Association between human papillomavirus DNA and temporal arteritis

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Association between human papillomavirus DNA and temporal arteritis
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Mohammadi, Amir
Pfeifer, John D
Lewis, James S. Jr.
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Abstract:
Background: To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the temporal artery. Methods: The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR) with INNO-LiPA HPV Genotyping Extra, 2) Cervista™ HPV HR. All cases were from the files of the Barnes-Jewish Hospital and Washington University in St. Louis. Results: HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only three of 15 (20%) were positive for HPV DNA. The second, independent method (CervistaTM) confirmed the aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which it was not possible to perform the test. The differences in HPV positivity between the histologically positive and negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls were both statistically significant (p = 0.001 and 0.002, respectively). Conclusions: The results of our study revealed a statistically significant association between HPV positivity and biopsy confirmed temporal giant cell arteritis GCA (p = 0.001). Further studies are necessary to elucidate the pathophysiology underlying this association. Keywords: Human papillomavirus, Giant cell arteritis, Polymerase chain reaction
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Mohammadi et al. BMC Musculoskeletal Disorders 2012, 13:132
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BMsculoskeletal Disorders
Musculoskeletal Disorders


Association between human papillomavirus DNA

and temporal arteritis

Amir Mohammadi*, John D Pfeifer2 and James S Lewis Jr2'3


Abstract
Background: To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the
temporal artery.
Methods: The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control
groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of
nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR)
with INNO-LiPA HPV Genotyping Extra, 2) Cervistam HPV HR. All cases were from the files of the Barnes-Jewish
Hospital and Washington University in St. Louis.
Results: HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and
in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only
three of 15 (20%) were positive for HPV DNA. The second, independent method (CervistaTM) confirmed the
aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which
it was not possible to perform the test. The differences in HPV positivity between the histologically positive and
negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls
were both statistically significant (p= 0.001 and 0.002, respectively).
Conclusions: The results of our study revealed a statistically significant association between HPV positivity and
biopsy confirmed temporal giant cell arteritis GCA (p= 0.001). Further studies are necessary to elucidate the
pathophysiology underlying this association.
Keywords: Human papillomavirus, Giant cell arteritis, Polymerase chain reaction


Background
Giant cell arteritis is one of the most common causes of
vasculitis involving the temporal artery. Other potential
causes are Wegener granulomatosis, polyarteritis nodosa
(PAN), and Buerger disease [1]. GCA generally affects
individuals over 55 years of age (with a mean age at
diagnosis of approximately 72 years) [2] with an annual
incidence of approximately 18 per 100,000 in persons
aged 50 years or older.
Histologically, GCA shows transmural inflammation
with mixed inflammatory cell infiltrate mostly consisting
of lymphocytes, histiocytes, plasma cells, occasional neu-
trophils and rarely eosinophils (Figure 1). This causes

*Correspondence amir mohammadi@jax ufl edu
Department of Pathology and Laboratory Medicine, University of Florida,
College of Medicine, Jacksonville, FI, USA
Full list of author information is available at the end of the article


destruction of the vessel's internal elastic lamina which
is best demonstrated with elastic stains such as
Verhoeff-Van Gieson (Figure 2) or Movat pentachrome.
The presence of giant cells, next to the elastic lamina in
particular, is the classic and pathognomonic feature of
GCA. However, this is present in only about 50% of
biopsy-proven cases.
Infectious agents have long been considered as a pos-
sible etiology of GCA. The concept is that GCA repre-
sents a chronic inflammatory response, triggered by an
infectious agent, with subsequent inappropriate tissue
response to injury. However, studies looking for organ-
isms have had conflicting results. Some have demon-
strated organism DNA, such as Herpes Simplex Virus,
while the vast majority have failed to demonstrate an as-
sociation. It is speculated that the inflammatory re-
sponse may be triggered by an infectious agent. If a


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distribution, and reproduction in any medium, provided the original work is properly cited.






Mohammadi et al. BMC Musculoskeletal Disorders 2012, 13:132
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Figure 1 Biopsy of temporal artery showing a transmural
mixed inflammatory cell infiltrate with intimal thickening, and
fragmentation and distortion of the internal elastic lamina (H &
E, original magnification x40).


connection between GCA and HPV infection were to be
established, the potential clinical implications are great,
as GCA, with its possible vision loss [3-5], polymyalgia
rheumatica and even eventually ischemic stroke [6-9],
might potentially be prevented by vaccination or other
strategies.
Human papillomavirus (HPV) is an increasingly com-
mon human pathogen in recent decades. It is a mucoso-
tropic virus which is not thought of as spreading
systemically. However, HPV genotype 16 has been found
integrated into the genome of bacteria isolated from cer-
vical cancer biopsies, and there is also published data
showing HPV viral particles within peripheral nerves



.
....















Figure 2 Elastic stains on the same case of histologically positive
temporal artery biopsy as shown in Figure 1, showing the
fragmentation, distortion and lack of continuity of the internal
elastic lamina, a characteristic feature of temporal arteritis
(Verhoeff-Van Gieson staining; original magnification, X40).


and small vascular endothelial cells adjacent to oral and
cervical cancers as demonstrated by transmission elec-
tron microscopy [7,10]. In this study, we sought to de-
termine if there is an association between GCA and
HPV.

Methods
With approval by the Washington University Human
Research Protection Office (HRPO), we searched the
Copath database of Barnes Jewish Hospital for temporal
artery biopsy specimens from 1995 to 2008 and retrieved
all specimens for which material was available. We iden-
tified approximately 60 cases, of which 43, (22 histologi-
cally positive and 21 histologically negative) had material
available for review. There were five males and 17
females in the histologically positive GCA group with a
mean age of 78.9 years. In the histologically negative
group, there were four males and 17 females with a
mean age of 67 years, and the control kidney vascular
margin group had six males and nine females with a
mean age of 38.2 years. There were no statistically sig-
nificant difference in gender among the groups
(p = 0.176). The clinical diagnosis of GCA was based on
the criteria of the American College of Rheumatology
[11]. However, for our study, we considered the gold
standard to be histologic evidence of temporal arteritis.
We also randomly selected 15 renal artery vascular re-
section margins from nephrectomy specimens in
patients without any history of vasculitis for use as the
negative controls.
Two methods were utilized to evaluate for HPV DNA.
The first, INNO-LiPA HPV Genotyping Extra, was used
to perform the testing in our research laboratory of the
division of anatomic and molecular Pathology, Depart-
ment of Pathology and Immunology, Washington Uni-
versity School of Medicine, St. Louis. All blocks were
then submitted to an outside laboratory (CPA Labora-
tory, Louisville, KY) for independent preparation of gen-
omic DNA and for testing employing the CervistaTM
HPV HR (Hologic, Madison, WI, USA) assay.
In both laboratories to avoid potential contamination,
a maximum physical separation between the pre- and
post-amplification steps was used. Separate pipettes and
other lab materials were used as a part of good labora-
tory practice. The FFPE blocks were cut and processed
under strict conditions to prevent DNA from being car-
ried over from one case to the next during microtomy.
Also a new blade was used for each case and the area
was cleaned. Ice cubes used to cool blocks were dis-
carded between cases.
With regards to the technical limitations of this study,
it is well known that formalin fixation randomly frag-
ments DNA in a duration-dependent manner, resulting
in a partial degradation. The degree of fragmentation


Page 2 of 9






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depends on the type and age of the sample and the con-
ditions used for fixation. Due to this degradation, FFPE
tissue is not suitable for amplification of large DNA seg-
ments. Nevertheless, PCR amplification of segments ran-
ging up to 1300 base pair has been reported. On the
other hand, incubation at an elevated temperature after
proteinase K digestion partially removes formalin cross-
linking of the DNA, improving yield as well as DNA
performance in assays. Furthermore, in one of the techni-
ques used in this study (INNO-LiPA HPV Genotyping),
short-PCR-fragment (SPF 10) primers are employed,
which amplify a 65 base pair segment of target DNA and
this testing is considered to be one of the most sensitive
PCR assays for the detection of HPV DNA.

Washington University testing
DNA extraction
DNA was extracted using PureGene Kit (Gentra, http://
www.Gentra.com) as per the manufacturer's instructions
from 10 pm sections cut from the paraffin blocks. The
concentration of the prepped DNA was measured spec-
trophotometrically using Nanodrop. Detailed procedure
information is available at their web site, but briefly, we
placed five 10 micron sections of tissue into a 1.5 mL
microcentrifuge tube and added 1.0 mL of xylene, vor-
texed, and incubated for five minutes with constant gen-
tle mixing. Then we centrifuged it for five minutes at
13,000-16,000 x g. In the fume hood, discarded by pipet-
ting the xylene supernatant and left behind the visible
pellet (tissue). We repeated this xylene wash twice. We
then added 1.0 mL 100% ethanol, vortexed, and incu-
bated five minutes with constant gentle mixing at room
temperature, centrifuged at 13,000-16,000 x g for five
minutes to pellet the tissue, and discarded the ethanol.
We repeated these ethanol washes twice. Subsequently
we added 1.0 mL 70% ethanol, gently mixed, and centri-
fuged at 13,000-16,000 x g for five minutes at 4oC,
removed all residual ethanol and allowed tissue pellet to
dry by centrifugation under vacuum for five minutes.
For cell lysis, we added 300 tl Cell Lysis Solution (Gen-
tra Puregene kit) and gently vortexed for 30 seconds.
Then we added three pl Puregene Proteinase K (20 mg/
ml), and mixed by inverting 25 times and incubated the
lysate at 55oC for three hours to overnight. We inverted
the tube periodically during the incubation. Then was
added three pl RNase A Solution to the cell lysate, and
mixed by inverting the tube 25 times and incubated at
37oC for 15 min to one hour. For Protein Precipitation
we cooled quickly the sample to room temperature by
placing on ice and added 100 pl Protein Precipitation
Solution (Gentra Puregene kit) to the cell lysate. The
volume should be 1/3 of the Cell Lysis Solution in the
tube. Subsequently, it is vortexed vigorously at high
speed for 20 seconds to mix the Protein Precipitation


Page 3 of 9


Solution uniformly with the cell lysate. Then, we centri-
fuged it at 13,000-16,000 x g for five minutes. The preci-
pitated proteins formed a tight pellet. If the protein
pellet was not tight, we vortexed vigorously for 20 sec-
onds at high speed, and then incubated on ice for 5 min.
We then centrifuged at 13,000-16,000 x g for three min-
utes. Using a pipette, we removed the supernatant con-
taining the DNA (leaving behind the precipitated protein
pellet) into a clean 1.5 mL microcentrifuge tube. We
added two tl (for 400 tl supernatant) of a DNA carrier
(glycogen; to final concentration of 50-150 mcg/pl) to
aid recovery of small DNA quantities and then vortexed
them. We then added 400 tl 100% of isopropanol. Sub-
sequently, it was mixed by inverting gently -50 times
until the white threads of DNA formed a visible clump,
and then it was centrifuged at 13,000-16,000 x g for 10
minutes and the supernatant was poured off. We added
500 pl of 70% ethanol and inverted the tube to wash the
DNA pellet. We centrifuged at 13,000-16,000 x g for 5
minutes, and carefully poured off the ethanol and
inverted and blotted the liquid from the tube on clean
absorbent paper and allowed to air dry for 10-15 min-
utes. Finally, we added 50 tl DNA Hydration Solution
and incubated at 65C for one hour to dissolve the
DNA. We incubated at room temperature overnight
with gentle shaking. Samples could then be centrifuged
briefly and transferred to a storage tube. The concentra-
tion of the DNA used for INNO-LiPA HPV Genotyping
Extra testing in each case was 50 ng.

INNO-LiPA HPV Genotyping testing
The INNO-LiPA HPV Genotyping Extra is based on the
principle of reverse hybridization. Part of the LI region
of the human papillomavirus (HPV) genome is amplified
using short-PCR-fragment assay (SPF10 primers), and
the resulting biotinylated amplicons are then denatured
and hybridized with specific oligonucleotide probes.
An additional primer pair for the amplification of the
human HLA-DPB1 gene is added to monitor sample
quality and extraction. The length of the HLA-DPB1
fragment is 280 base pairs. All probes are immobilized
as parallel lines on membrane strips. After hybridization
and stringent washing, streptavidin-conjugated alkaline
phosphatase is added, which binds to any biotinylated
hybrid previously formed.
Incubation with BCIP (5-Bromo-4-Chloro-3'-Indoly-
phosphate p-Toluidine Salt)/NBT (Nitro-Blue Tetrazo-
lium Chloride) chromogen yields a purple precipitate,
and the results are visually interpreted using the refer-
ence guide provided. An amplification kit (INNO-LiPA
HPV Genotyping Extra Amp) is used for standardized
preparation of biotinylated amplified material. This amp-
lification kit is based on the polymerase chain reaction
(PCR) using SPF10 primers.







Mohammadi et al. BMC Musculoskeletal Disorders 2012, 13:132
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Amplification products are subsequently hybridized
using a single typing strip on which 28 sequence-specific
DNA probe lines and 4 control lines are fixed, which
permits specific detection of 28 HPV genotypes, includ-
ing all 18 high-risk genotypes, and 10 low-risk genotypes
(HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44,
45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74,
and 82) as described by the manufacturer (Figure 3).

CPA Laboratory testing
DNA extraction
Slides were cut from the original blocks, and The
QIAamp DNA FFPE Tissue kit (QIAGEN, www.qiagen.
com) was used for purification of genomic DNA from
formalin-fixed, paraffin-embedded tissues according to
the manufacturer's instructions with exception of incu-
bation time. Overnight incubation in proteinase K for di-
gestion of proteins/contaminants is not recommended
by Qiagen but is something that CPA Laboratory has
found useful to increase the nucleic acid elution.
Briefly, the QIAamp DNA FFPE Tissue procedure
consisted of several steps including removal of paraffin
from slides using xylene, subsequent specimen lysis
under denaturing conditions with proteinase K, incuba-
tion at 90C to reverse all formalin cross-linking, and


Page 4 of 9


DNA binding to the membrane for removal of contami-
nants. Residual contaminants were washed away and
pure, concentrated DNA was eluted from the
membrane.


CervistaTM HPV HR testing
CervistaTm HPV HR [12] is a qualitative, diagnostic test
for the detection of DNA from 14 high-risk HPV types
(i.e., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,
66, and 68). The CervistaTm HPV HR test uses the Inva-
der chemistry which is a signal amplification technique
for detection of specific nucleic acid sequences. In this
method two types of isothermal reactions are used: a
primary reaction that occurs on the targeted DNA se-
quence and a secondary reaction that produces a fluor-
escent signal. During the primary reaction, two types of
sequence specific oligonucleotides (i.e. a probe oligo-
nucleotide and an Invader oligonucleotide) bind to the
target DNA recognition site. When those sequence spe-
cific oligonucleotides overlap by at least one base pair
on the target sequence, an invasive structure forms that
acts as a substrate for the Cleavase enzyme. The en-
zyme cleaves the 5' portion (flap) of the probe at the
position of the overlap.


INNO-LiPA HPV Genotyping Extra
Data reporting sheet
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20


"- -" m N -

2r3 .r I I I I I I i 2i






24














correspond with HPV type 16.






Mohammadi et al. BMC Musculoskeletal Disorders 2012, 13:132
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The probes are present in large molar excess and cycle
rapidly on and off the target sequence generating many
cleaved 5' flaps per target sequence. The cleaved flaps
then bind to a universal hairpin fluorescence resonance
energy transfer (FRET) oligonucleotide creating another
invasive structure that the Cleavase enzyme recognizes
as a substrate. The enzyme cleaves the FRET oligonu-
cleotides between the fluorophore and quencher mol-
ecule and produces a fluorescence signal as the cleaved
flaps cycle on and off. For each copy of target, the com-
bined primary and secondary reactions result in 106-
107 fold signal amplification per hour. The reagents for
this test are provided as three oligonucleotide master
mixtures, which identify the 14 types of HPV arranged
according to phylogenetic relatedness. Master mixture 1
identifies the positivity of genotypes 51, 56 and 66
(MM1: HPV 51, 56 and 66). Master mixture 2 (MM2)
shows the positivity of genotypes HPV 18, 39, 45, 59 and
68 and master mixture 3 (MM3) reveals the positivity of
HPV genotypes 16, 31, 33, 35, 52 and 58. By design, the
released 5'-flaps bind only to their respective FRET oli-
gonucleotides to generate target-specific signal. A posi-
tive result indicates that at least one of the 14 high-risk
types is present in the DNA sample. For each case 150
to 200 ng of DNA prepped from the FFPE tissues at
CPA Labs were utilized for the Cervista HPV testing.
The sensitivity of this test is set at 5000 copies of HPV
DNA. The Cervista assay has an internal control to ver-
ify if the assay worked. If the internal control does not
pass, it suggests either the sample is degraded or extrac-
tion failed or the PCR did not work.

Statistics
Statistical analyses were performed using the Chi-Square,
T-test and Fisher's exact tests to evaluate the correlation
between the presence of HPV DNA and other variables.
P values < 0.05 were considered significant. All statistical
analyses were performed using the SAS v.9.1 system soft-
ware (SAS Institute Inc., Cary, NC).

Results
Using the INNO-LiPA HPV Genotyping Extra, 16 of the
22 (17 female and 5 male) biopsy-confirmed GCA cases
were positive for HPV. Twelve out of the 16 (75%) HPV-
positive GCA-positive patients were female. Among
these 16 positive cases, 5 had a HPV 6 alone (31%), 3
cases had HPV 16 alone (19%), 7 cases had both types 6
and 16 (44%), and only one case was extensively "multi-
genotype" showing types 6, 16, 31, 33, and 40 (6%) (Test-
ing results are presented in Tables 1 and 2). In all cases
where HPV was detected, type 6, type 16, or both were
present (100%). The cases were also tested by the Cer-
vistaT HPV HR assay for confirmation, which only
evaluates for high risk HPV types (not for HPVs 6 and


Page 5 of 9


11). Two cases which were HPV type 6 by INNO-LiPA
(cases # 4 and 6) had low genomic DNA when prepped
for the CervistaTM HPV HR testing, so could not be
evaluated by this method. All positive cases of the Cer-
vistaTm HPV HR were from the Master Mix 3 which
includes the HPV genotypes 16, 31, 33, 35, 52 and 58.
As expected, all cases for which genotype 6 was present
in the INNO-LiPA method were negative with the Cer-
vista method since low risk types are not evaluated in
that assay. All negative cases with INNO-LiPA HPV
Genotyping Extra were negative also with CervistaTM
HPV HR testing. As such, there was 100% agreement be-
tween the two testing methods on the histologically
positive temporal arteritis cases.
Among the 21 cases of GCA histologically negative bi-
opsies, 5 were positive for HPV by INNO-LiPA HPV
Genotyping Extra, of which 3 (60%) were genotype 16,
one (20%) was genotype 6 and one (20%) was genotype
52. CervistaTM HPV HR testing revealed positivity of
Master Mix 3 for all 4 cases which were positive by
INNO-LiPA HPV Genotyping Extra for high risk HPV.
Case number 2 which was negative with INNO-LiPA
was of low genomic DNA with Cervista method. As
such, there was 100% agreement between the two testing
methods on the histologically negative temporal artery
biopsy cases.
Among the 15 nephrectomy vascular resection mar-
gins (controls), 3 were positive for HPV by INNO-LiPA
HPV Genotyping Extra. Two cases showed genotype 16
(1 case of Wilms' tumor and 1 of acute and chronic py-
elonephritis), and one genotype 6 (a case of papillary
urothelial carcinoma). CervistaTM HPV HR testing
revealed positivity of Master Mix 3 for both cases which
were positive for high risk HPV by INNO-LiPA HPV
Genotyping Extra. As such, there was 100% agreement
between the two testing methods on the 15 vascular
margin control cases.
The association between HPV positivity and histologi-
cally confirmed GCA was statistically significant
(p = 0.0013). There was also a statistically significant as-
sociation between HPV positivity and Caucasian race
(p =0.0339). No other associations were statistically
significant.
In multivariate analysis (Table 3), subjects with histo-
logically confirmed GCA had a 56-fold higher likelihood
of having HPV positivity adjusted by gender, age and
race (OR, point estimate 56.01; 95% CI 3.5-895.68).

Discussion
Giant cell (temporal) arteritis is a chronic vasculitis in-
volving medium and large size arteries that typically
affects individuals older than 50 years of age.[1,3,4] Al-
though it usually affects the superficial temporal arteries,
it can also affect the aorta, carotid, subclavian, vertebral,







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Table 1 Results of the HPV testing by both different methods.
Histologically INNO LiPA Cervista Histologically Negative
Positive GCA cases Results Results GCA cases
1 Neg Neg 1
2 6 Neg 2
3 Neg Neg 3
4 6 LGD 4
5 6,16,31,33,40 pos MM3 5
6 6 LGD 6
7 6 Neg 7
8 6 Neg 8
9 Neg Neg 9
10 6,16 pos MM3 10
11 16 pos MM3 11
12 6,16 pos MM3 12
13 16 pos MM3 13
14 6,16 pos MM3 14
15 Neg Neg 15
16 Neg Neg 16
17 Neg Neg 17
18 6,16 pos MM3 18
19 6,16 pos MM3 19
20 6,16 pos MM3 20
21 16 pos MM3 21
22 6,16 pos MM3
LGD: Low Genomic DNA.
POS MM3: Positive in Master Mix 3.
HPV= Human papillomavirus.
MM1: HPV types 51, 56 and 66.
MM2: HPV types 18, 39, 45, 59 and 68.
MM3: HPV types 16, 31, 33, 35, 52 and 58.


Page 6 of 9


INNO LiPA
Results
Neg
Neg
Neg
6


and iliac arteries. The classical picture of granulomatous clearly der
inflammation with multinucleated giant cells is observed fibers (Figu
in approximately 50% of the patients. The etiol
Histologically, the disease progresses from minimal has not be
involvement of vessels, with only collections of lym- strated tha
phocytes confined to the internal or external elastic tissue-infilt
lamina or adventitia to a panarteritis with segmental that they ar
areas of necrosis of the arterial wall and extensive de- the affected
struction of the elastic laminae, a feature that can be ease [13].


Table 2 Correlation between the three study groups for HPV test results
Biopsy Positive GCA* Biopsy Negative GCA


PV Positive
SPV Neqative


16 (73%)
6 (27%)


5 (24%)
16 (76%)


Cervista
Results
Neg
LGD
Neg
Neg
Neg
pos MM3
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
pos MM3
Neg
pos MM3
Neg
pos MM3
Neg


Kidney Vascular INNO LiPA Cervista
margin Results Results


Neg
Neg
Neg
Neg
pos MM3
Neg
pos MM3
Neg
Neg
Neg
Neg


nonstrated with special stains for elastic
res 1 and 2).
ogy of the inflammatory reaction in the GCA
en identified. However, it has been demon-
t there is a restricted clonal expansion of
rating T cells in these lesions, which suggests
re reacting to a specific antigen located within
1 arterial wall which is thus eliciting the dis-
The most recent hypothesis regarding the


All Temporal Artery
Biopsies
21(49%)
22 (51%)


Renal Artery
Controls**


Total 22 21 43 15
*Differences between biopsy positive and biopsy negative cases and between biopsy positive and control cases were statistically significant ((p= 0.001 and 0.002,
respectively).
*"Difference between biopsy negative and control cases was not statistically significant (p= 0.79).
GCA= giant cell arteritis; HPV= human papillomavirus.







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Table 3 Odds Ratios for variable and presence of
histologically positive temporal artery biopsies


Variable

Gender F vs. M
Race:
African American vs.
Caucasian
HPV
Age


Point
Estimate
2.762
0.113


56.014
1.196


95% Wald Confidence
Interval
0.329 23.222
0.006 2.199


3.503 895.681
1.053 1.358


HPV= human papillomavirus.


etiology of GCA contends that a response to endothelial
injury (maybe due to infection) leads to an inappropriate
activation of T-cell-mediated immunity [14,15]. The sub-
sequent release of inflammatory mediators within the ar-
terial vessel wall can attract macrophages which then
become multinucleated giant cells, creating the charac-
teristic histology of this disease and also leading to an
oligoclonal expansion of T-cells directed against antigens
within or near the elastic lamina. This cascade of events
in due course results in vessel wall damage, intimal
hyperplasia, and eventual stenotic occlusion.
Infectious agents have been considered in the past as
the etiology of GCA. Elling et al., by indirect serological
evidence, found high incidence of GCA within a popula-
tion associated with Chlamydia pneumoniae, Myco-
plasma pneumoniae, and Parvovirus B19 [16]. Published
data from Wagner et al. [15] and Powers et al. [17]
reported a relationship between GCA, C. pneumonia
and herpes simplex virus (HSV). However, further stud-
ies were unable to detect the presence of these infectious
agents. Cooper et al. [14] and Cankovic and Zarbo [18],
could not confirm the association between GCA and C.
pneumonia, HSV, varicella zoster virus (VZV), Epstein-
Barr virus (EBV), or human herpesvirus 7 (HHV7). In
addition, Rodriguez-Pla et al. [19], Helweg-Larsen [20]
and other authors [21-23] could not confirm the pres-
ence of herpes viruses, varicella zoster virus, parvovirus,
or C. pneumoniae in temporal artery GCA. Due to this
conflicting and contradictory data, no conclusive link
has, to date, been demonstrated between a GCA and a
specific infectious agent.
The role of human papillomavirus (HPV) in the devel-
opment of cervical, head and neck, anal and skin cancers
is well known. Molecular and epidemiological studies
have shown that persistent HPV infection is the most
important risk factor for cervical cancer [24,25]. High
risk HPV also has an important role in anogenital and
oropharyngeal squamous cell carcinoma [26]. They are
also implicated with skin cancer in individuals with epi-
dermodysplasia verruciformis [27] and can induce a var-
iety of proliferative lesions, such as warts and laryngeal


Page 7 of 9


papillomas. There are very few associations between
HPV and non-neoplastic diseases.
Interestingly, Ma et al. [10] reported the presence of
HPV type 16 in the genome of bacterial strains (Entero-
coccus, Staphylococcus, Bacillus and Corynebacterium)
and demonstrated the HPV viral particles by transmis-
sion electron microscopy in those bacteria. In addition,
HPV type 16 has been detected in peripheral nerves,
and vascular endothelial cells [7]. Another possible path-
way is for HPV to disseminate systemically. This has
long been debated [28]. The role of viremia in the
pathogenesis of HPV-related diseases is still unclear, al-
though HPV DNA has been detected in peripheral blood
in some studies, though in varying amounts [29-32].
In this study, we found that HPV DNA, of both high
and low risk types, is present in formalin-fixed,
paraffin-embedded temporal artery biopsy specimens,
and in statistically significantly higher numbers in the
arteritis specimens compared to histologically-negative
temporal artery biopsies and nephrectomy vascular mar-
gin controls. The validity of these results was confirmed
with exactly matching results by an outside laboratory
that independently prepped DNA from the paraffin
blocks and utilized a completely different detection
method. Our results are admittedly surprising and bring
many questions about what HPV's role, if any, would be
in temporal arteritis. HPV has been associated with
tumorigenesis, and many studies have investigated HPV-
related modification of the immune system to establish
infection. However, we are not aware of any literature
citing HPV as an inciting agent for inflammatory dis-
eases. Our findings do support the notion that HPV can
disseminate systemically, but do not, by themselves, tell
us anything specific about the pathophysiological rela-
tionship between the virus and temporal arteritis, if
there even is one. Further studies are necessary to ad-
dress this.
It is important to mention that a negative temporal ar-
tery biopsy does not rule out the diagnosis of temporal
artery GCA, since the changes can be patchy with skip
areas of uninvolved artery. Although serial sectioning of
the temporal artery biopsy is recommended, not all cases
will be diagnosed histologically [33]. Actually, after re-
view of the medical records of five of our biopsy-
negative HPV-positive cases, in one case, the patient
showed dramatic improvement after treatment with ster-
oids (possible false negative case which was not included
as a HPV-positive case in our statistical analysis).
It is also worth noting that of the 16 histologically-
proven GCA cases that had HPV, 11 were high risk
genotype 16. Only five cases were positive for low risk
HPV genotype 6. Although, the importance of distin-
guishing low risk from high risk HPV genotypes has
been established in the pathogenesis of neoplasms, what







Mohammadi et al. BMC Musculoskeletal Disorders 2012, 13:132
http://www.biomedcentral.com/1471-2474/13/132




its significance might be in inflammatory disorders such
as GCA is currently unknown.


Conclusion
In summary, we have identified HPV DNA in the major-
ity of histologically-proven giant cell (temporal) arteritis
specimens and validated these results by a completely
independent outside laboratory assay. The association
raises questions regarding the biology of HPV infections,
when dissemination occurs, and what this dissemination
means clinically. Such studies are required to understand
the significance of the association in our series.

Competing interests
The authors declare that they have no competing interests

Authors' contributions
AM conceived the study, carried out the molecular testing, analyzed the
data, and drafted the manuscript JDP and JSL, designed the study, collected
data, helped interpret findings and critically revised the manuscript All
authors read and approved the final manuscript

Acknowledgment
The authors would like to thank Jean (Qin) Zhang from the Division of
Biostatistics of Washington University in St Louis for her expert statistical
analysis of our data We would also like to thank to Dr Xiaopei Zhu for
outstanding expert technical assistance with the INNO LiPA PCR testing
Finally, we especially thank Sameer S Talwalkar, MD, FCAP, Medical Director,
Molecular Diagnostics of CPA Lab in Louisville, KY, for excellent assistance
with Cervista testing

Author details
Department of Pathology and Laboratory Medicine, University of Florida,
College of Medicine, Jacksonville, FI, USA 2Lauren V Ackerman Laboratory of
Surgical Pathology, Department of Pathology and Immunology, Division of
Anatomic and Molecular Pathology, Department of Pathology and
Immunology, Washington University School of Medicine, St Louis, MO, USA
Department of Otolaryngology Head and Neck Surgery, Washington
University School of Medicine, St Louis, MO, USA

Received: 14 December 2011 Accepted: 25 July 2012
Published: 25 July 2012

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doi:10.1186/1471-2474-13-132
Cite this article as: Mohammadi et al Association between human
papillomavirus DNA and temporal arteritis. BMC Musculoskeletal Disorders
2012 13132


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RESEARCHARTICLEOpenAccessAssociationbetweenhumanpapillomavirusDNA andtemporalarteritisAmirMohammadi1*,JohnDPfeifer2andJamesSLewisJr2,3AbstractBackground: Toexaminetherelationshipbetweenhumanpapillomavirus(HPV)andgiantcellarteritis(GCA)ofthe temporalartery. Methods: Thestudygroupconsistedof22casesofhistologicallypositive/biopsyconfirmedGCA.Thecontrol groupsconsistedof21histologicallynegativetemporalarterybiopsiesandfifteencasesofvascularmarginsof nephrectomies.FordetectionofthepresenceofHPV,twomethodswereused:1)polymerasechainreaction(PCR) withINNO-LiPAHPVGenotypingExtra,2)Cervista ™ HPVHR.AllcaseswerefromthefilesoftheBarnes-Jewish HospitalandWashingtonUniversityinSt.Louis. Results: HPVDNAwasdetectedbyPCRandgenotypingin16of22(73%)histologicallypositivecasesofGCAand inonlyfiveof21(24%)histologicallynegativetemporalarterybiopsies.Amongthevascularmargincontrols,only threeof15(20%)werepositiveforHPVDNA.Thesecond,independentmethod(CervistaTM)confirmedthe aforesaidresultswith100%concordancewiththeexceptionofthreecaseswhichhadlowgenomicDNAforwhich itwasnotpossibletoperformthetest.ThedifferencesinHPVpositivitybetweenthehistologicallypositiveand negativetemporalarterybiopsiesandbetweenthehistologicallypositivetemporalarterybiopsiesandcontrols werebothstatisticallysignificant(p=0.001and0.002,respectively). Conclusions: TheresultsofourstudyrevealedastatisticallysignificantassociationbetweenHPVpositivityand biopsyconfirmedtemporalgiantcellarteritisGCA(p=0.001).Furtherstudiesarenecessarytoelucidatethe pathophysiologyunderlyingthisassociation. Keywords: Humanpapillomavirus,Giantcellarteritis,PolymerasechainreactionBackgroundGiantcellarteritisisoneofthemostcommoncausesof vasculitisinvolvingthetemporalartery.Otherpotential causesareWegenergranulomatosis,polyarteritisnodosa (PAN),andBuergerdisease[1].GCAgenerallyaffects individualsover55yearsofage(withameanageat diagnosisofapproximately72years)[2]withanannual incidenceofapproximately18per100,000inpersons aged50yearsorolder. Histologically,GCAshowstransmuralinflammation withmixedinflammatorycellinfiltratemostlyconsisting oflymphocytes,histiocytes,plasmacells,occasionalneutrophilsandrarelyeosinophils(Figure1).Thiscauses destructionofthevessel'sinternalelasticlaminawhich isbestdemonstratedwithelasticstainssuchas Verhoeff-VanGieson(Figure2)orMovatpentachrome. Thepresenceofgiantcells,nexttotheelasticlaminain particular,istheclassicandpathognomonicfeatureof GCA.However,thisispresentinonlyabout50%of biopsy-provencases. InfectiousagentshavelongbeenconsideredasapossibleetiologyofGCA.TheconceptisthatGCArepresentsachronicinflammatoryresponse,triggeredbyan infectiousagent,withsubsequentinappropriatetissue responsetoinjury.However,studieslookingfororganismshavehadconflictingresults.SomehavedemonstratedorganismDNA,suchasHerpesSimplexVirus, whilethevastmajorityhavefailedtodemonstrateanassociation.Itisspeculatedthattheinflammatoryresponsemaybetriggeredbyaninfectiousagent.Ifa *Correspondence: amir.mohammadi@jax.ufl.edu1DepartmentofPathologyandLaboratoryMedicine,UniversityofFlorida, CollegeofMedicine,Jacksonville,Fl,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Mohammadietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132 http://www.biomedcentral.com/1471-2474/13/132

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connectionbetweenGCAandHPVinfectionweretobe established,thepotentialclinicalimplicationsaregreat, asGCA,withitspossiblevisionloss[3-5],polymyalgia rheumaticaandeveneventuallyischemicstroke[6-9], mightpotentiallybepreventedbyvaccinationorother strategies. Humanpapillomavirus(HPV)isanincreasinglycommonhumanpathogeninrecentdecades.Itisamucosotropicviruswhichisnotthoughtofasspreading systemically.However,HPVgenotype16hasbeenfound integratedintothegenomeofbacteriaisolatedfromcervicalcancerbiopsies,andthereisalsopublisheddata showingHPVviralparticleswithinperipheralnerves andsmallvascularendothelialcellsadjacenttooraland cervicalcancersasdemonstratedbytransmissionelectronmicroscopy[7,10].Inthisstudy,wesoughttodetermineifthereisanassociationbetweenGCAand HPV.MethodsWithapprovalbytheWashingtonUniversityHuman ResearchProtectionOffice(HRPO),wesearchedthe CopathdatabaseofBarnesJewishHospitalfortemporal arterybiopsyspecimensfrom1995to2008andretrieved allspecimensforwhichmaterialwasavailable.Weidentifiedapproximately60cases,ofwhich43,(22histologicallypositiveand21histologicallynegative)hadmaterial availableforreview.Therewerefivemalesand17 femalesinthehistologicallypositiveGCAgroupwitha meanageof78.9years.Inthehistologicallynegative group,therewerefourmalesand17femaleswitha meanageof67years,andthecontrolkidneyvascular margingrouphadsixmalesandninefemaleswitha meanageof38.2years.Therewerenostatisticallysignificantdifferenceingenderamongthegroups (p=0.176).TheclinicaldiagnosisofGCAwasbasedon thecriteriaoftheAmericanCollegeofRheumatology [11].However,forourstudy,weconsideredthegold standardtobehistologicevidenceoftemporalarteritis. Wealsorandomlyselected15renalarteryvascularresectionmarginsfromnephrectomyspecimensin patientswithoutanyhistoryofvasculitisforuseasthe negativecontrols. TwomethodswereutilizedtoevaluateforHPVDNA. Thefirst,INNO-LiPAHPVGenotypingExtra,wasused toperformthetestinginourresearchlaboratoryofthe divisionofanatomicandmolecularPathology,DepartmentofPathologyandImmunology,WashingtonUniversitySchoolofMedicine,St.Louis.Allblockswere thensubmittedtoanoutsidelaboratory(CPALaboratory,Louisville,KY)forindependentpreparationofgenomicDNAandfortestingemployingtheCervistaTMHPVHR(Hologic,Madison,WI,USA)assay. Inbothlaboratoriestoavoidpotentialcontamination, amaximumphysicalseparationbetweenthepre-and post-amplificationstepswasused.Separatepipettesand otherlabmaterialswereusedasapartofgoodlaboratorypractice.TheFFPEblockswerecutandprocessed understrictconditionstopreventDNAfrombeingcarriedoverfromonecasetothenextduringmicrotomy. Alsoanewbladewasusedforeachcaseandthearea wascleaned.Icecubesusedtocoolblockswerediscardedbetweencases. Withregardstothetechnicallimitationsofthisstudy, itiswellknownthatformalinfixationrandomlyfragmentsDNAinaduration-dependentmanner,resulting inapartialdegradation.Thedegreeoffragmentation Figure1 Biopsyoftemporalarteryshowingatransmural mixedinflammatorycellinfiltratewithintimalthickening,and fragmentationanddistortionoftheinternalelasticlamina(H& E,originalmagnificationx40). Figure2 Elasticstainsonthesamecaseofhistologicallypositive temporalarterybiopsyasshowninFigure1,showingthe fragmentation,distortionandlackofcontinuityoftheinternal elasticlamina,acharacteristicfeatureoftemporalarteritis (Verhoeff-VanGiesonstaining;originalmagnification,X40). Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page2of9 http://www.biomedcentral.com/1471-2474/13/132

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dependsonthetypeandageofthesampleandtheconditionsusedforfixation.Duetothisdegradation,FFPE tissueisnotsuitableforamplificationoflargeDNAsegments.Nevertheless,PCRamplificationofsegmentsrangingupto1300basepairhasbeenreported.Onthe otherhand,incubationatanelevatedtemperatureafter proteinaseKdigestionpartiallyremovesformalincrosslinkingoftheDNA,improvingyieldaswellasDNA performanceinassays.Furthermore,inoneofthetechniquesusedinthisstudy(INNO-LiPAHPVGenotyping), short-PCR-fragment(SPF10)primersareemployed, whichamplifya65basepairsegmentoftargetDNAand thistestingisconsideredtobeoneofthemostsensitive PCRassaysforthedetectionofHPVDNA.WashingtonUniversitytesting DNAextractionDNAwasextractedusingPureGeneKit(Gentra,http:// www.Gentra.com)asperthemanufacturer'sinstructions from10 msectionscutfromtheparaffinblocks.The concentrationofthepreppedDNAwasmeasuredspectrophotometricallyusingNanodrop.Detailedprocedure informationisavailableattheirwebsite,butbriefly,we placedfive10micronsectionsoftissueintoa1.5mL microcentrifugetubeandadded1.0mLofxylene,vortexed,andincubatedforfiveminuteswithconstantgentlemixing.Thenwecentrifugeditforfiveminutesat 13,000-16,000xg.Inthefumehood,discardedbypipettingthexylenesupernatantandleftbehindthevisible pellet(tissue).Werepeatedthisxylenewashtwice.We thenadded1.0mL100%ethanol,vortexed,andincubatedfiveminuteswithconstantgentlemixingatroom temperature,centrifugedat13,000-16,000xgforfive minutestopelletthetissue,anddiscardedtheethanol. Werepeatedtheseethanolwashestwice.Subsequently weadded1.0mL70%ethanol,gentlymixed,andcentrifugedat13,000-16,000xgforfiveminutesat4C, removedallresidualethanolandallowedtissuepelletto drybycentrifugationundervacuumforfiveminutes. Forcelllysis,weadded300 lCellLysisSolution(GentraPuregene ™ kit)andgentlyvortexedfor30seconds. Thenweaddedthree lPuregeneProteinaseK(20mg/ ml),andmixedbyinverting25timesandincubatedthe lysateat55Cforthreehourstoovernight.Weinverted thetubeperiodicallyduringtheincubation.Thenwas addedthree lRNaseASolutiontothecelllysate,and mixedbyinvertingthetube25timesandincubatedat 37Cfor15mintoonehour.ForProteinPrecipitation wecooledquicklythesampletoroomtemperatureby placingoniceandadded100 lProteinPrecipitation Solution(GentraPuregene ™ kit)tothecelllysate.The volumeshouldbe1/3oftheCellLysisSolutioninthe tube.Subsequently,itisvortexedvigorouslyathigh speedfor20secondstomixtheProteinPrecipitation Solutionuniformlywiththecelllysate.Then,wecentrifugeditat13,000-16,000xgforfiveminutes.Theprecipitatedproteinsformedatightpellet.Iftheprotein pelletwasnottight,wevortexedvigorouslyfor20secondsathighspeed,andthenincubatedonicefor5min. Wethencentrifugedat13,000 – 16,000x g forthreeminutes.Usingapipette,weremovedthesupernatantcontainingtheDNA(leavingbehindtheprecipitatedprotein pellet)intoaclean1.5mLmicrocentrifugetube.We addedtwo l(for400 lsupernatant)ofaDNAcarrier (glycogen;tofinalconcentrationof50-150mcg/ l)to aidrecoveryofsmallDNAquantitiesandthenvortexed them.Wethenadded400 l100%ofisopropanol.Subsequently,itwasmixedbyinvertinggently~50times untilthewhitethreadsofDNAformedavisibleclump, andthenitwascentrifugedat13,000-16,000xgfor10 minutesandthesupernatantwaspouredoff.Weadded 500 lof70%ethanolandinvertedthetubetowashthe DNApellet.Wecentrifugedat13,000-16,000xgfor5 minutes,andcarefullypouredofftheethanoland invertedandblottedtheliquidfromthetubeonclean absorbentpaperandallowedtoairdryfor10-15minutes.Finally,weadded50 lDNAHydrationSolution andincubatedat65Cforonehourtodissolvethe DNA.Weincubatedatroomtemperatureovernight withgentleshaking.Samplescouldthenbecentrifuged brieflyandtransferredtoastoragetube.TheconcentrationoftheDNAusedforINNO-LiPAHPVGenotyping Extra testingineachcasewas50ng.INNO-LiPAHPVGenotypingtestingTheINNO-LiPAHPVGenotyping Extra isbasedonthe principleofreversehybridization.PartoftheL1region ofthehumanpapillomavirus(HPV)genomeisamplified usingshort-PCR-fragmentassay(SPF10primers),and theresultingbiotinylatedampliconsarethendenatured andhybridizedwithspecificoligonucleotideprobes. Anadditionalprimerpairfortheamplificationofthe humanHLA-DPB1geneisaddedtomonitorsample qualityandextraction.ThelengthoftheHLA-DPB1 fragmentis280basepairs.Allprobesareimmobilized asparallellinesonmembranestrips.Afterhybridization andstringentwashing,streptavidin-conjugatedalkaline phosphataseisadded,whichbindstoanybiotinylated hybridpreviouslyformed. IncubationwithBCIP(5-Bromo-4-Chloro-3 ’ -Indolyphosphatep-ToluidineSalt)/NBT(Nitro-BlueTetrazoliumChloride)chromogenyieldsapurpleprecipitate, andtheresultsarevisuallyinterpretedusingthereferenceguideprovided.Anamplificationkit(INNO-LiPA HPVGenotyping Extra Amp)isusedforstandardized preparationofbiotinylatedamplifiedmaterial.Thisamplificationkitisbasedonthepolymerasechainreaction (PCR)usingSPF10primers.Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page3of9 http://www.biomedcentral.com/1471-2474/13/132

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Amplificationproductsaresubsequentlyhybridized usingasingletypingstriponwhich28sequence-specific DNAprobelinesand4controllinesarefixed,which permitsspecificdetectionof28HPVgenotypes,includingall18high-riskgenotypes,and10low-riskgenotypes (HPVtypes6,11,16,18,26,31,33,35,39,40,43,44, 45,51,52,53,54,56,58,59,66,68,69,70,71,73,74, and82)asdescribedbythemanufacturer(Figure3).CPALaboratorytesting DNAextractionSlideswerecutfromtheoriginalblocks,andThe QIAampDNAFFPETissuekit(QIAGEN,www.qiagen. com)wasusedforpurificationofgenomicDNAfrom formalin-fixed,paraffin-embeddedtissuesaccordingto themanufacturer ’ sinstructionswithexceptionofincubationtime.OvernightincubationinproteinaseKfordigestionofproteins/contaminantsisnotrecommended byQiagenbutissomethingthatCPALaboratoryhas foundusefultoincreasethenucleicacidelution. Briefly,theQIAampDNAFFPETissueprocedure consistedofseveralstepsincludingremovalofparaffin fromslidesusingxylene,subsequentspecimenlysis underdenaturingconditionswithproteinaseK,incubationat90Ctoreverseallformalincross-linking,and DNAbindingtothemembraneforremovalofcontaminants.Residualcontaminantswerewashedawayand pure,concentratedDNAwaselutedfromthe membrane.CervistaTMHPVHRtestingCervistaTMHPVHR[12]isaqualitative,diagnostictest forthedetectionofDNAfrom14high-riskHPVtypes (i.e.,types16,18,31,33,35,39,45,51,52,56,58,59, 66,and68).TheCervistaTMHPVHRtestusestheInvaderWchemistrywhichisasignalamplificationtechnique fordetectionofspecificnucleicacidsequences.Inthis methodtwotypesofisothermalreactionsareused:a primaryreactionthatoccursonthetargetedDNAsequenceandasecondaryreactionthatproducesafluorescentsignal.Duringtheprimaryreaction,twotypesof sequencespecificoligonucleotides(i.e.aprobeoligonucleotideandanInvaderWoligonucleotide)bindtothe targetDNArecognitionsite.Whenthosesequencespecificoligonucleotidesoverlapbyatleastonebasepair onthetargetsequence,aninvasivestructureformsthat actsasasubstratefortheCleavaseWenzyme.Theenzymecleavesthe5 ’ portion(flap)oftheprobeatthe positionoftheoverlap. Figure3 INNO-LiPAtypingon16patients.Strips#5,9,11,14,15and16arepositivebecausetheyshowclearlyvisiblelinesof hybridization. Thelinepatterns(correspondingto#3oftheprobesidebars)werecomparedtotheinterpretationchartsuppliedwiththekit correspondwithHPVtype16. Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page4of9 http://www.biomedcentral.com/1471-2474/13/132

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Theprobesarepresentinlargemolarexcessandcycle rapidlyonandoffthetargetsequencegeneratingmany cleaved5 ’ flapspertargetsequence.Thecleavedflaps thenbindtoauniversalhairpinfluorescenceresonance energytransfer(FRET)oligonucleotidecreatinganother invasivestructurethattheCleavaseWenzymerecognizes asasubstrate.TheenzymecleavestheFREToligonucleotidesbetweenthefluorophoreandquenchermoleculeandproducesafluorescencesignalasthecleaved flapscycleonandoff.Foreachcopyoftarget,thecombinedprimaryandsecondaryreactionsresultin106– 107foldsignalamplificationperhour.Thereagentsfor thistestareprovidedasthreeoligonucleotidemaster mixtures,whichidentifythe14typesofHPVarranged accordingtophylogeneticrelatedness.Mastermixture1 identifiesthepositivityofgenotypes51,56and66 (MM1:HPV51,56and66).Mastermixture2(MM2) showsthepositivityofgenotypesHPV18,39,45,59and 68andmastermixture3(MM3)revealsthepositivityof HPVgenotypes16,31,33,35,52and58.Bydesign,the released5'-flapsbindonlytotheirrespectiveFREToligonucleotidestogeneratetarget-specificsignal.Apositiveresultindicatesthatatleastoneofthe14high-risk typesispresentintheDNAsample.Foreachcase150 to200ngofDNApreppedfromtheFFPEtissuesat CPALabswereutilizedfortheCervistaHPVtesting. Thesensitivityofthistestissetat5000copiesofHPV DNA.TheCervistaassayhasaninternalcontroltoverifyiftheassayworked.Iftheinternalcontroldoesnot pass,itsuggestseitherthesampleisdegradedorextractionfailedorthePCRdidnotwork.StatisticsStatisticalanalyseswereperformedusingtheChi-Square, T -testandFisher'sexactteststoevaluatethecorrelation betweenthepresenceofHPVDNAandothervariables. P values < 0.05wereconsideredsignificant.Allstatistical analyseswereperformedusingtheSASv.9.1systemsoftware(SASInstituteInc.,Cary,NC).ResultsUsingtheINNO-LiPAHPVGenotyping Extra, 16ofthe 22(17femaleand5male)biopsy-confirmedGCAcases werepositiveforHPV.Twelveoutofthe16(75%)HPVpositiveGCA-positivepatientswerefemale.Among these16positivecases,5hadaHPV6alone(31%),3 caseshadHPV16alone(19%),7caseshadbothtypes6 and16(44%),andonlyonecasewasextensively"multigenotype"showingtypes6,16,31,33,and40(6%)(TestingresultsarepresentedinTables1and2).Inallcases whereHPVwasdetected,type6,type16,orbothwere present(100%).ThecaseswerealsotestedbytheCervistaTMHPVHRassayforconfirmation,whichonly evaluatesforhighriskHPVtypes(notforHPVs6and 11).TwocaseswhichwereHPVtype6byINNO-LiPA (cases#4and6)hadlowgenomicDNAwhenprepped fortheCervistaTMHPVHRtesting,socouldnotbe evaluatedbythismethod.AllpositivecasesoftheCervistaTMHPVHRwerefromtheMasterMix3which includestheHPVgenotypes16,31,33,35,52and58. Asexpected,allcasesforwhichgenotype6waspresent intheINNO-LiPAmethodwerenegativewiththeCervistamethodsincelowrisktypesarenotevaluatedin thatassay.AllnegativecaseswithINNO-LiPAHPV Genotyping Extra werenegativealsowithCervistaTMHPVHRtesting.Assuch,therewas100%agreementbetweenthetwotestingmethodsonthehistologically positivetemporalarteritiscases. Amongthe21casesofGCAhistologicallynegativebiopsies,5werepositiveforHPVbyINNO-LiPAHPV Genotyping Extra ,ofwhich3(60%)weregenotype16, one(20%)wasgenotype6andone(20%)wasgenotype 52.CervistaTMHPVHRtestingrevealedpositivityof MasterMix3forall4caseswhichwerepositiveby INNO-LiPAHPVGenotyping Extra forhighriskHPV. Casenumber2whichwasnegativewithINNO-LiPA wasoflowgenomicDNAwithCervistamethod.As such,therewas100%agreementbetweenthetwotesting methodsonthehistologicallynegativetemporalartery biopsycases. Amongthe15nephrectomyvascularresectionmargins(controls),3werepositiveforHPVbyINNO-LiPA HPVGenotyping Extra .Twocasesshowedgenotype16 (1caseofWilms'tumorand1ofacuteandchronicpyelonephritis),andonegenotype6(acaseofpapillary urothelialcarcinoma).CervistaTMHPVHRtesting revealedpositivityofMasterMix3forbothcaseswhich werepositiveforhighriskHPVbyINNO-LiPAHPV Genotyping Extra. Assuch,therewas100%agreement betweenthetwotestingmethodsonthe15vascular margincontrolcases. TheassociationbetweenHPVpositivityandhistologicallyconfirmedGCAwasstatisticallysignificant (p=0.0013).TherewasalsoastatisticallysignificantassociationbetweenHPVpositivityandCaucasianrace (p=0.0339).Nootherassociationswerestatistically significant. Inmultivariateanalysis(Table3),subjectswithhistologicallyconfirmedGCAhada56-foldhigherlikelihood ofhavingHPVpositivityadjustedbygender,ageand race(OR,pointestimate56.01;95%CI3.5-895.68).DiscussionGiantcell(temporal)arteritisisachronicvasculitisinvolvingmediumandlargesizearteriesthattypically affectsindividualsolderthan50yearsofage.[1,3,4]Althoughitusuallyaffectsthesuperficialtemporalarteries, itcanalsoaffecttheaorta,carotid,subclavian,vertebral,Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page5of9 http://www.biomedcentral.com/1471-2474/13/132

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andiliacarteries.Theclassicalpictureofgranulomatous inflammationwithmultinucleatedgiantcellsisobserved inapproximately50%ofthepatients. Histologically,thediseaseprogressesfromminimal involvementofvessels,withonlycollectionsoflymphocytesconfinedtotheinternalorexternalelastic laminaoradventitiatoapanarteritiswithsegmental areasofnecrosisofthearterialwallandextensivedestructionoftheelasticlaminae,afeaturethatcanbe clearlydemonstratedwithspecialstainsforelastic fibers(Figures1and2). TheetiologyoftheinflammatoryreactionintheGCA hasnotbeenidentified.However,ithasbeendemonstratedthatthereisarestrictedclonalexpansionof tissue-infiltratingTcellsintheselesions,whichsuggests thattheyarereactingtoaspecificantigenlocatedwithin theaffectedarterialwallwhichisthuselicitingthedisease[13].Themostrecenthypothesisregardingthe Table1ResultsoftheHPVtestingbybothdifferentmethods.Histologically PositiveGCAcases INNOLiPA Results Cervista Results HistologicallyNegative GCAcases INNOLiPA Results Cervista Results KidneyVascular margin INNOLiPA Results Cervista Results 1NegNeg1NegNeg1NegNeg 26Neg2NegLGD2NegNeg 3NegNeg3NegNeg3NegNeg 46LGD46Neg4NegNeg 56,16,31,33,40posMM35NegNeg5NegNeg 66LGD652posMM36NegNeg 76Neg7NegNeg7NegNeg 86Neg8NegNeg86Neg 9NegNeg9NegNeg916posMM3 106,16posMM310NegNeg10NegNeg 1116posMM311NegNeg1116posMM3 126,16posMM312NegNeg12NegNeg 1316posMM313NegNeg13NegNeg 146,16posMM314NegNeg14NegNeg 15NegNeg15NegNeg15NegNeg 16NegNeg1616posMM3 17NegNeg17NegNeg 186,16posMM31816posMM3 196,16posMM319NegNeg 206,16posMM32016posMM3 2116posMM321NegNeg 226,16posMM3LGD :LowGenomicDNA. POSMM3 :PositiveinMasterMix3. HPV =Humanpapillomavirus. MM1:HPVtypes51,56and66. MM2:HPVtypes18,39,45,59and68. MM3:HPVtypes16,31,33,35,52and58. Table2CorrelationbetweenthethreestudygroupsforHPVtestresultsBiopsyPositiveGCA*BiopsyNegativeGCAAllTemporalArtery Biopsies RenalArtery Controls** HPVPositive16(73%)5(24%)21(49%)3 HPVNegative6(27%)16(76%)22(51%)12 Total22214315*Differencesbetweenbiopsypositiveandbiopsynegativecasesandbetweenbiopsypositiveandcontrolcaseswerestatisticallysignificant((p=0 .001and0.002, respectively). **Differencebetweenbiopsynegativeandcontrolcaseswasnotstatisticallysignificant(p=0.79). GCA =giantcellarteritis; HPV =humanpapillomavirus.Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page6of9 http://www.biomedcentral.com/1471-2474/13/132

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etiologyofGCAcontendsthataresponsetoendothelial injury(maybeduetoinfection)leadstoaninappropriate activationofT-cell-mediatedimmunity[14,15].Thesubsequentreleaseofinflammatorymediatorswithinthearterialvesselwallcanattractmacrophageswhichthen becomemultinucleatedgiantcells,creatingthecharacteristichistologyofthisdiseaseandalsoleadingtoan oligoclonalexpansionofT-cellsdirectedagainstantigens withinorneartheelasticlamina.Thiscascadeofevents induecourseresultsinvesselwalldamage,intimal hyperplasia,andeventualstenoticocclusion. Infectiousagentshavebeenconsideredinthepastas theetiologyofGCA.Ellingetal.,byindirectserological evidence,foundhighincidenceofGCAwithinapopulationassociatedwith Chlamydiapneumoniae Mycoplasmapneumoniae ,andParvovirusB19[16].Published datafromWagneretal.[15]andPowersetal.[17] reportedarelationshipbetweenGCA, C.pneumonia andherpessimplexvirus(HSV).However,furtherstudieswereunabletodetectthepresenceoftheseinfectious agents.Cooperetal.[14]andCankovicandZarbo[18], couldnotconfirmtheassociationbetweenGCAand C. pneumonia ,HSV,varicellazostervirus(VZV),Epstein – Barrvirus(EBV),orhumanherpesvirus7(HHV7).In addition,Rodriguez-Plaetal.[19],Helweg-Larsen[20] andotherauthors[21-23]couldnotconfirmthepresenceofherpesviruses,varicellazostervirus,parvovirus, or C.pneumoniae intemporalarteryGCA.Duetothis conflictingandcontradictorydata,noconclusivelink has,todate,beendemonstratedbetweenaGCAanda specificinfectiousagent. Theroleofhumanpapillomavirus(HPV)inthedevelopmentofcervical,headandneck,analandskincancers iswellknown.Molecularandepidemiologicalstudies haveshownthatpersistentHPVinfectionisthemost importantriskfactorforcervicalcancer[24,25].High riskHPValsohasanimportantroleinanogenitaland oropharyngealsquamouscellcarcinoma[26].Theyare alsoimplicatedwithskincancerinindividualswithepidermodysplasiaverruciformis[27]andcaninduceavarietyofproliferativelesions,suchaswartsandlaryngeal papillomas.Thereareveryfewassociationsbetween HPVandnon-neoplasticdiseases. Interestingly,Maetal.[10]reportedthepresenceof HPVtype16inthegenomeofbacterialstrains( Enterococcus Staphylococcus Bacillus and Corynebacterium ) anddemonstratedtheHPVviralparticlesbytransmissionelectronmicroscopyinthosebacteria.Inaddition, HPVtype16hasbeendetectedinperipheralnerves, andvascularendothelialcells[7].AnotherpossiblepathwayisforHPVtodisseminatesystemically.Thishas longbeendebated[28].Theroleofviremiainthe pathogenesisofHPV-relateddiseasesisstillunclear,althoughHPVDNAhasbeendetectedinperipheralblood insomestudies,thoughinvaryingamounts[29-32]. Inthisstudy,wefoundthatHPVDNA,ofbothhigh andlowrisktypes,ispresentinformalin-fixed, paraffin-embeddedtemporalarterybiopsyspecimens, andinstatisticallysignificantlyhighernumbersinthe arteritisspecimenscomparedtohistologically-negative temporalarterybiopsiesandnephrectomyvascularmargincontrols.Thevalidityoftheseresultswasconfirmed withexactlymatchingresultsbyanoutsidelaboratory thatindependentlypreppedDNAfromtheparaffin blocksandutilizedacompletelydifferentdetection method.Ourresultsareadmittedlysurprisingandbring manyquestionsaboutwhatHPV'srole,ifany,wouldbe intemporalarteritis.HPVhasbeenassociatedwith tumorigenesis,andmanystudieshaveinvestigatedHPVrelatedmodificationoftheimmunesystemtoestablish infection.However,wearenotawareofanyliterature citingHPVasanincitingagentforinflammatorydiseases.OurfindingsdosupportthenotionthatHPVcan disseminatesystemically,butdonot,bythemselves,tell usanythingspecificaboutthepathophysiologicalrelationshipbetweenthevirusandtemporalarteritis,if thereevenisone.Furtherstudiesarenecessarytoaddressthis. Itisimportanttomentionthatanegativetemporalarterybiopsydoesnotruleoutthediagnosisoftemporal arteryGCA,sincethechangescanbepatchywithskip areasofuninvolvedartery.Althoughserialsectioningof thetemporalarterybiopsyisrecommended,notallcases willbediagnosedhistologically[33].Actually,afterreviewofthemedicalrecordsoffiveofourbiopsynegativeHPV-positivecases,inonecase,thepatient showeddramaticimprovementaftertreatmentwithsteroids(possiblefalsenegativecasewhichwasnotincluded asaHPV-positivecaseinourstatisticalanalysis). Itisalsoworthnotingthatofthe16histologicallyprovenGCAcasesthathadHPV,11werehighrisk genotype16.Onlyfivecaseswerepositiveforlowrisk HPVgenotype6.Although,theimportanceofdistinguishinglowriskfromhighriskHPVgenotypeshas beenestablishedinthepathogenesisofneoplasms,what Table3OddsRatiosforvariableandpresenceof histologicallypositivetemporalarterybiopsiesVariablePoint Estimate 95%WaldConfidence Interval GenderF vs. M2.7620.32923.222 Race: AfricanAmerican vs. Caucasian 0.1130.0062.199 HPV56.0143.503895.681 Age1.1961.0531.358HPV =humanpapillomavirus.Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page7of9 http://www.biomedcentral.com/1471-2474/13/132

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itssignificancemightbeininflammatorydisorderssuch asGCAiscurrentlyunknown.ConclusionInsummary,wehaveidentifiedHPVDNAinthemajorityofhistologically-provengiantcell(temporal)arteritis specimensandvalidatedtheseresultsbyacompletely independentoutsidelaboratoryassay.Theassociation raisesquestionsregardingthebiologyofHPVinfections, whendisseminationoccurs,andwhatthisdissemination meansclinically.Suchstudiesarerequiredtounderstand thesignificanceoftheassociationinourseries.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions AMconceivedthestudy,carriedoutthemoleculartesting,analyzedthe data,anddraftedthemanuscript.JDPandJSL,designedthestudy,collected data,helpedinterpretfindingsandcriticallyrevisedthemanuscript.All authorsreadandapprovedthefinalmanuscript. Acknowledgment TheauthorswouldliketothankJean(Qin)ZhangfromtheDivisionof BiostatisticsofWashingtonUniversityinSt.Louisforherexpertstatistical analysisofourdata.WewouldalsoliketothanktoDr.XiaopeiZhufor outstandingexperttechnicalassistancewiththeINNOLiPAPCRtesting. Finally,weespeciallythankSameerS.Talwalkar,MD,FCAP,MedicalDirector, MolecularDiagnosticsofCPALabinLouisville,KY,forexcellentassistance withCervistatesting. Authordetails1DepartmentofPathologyandLaboratoryMedicine,UniversityofFlorida, CollegeofMedicine,Jacksonville,Fl,USA.2LaurenV.AckermanLaboratoryof SurgicalPathology,DepartmentofPathologyandImmunology,Divisionof AnatomicandMolecularPathology,DepartmentofPathologyand Immunology,WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA.3DepartmentofOtolaryngology – HeadandNeckSurgery,Washington UniversitySchoolofMedicine,St.Louis,MO,USA. Received:14December2011Accepted:25July2012 Published:25July2012 References1.BurkeA,VirmaniR: BloodVessels .In Sternberg'sDiagnosticSurgical Pathology.Volume1.5thedition .EditedbyMillsSE.Philadelphia:Lippincott Williams&Wilkins;2009:1236 – 1238. 2.SmetanaGW,ShmerlingRH: Doesthispatienthavetemporalarteritis? JAMA 2002, 287 (1):92 – 101. 3.GordonLK,LevinLA: Visuallossingiantcellarteritis. JAMA 1998, 280 (4):385 – 386. 4.FontC,CidMC,Coll-VinentB,Lopez-SotoA,GrauJM: Clinicalfeaturesin patientswithpermanentvisuallossduetobiopsy-provengiantcell arteritis. BrJRheumatol 1997, 36 (2):251 – 254. 5.LiuGT,GlaserJS,SchatzNJ,SmithJL: Visualmorbidityingiantcell arteritis. Clinicalcharacteristicsandprognosisforvision.Ophthalmology 1994, 101 (11):1779 – 1785. 6.HellmannDB: Temporalarteritis:acough,toothache,andtongue infarction. JAMA 2002, 287 (22):2996 – 3000. 7.FuleT,MatheM,SubaZ,CsapoZ,SzarvasT,TatraiP,PakuS,KovalszkyI: Thepresenceofhumanpapillomavirus16inneuralstructuresand vascularendothelialcells. Virology 2006, 348 (2):289 – 296. 8.Gonzalez-GayMA,Vazquez-RodriguezTR,Lopez-DiazMJ,Miranda-FilloyJA, Gonzalez-JuanateyC,MartinJ,LlorcaJ: Epidemiologyofgiantcellarteritis andpolymyalgiarheumatica. ArthritisRheum 2009, 61 (10):1454 – 1461. 9.ElkindMS: Inflammatorymarkersandstroke. CurrCardiolRep 2009, 11 (1):12 – 20. 10.MaZ,LiuL,ZhangF,YuM,WangK,LuoJ,LiuK,ChenB,XuL: Human papillomavirustype16existsinbacteriaisolatedfromcervicalcancer biopsies. JIntMedRes 2009, 37 (4):1065 – 1074. 11.HunderGG,BlochDA,MichelBA,StevensMB,ArendWP,CalabreseLH, EdworthySM,FauciAS,LeavittRY,LieJT, etal : TheAmericanCollegeof Rheumatology1990criteriafortheclassificationofgiantcellarteritis. ArthritisRheum 1990, 33 (8):1122 –1128. 12. 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JRheumatol 1996, 23 (1):112 – 119. 17.PowersJF,BedriS,HusseinS,SalomonRN,TischlerAS: Highprevalenceof herpessimplexvirusDNAintemporalarteritisbiopsyspecimens. AmJ ClinPathol 2005, 123 (2):261 – 264. 18.CankovicM,ZarboRJ: Failuretodetecthumanherpessimplexvirus, cytomegalovirus,andEpstein-Barrvirusviralgenomesingiantcell arteritisbiopsyspecimensbyreal-timequantitativepolymerasechain reaction. CardiovascPathol 2006, 15 (5):280 – 286. 19.Rodriguez-PlaA,Bosch-GilJA,Echevarria-MayoJE,Rossello-UrgellJ, Solans-LaqueR,Huguet-RedecillaP,StoneJH,Vilardell-TarresM: No detectionofparvovirusB19orherpesvirusDNAingiantcellarteritis. JClinVirol 2004, 31 (1):11 – 15. 20.Helweg-LarsenJ,TarpB,ObelN,BaslundB: Noevidenceofparvovirus B19,Chlamydiapneumoniaeorhumanherpesvirusinfectionin temporalarterybiopsiesinpatientswithgiantcellarteritis. Rheumatology(Oxford) 2002, 41 (4):445 – 449. 21.NjauF,NessT,WittkopU,PancratzT,EickhoffM,HudsonAP,HallerH, WagnerAD: NocorrelationbetweengiantcellarteritisandChlamydia pneumoniaeinfection:investigationof189patientsbystandardand improvedPCRmethods. JClinMicrobiol 2009, 47 (6):1899 – 1901. 22.HaugebergG,BieR,NordboSA: Chlamydiapneumoniaenotdetectedin temporalarterybiopsiesfrompatientswithtemporalarteritis. ScandJ Rheumatol 2000, 29 (2):127 –128. 23.NordborgC,NordborgE,PetursdottirV,LaGuardiaJ,MahalingamR, WellishM,GildenDH: Searchforvaricellazostervirusingiantcell arteritis. AnnNeurol 1998, 44 (3):413 – 414. 24.CuschieriKS,CubieHA,WhitleyMW,GilkisonG,ArendsMJ,GrahamC, McGooganE: PersistenthighriskHPVinfectionassociatedwith developmentofcervicalneoplasiainaprospectivepopulationstudy. JClinPathol 2005, 58 (9):946 – 950. 25.ZurHausenH: Papillomavirusesandcancer:frombasicstudiestoclinical application. NatRevCancer 2002, 2 (5):342 – 350. 26.GillisonML: Humanpapillomavirus-associatedheadandneckcancerisa distinctepidemiologic,clinical,andmolecularentity. SeminOncol 2004, 31 (6):744 – 754. 27.MasiniC,FuchsPG,GabrielliF,StarkS,SeraF,PlonerM,MelchiCF, PrimaveraG,PirchioG,PicconiO,PetaseccaP,CattaruzzaMS,PfisterHJ, AbeniD: Evidencefortheassociationofhumanpapillomavirusinfection andcutaneoussquamouscellcarcinomainimmunocompetent individuals. ArchDermatol 2003, 139 (7):890 – 894. 28.GnanamonyM,PeedicayilA,SubhashiniJ,RamTS,RajasekarA, GravittP,AbrahamP: DetectionandquantitationofHPV16and18 inplasmaofIndianwomenwithcervicalcancer. GynecolOncol 2010, 116 (3):447 – 451. 29.HoCM,YangSS,ChienTY,HuangSH,JengCJ,ChangSF: Detectionand quantitationofhumanpapillomavirustype16,18and52DNAinthe peripheralbloodofcervicalcancerpatients. GynecolOncol 2005, 99 (3):615 – 621.Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page8of9 http://www.biomedcentral.com/1471-2474/13/132

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30.WeiYC,ChouYS,ChuTY: Detectionandtypingofminimalhuman papillomavirusDNAinplasma. IntJGynaecolObstet 2007, 96 (2):112 – 116. 31.DongSM,PaiSI,RhaSH,HildesheimA,KurmanRJ,SchwartzPE,MortelR, McGowanL,GreenbergMD,BarnesWA,SidranskyD: Detectionand quantitationofhumanpapillomavirusDNAintheplasmaofpatients withcervicalcarcinoma. CancerEpidemiolBiomarkersPrev 2002, 11 (1):3 – 6. 32.YangHJ,LiuVW,TsangPC,YipAM,TamKF,WongLC,NgTY,NganHY: QuantificationofhumanpapillomavirusDNAintheplasmaofpatients withcervicalcancer. IntJGynecolCancer 2004, 14 (5):903 – 910. 33.BrackA,Martinez-TaboadaV,StansonA,GoronzyJJ,WeyandCM: Disease patternincranialandlarge-vesselgiantcellarteritis. ArthritisRheum 1999, 42 (2):311 – 317.doi:10.1186/1471-2474-13-132 Citethisarticleas: Mohammadi etal. : Associationbetweenhuman papillomavirusDNAandtemporalarteritis. BMCMusculoskeletalDisorders 2012 13 :132. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Mohammadi etal.BMCMusculoskeletalDisorders 2012, 13 :132Page9of9 http://www.biomedcentral.com/1471-2474/13/132


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p Association between human papillomavirus DNA and temporal arteritis
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au id A1 ca yes snm Mohammadifnm Amirinsr iid I1 email amir.mohammadi@jax.ufl.edu
A2 Pfeifermi DJohnI2 pfeifer@path.wustl.edu
A3 LewisSJamessuf JrI3 jlewis@path.wustl.edu
insg
ins Department of Pathology and Laboratory Medicine, University of Florida, College of Medicine, Jacksonville, Fl, USA
Lauren V. Ackerman Laboratory of Surgical Pathology, Department of Pathology and Immunology, Division of Anatomic and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA
Department of Otolaryngology – Head and Neck Surgery, Washington University School of Medicine, St. Louis, MO, USA
source BMC Musculoskeletal Disorders
issn 1471-2474
pubdate 2012
volume 13
issue 1
fpage 132
url http://www.biomedcentral.com/1471-2474/13/132
xrefbib pubidlist pubid idtype doi 10.1186/1471-2474-13-132pmpid 22831396
history rec date day 14month 12year 2011acc 1072012pub 2572012
cpyrt 2012collab Mohammadi et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Human papillomavirus
Giant cell arteritis
Polymerase chain reaction
abs
sec
st
Abstract
Background
To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the temporal artery.
Methods
The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR) with INNO-LiPA HPV Genotyping Extra, 2) Cervista™ HPV HR. All cases were from the files of the Barnes-Jewish Hospital and Washington University in St. Louis.
Results
HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only three of 15 (20%) were positive for HPV DNA. The second, independent method (Cervistasup TM) confirmed the aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which it was not possible to perform the test. The differences in HPV positivity between the histologically positive and negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls were both statistically significant (p = 0.001 and 0.002, respectively).
Conclusions
The results of our study revealed a statistically significant association between HPV positivity and biopsy confirmed temporal giant cell arteritis GCA (p = 0.001). Further studies are necessary to elucidate the pathophysiology underlying this association.
bdy
Background
Giant cell arteritis is one of the most common causes of vasculitis involving the temporal artery. Other potential causes are Wegener granulomatosis, polyarteritis nodosa (PAN), and Buerger disease abbrgrp
abbr bid B1 1
. GCA generally affects individuals over 55 years of age (with a mean age at diagnosis of approximately 72 years)
B2 2
with an annual incidence of approximately 18 per 100,000 in persons aged 50 years or older.Histologically, GCA shows transmural inflammation with mixed inflammatory cell infiltrate mostly consisting of lymphocytes, histiocytes, plasma cells, occasional neutrophils and rarely eosinophils (Figure figr fid F1 1). This causes destruction of the vessel's internal elastic lamina which is best demonstrated with elastic stains such as Verhoeff-Van Gieson (Figure F2 2) or Movat pentachrome. The presence of giant cells, next to the elastic lamina in particular, is the classic and pathognomonic feature of GCA. However, this is present in only about 50% of biopsy-proven cases.
fig Figure 1caption Biopsy of temporal artery showing a transmural mixed inflammatory cell infiltrate with intimal thickening, and fragmentation and distortion of the internal elastic lamina (H & E, original magnification x40)text
b Biopsy of temporal artery showing a transmural mixed inflammatory cell infiltrate with intimal thickening, and fragmentation and distortion of the internal elastic lamina (H & E, original magnification x40).
graphic file 1471-2474-13-132-1
Figure 2Elastic stains on the same case of histologically positive temporal artery biopsy as shown in Figure 1, showing the fragmentation, distortion and lack of continuity of the internal elastic lamina, a characteristic feature of temporal arteritis (Verhoeff-Van Gieson staining; original magnification, X40).
Elastic stains on the same case of histologically positive temporal artery biopsy as shown in Figure
1
, showing the fragmentation, distortion and lack of continuity of the internal elastic lamina, a characteristic feature of temporal arteritis (Verhoeff-Van Gieson staining; original magnification, X40).
1471-2474-13-132-2 Infectious agents have long been considered as a possible etiology of GCA. The concept is that GCA represents a chronic inflammatory response, triggered by an infectious agent, with subsequent inappropriate tissue response to injury. However, studies looking for organisms have had conflicting results. Some have demonstrated organism DNA, such as Herpes Simplex Virus, while the vast majority have failed to demonstrate an association. It is speculated that the inflammatory response may be triggered by an infectious agent. If a connection between GCA and HPV infection were to be established, the potential clinical implications are great, as GCA, with its possible vision loss
B3 3
B4 4
B5 5
, polymyalgia rheumatica and even eventually ischemic stroke
B6 6
B7 7
B8 8
B9 9
, might potentially be prevented by vaccination or other strategies.Human papillomavirus (HPV) is an increasingly common human pathogen in recent decades. It is a mucosotropic virus which is not thought of as spreading systemically. However, HPV genotype 16 has been found integrated into the genome of bacteria isolated from cervical cancer biopsies, and there is also published data showing HPV viral particles within peripheral nerves and small vascular endothelial cells adjacent to oral and cervical cancers as demonstrated by transmission electron microscopy
7
B10 10
. In this study, we sought to determine if there is an association between GCA and HPV.
Methods
With approval by the Washington University Human Research Protection Office (HRPO), we searched the Copath database of Barnes Jewish Hospital for temporal artery biopsy specimens from 1995 to 2008 and retrieved all specimens for which material was available. We identified approximately 60 cases, of which 43, (22 histologically positive and 21 histologically negative) had material available for review. There were five males and 17 females in the histologically positive GCA group with a mean age of 78.9 years. In the histologically negative group, there were four males and 17 females with a mean age of 67 years, and the control kidney vascular margin group had six males and nine females with a mean age of 38.2 years. There were no statistically significant difference in gender among the groups (p = 0.176). The clinical diagnosis of GCA was based on the criteria of the American College of Rheumatology
B11 11
. However, for our study, we considered the gold standard to be histologic evidence of temporal arteritis. We also randomly selected 15 renal artery vascular resection margins from nephrectomy specimens in patients without any history of vasculitis for use as the negative controls.Two methods were utilized to evaluate for HPV DNA. The first, INNO-LiPA HPV Genotyping Extra, was used to perform the testing in our research laboratory of the division of anatomic and molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis. All blocks were then submitted to an outside laboratory (CPA Laboratory, Louisville, KY) for independent preparation of genomic DNA and for testing employing the CervistaTM HPV HR (Hologic, Madison, WI, USA) assay.In both laboratories to avoid potential contamination, a maximum physical separation between the pre- and post-amplification steps was used. Separate pipettes and other lab materials were used as a part of good laboratory practice. The FFPE blocks were cut and processed under strict conditions to prevent DNA from being carried over from one case to the next during microtomy. Also a new blade was used for each case and the area was cleaned. Ice cubes used to cool blocks were discarded between cases.With regards to the technical limitations of this study, it is well known that formalin fixation randomly fragments DNA in a duration-dependent manner, resulting in a partial degradation. The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation. Due to this degradation, FFPE tissue is not suitable for amplification of large DNA segments. Nevertheless, PCR amplification of segments ranging up to 1300 base pair has been reported. On the other hand, incubation at an elevated temperature after proteinase K digestion partially removes formalin cross-linking of the DNA, improving yield as well as DNA performance in assays. Furthermore, in one of the techniques used in this study (INNO-LiPA HPV Genotyping), short-PCR-fragment (SPF 10) primers are employed, which amplify a 65 base pair segment of target DNA and this testing is considered to be one of the most sensitive PCR assays for the detection of HPV DNA.
Washington University testing
DNA extraction
DNA was extracted using PureGene Kit (Gentra, http://www.Gentra.com) as per the manufacturer's instructions from 10 μm sections cut from the paraffin blocks. The concentration of the prepped DNA was measured spectrophotometrically using Nanodrop. Detailed procedure information is available at their web site, but briefly, we placed five 10 micron sections of tissue into a 1.5 mL microcentrifuge tube and added 1.0 mL of xylene, vortexed, and incubated for five minutes with constant gentle mixing. Then we centrifuged it for five minutes at 13,000-16,000 x g. In the fume hood, discarded by pipetting the xylene supernatant and left behind the visible pellet (tissue). We repeated this xylene wash twice. We then added 1.0 mL 100% ethanol, vortexed, and incubated five minutes with constant gentle mixing at room temperature, centrifuged at 13,000-16,000 x g for five minutes to pellet the tissue, and discarded the ethanol. We repeated these ethanol washes twice. Subsequently we added 1.0 mL 70% ethanol, gently mixed, and centrifuged at 13,000-16,000 x g for five minutes at 4°C, removed all residual ethanol and allowed tissue pellet to dry by centrifugation under vacuum for five minutes. For cell lysis, we added 300 μl Cell Lysis Solution (Gentra Puregene™ kit) and gently vortexed for 30 seconds. Then we added three μl Puregene Proteinase K (20 mg/ml), and mixed by inverting 25 times and incubated the lysate at 55°C for three hours to overnight. We inverted the tube periodically during the incubation. Then was added three μl RNase A Solution to the cell lysate, and mixed by inverting the tube 25 times and incubated at 37°C for 15 min to one hour. For Protein Precipitation we cooled quickly the sample to room temperature by placing on ice and added 100 μl Protein Precipitation Solution (Gentra Puregene™ kit) to the cell lysate. The volume should be 1/3 of the Cell Lysis Solution in the tube. Subsequently, it is vortexed vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate. Then, we centrifuged it at 13,000-16,000 x g for five minutes. The precipitated proteins formed a tight pellet. If the protein pellet was not tight, we vortexed vigorously for 20 seconds at high speed, and then incubated on ice for 5 min. We then centrifuged at 13,000–16,000 x it g for three minutes. Using a pipette, we removed the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a clean 1.5 mL microcentrifuge tube. We added two μl (for 400 μl supernatant) of a DNA carrier (glycogen; to final concentration of 50-150 mcg/μl) to aid recovery of small DNA quantities and then vortexed them. We then added 400 μl 100% of isopropanol. Subsequently, it was mixed by inverting gently ~50 times until the white threads of DNA formed a visible clump, and then it was centrifuged at 13,000-16,000 x g for 10 minutes and the supernatant was poured off. We added 500 μl of 70% ethanol and inverted the tube to wash the DNA pellet. We centrifuged at 13,000-16,000 x g for 5 minutes, and carefully poured off the ethanol and inverted and blotted the liquid from the tube on clean absorbent paper and allowed to air dry for 10-15 minutes. Finally, we added 50 μl DNA Hydration Solution and incubated at 65°C for one hour to dissolve the DNA. We incubated at room temperature overnight with gentle shaking. Samples could then be centrifuged briefly and transferred to a storage tube. The concentration of the DNA used for INNO-LiPA HPV Genotyping Extra testing in each case was 50 ng.
INNO-LiPA HPV Genotyping testing
The INNO-LiPA HPV Genotyping Extra is based on the principle of reverse hybridization. Part of the L1 region of the human papillomavirus (HPV) genome is amplified using short-PCR-fragment assay (SPF10 primers), and the resulting biotinylated amplicons are then denatured and hybridized with specific oligonucleotide probes.An additional primer pair for the amplification of the human HLA-DPB1 gene is added to monitor sample quality and extraction. The length of the HLA-DPB1 fragment is 280 base pairs. All probes are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added, which binds to any biotinylated hybrid previously formed.Incubation with BCIP (5-Bromo-4-Chloro-3’-Indolyphosphate p-Toluidine Salt)/NBT (Nitro-Blue Tetrazolium Chloride) chromogen yields a purple precipitate, and the results are visually interpreted using the reference guide provided. An amplification kit (INNO-LiPA HPV Genotyping Extra Amp) is used for standardized preparation of biotinylated amplified material. This amplification kit is based on the polymerase chain reaction (PCR) using SPF10 primers.Amplification products are subsequently hybridized using a single typing strip on which 28 sequence-specific DNA probe lines and 4 control lines are fixed, which permits specific detection of 28 HPV genotypes, including all 18 high-risk genotypes, and 10 low-risk genotypes (HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74, and 82) as described by the manufacturer (Figure F3 3).
Figure 3INNO-LiPA typing on 16 patients. Strips # 5, 9, 11, 14, 15 and 16 are positive because they show clearly visible lines of hybridization
INNO-LiPA typing on 16 patients. Strips # 5, 9, 11, 14, 15 and 16 are positive because they show clearly visible lines of hybridization. The line patterns (corresponding to # 3 of the probe side bars) were compared to the interpretation chart supplied with the kit correspond with HPV type 16.
1471-2474-13-132-3
CPA Laboratory testing
DNA extraction
Slides were cut from the original blocks, and The QIAamp DNA FFPE Tissue kit (QIAGEN, http://www.qiagen.com) was used for purification of genomic DNA from formalin-fixed, paraffin-embedded tissues according to the manufacturer’s instructions with exception of incubation time. Overnight incubation in proteinase K for digestion of proteins/contaminants is not recommended by Qiagen but is something that CPA Laboratory has found useful to increase the nucleic acid elution.Briefly, the QIAamp DNA FFPE Tissue procedure consisted of several steps including removal of paraffin from slides using xylene, subsequent specimen lysis under denaturing conditions with proteinase K, incubation at 90°C to reverse all formalin cross-linking, and DNA binding to the membrane for removal of contaminants. Residual contaminants were washed away and pure, concentrated DNA was eluted from the membrane.
CervistaTM HPV HR testing
CervistaTM HPV HR
B12 12
is a qualitative, diagnostic test for the detection of DNA from 14 high-risk HPV types (i.e., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The CervistaTM HPV HR test uses the Invader® chemistry which is a signal amplification technique for detection of specific nucleic acid sequences. In this method two types of isothermal reactions are used: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal. During the primary reaction, two types of sequence specific oligonucleotides (i.e. a probe oligonucleotide and an Invader® oligonucleotide) bind to the target DNA recognition site. When those sequence specific oligonucleotides overlap by at least one base pair on the target sequence, an invasive structure forms that acts as a substrate for the Cleavase® enzyme. The enzyme cleaves the 5’ portion (flap) of the probe at the position of the overlap.The probes are present in large molar excess and cycle rapidly on and off the target sequence generating many cleaved 5’ flaps per target sequence. The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide creating another invasive structure that the Cleavase® enzyme recognizes as a substrate. The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces a fluorescence signal as the cleaved flaps cycle on and off. For each copy of target, the combined primary and secondary reactions result in 106– 107 fold signal amplification per hour. The reagents for this test are provided as three oligonucleotide master mixtures, which identify the 14 types of HPV arranged according to phylogenetic relatedness. Master mixture 1 identifies the positivity of genotypes 51, 56 and 66(MM1: HPV 51, 56 and 66). Master mixture 2 (MM2) shows the positivity of genotypes HPV 18, 39, 45, 59 and 68 and master mixture 3 (MM3) reveals the positivity of HPV genotypes 16, 31, 33, 35, 52 and 58. By design, the released 5'-flaps bind only to their respective FRET oligonucleotides to generate target-specific signal. A positive result indicates that at least one of the 14 high-risk types is present in the DNA sample. For each case 150 to 200 ng of DNA prepped from the FFPE tissues at CPA Labs were utilized for the Cervista HPV testing. The sensitivity of this test is set at 5000 copies of HPV DNA. The Cervista assay has an internal control to verify if the assay worked. If the internal control does not pass, it suggests either the sample is degraded or extraction failed or the PCR did not work.
Statistics
Statistical analyses were performed using the Chi-Square, T-test and Fisher's exact tests to evaluate the correlation between the presence of HPV DNA and other variables. P values < 0.05 were considered significant. All statistical analyses were performed using the SAS v.9.1 system software (SAS Institute Inc., Cary, NC).
Results
Using the INNO-LiPA HPV Genotyping Extra, 16 of the 22 (17 female and 5 male) biopsy-confirmed GCA cases were positive for HPV. Twelve out of the 16 (75%) HPV-positive GCA-positive patients were female. Among these 16 positive cases, 5 had a HPV 6 alone (31%), 3 cases had HPV 16 alone (19%), 7 cases had both types 6 and 16 (44%), and only one case was extensively "multi-genotype" showing types 6, 16, 31, 33, and 40 (6%) (Testing results are presented in Tables tblr tid T1 1 and T2 2). In all cases where HPV was detected, type 6, type 16, or both were present (100%). The cases were also tested by the CervistaTM HPV HR assay for confirmation, which only evaluates for high risk HPV types (not for HPVs 6 and 11). Two cases which were HPV type 6 by INNO-LiPA (cases # 4 and 6) had low genomic DNA when prepped for the CervistaTM HPV HR testing, so could not be evaluated by this method. All positive cases of the CervistaTM HPV HR were from the Master Mix 3 which includes the HPV genotypes 16, 31, 33, 35, 52 and 58. As expected, all cases for which genotype 6 was present in the INNO-LiPA method were negative with the Cervista method since low risk types are not evaluated in that assay. All negative cases with INNO-LiPA HPV Genotyping Extra were negative also with CervistaTM HPV HR testing. As such, there was 100% agreement between the two testing methods on the histologically positive temporal arteritis cases.
table
Table 1
Results of the HPV testing by both different methods.
tgroup align left cols 9
colspec center colname c1 colnum 1 colwidth 1*
c2 2
c3 3
c4 4
c5 5
c6 6
c7 7
c8 8
c9
thead valign top
row rowsep
entry
Histologically Positive GCA cases
INNO LiPA Results
Cervista Results
Histologically Negative GCA cases
INNO LiPA Results
Cervista Results
Kidney Vascular margin
INNO LiPA Results
Cervista Results
tfoot
LGD: Low Genomic DNA.
POS MM3: Positive in Master Mix 3.
HPV = Human papillomavirus.MM1: HPV types 51, 56 and 66.MM2: HPV types 18, 39, 45, 59 and 68.MM3: HPV types 16, 31, 33, 35, 52 and 58.
tbody
1
Neg
Neg
1
Neg
Neg
1
Neg
Neg
2
6
Neg
2
Neg
LGD
2
Neg
Neg
3
Neg
Neg
3
Neg
Neg
3
Neg
Neg
4
6
LGD
4
6
Neg
4
Neg
Neg
5
6, 16, 31, 33, 40
pos MM3
5
Neg
Neg
5
Neg
Neg
6
6
LGD
6
52
pos MM3
6
Neg
Neg
7
6
Neg
7
Neg
Neg
7
Neg
Neg
8
6
Neg
8
Neg
Neg
8
6
Neg
9
Neg
Neg
9
Neg
Neg
9
16
pos MM3
10
6, 16
pos MM3
10
Neg
Neg
10
Neg
Neg
11
16
pos MM3
11
Neg
Neg
11
16
pos MM3
12
6, 16
pos MM3
12
Neg
Neg
12
Neg
Neg
13
16
pos MM3
13
Neg
Neg
13
Neg
Neg
14
6, 16
pos MM3
14
Neg
Neg
14
Neg
Neg
15
Neg
Neg
15
Neg
Neg
15
Neg
Neg
16
Neg
Neg
16
16
pos MM3
17
Neg
Neg
17
Neg
Neg
18
6, 16
pos MM3
18
16
pos MM3
19
6, 16
pos MM3
19
Neg
Neg
20
6, 16
pos MM3
20
16
pos MM3
21
16
pos MM3
21
Neg
Neg
22
6, 16
pos MM3
Table 2
Correlation between the three study groups for HPV test results
Biopsy Positive GCA*
Biopsy Negative GCA
All Temporal Artery Biopsies
Renal Artery Controls**
*Differences between biopsy positive and biopsy negative cases and between biopsy positive and control cases were statistically significant ((p = 0.001 and 0.002, respectively).**Difference between biopsy negative and control cases was not statistically significant (p = 0.79).
GCA = giant cell arteritis; HPV = human papillomavirus.
HPV Positive
16 (73%)
5 (24%)
21(49%)
3
HPV Negative
6 (27%)
16 (76%)
22 (51%)
12
Total
22
21
43
15
Among the 21 cases of GCA histologically negative biopsies, 5 were positive for HPV by INNO-LiPA HPV Genotyping Extra, of which 3 (60%) were genotype 16, one (20%) was genotype 6 and one (20%) was genotype 52. CervistaTM HPV HR testing revealed positivity of Master Mix 3 for all 4 cases which were positive by INNO-LiPA HPV Genotyping Extra for high risk HPV. Case number 2 which was negative with INNO-LiPA was of low genomic DNA with Cervista method. As such, there was 100% agreement between the two testing methods on the histologically negative temporal artery biopsy cases.Among the 15 nephrectomy vascular resection margins (controls), 3 were positive for HPV by INNO-LiPA HPV Genotyping Extra. Two cases showed genotype 16 (1 case of Wilms' tumor and 1 of acute and chronic pyelonephritis), and one genotype 6 (a case of papillary urothelial carcinoma). CervistaTM HPV HR testing revealed positivity of Master Mix 3 for both cases which were positive for high risk HPV by INNO-LiPA HPV Genotyping Extra. As such, there was 100% agreement between the two testing methods on the 15 vascular margin control cases.The association between HPV positivity and histologically confirmed GCA was statistically significant (p = 0.0013). There was also a statistically significant association between HPV positivity and Caucasian race (p = 0.0339). No other associations were statistically significant.In multivariate analysis (Table T3 3), subjects with histologically confirmed GCA had a 56-fold higher likelihood of having HPV positivity adjusted by gender, age and race (OR, point estimate 56.01; 95% CI 3.5-895.68).
Table 3
Odds Ratios for variable and presence of histologically positive temporal artery biopsies
Variable
Point Estimate
nameend namest
95% Wald Confidence Interval
HPV = human papillomavirus.
Gender F
vs.
M
char .
2.762
0.329
23.222
Race: African American
vs.
Caucasian
0.113
0.006
2.199
HPV
56.014
3.503
895.681
Age
1.196
1.053
1.358
Discussion
Giant cell (temporal) arteritis is a chronic vasculitis involving medium and large size arteries that typically affects individuals older than 50 years of age.
1
3
4
Although it usually affects the superficial temporal arteries, it can also affect the aorta, carotid, subclavian, vertebral, and iliac arteries. The classical picture of granulomatous inflammation with multinucleated giant cells is observed in approximately 50% of the patients.Histologically, the disease progresses from minimal involvement of vessels, with only collections of lymphocytes confined to the internal or external elastic lamina or adventitia to a panarteritis with segmental areas of necrosis of the arterial wall and extensive destruction of the elastic laminae, a feature that can be clearly demonstrated with special stains for elastic fibers (Figures 1 and 2).The etiology of the inflammatory reaction in the GCA has not been identified. However, it has been demonstrated that there is a restricted clonal expansion of tissue-infiltrating T cells in these lesions, which suggests that they are reacting to a specific antigen located within the affected arterial wall which is thus eliciting the disease
B13 13
. The most recent hypothesis regarding the etiology of GCA contends that a response to endothelial injury (maybe due to infection) leads to an inappropriate activation of T-cell-mediated immunity
B14 14
B15 15
. The subsequent release of inflammatory mediators within the arterial vessel wall can attract macrophages which then become multinucleated giant cells, creating the characteristic histology of this disease and also leading to an oligoclonal expansion of T-cells directed against antigens within or near the elastic lamina. This cascade of events in due course results in vessel wall damage, intimal hyperplasia, and eventual stenotic occlusion.Infectious agents have been considered in the past as the etiology of GCA. Elling et al., by indirect serological evidence, found high incidence of GCA within a population associated with Chlamydia pneumoniae, Mycoplasma pneumoniae and Parvovirus B19
B16 16
. Published data from Wagner et al.
15
and Powers et al.
B17 17
reported a relationship between GCA, C. pneumonia and herpes simplex virus (HSV). However, further studies were unable to detect the presence of these infectious agents. Cooper et al.
14
and Cankovic and Zarbo
B18 18
, could not confirm the association between GCA and C. pneumonia, HSV, varicella zoster virus (VZV), Epstein–Barr virus (EBV), or human herpesvirus 7 (HHV7). In addition, Rodriguez-Pla et al.
B19 19
, Helweg-Larsen
B20 20
and other authors
B21 21
B22 22
B23 23
could not confirm the presence of herpes viruses, varicella zoster virus, parvovirus, or C. pneumoniae in temporal artery GCA. Due to this conflicting and contradictory data, no conclusive link has, to date, been demonstrated between a GCA and a specific infectious agent.The role of human papillomavirus (HPV) in the development of cervical, head and neck, anal and skin cancers is well known. Molecular and epidemiological studies have shown that persistent HPV infection is the most important risk factor for cervical cancer
B24 24
B25 25
. High risk HPV also has an important role in anogenital and oropharyngeal squamous cell carcinoma
B26 26
. They are also implicated with skin cancer in individuals with epidermodysplasia verruciformis
B27 27
and can induce a variety of proliferative lesions, such as warts and laryngeal papillomas. There are very few associations between HPV and non-neoplastic diseases.Interestingly, Ma et al.
10
reported the presence of HPV type 16 in the genome of bacterial strains (Enterococcus Staphylococcus Bacillus and Corynebacterium) and demonstrated the HPV viral particles by transmission electron microscopy in those bacteria. In addition, HPV type 16 has been detected in peripheral nerves, and vascular endothelial cells
7
. Another possible pathway is for HPV to disseminate systemically. This has long been debated
B28 28
. The role of viremia in the pathogenesis of HPV-related diseases is still unclear, although HPV DNA has been detected in peripheral blood in some studies, though in varying amounts
B29 29
B30 30
B31 31
B32 32
.In this study, we found that HPV DNA, of both high and low risk types, is present in formalin-fixed, paraffin-embedded temporal artery biopsy specimens, and in statistically significantly higher numbers in the arteritis specimens compared to histologically-negative temporal artery biopsies and nephrectomy vascular margin controls. The validity of these results was confirmed with exactly matching results by an outside laboratory that independently prepped DNA from the paraffin blocks and utilized a completely different detection method. Our results are admittedly surprising and bring many questions about what HPV's role, if any, would be in temporal arteritis. HPV has been associated with tumorigenesis, and many studies have investigated HPV-related modification of the immune system to establish infection. However, we are not aware of any literature citing HPV as an inciting agent for inflammatory diseases. Our findings do support the notion that HPV can disseminate systemically, but do not, by themselves, tell us anything specific about the pathophysiological relationship between the virus and temporal arteritis, if there even is one. Further studies are necessary to address this.It is important to mention that a negative temporal artery biopsy does not rule out the diagnosis of temporal artery GCA, since the changes can be patchy with skip areas of uninvolved artery. Although serial sectioning of the temporal artery biopsy is recommended, not all cases will be diagnosed histologically
B33 33
. Actually, after review of the medical records of five of our biopsy-negative HPV-positive cases, in one case, the patient showed dramatic improvement after treatment with steroids (possible false negative case which was not included as a HPV-positive case in our statistical analysis).It is also worth noting that of the 16 histologically-proven GCA cases that had HPV, 11 were high risk genotype 16. Only five cases were positive for low risk HPV genotype 6. Although, the importance of distinguishing low risk from high risk HPV genotypes has been established in the pathogenesis of neoplasms, what its significance might be in inflammatory disorders such as GCA is currently unknown.
Conclusion
In summary, we have identified HPV DNA in the majority of histologically-proven giant cell (temporal) arteritis specimens and validated these results by a completely independent outside laboratory assay. The association raises questions regarding the biology of HPV infections, when dissemination occurs, and what this dissemination means clinically. Such studies are required to understand the significance of the association in our series.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AM conceived the study, carried out the molecular testing, analyzed the data, and drafted the manuscript. JDP and JSL, designed the study, collected data, helped interpret findings and critically revised the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgment
The authors would like to thank Jean (Qin) Zhang from the Division of Biostatistics of Washington University in St. Louis for her expert statistical analysis of our data. We would also like to thank to Dr. Xiaopei Zhu for outstanding expert technical assistance with the INNO LiPA PCR testing. Finally, we especially thank Sameer S. Talwalkar, MD, FCAP, Medical Director, Molecular Diagnostics of CPA Lab in Louisville, KY, for excellent assistance with Cervista testing.
refgrp Blood VesselsBurkeAVirmaniRSternberg's Diagnostic Surgical Pathology. Volume 1. 5th editionpublisher Philadelphia: Lippincott Williams & Wilkinseditor Mills SE20091236lpage 1238pmcid 3445545link fulltext 23028547Does this patient have temporal arteritis?SmetanaGWShmerlingRHJAMA200228719210110.1001/jama.287.1.9211754714Visual loss in giant cell arteritisGordonLKLevinLAJAMA1998280438538610.1001/jama.280.4.3859686559Clinical features in patients with permanent visual loss due to biopsy-proven giant cell arteritisFontCCidMCColl-VinentBLopez-SotoAGrauJMBr J Rheumatol199736225125410.1093/rheumatology/36.2.2519133940Visual morbidity in giant cell arteritisLiuGTGlaserJSSchatzNJSmithJLClinical characteristics and prognosis for vision. Ophthalmology19941011117791785Temporal arteritis: a cough, toothache, and tongue infarctionHellmannDBJAMA2002287222996300010.1001/jama.287.22.299612052130The presence of human papillomavirus 16 in neural structures and vascular endothelial cellsFuleTMatheMSubaZCsapoZSzarvasTTatraiPPakuSKovalszkyIVirology2006348228929610.1016/j.virol.2005.12.04316499942Epidemiology of giant cell arteritis and polymyalgia rheumaticaGonzalez-GayMAVazquez-RodriguezTRLopez-DiazMJMiranda-FilloyJAGonzalez-JuanateyCMartinJLlorcaJArthritis Rheum200961101454146110.1002/art.2445919790127Inflammatory markers and strokeElkindMSCurr Cardiol Rep2009111122010.1007/s11886-009-0003-219091170Human papillomavirus type 16 exists in bacteria isolated from cervical cancer biopsiesMaZLiuLZhangFYuMWangKLuoJLiuKChenBXuLJ Int Med Res20093741065107419761689The American College of Rheumatology 1990 criteria for the classification of giant cell arteritisHunderGGBlochDAMichelBAStevensMBArendWPCalabreseLHEdworthySMFauciASLeavittRYLieJTetal Arthritis Rheum1990338112211282202311Third Wave Technologies, The Cervista TM HPV HR
http://www.cervistahpv.com/pdf/Cervista_HPV_HR_PI_15-3100_Rev_C.pdf
Distinct vascular lesions in giant cell arteritis share identical T cell clonotypesWeyandCMSchonbergerJOppitzUHunderNNHicokKCGoronzyJJJ Exp Med1994179395196010.1084/jem.179.3.95121914128113687Infection and temporal arteritis: a PCR-based study to detect pathogens in temporal artery biopsy specimensCooperRJD'ArcySKirbyMAl-BuhtoriMRahmanMJProctorLBonshekREJ Med Virol200880350150510.1002/jmv.2109218205226Detection of Chlamydia pneumoniae in giant cell vasculitis and correlation with the topographic arrangement of tissue-infiltrating dendritic cellsWagnerADGerardHCFresemannTSchmidtWAGromnica-IhleEHudsonAPZeidlerHArthritis Rheum20004371543155110.1002/1529-0131(200007)43:7<1543::AID-ANR19>3.0.CO;2-810902759Synchronous variations of the incidence of temporal arteritis and polymyalgia rheumatica in different regions of Denmark; association with epidemics of Mycoplasma pneumoniae infectionEllingPOlssonATEllingHJ Rheumatol19962311121198838518High prevalence of herpes simplex virus DNA in temporal arteritis biopsy specimensPowersJFBedriSHusseinSSalomonRNTischlerASAm J Clin Pathol2005123226126410.1309/2996TT2CTLTKN0KT15842052Failure to detect human herpes simplex virus, cytomegalovirus, and Epstein-Barr virus viral genomes in giant cell arteritis biopsy specimens by real-time quantitative polymerase chain reactionCankovicMZarboRJCardiovasc Pathol200615528028610.1016/j.carpath.2006.05.00716979035No detection of parvovirus B19 or herpesvirus DNA in giant cell arteritisRodriguez-PlaABosch-GilJAEchevarria-MayoJERossello-UrgellJSolans-LaqueRHuguet-RedecillaPStoneJHVilardell-TarresMJ Clin Virol2004311111510.1016/j.jcv.2004.05.00315288607No evidence of parvovirus B19, Chlamydia pneumoniae or human herpes virus infection in temporal artery biopsies in patients with giant cell arteritisHelweg-LarsenJTarpBObelNBaslundBRheumatology (Oxford)200241444544910.1093/rheumatology/41.4.445No correlation between giant cell arteritis and Chlamydia pneumoniae infection: investigation of 189 patients by standard and improved PCR methodsNjauFNessTWittkopUPancratzTEickhoffMHudsonAPHallerHWagnerADJ Clin Microbiol20094761899190110.1128/JCM.02438-08269111919386842Chlamydia pneumoniae not detected in temporal artery biopsies from patients with temporal arteritisHaugebergGBieRNordboSAScand J Rheumatol200029212712810.1080/03009740075000194110777127Search for varicella zoster virus in giant cell arteritisNordborgCNordborgEPetursdottirVLaGuardiaJMahalingamRWellishMGildenDHAnn Neurol199844341341410.1002/ana.4104403239749614Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population studyCuschieriKSCubieHAWhitleyMWGilkisonGArendsMJGrahamCMcGooganEJ Clin Pathol200558994695010.1136/jcp.2004.022863177081216126875Papillomaviruses and cancer: from basic studies to clinical applicationZur HausenHNat Rev Cancer20022534235010.1038/nrc79812044010Human papillomavirus-associated head and neck cancer is a distinct epidemiologic, clinical, and molecular entityGillisonMLSemin Oncol200431674475410.1053/j.seminoncol.2004.09.01115599852Evidence for the association of human papillomavirus infection and cutaneous squamous cell carcinoma in immunocompetent individualsMasiniCFuchsPGGabrielliFStarkSSeraFPlonerMMelchiCFPrimaveraGPirchioGPicconiOPetaseccaPCattaruzzaMSPfisterHJAbeniDArch Dermatol2003139789089410.1001/archderm.139.7.89012873884Detection and quantitation of HPV 16 and 18 in plasma of Indian women with cervical cancerGnanamonyMPeedicayilASubhashiniJRamTSRajasekarAGravittPAbrahamPGynecol Oncol2010116344745110.1016/j.ygyno.2009.10.08119922992Detection and quantitation of human papillomavirus type 16, 18 and 52 DNA in the peripheral blood of cervical cancer patientsHoCMYangSSChienTYHuangSHJengCJChangSFGynecol Oncol200599361562110.1016/j.ygyno.2005.07.00416099020Detection and typing of minimal human papillomavirus DNA in plasmaWeiYCChouYSChuTYInt J Gynaecol Obstet200796211211610.1016/j.ijgo.2006.08.01217250836Detection and quantitation of human papillomavirus DNA in the plasma of patients with cervical carcinomaDongSMPaiSIRhaSHHildesheimAKurmanRJSchwartzPEMortelRMcGowanLGreenbergMDBarnesWASidranskyDCancer Epidemiol Biomarkers Prev20021113611815394Quantification of human papillomavirus DNA in the plasma of patients with cervical cancerYangHJLiuVWTsangPCYipAMTamKFWongLCNgTYNganHYInt J Gynecol Cancer200414590391010.1111/j.1048-891X.2004.014528.x15361202Disease pattern in cranial and large-vessel giant cell arteritisBrackAMartinez-TaboadaVStansonAGoronzyJJWeyandCMArthritis Rheum199942231131710.1002/1529-0131(199902)42:2<311::AID-ANR14>3.0.CO;2-F10025926
Pre-publication historyThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/132/prepub


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Abstract
Background
To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the temporal artery.
Methods
The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR) with INNO-LiPA HPV Genotyping Extra, 2) Cervista™ HPV HR. All cases were from the files of the Barnes-Jewish Hospital and Washington University in St. Louis.
Results
HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only three of 15 (20%) were positive for HPV DNA. The second, independent method (CervistaTM) confirmed the aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which it was not possible to perform the test. The differences in HPV positivity between the histologically positive and negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls were both statistically significant (p = 0.001 and 0.002, respectively).
Conclusions
The results of our study revealed a statistically significant association between HPV positivity and biopsy confirmed temporal giant cell arteritis GCA (p = 0.001). Further studies are necessary to elucidate the pathophysiology underlying this association.
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Lewis, James S Jr
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Amir Mohammadi et al.; licensee BioMed Central Ltd.
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BMC Musculoskeletal Disorders. 2012 Jul 25;13(1):132
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