Patient 10, without
known HIV infection and without detectable HIV
DNA, had rare mononuclear cells with positive signal
for HIV RNA, for unclear reasons. The remaining
HIV-negative control patients 11 to I 5 had no detectable HIV RNA signal.
The large nerve fascicles seen in the DRG sections
were always negative for HIV DNA and RNA (Fig B),
even in the presence of many surrounding HIVpositive cells in the DRG region.
Discussion
The cause of the predominantly sensory axonal neuropathy associated with HIV infection is unknown, although in some cases it may be caused by vitamin B,,
deficiency, drug toxicity, or other infections including
CMV (cytomegalovirus) and HTLV-I1 (human T-cell
lymphotropic virus type 11) [ 1, 91. Pathological studies
demonstrate nodules of Nageotte and rostra1 gracile
tract degeneration, which support that the DRG may
be the primary site of damage for the HIV-associated
predominantly sensory neuropathy [5, 101. The lack of
regenerating nerve fibers [I] is also compatible with
primary degeneration of nerve cell bodies. The bloodnerve barrier is relatively permeable at the sire of the
DRG compared with the peripheral nerve, which may
allow entry of HIV-I. In previous studies using immunohistochemistry and in situ hybridization, HIV has
been detected in DRG tissue in macrophages, but not
in neurons [4, 51. In these studies, low levels of virus
Brief Communication: Brannagan et al: HIV Infection of DRG Neurons
369
Table. PCR In Situ Hybridization and RT In Situ PCR for HIV in DRG
HIV DNA
Patient
No.
Clinical
1
AIDS,
neuropathy
2
AIDS,
neuropath y
3
AIDS,
neuropathy
4
6
AIDS,
neuropathy
AIDS,
neuropathy
AIDS
7
AIDS
8
AIDS
9
AIDS
5
10
11
12
13
14
15
Pathology
Satellite Cells
Interstitial Cells HIV RNA
1/48 (2%)
16/94 (17%)
lOill0 (9%)
gag message in same pattern as
DNA in serial sections
13/55 (24%)
27/90 (30%)
14/95 (15%)
gag message in same pattern as
DNA in serial sections
1/49 (2%)
12/112 (11%)
6/88 (7%)
gag message in same pattern as
DNA in serial sections
4/37 (1 1%)
151103 (15%)
12/84 (14%)
1/47 (2"/0)
5/89 (6%)
2/71 (3%)
0
4/78 (5%)
2/79 (3%)
0
3/80 (4%)
3/77 (4%)
gag message in same pattern as
DNA in serial sections
gag message in rare interstitial
cells
gag message, occasional interstitial cells
None
Neurons
Ganglionitis
lymphocytes
and plasma
cells
1 focus of inflammation
with plasma
cells
Inflammatory
infiltrates,
iioduies of
Nageotte,
CMV inclusions
Normal
Nodules of
Nageotte
Normal
Rare nodules of
Nageo tte
1 focus of lymphocytic inflammation,
rare nodule
of Nageotte
Numerous
nodules of
Nageotte
Normal
Adenocarcinoma of
colon
Cardiac arrest Normal
Acute lymNormal
phocytic
leukemia
Minimal
Werdnignodules of
Hoffmann
Nageotte
Normal
Multisystem
atrophy
Normal
ALS
0
0
0
1/72 (1%)
0
1/69 (1%)
None
None
0
0
0
gag message, rare mononuclear
cells
0
0
0
0
0
0
None
None
0
0
0
None
0
0
0
None
0
0
0
None
Cell counts were done in one or two representative sections at ZOOX. 0 indicates that no signal was seen in 50 to 100 cells.
PCR = polymerase chain reaction; RT = reverse transcription; HIV = human immunodeficiency virus; DRG = dorsal root gangliodganglia;
AIDS = acquired immunodeficiency syndrome; CMV = cytomegalovirus; ALS = amyotrophic lateral sclerosis.
below the threshold of 10 to 20 copies/cell may have
been missed [6].
Our studies indicate that HIV-1 is present in DRG
neurons and surrounding satellite cells in patients with
peripheral neuropathy. The infection is productive
with the presence of viral RNA corresponding to the
gag protein. These results parallel recent studies demonstrating productive HIV infection of neurons in the
brain in HIV-associated dementia that could not be
370 Annals of Neurology
Vol 42
No 3
September 1997
detected without using PCR-amplified techniques [7,
1 1, 121. Previous studies have focused on HIV-infected
macrophages and neurotoxic products produced by activated macrophages [l]. Some of these same products
such as tumor necrosis factor-a and interleukin-1 p can
also activate HIV viral production of latently infected
neural cells [13, 141. The HIV viral protein tat, which
can be secreted from infected macrophages, can play a
similar role [15].
D
C
Fig. Molecular analyses of dorsal root ganglia. In situ polymerase chain reaction (PCR)-amplified human immunodeficiency virus
type 1 (HIV-1) DNA was detected in many cells in the dorsal root ganglia in a person who had a sensory neuropathy (A). Many
satellite cells and occasional neurons were viral infcted. The signal was not evident in the adjoining nerve trunk (B). The signal
was lost .f the HIV-1 primers, magnesium, or Tag polymerase was omitted (C).PCR in situ hybridization f . r the provirus revealed
rare positive satellite cells in the dorsal root ganglia in a person with acquired immunodeficiency syndrome without clinical peripheral neuropathy (0).
Our studies do not prove that HIV infection of
DRG neurons results in neuropathy. The correlation,
however, between clinical neuropathy and the presence
of infection in the DRG, suggests that HIV infection
of DRG neurons and adjacent cells may contribute to
the associated neuropathy.
Presented in part in abstract form at the 121st Annual Meeting of
the American Neurological Association, October 13-16, 1396, Miami, FL.
References
1. Griffin JW, Wesselingh SL, Griffin DE, et al. Peripheral nerve
disorders in HIV infection. In: Price RW, Periy SW, eds. HIV,
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sensory ganglion neurons. Ann Neurol 1993;34:855-863
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Brief Communication: Brannagan e t al: HIV Infection o f DRG Neurons
371
7. Nuovo GJ, Alfieri ML. AIDS dementia is associated wirh massive, activated HIV-1 infection and concomitant expression of
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Factor V Leiden Mutation:
An Unrecognized Cause of
Hemiplegic Cerebral Palsy,
Neonatal Stroke, and
Placental Thrombosis
Olafur 'Thorarensen, MD,' Stephen Ryan, MD,"
Jill Hunter, M D , t and Donald P. Younkin, MD*
~
Thorarensen 0, Ryan S, Hunter 1, Younkin DP.
Factor V Leiden mutation: an unrecognized cause
of hemiplegic cerebral palsy, neonatal
stroke, and placental thrombosis.
Ann Neurol 1997;42:372-375
Activated protein C (APC) resistance due to factor V
Leiden mutation is the most common cause of familial
thrombosis [I]. It has a prevalence of 2 to 7% [2-41, is
found in all ethnic groups studied to date, and is 10
times as common as protein C, protein S, and antithrombin 111 deficiencies [3]. Although APC resistance
is associated with thromboembolic disease [4] including stroke in childhood and young adults [5-71, its association with hemiplegic cerebral palsy or placental
thrombosis has not been reported. We report 3 babies
who are heterozygous for factor V Leiden mutation
and had neonatal cerebrovascular disorders, including
ischemic infarction and hemorrhagic stroke. Multiple
placental thrombi were present on the fetal side in one
of these infants. Recent studies report that 40% of cerebral palsy (CP) is related to vascular factors, including infarction and hemorrhage [S]. We suspect that
factor V Leiden mutation may be an important cause
of neonatal stroke, CP, and placental thrombosis.
Case Reports
Case 1
A male infant presented at 6 months of age with right hemiplegia, which was first observed a t 3 months. The last week
~~
Activated protein C resistance caused by an Arg506Gln
mutation in the factor V gene (factor V Leiden mutation)
is the most common cause of familial thrombosis. This
From rhe Divisions of *Neurology and tNeuroradioloLy, Children's
Hospital of Philadelphia, and Departments of *Neurology, *Pediatrics, and tRadiology, University of Pennsylvania, Philadelphia, PA.
Received Jan 2, 1397, and in revised form Mar
publication Mar 10, 1997.
4.Accepted for
Address correspondencc to Dr Younkin, Division of Neurology, 6th
Floor, Wood Bldg. Children's Hospital of Philadelphia, 34th St and
Civic Center Blvd, Philadelphia, PA 19104.
372
mutation is associated with arterial and venous thromboembolic disease in neonates, infants, and children, but is
not a significant risk factor for ischemic stroke in adults.
We report on 3 babies with different neonatal cerebrovascular disorders including ischemic infarction and hemorrhagic stroke who are heterozygous for factor V Leiden
mutation. One infant had multiple thrombi in the fetal
placental vasculature. This is the first reported association between hemiplegic cerebral palsy, placental thrombosis, and factor V Leiden mutation. We suspect that activated protein C resistance may be an important cause of
in utero cerebrovascular disease and hemiplegic cerebral
Palsy.
Copyrighr
of the pregnancy was complicated by roxemia, which was
treated with magnesium sulfate. He was born at term by
spontaneous vaginal delivery to a 30-year-old G2P1 (two
pregnancies, one viable birth) mother. Birth weight was 7 Ib
12 oz. Apgar score was 8 and 8 after 1 and 5 minutes. O n
the firsr day of life, he had mild respiratory problems, possibly related to magnesium-induced weakness, and required
supplemental oxygen. He did not have other neurologic
problems in the nursery and was discharged on day 3 of life.
Early development was normal, but by 3 months he had a
left-hand preference. His father had a history of deep vein
thrombosis treated with warfarin. Physical examination revealed right spastic emiparesis with the arm more involved
than the leg, and mild right facial weakness. Magnetic reso-
0 1997 by the American Neurological Association
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detected, immunodeficiency, dorsal, reaction, chains, virus, human, roots, ganglion, hybridization, neurons, infectious, polymerase, situ
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