ARTICLE
Auteur(s) : Andrea L Neimann1,2,4, Richard L
Hodinka2,3, Yagnya B Joshi2,3, Michael
Elkan2,3, Abby S Van Voorhees1,2,4, Joel M
Gelfand1,2,4
1Department of Dermatology, University of
Pennsylvania, 3600 Spruce Street, 2 Maloney Building, Philadelphia,
PA 19104, USAFax: (+1) 215-615-4966
2University of Pennsylvania School of Medicine,
Philadelphia, PA, USA
3Departments of Pediatrics and Pathology, Children’s
Hospital of Philadelphia (CHOP), Philadelphia, PA, USA
4Center for Clinical Epidemiology and Biostatistics,
University of Pennsylvania, Philadelphia, PA, USA
accepté le 15 Mai 2006
Psoriasis is a common chronic immune mediated disease that affects
2% of the general population [1-5]. The pathophysiology of
psoriasis is characterized by increases in antigen presentation, up
regulation of T cells, and TH-1 inflammatory cytokines resulting in
epidermal hyper-proliferation and in some patients an inflammatory
arthropathy [6]. The cause of the increased inflammatory activity
in psoriasis is unknown. Interestingly, transgenic mouse models
demonstrate that skin infected with human herpesviruses (HHV) such
as Epstein-Barr virus (EBV) resembles the phenotype of psoriasis
[7]. The role of infection with human herpesviruses in psoriasis
patients, however, has not been well characterized.Since psoriasis
is mediated by abnormal T cell function, this immune dysregulation
may be associated with an inadequacy in controlling latent EBV
and/or HHV-6 infection, given that T cells have been shown to be
critical to controlling latent infection with EBV and HHV-6 [8-10].
Also, chronic viral infections such as EBV have been shown to
augment the production of cytokines such as TNF-alpha by human
monocytes and macrophages which are known to be associated with the
psoriasis phenotype [8].Patients with severe psoriasis are often
exposed to medications which are immunosuppressive.
Immunosuppressive therapies are well known to increase the risk of
EBV-associated lymphomas, with the magnitude of risk being related
to the degree of immunosuppression. In psoriasis patients there
have been case reports of EBV-associated lymphomas in patients
treated with either low dose methotrexate or cyclosporine, and
reports of atypical blood lymphocytes in psoriasis patients treated
with low dose cyclosporine therapy [11-13]. These case reports
suggest that immunosuppressive therapies used for psoriasis may
induce lymphoma through decreased immunologic control of latent EBV
infection.Although studies have indicated a potential association
of EBV and psoriasis as well as the potential for psoriasis
therapies to induce EBV-associated lymphoma, there have been no
studies examining the activity of EBV or similar viruses such as
HHV-6 using sensitive techniques in these patients. The objective
of this study was therefore to determine if patients with severe
psoriasis and patients with severe psoriasis being treated with
immunosuppressive therapies have higher rates of EBV and HHV-6
replication in their peripheral white blood cells than patients who
are otherwise healthy as measured by real-time polymerase chain
reaction (PCR).
Materials and methods
Patient selection
Study subjects were recruited from the population of patients seen
in the outpatient practices of the Department of Dermatology at the
Hospital of the University of Pennsylvania. The Internal Review
Board (IRB) of the Hospital of the University of Pennsylvania
approved the protocol prior to the start of the study. We included
only patients between the ages of 30 and 60 and excluded all
patients with a history of recent immunization or a clinically
significant infection within 2 months of study entry. Patients
being treated with phototherapy or oral retinoids needed to be off
these medications for a period of at least 4 weeks in order to be
eligible for the study. We also excluded pregnant women, minors and
patients unable to give voluntary consent.
Study groups were comprised of healthy controls, patients with
severe psoriasis, and patients with psoriasis being treated with
immunosuppressive medications. Healthy controls were defined as
ambulatory dermatology patients with no history of an immune
mediated disease and no history of treatment with an
immunosuppressive therapy. Severe psoriasis was defined as patients
with greater than 10% body surface area (BSA) affected with
psoriasis, and with typical plaques being at least moderately red
in color, having moderate thickness or with moderate scaling as
defined by the Psoriasis Activity Severity Index. These patients
must not have been on immunosuppressive medications within the
preceding 2 months. Finally, psoriasis patients treated with
immunosuppressive medications were required to be on therapy for at
least 8 weeks. Immunosuppressive mediations that qualified for
inclusion in the study were etanercept, efalizumab, infliximab,
methotrexate (at least 10 mg/wk), cyclosporine (at least
3 mg/kg), and alefacept.
Specimen collection
A total of 4-7 mL of whole blood was collected in sterile tubes
containing EDTA as the anticoagulant. Leukocytes were isolated from
2.0 mL of the whole blood using 6.0 mL of Puregene
(Gentra Systems, Minneapolis, MN) red blood cell lysis solution.
The white blood cells were then pelleted by centrifugation for
2 min at 2,000 × g, washed once in 15 ml of PBS w/o
Ca++ or Mg++, and resuspended in 2.0 ml of
PBS. The cells were counted using a hemocytometer and the
concentration of cells was adjusted to 2.0 × 106
cells/mL by diluting in PBS. Single-use aliquots of 250 μL of
suspended leukocytes from each specimen were stored at
– 70 oC in Sarstedt cryovials.
Nucleic Acid Isolation
The MagNA Pure LC instrument (Roche Diagnostics, Indianapolis, IN)
and Total Nucleic Acid Isolation Kit (Roche Diagnostics) were used
for the automated extraction and isolation of EBV or HHV-6 DNA from
200 μL of purified white blood cells. The samples were dissolved
and simultaneously stabilized by incubation with a buffer
containing denaturing agents and proteinase K. Nucleic acids
released from the samples were bound to the surface of magnetic
glass particles and unbound substances were removed by several
washes. The purified nucleic acids were then eluted in a low salt
buffer. The isolation process was performed within a plastic
cartridge that contained 32 sample wells. Isolated total nucleic
acids was diluted in a final volume of 66 μl of elution buffer and
immediately processed for real-time PCR.
Primers and Probe for EBV and HHV-6 DNA Real-Time PCR
The sequences for the forward and reverse primers and corresponding
fluorogenic probes used for the individual EBV and HHV-6 DNA
real-time TaqMan PCR assays are shown in table 1( Table 1 ).
The primers amplify a 74 bp fragment of the nonglycosylated
membrane protein BNRF1 p143 of EBV and a 173-176 bp product of the
U65-U66 genes of HHV-6, respectively [14, 15].
Table 1 Sequences for primers and probes used for
realtime TaqMan PCR assays
|
Primer/Probe
|
Sequence
|
|
EBV
|
|
|
Forward Primer
|
5′-GGA ACC TGG TCA TCC TTG C-3′
|
|
Reverse Primer
|
5′-ACG TGC ATG GAC CGG TTA AT-3′
|
|
TaqMan Probe
|
5’-(FAM)-CGC AGG CAC TCG TAC TGC TCG CT -(TAMRA)-3’
|
|
HHV-6
|
|
|
Forward Primer
|
5′-GAC AAT CAC ATG CCT GGA TAA TG-3′
|
|
Reverse Primer
|
5′-TGT AAG CGT GTG GTA ATG GAC TAA-3′
|
|
TaqMan Probe
|
5′-(FAM)-AGC AGC TGG CGA AAA GTG CTG TGC-(TAMRA)-3′
|
Real-Time TaqMan PCR
Individual EBV and HHV-6 DNA real-time TaqMan PCR assays were
performed in 50 μL volumes containing 1X TaqMan Universal Master
Mix (Applied Biosystems, Foster City, CA), 900 nM of forward and
reverse primers, 200 nM of the fluorogenic probe, nuclease-free
water with yeast tRNA (60 ng/mL; Roche Molecular Biochemicals,
Indianapolis, IN) and 5 μL of isolated total nucleic acid. The
mixtures were added to individual wells of 96-well reaction plates,
the plates were sealed with adhesive optical covers, and then
rotated on an orbital shaker for 30 sec to thoroughly mix and
remove air bubbles in the wells. The amplification and detection
were completed using an ABI Prism 7000 Sequence Detection System
(Applied Biosystems). The thermal cycling parameters consisted of 1
cycle for 2 min at 50 oC to activate the
uracil N’-glycosylase to prevent possible PCR contamination from
previous reactions, 1 cycle for 10 min at
95 oC to inactivate the uracil N’-glycosylase and
to activate the AmpliTaq Gold DNA polymerase, and 45 two-step
cycles of 15 sec at 95 oC and 60 sec at
60 oC. Positive and negative controls were
processed with each batch of clinical specimens from extraction of
nucleic acids through the detection of amplified product. Negative
controls consisted of purified human white blood cells negative for
EBV and HHV-6 DNA by real-time PCR. Following purification as
described above, the negative control cells were counted using a
hemocytometer and the concentration was adjusted to 2.0 ×
106 cells/mL by diluting in PBS. EBV-infected Raji cells
(CCL-86, American Type Culture Collection, Rockville, MD) at 2.0 ×
106 cells/mL and 2 × 107 viral particles/ml
of HHV-6 strain Z-29 Variant B (Advanced Biotechnologies, Inc.,
Columbia, MD) were used as positive controls. Single-use aliquots
of 250 μL of suspended material from each control were stored at
– 70 oC in Sarstedt cryovials. Specimens and
controls were tested in singlet and were considered positive when
the generated fluorescence signal at the threshold cycle
(Ct) value exceeded a defined threshold limit.
Statistical analysis
Our primary analysis compared the rate of being positive for EBV or
HHV-6 DNA by PCR in severe psoriasis patients or severe psoriasis
patients treated with immunosuppressive medications, to that in the
healthy control population using the Fishers exact test.
This study was powered to rule out the presence of EBV
replication in greater than 11% of patients in the psoriasis
groups. For example, if no patients within a particular psoriasis
study group have a positive PCR level of EBV then that would rule
out a true rate of EBV replication in the blood of 11% or greater
based on a 95% confidence interval. Based on the published
literature, we expected that healthy controls would not have
measurable PCR levels of EBV or HHV-6.
Results
Patient characteristics are summarized in table 2. The mean age for
healthy controls, patients with severe psoriasis not on
immunosuppressive treatment, and patients with severe psoriasis on
immunosuppressive treatment was 39, 45 and 46 respectively (P =
0.1, Kruskall Wallis). Women made up sixty percent of healthy
controls, thirty-six percent of patients with severe psoriasis, and
thirty-eight percent of patients on immunosuppressive treatment (P
= 0.45). Median body surface area of involvement was 11.5%
(interquartile range 2.5-20%) for patients with severe psoriasis on
immunosuppressive therapy and was 20% (interquartile range 15-35%)
in patients with severe psoriasis, not on immunosuppressive
therapy. In patients on immunosuppressive therapy, 9 were on
methotrexate (34.62%), 2 were or cyclosporine (7.69%), 14 were on
etanercept (53.85%), and 1 was on alefacept (3.85%) (table 2( Table 2 )).
EBV and HHV-6 DNA in patients and controls
Using real-time Taqman PCR we detected no HHV-6 or EBV DNA in any
of the healthy controls (0/10; 95% confidence interval 0-0.26). We
also found no evidence of HHV-6 or EBV DNA in purified peripheral
white blood cells of patients in either of the psoriasis groups
(0/26 in patients on immunosuppressive therapy; 95% confidence
interval 0-0.11 and 0/25 in patients with severe psoriasis not on
immunosuppressive therapy; 95% confidence interval 0-0.11). Results
are summarized in table 3( Table 3
).
Table 2 Patient characteristics
|
Healthy control
|
Psoriasis on treatment
|
Psoriasis not on treatment
|
|
Total number patients (N)
|
10
|
26
|
25
|
|
Mean age
|
39
|
46
|
45
|
|
Females
|
6(60%)
|
10(38%)
|
9(36%)
|
|
Median BSA
|
|
11.5% (IQR 2.5-20%)
|
20% (IQR 15-35%)
|
|
Medications:
|
|
|
|
|
Methotrexate
|
|
9(34.62%)
|
|
|
Cyclosporine
|
|
2(7.69%)
|
|
|
Etanercept
|
|
14(53.85%)
|
|
|
Alefacept
|
|
1(3.85%)
|
|
Table 3 HHV-6 AND EBV DNA levels in psoriasis patients
and controls
|
|
|
Total (N)
|
95% Confidence interval
|
|
Healthy controls
|
0
|
0
|
10
|
0-0.26
|
|
Psoriasis on treatment
|
0
|
0
|
26
|
0-0.11
|
|
Psoriasis not on treatment
|
0
|
0
|
25
|
0-0.11
|
Discussion
More than 90% of adults have been infected with one or more members
of the human herpesvirus family, including EBV and HHV-6 [16-19].
Primary infection with these viruses results in the establishment
of a latent infection; reactivation can occur in response to
different stimuli, particularly immunosuppression [16, 19, 20]. The
immunological nature of psoriasis suggests a potential association
with latent viral infections such as EBV or HHV-6.
The results of this study demonstrate that having severe
psoriasis is not associated with increases in EBV and HHV-6
replication. This finding is important as it suggests that the
immunologic abnormalities that create the psoriasis phenotype do
not affect the patient’s ability to control latent viral
infections. Additionally, the results indicate that unlike other
TH-1 mediated diseases, psoriasis is not associated with
abnormalities in EBV or HHV-6 infection. For example, HHV-6 has
been associated with Sjogren’s disease and systemic lupus
erythematosis, whereas EBV has been associated with rheumatoid
arthritis, Sjogren’s disease, and multiple sclerosis [21-31].
The results of this study also suggest that immunosuppressive
therapies commonly used to treat severe psoriasis do not alter the
patients’ ability to control latent EBV or HHV-6 infection. These
results are important given that EBV and HHV-6 infections are
associated with numerous other disease states, including lymphomas.
For example, previous studies have indicated that latent viral
infections with Epstein Barr virus (EBV) and HHV-6 and may be
associated with Non Hodgkin’s lymphoma (both B cell and T cell) and
Hodgkin’s lymphoma [19, 21, 32-37] in those undergoing
immunosuppressive treatment as well as in otherwise immune
competent individuals [21, 26]. Although some large epidemiologic
studies have demonstrated an increased risk of lymphoma in
psoriasis patients, our current study does not support that this
observed increased risk is due to abnormalities in control of
latent EBV or HHV-6 infection [4, 35, 38].
To the best of our knowledge, this study is the first systematic
investigation of EBV and HHV-6 infection in patients with
psoriasis. A particular strength of this study is the use of an
ultra-sensitive PCR assay of EBV and HHV-6 activity. The assay we
performed is routinely used by our clinical lab for the measurement
of human herpes viruses in children at risk for post transplant
lymphoproliferative disease and therefore our assay has high
quality control in order to ensure the identification of HHV with a
high degree of sensitivity and specificity. None of the patients in
our study demonstrated any evidence of active EBV or HHV-6
replication. This observation is consistent with previous studies
which have demonstrated that in healthy volunteers EBV and HHV-6
replication is generally not detectable in significant levels in
the plasma, serum or peripheral blood mononuclear cells based on
PCR [14, 17, 33, 39]. In immunosuppressed transplant recipients
without EBV related disease, observed circulatory EBV DNA levels
are generally higher than those in healthy sero-positive donors
(mean load of 440 copies per ml of plasma versus 0 copies per mL of
plasma respectively) and in those with posttransplant
lymphoproliferative disorders PCR levels average 544,570 copies per
mL [14, 40].
As with all studies, there are limitations to consider. First,
although the sample size was relatively large, we could not rule
out that a small percentage of patients with psoriasis or those
treated with immunosuppressive regimens do have an increased rate
of EBV or HHV-6 replication. This limitation is based on our 95%
confidence intervals that would rule out a true rate of EBV
replication in the blood of 11% (of psoriasis patients) or greater.
Additionally, we examined immunosuppressive therapies as a class
effect. It is possible that only certain types of immunosuppressive
therapies increase EBV or HHV-6 replication. This possibility would
be consistent with a recent study that showed that in patients with
rheumatoid arthritis and polymyositis on methotrexate therapy, mean
EBV viral loads were statistically significantly higher than in
those patients treated with immunosuppressive regimens not
including methotrexate [41]. In our study, only nine (34,62%)
patients with psoriasis on immunosuppressive therapy received
methotrexate, which provides limited statistical power for studying
the impact of methotrexate on HHV replication. Therefore,
additional studies are necessary to further determine if individual
systemic therapies used for psoriasis have the capacity to increase
the replication of EBV or HHV-6 through their associated immune
suppressing effects.
In conclusion, this study found no abnormalities in EBV or HHV-6
replication in patients with severe psoriasis and patients with
severe psoriasis being treated with immunosuppressive medication
compared to healthy controls. These findings are important as they
indicate that the pathophysiology of psoriasis and the
immunosuppressive treatments in general used for psoriasis do not
predispose the patient to EBV or HHV-6 related disease.
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