Erythroid Disturbances Before and After Treatment of Portuguese Psoriasis Vulgaris Patients

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Erythroid Disturbances Before and After Treatmentof Portuguese Psoriasis Vulgaris PatientsA Cross-Sectional and Longitudinal Study

Susana Coimbra,1,2,3 Hugo Oliveira,4 Flavio Reis,5 Luıs Belo,1,2 Susana Rocha,1,2 Alexandre Quintanilha,2,6

Americo Figueiredo,4 Frederico Teixeira,5 Elisabeth Castro,1,2 Petronila Rocha-Pereira2,7 and Alice Santos-Silva1,2

1 Department of Biological Sciences, Biochemistry, Faculty of Pharmacy, University of Porto, Porto, Portugal

2 Institute of Cellular and Molecular Biology (IBMC), University of Porto, Porto, Portugal

3 Center of Investigation of Technologies of Health (CITS) – IPSN-CESPU, Gandra-Paredes, Portugal

4 Dermatology Service, University Hospitals of Coimbra, Coimbra, Portugal

5 Institute of Experimental Pharmacology and Therapeutics, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

6 Institute for Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal

7 Center of Investigation of Health Sciences (CICS), University of Beira Interior, Covilha, Portugal

Abstract Background: A few studies in psoriasis vulgaris patients have reported changes suggesting red blood cell

(RBC) damage is linked to neutrophil activation, oxidative stress, and psoriasis worsening.

Objective: The aim of this study was to evaluate erythroid disturbances in Portuguese psoriasis vulgaris

patients, before, during, and after treatment.

Methods:Across-sectional study (n= 73 patients vs 40 healthy control subjects) followed by a longitudinal study

(n= 47 patients) was performed, with assessments before, and at 3, 6, and 12weeks of therapy (10 patients started

topical treatment, 17 narrow-band UVB, and 20 photochemotherapy [psoralen plus UVA; PUVA]).

Evaluations included hematologic data, total bilirubin levels, membrane-bound hemoglobin (MBH), membrane

protein band 3 profile, total plasma antioxidant status (TAS), lipid peroxidation (thiobarbituric acid [TBA]

assay), elastase, lactoferrin, and C-reactive protein (CRP).

Results: Before treatment, patients presented with higher leukocyte/neutrophil and reticulocyte counts,

elastase, lactoferrin, TBA, TBA/TAS, reticulocyte production index, total bilirubin andMBH values, lower

RBC and hematocrit, higher percentages of high-molecular-weight aggregates, and lower percentages of

band 3 monomer. After treatment, we observed a reversal in most of the parameters. However, patients still

presented with values suggestive of accelerated RBC damage, removal, and production, as most of the

parameters were still higher than those in the control group; the same occurred with CRP.

Conclusion: Our data suggest that psoriasis vulgaris triggers an inflammatory response, with release of acute-

phase reactants, reactive oxygen species, cationic proteins, and proteases, leading to enhanced RBC damage/aging and, ultimately, to enhancedRBC removal. These assumptions were strengthened by the observation that,

with treatment, all of these changeswere reversed, the inflammationwas reduced, the production of reticulocytes

was increased, and the RBCs presented changes usually observed in younger/less damaged RBCs. These

erythroid changes were enhanced with PUVA therapy, probably due to the more pronounced clearing of the

lesions, as suggested by Psoriasis Area and Severity Index (PASI) scores. Finally, after treatment, a residual

inflammation still persisted that might contribute to the observed erythroid disturbances.

Introduction

Psoriasis vulgaris is a chronic and recurrent inflammatory

skin disease that has been associated with oxidative stress and

with marked inflammatory changes in the epidermis and der-

mis, as well as in plasmatic markers of inflammatory re-

sponse.[1] This response is linked to the release of chemotactic

substances that trigger the mobilization and activation of

Approval for publication Signed Date Number of amended pages returned

ORIGINAL RESEARCH ARTICLEAm J Clin Dermatol 2012; 13 (1): 1-11

1175-0561/12/0001-0001/$49.95/0

ª 2012 Adis Data Information BV. All rights reserved.

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inflammatory cells, such as neutrophils, which seem to play a

crucial role in the evolution of psoriasis.[2,3] A significant rise in

leukocytes, due to an increase in neutrophils and lymphocytes,

has been reported in the earliest stages of psoriatic lesions.[4]

This rise has been associated with an increase in elastase and

lactoferrin, both products of neutrophil activation, and with

the severity of the disease, as defined by the Psoriasis Area and

Severity Index (PASI).[1,5] Along the inflammatory process,

several cytokines are released by inflammatory cells that lead to

a rise in C-reactive protein (CRP).[6] This acute-phase protein

has emerged as a risk factor for atherosclerosis and it has been

proposed to also have an active role in inflammation,[7-10] by

inhibiting neutrophil chemotaxis and endothelial cell adhesion,

reducing transmigration to the site of inflammation and, there-

fore, increasing the number of circulating activated neutrophils.

When activated, neutrophils are able to release oxygen me-

tabolites and their granular constituents, both of which may

induce oxidative and proteolytic modifications to plasma

constituents and to other blood cells, namely erythrocytes.[11,12]

A strong association between psoriasis, overweight/obesity,and cardiovascular co-morbidities has been proposed.[13-16]

Obesity is a proinflammatory state, presenting as a low-grade,

chronic inflammatory condition that might further enhance

inflammation in psoriatic patients;[17,18] it might also stimulate

erythropoiesis to increase the number of circulating red cells[19]

to face the higher oxygen requirements, due to the increase in

body mass.

The erythrocyte has a limited biosynthesis capacity, suffer-

ing and accumulating physical and chemical changes, that

become more pronounced with cell aging, and whenever an un-

usual physical or chemical stress develops.[20] The inflammatory

process in psoriasis, in part the result of leukocyte activation,

seems to impose oxidative and proteolytic changes in red blood

cells (RBCs) that lead to accelerated RBC aging and premature

removal.[12] The removal of senescent or damaged RBCs

involves the development of a neoantigen on the membrane

surface that is immunologically related to band 3,[21-23] a

transmembrane RBC protein that seems to have a key role in

erythrocyte function, and membrane deformability.[24] Mod-

ifications in this protein trigger the binding of specific natural

anti-band 3 autoantibodies and complement activation, mark-

ing the cell for death.[25,26] Actually, the degradation of the

RBC metabolism and failure of the antioxidant defences in

damaged or senescent RBCs favours the development of oxi-

dative stress within the cell, allowing hemoglobin oxidation

and its linkage to the cytoplasmatic domain of band 3, pro-

moting the aggregation of band 3 and the binding of anti-band 3

autoantibodies.[27] Membrane-bound hemoglobin (MBH) and

the band 3 profile were proposed as good cumulative markers

of erythrocyte aging and/or damage in several inflammatory

and oxidative stress conditions.[28-35] Differences in the band 3

profile (high-molecular-weight aggregates [HMWAg], band 3

monomer, and proteolytic fragments) have been associated

with the age and condition of RBCs.[12,29,30,35,36] Older and

damaged RBCs have higher band 3 aggregation and lower

fragmentation, whereas younger RBCs showed reduced ag-

gregation and higher fragmentation.

Other studies have reported erythrocyte membrane changes

in psoriasis, namely, in membrane lipid peroxidation,[37,38] in

the sodium-potassium adenosine triphosphatase pump,[39] in the

bindingof cyclic adenosinemonophosphate to proteinkinaseA,[40]

and in the expression and distribution ofmembrane protein 4.1.[41]

Previous studies in our laboratory[12] showed that leukocyte

activation seems to induce, especially in the active forms of

psoriasis, an altered erythrocyte band 3 profile, similar to that

observed in older and/or damaged erythrocytes; we also ob-

served a lower RBC count accompanied by changes suggesting

a slight increase in RBC removal and RBC production. How-

ever, few longitudinal studies have been performed to examine

the erythrocyte changes and their relationship with neutrophil

activation, oxidative stress, and inflammation, before, during,

and after treatment for psoriasis. These studies could contrib-

ute to a better understanding of psoriasis pathogenesis, and of

the efficacy of currently used therapies.

Topical agents, appropriate wavelengths of UV radiation,

and systemic therapies are treatments commonly used, singly or

in combination, in psoriasis vulgaris.[42] Topical agents are

usually sufficient to control mild psoriasis, while moderate to

severe psoriasis often requires phototherapy (narrow-band

UVB [NB-UVB], photochemotherapy [psoralen plus UVA;

PUVA]) or even systemic agents.

Our aim was to study erythrocyte changes alongside neu-

trophil activation, oxidative stress, and inflammatory markers

in Portuguese psoriasis vulgaris patients, before, during, and

after treatment, by performing a cross-sectional and a longi-

tudinal study (before and 3, 6, and 12 weeks after therapy).

Indeed, by performing a longitudinal study, we aimed to eval-

uate the changes occurring during the course of treatment, with

improvement of the disease and clearing of the lesions. The

results were also analyzed according to the type of therapy used,

i.e. topical, NB-UVB, and PUVA.

Materials and Methods

The protocol was approved by the Committee on Ethics of

the University Hospitals of Coimbra, Coimbra, Portugal.

2 Coimbra et al.

ª 2012 Adis Data Information BV. All rights reserved. Am J Clin Dermatol 2012; 13 (1)

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Subjects and Treatment

All of the 73 patients presented with chronic psoriasis vulgaris

during an exacerbation phase (33 women and 40 men, with a

median PASI score of 18.0 [interquartile range: 10.8–29.1]) and

gave informed consent to participate in the cross-sectional

study; 47 of the 73 patients gave their informed consent to

participate in a 12-week follow-up study.

Clinical evaluation of psoriasis was performed using the

PASI score[43] and was measured by the same dermatologist to

diminish subjectivity. Psoriasis was diagnosed between 2 months

and 55 years before the study (duration of the disease) and the

age at onset of psoriasis was 3–65 years. All patients were

clinically and analytically studied in the active phase of the

disease, meaning during an exacerbation of psoriatic lesions,

before starting therapy. The 47 patients who agreed to partic-

ipate in the follow-up study were also evaluated 3, 6, and

12 weeks after starting treatment. Of these, 20 patients received

PUVA therapy, 17 patients were treated with NB-UVB radia-

tion, and 10 patients received topical treatment (calcipotriene

[calcipotriol] or betamethasone dipropionate, or a combination

of the two). The type of treatment was decided by the patient’s

dermatologist, according to the severity of the disease pre-

sentation and the clinical and therapeutic history of the patient.

After a treatment had been chosen, patients were invited to

participate in the study.

NB-UVB irradiation (311 – 2 nm) was administered using

a Waldmann 7001K cabin (UVA/UVB-TL01; Waldmann

Medizintechnik, Villigen-Schwenningen, Germany). The initial

dose, dependent on the phototype of the patient, was 0.1–

0.3 J/cm2; an increasing dose schedule was used, based on an

increase of 0.1 J/cm2 at every session (three times weekly) until a

maximum dose of 2.5 J/cm2 was reached. UVA irradiation (320–

400 nm) was administered using the same cabin; 2 hours earlier,

methoxsalen (8-methoxypsoralen) was administered (0.6mg/kgbodyweight). The initial dose of UVAwas 2–3 J/cm2, according

to the patient’s phototype; an increasing dose schedule was used,

based on an increase of 0.5 J/cm2 at every session (three times

weekly) until a maximum dose of 12 J/cm2 was reached. Eyes and

genitals were shielded during the irradiation procedures.

Patients in the topical, NB-UVB, and PUVA groups pre-

sented with similar baseline characteristics with regard to age,

duration of the disease, sex, and body mass index (BMI) [data

not shown]. However, patients in the topical treatment group

differed from those in the NB-UVB (p £ 0.001) and PUVA

(p £ 0.001) groups with regard to the PASI score; NB-UVB and

PUVA groups did not present with significantly different PASI

values (p = 0.125).

The control group included 40 apparently healthy volunteers

(18 women and 22 men) without psoriasis or other skin disease,

with normal hematologic and biochemical values, andmatched

with patients for sex, age (41 – 14 and 45 – 15 years in control

and patient groups, respectively; p = 0.147), and smoking habits

(20% and 19% smokers, respectively).

Patients and control subjects presenting at the start of the

study with other skin diseases, anemia, diabetes mellitus, or

cardiovascular, liver, kidney, inflammatory, or infectious

diseases were excluded from the study. None of the selected

patients received any psoriatic treatment for at least 1 month

prior to the start of the study. None of the selected patients was

receiving regular therapy that could interfere with our results or

that could result in a flare of psoriasis (e.g. ACE inhibitors,

b-adrenoceptor antagonists [b-blockers], lithium, andNSAIDs).

Additionally, we evaluated in all subjects the serum levels of

vitamin B12, folic acid, and iron, and excluded all individuals

showing any hematologic condition. Patients were also asked

about their alcohol consumption and excluded when it was high.

Moreover, patients were asked tomaintain their nutritional habits

and physical exercise activity during the treatment.

Collection and Preparation of Blood Samples

Blood from non-fasted subjects was collected in order

to obtain whole blood, plasma (the anticoagulant used was

edetic acid [ethylenediaminetetraacetic acid or EDTA]), and

serum. None of the collected samples was icteric or he-

molysed. Blood samples were processed within 2 hours after

collection.

To isolate and prepare RBC membranes, leukocytes and

plasma were isolated from RBCs and discarded after cen-

trifugation on a double-density gradient (700 g for 30 minutes

at room temperature; Histopaque�-1.077; Histopaque�-1.119;

Sigma-Aldrich Co. LLC, St. Louis, MO, USA). RBCs were

washed with saline solution and submitted to hypotonic lysis,

according to the method by Dodge et al.[44] The membrane

suspensions were carefully washed inDodge buffer, using in the

first twowashes a protease inhibitor, phenylmethylsulfonylfluoride

(final concentration of 0.1mmol/L in Dodge buffer). The pro-

tein concentration of membrane suspensions was determined

by the Bradford method.[45]

Assays

Total and differential leukocyte count, RBC count, hemo-

globin level, hematocrit, and hematimetric indices were evaluated

by using an automatic blood cell counter (Sysmex XT-1800i;

Erythroid Disturbances: Psoriasis Vulgaris Therapy 3


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Sysmex Europe GMBH, Norderstedt, Germany). Cell mor-

phology was evaluated in Wright-stained blood films.

Reticulocyte count was performed in supravital newmethylene

blue-stained blood films (reticulocyte stain; Sigma, St Louis,

MO, USA) and the reticulocyte production index (RPI) was

calculated.

As an index ofRBC removal and destruction, the serum level

of total bilirubin was evaluated by using a commercially

available kit (Bil-T; Roche, Basel, Switzerland).

Plasma levels of elastase and lactoferrin were evaluated by

enzyme immunoassays (human PMN elastase ELISA; Bender

MedSystems, Vienna, Austria; and Bioxytech Lactof EIA;

OxisResearch, Foster City, CA, USA, respectively).

The serum levels of CRP were evaluated by immuno-

turbidimetry (CRP [latex] High-Sensitivity; RocheDiagnostics,

Basel, Switzerland).

To study the oxidative stress we evaluated total antioxidant

status (TAS) [colorimetric assay; Randox Laboratories, Crumlin,

UK] and lipid peroxidation (thiobarbituric acid reactivity

[TBA] assay),[46] and calculated the ratio between the two

parameters.

MBH was measured spectrophotometrically after protein

dissociation of membrane components with octoxynol (Triton

X-100; Sigma, St. Louis, MO, USA) [5% in Dodge buffer] at

415 nm, and this value was corrected by subtracting the ab-

sorbance of the background at 700 nm; the value obtained and

the membrane protein concentration were used to calculate the

percentage of MBH.

To study the erythrocyte membrane band 3 profile, RBC

membranes were treated with an equal volume of a solubiliza-

tion buffer containing 0.125mol/LTris HCl pH 6.8, 4% sodium

laurilsulfate (sodium dodecyl sulfate, SDS), 20% glycerol, 10%2-mercaptoethanol, heat denatured and submitted to poly-

acrylamide gel electrophoresis (SDS-PAGE) [20 mg protein/lane], using the discontinuous Laemmli system[47] (a 9% sepa-

rating gel and a 4.5% stacking gel). Membrane proteins

were electrophoretically transferred from SDS gels to a nitro-

cellulose sheet with a porosity of 0.2 mm (Sigma).[48] Additional

reactive sites on the nitrocellulose were blocked by incubation

in a low-fat milk phosphate buffered solution (PBS) pH 7 (5%in 0.1% octoxynol) for 1 hour at room temperature and under

gentle rotation. Band 3 immunoblot was then performed to

determine whether erythrocyte band 3 was modified; mono-

clonal antibodies anti-human band 3, produced in mouse,

recognizing an epitope located in the cytoplasmic pole of the

band 3 molecule[49] (Sigma) were added (dilution 1:3000) and

incubated for 4 hours; washing of the nitrocellulose was fol-

lowed by the addition and incubation with anti-mouse Ig per-

oxidase linked (Sigma) for 1 hour (dilution 1:4000). The

incubations were carried out at room temperature; the dilutions

of the antibodies were prepared with PBS pH 7.0 containing

0.1% octoxynol and 0.5% low-fat drymilk. The washes used the

same buffer without low-fat dry milk. Hydrogen peroxide and

a-cloronaphtol (Sigma) were used to develop the immunoblot.[35]

The immunoblots were scanned (Darkroom CN UV/wl,BioCaptMW version 99; Vilber Lourmat, Marne-La-Vallee,

France) and the relative amount of band 3 monomer, of

HMWAg and of proteolytic fragments (as a percentage of

total), was quantified by densitometry (Bio1D++ version 99;

Vilber Lourmat, Marne-La-Vallee, France).

Statistical Analysis

The statistical analysis was performed using the Statistical

Package for Social Sciences (SPSS, version 16 for Windows,

Chicago, IL, USA). To evaluate the differences between con-

trol and psoriatic groups, at baseline (T0) or after 12 weeks of

treatment (T12), we used the unpaired Student’s t-test for the

determinations presenting aGaussian distribution, and theMann-

Whitney U test for those presenting a non-Gaussian dis-

tribution. To evaluate the differences between the active stage

of disease and the end of treatment we used the paired Student’s

t-test for the determinations presenting a Gaussian distribu-

tion, and the Wilcoxon signed-rank test for those presenting a

non-Gaussian distribution. A p-value lower than 0.05 was con-

sidered statistically significant. The adjustment for confounding

factors used analysis of covariance, after transformation of var-

iables (when necessary). The correlation analysis was performed

by calculating the Pearson or the Spearman coefficient correla-

tion, according to Gaussian distribution of the substances.

Results

The weight of patients did not change during and after

treatment (data not shown). Psoriatic patients had significantly

higher median [interquartile range] BMI values, when com-

pared with matched control subjects (27.18 [23.79–29.73] and

25.26 [21.83–28.52], respectively; p = 0.046). Thus, psoriatic

patients seem to be more prone to overweight and obesity,

because 67% of them presented with a BMI >25 kg/m2 com-

pared with 53% in the control group.

We observed that psoriatic patients presented with signif-

icantly higher leukocyte and neutrophil counts, as well as sig-

nificantly higher elastase, lactoferrin, and CRP levels (table I).

Concerning the oxidative stress markers, psoriatic patients

presented with significantly higher TBA and TBA/TAS values;

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however, after BMI adjustment, TBA/TAS results lost sta-

tistical significance. No statistically significant difference was

observed between groups in TAS.

In table II we present the qualitative and quantitative RBC

studies that were performed, the plasma levels of total bilirubin

(as a marker of RBC removal), and the values of reticulocytes

and RPI (as markers of erythroid bone marrow production).

Psoriatic patients presented with significantly lower values of

RBC and hematocrit, and higher values of mean cell hemo-

globin (MCH) andMCH concentration (MCHC) than control

subjects. The reticulocyte count, RPI, and the total bilirubin

levels were also significantly higher in psoriatic patients. Con-

cerning erythrocyte damage,MBH (amarker of oxidative stress

in RBCs) showed significantly higher values, and the band 3

profile presented a significantly different pattern for psoriatic

patients, with higher percentages of HMWAg and lower per-

centages of band 3 monomer. All the statistical significances

observed persisted after adjustment for BMI.

For psoriatic patients we observed a significant positive

correlation of BMI with RBC count (r = 0.279, p= 0.017) and

with hemoglobin level (r= 0.232, p = 0.048). Furthermore, we

found that CRP presented positive significant correlations with

PASI score (r = 0.310, p= 0.008), leukocyte count (r = 0.489,

p < 0.001), neutrophil count (r = 0.522, p< 0.001), elastase

(r = 0.455, p< 0.001), and lactoferrin level (r = 0.323, p= 0.005).

Concerning the follow-up study (n = 47), we observed slight

changes in the studied parameters during the course of treat-

ment, which reached statistical significance at the end of treat-

ment (table III). Indeed, we found a significant decrease in

leukocyte and neutrophil counts, and in the neutrophil acti-

vation products, elastase and lactoferrin. CRP also showed a

significant decrease, as did TBA and the TBA/TAS ratio. We

should note that at the end of treatment, with clearing of the

lesions (as shown by PASI), the CRP and lactoferrin levels were

still statistically significantly higher than in the control group

(table III).

The erythrocyte parameters also showed slight changes with

treatment, some of them reaching statistical significance at the

end of treatment (table IV), namely, mean cell volume (MCV)

and MCH decreased significantly and reticulocyte count and

RPI showed a significant rise. No significant changes were

observed for total bilirubin levels. Considering the erythrocyte

damagemarkers, a significant decrease was observed forMBH,

and a different band 3 profile was found, with a decrease in

HMWAg and a rise in proteolytic fragments at the end of

treatment.

As shown, several changes occurred during treatment, with

improvement of the disease; however, some of the changes in-

duced by treatment did not reach values similar to those pre-

sented by the control group. Thus, the RBC count, hematocrit,

MCH,MCHC, reticulocyte count, RPI and bilirubin level were

still significantly higher than in the control subjects. MBH was

also still significantly higher, and the band 3 profile was also

different, with a decreased percentage of band 3 monomer and

increased percentage of proteolytic fragments compared with

control subjects (that lost significance after statistical adjust-

ment for BMI).

Analysing the results according to the therapy used, we

found that patients receiving PUVA treatment presented with a

significant reduction in leukocyte and neutrophil counts, elas-

tase, lactoferrin, CRP, TBA, and TBA/TAS (table V). Patients

treated with NB-UVB showed a significant decrease in the

values of elastase, lactoferrin, CRP, TBA, and TBA/TAS.With

topical therapy, only a trend towards reduced values of elastase

Table I. Leukocyte activation and redox status for control subjects and psoriatic patientsa

Parameter Control subjects (n = 38) Psoriatic patients (n = 73) p-Value

WBCs (·109/L) 6.70 [5.20–7.50] 7.00 [5.75–8.70] 0.043

Neutrophils (·109/L) 3.95 [2.98–4.60] 4.30 [3.40–5.95] 0.016

Elastase (ng/mL) 40.5 [32.8–45.6] 52.5 [42.8–67.8] <0.001

Lactoferrin (ng/mL) 182.5 [128.0–203.8] 239.0 [201.0–349.5] <0.001

CRP (mg/L) 1.51 [0.60–2.75] 4.18 [1.59–9.12] <0.001

TBA (mmol/L) 1.18 [1.00–1.41]b 1.36 [1.21–1.64] 0.004

TAS (mmol/L) 1.30 [1.09–1.61]b 1.30 [1.22–1.43] 0.228

TBA/TAS (·10-3) 0.90 [0.73–1.13]b 1.05 [0.89–1.31] 0.047c

a Values expressed as median [interquartile range].

b n = 37.

c Loss of significance when adjusted for body mass index.

CRP = C-reactive protein; TAS = total antioxidant status; TBA = thiobarbituric acid reactivity; WBCs = white blood cells.



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and a significant reduction in TBA was found. Considering the

erythrocyte parameters, the group receiving topical therapy did

not present with any significant changes (table VI). In patients

receivingNB-UVB and PUVA therapy, a significantly different

band 3 profile was observed after treatment; PUVA patients

presented with a significant reduction in HMWAg and a sig-

nificant rise in proteolytic fragments; NB-UVB patients pre-

sented with, besides these changes, a significant increase in

band 3 monomers. Moreover, both PUVA and NB-UVB pa-

tients presented with a significant decrease in MBH. After

PUVA therapy, a significant increase was observed for retic-

ulocyte count and RPI. Some morphologic changes were also

observed after PUVA therapy, namely, a reduction in MCV

and an increase in MCH.

After treatment, the PASI score differed significantly be-

tween the topical and PUVA groups (p = 0.013), but not be-

tween topical and NB-UVB patients (p = 0.443); PUVA

patients showed a trend towards lower PASI values than those

treated with NB-UVB (p = 0.074).

Discussion

A few studies in patients in the active phase of psoriasis

vulgaris have reported a reduced number of erythrocytes com-

pared with a healthy control group, and changes denoting an

enhanced damage or aging process, which seems to be strongly

connected with neutrophil activation, oxidative stress, and

worsening of psoriasis.[12,37,38] In recent years, an association of

psoriasis with overweight/obesity has been proposed[15] that

might lead to enhanced inflammation and an increase in car-

diovascular co-morbidities. Moreover, the number of circulating

erythrocytes would also be expected to increase, to perfuse a

higher body mass. Our psoriatic patients presented with sig-

nificantly higher BMI values compared with matched control

subjects, 67% of them presenting with a BMI >25 kg/m2. In-

deed, significant correlations of BMI with RBC count and

hemoglobin levels were observed. However, the RBC count,

hematocrit and hemoglobin levels were significantly lower in

our patients, even after adjustment for BMI, suggesting that

these changes are related to psoriasis. Our data suggest that this

reduction in circulating RBCs might result from accelerated

erythrocyte damage/aging and removal, which is not ad-

equately compensated by an increased erythropoiesis. Indeed,

patients’ erythrocytes presented with a significantly higher

MBH, linked to a significantly higher band 3 aggregation, both

changes denoting RBC damage. This damage seems to lead to

RBC removal, as suggested by the significantly higher bilirubin

levels that were almost double the values in the control group.

The associated rise in reticulocytes and RPI supports the de-

velopment of a higher erythropoietic stimulus to face a higher

rate of RBC damage and removal. We also found that patients

presented with a significantly higher number of total leukocytes,

Table II. Erythrocyte parameters, total bilirubin, membrane-bound hemoglobin (MBH), and band 3 profile for control subjects and psoriatic patientsa

Parameter Control subjects (n = 40) Psoriatic patients (n = 73) p-Valueb

RBCs (·1012/L) 4.89 [4.58–5.14] 4.58 [4.29–5.03] 0.008

Hematocrit (%) 44.2 [41.1–46.4] 41.6 [38.7–44.6] 0.002

Hemoglobin (g/dL) 14.4 [13.0–15.1] 14.4 [13.5–15.3] 0.530

MCV (fL) 90.1 [87.9–91.9] 88.9 [86.2–94.0] 0.760

MCH (pg) 28.9 [28.2–29.8] 30.7 [29.7–32.6] <0.001

MCHC (g/dL) 32.4 [31.6–32.8] 34.6 [34.1–35.0] <0.001

Reticulocytes (·109/L) 43.4 [27.6–54.8] 54.2 [39.0–75.7] 0.003

RPI 0.87 [0.53–1.11] 1.07 [0.78–1.52] 0.004

Total bilirubin (mg/dL) 0.36 [0.25–0.61] 0.66 [0.49–0.80] <0.001

MBH (% ·10-2) 0.41 [0.34–0.52] 0.68 [0.55–0.90] <0.001

Band 3 profile (%)

HMWAg 13.7 [10.9–15.9] 18.6 [14.9–20.7] <0.001

band 3 64.3 [61.9–66.5] 59.3 [55.6–63.4] <0.001

total Pfrag 22.9 [19.5–24.8] 22.3 [20.0–25.4] 0.741


b p < 0.05 considered statistically significant.

HMWAg = high-molecular-weight aggregates; MCH = mean cell hemoglobin; MCHC = MCH concentration; MCV = mean cell volume; Pfrag = proteolytic

fragments; RBCs = red blood cells; RPI = reticulocyte production index.

6 Coimbra et al.


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and this rise was mainly due to an increase in neutrophils. It is

known that neutrophil activation is linked to metabolic acti-

vation, with production of oxygen metabolites, and to de-

granulation, with release of granule constituents. Elastase and

lactoferrin are components of the primary and secondary

granules, respectively, and both presented significantly higher

values in psoriatic patients, as compared with the control

group, suggesting a neutrophil activation process.

Inflammation is a hallmark of psoriasis and, in accordance,

our patients presented with significantly higher CRP levels than

the control group and, moreover, the CRP level was signif-

icantly and positively correlated with leukocyte count, neutrophil

count, elastase, and lactoferrin levels, further strengthening the

linkage between psoriasis and the inflammatory response. The

activation of inflammatory cells is also known to be associated

with the release of several cytokines, some of which are known

Table III. Follow-up study of leukocyte activation and redox status for psoriatic patients (n = 47)a

Parameter Baseline (T0) 3rd week (T3) 6th week (T6) 12th week (T12)

WBCs (·109/L) 6.90 [5.80–8.00] 6.60 [5.30–7.90] 6.40 [5.30–7.60] 6.4 [5.4–7.2]*

Neutrophils (·109/L) 4.00 [3.30–5.80] 4.00 [2.90–5.30] 3.90 [2.90–4.90] 3.90 [3.10–4.70]*

Elastase (ng/mL) 47.8 [37.0–58.2] 39.3 [34.7–51.3] 40.3 [33.4–48.5] 37.5 [31.8–45.0]*

Lactoferrin (ng/mL) 239.0 [199.0–300.0] 231.0 [197.0–297.0] 216.0 [193.0–277.0] 203.0 [157.0–266.0]***-

CRP (mg/L) 3.15 [1.25–6.80] 2.80 [1.31–6.27] 2.65 [1.06–7.03] 2.32 [1.10–5.40]**-

TBA (mmol/L) 1.36 [1.21–1.62] 1.26 [1.05–1.51] 1.15 [1.03–1.51] 1.21 [0.97–1.41]***

TAS (mmol/L) 1.30 [1.21–1.40] 1.28 [1.19–1.40] 1.30 [1.20–1.37] 1.30 [1.20–1.40]

TBA/TAS (·10-3) 1.06 [0.91–1.30] 1.02 [0.82–1.26] 0.89 [0.79–1.21] 0.89 [0.76–1.11]***


CRP = C-reactive protein; TAS = total antioxidant status; TBA = thiobarbituric acid reactivity; WBCs = white blood cells; * p (T0 vs T12) < 0.05, ** p (T0 vs

T12) £ 0.01; *** p (T0 vs T12) £ 0.001; - p (control vs T12) £ 0.01.

Table IV. Follow-up study of Psoriasis Area and Severity Index (PASI), erythrocyte parameters, total bilirubin, membrane-bound hemoglobin (MBH), and

band 3 profile for psoriatic patients (n = 47)a

Parameter Baseline (T0) 3rd week (T3) 6th week (T6) 12th week (T12)

PASI 16.3 [10.5–26.5] 7.2 [3.7–16.2] 4.4 [2.8–8.1] 2.8 [1.4–4.9]***

RBCs (·1012/L) 4.56 [4.29–5.01] 4.58 [4.25–4.90] 4.53 [4.21–4.90] 4.68 [4.28–4.95]--

Hematocrit (%) 41.3 [38.7–44.0] 41.3 [38.2–42.9] 40.4 [38.1–43.6] 41.4 [38.7–43.8]---

Hemoglobin (g/dL) 14.4 [13.5–15.1] 14.1 [13.4–14.9] 14.1 [13.1–14.9] 14.1 [13.3–14.9]

MCV (fL) 88.9 [86.3–93.4] 88.6 [86.3–91.1] 89.2 [85.7–92.4] 88.4 [85.4–92.0]*

MCH (pg) 30.8 [29.8–31.7] 30.8 [30.0–31.8] 31.0 [29.5–31.7] 30.8 [29.5–31.7]*---

MCHC (g/dL) 34.6 [34.3–35.0] 34.6 [34.2–35.0] 34.5 [34.0–35.0] 34.5 [34.0–35.0]---

Reticulocytes (·109/L) 55.7 [39.1–74.5] 62.0 [41.2–91.6] 57.1 [41.0–92.4] 58.9 [40.6–105.8]**---

RPI 1.03 [0.75–1.47] 1.19 [0.82–1.82] 1.04 [0.79–1.78] 1.19 [0.80–2.06]**---

Total bilirubin (mg/dL) 0.60 [0.46–0.80] 0.61 [0.45–0.80] 0.57 [0.39–0.80] 0.54 [0.38–0.80]---

MBH (% ·10-2) 0.73 [0.56–0.90] 0.53 [0.44–0.75] 0.58 [0.42–0.75] 0.53 [0.43–0.73]***--

Band 3 profile (%)

HMWAg 18.6 [13.8–20.2] 16.1 [13.0–19.5] 14.5 [11.0–18.5] 13.3 [10.7–17.4]***

band 3 59.3 [54.1–63.3] 59.9 [56.2–63.8] 60.2 [55.0–63.7] 61.5 [55.5–65.4]--

total Pfrag 22.6 [20.6–25.4] 24.1 [19.4–28.9] 25.1 [21.3–30.0] 25.1 [20.6–31.1]***- b


b Loss of significance when adjusted for body mass index.

HMWAg = high-molecular-weight aggregates; MCH = mean cell hemoglobin; MCHC = MCH concentration; MCV = mean cell volume; Pfrag = proteolytic

fragments; RBCs = red blood cells; RPI = reticulocyte production index; * p (T0 vs T12) < 0.05, ** p (T0 vs T12) £ 0.01, *** p (T0 vs T12) £ 0.001; - p (control vs

T12) < 0.05, -- p (control vs T12) £ 0.01, --- p (control vs T12) £ 0.001.



AU

TH

OR

PR

OO

F

to inhibit erythropoiesis, such as tumor necrosis factor-a and

interferon-g.[50,51] We should not exclude, therefore, that these

cytokines could inhibit to some degree the erythropoietic

stimulus triggered by the enhanced RBC removal. Actually, the

physiologic mechanism to maintain a normal value of circu-

lating RBCs was triggered in our patients, as shown by the

increase in RPI and circulating reticulocyes; however, that

stimulus was not sufficient to reach control values.

Furthermore, as already mentioned, neutrophil activation is

linked to the production of oxygen metabolites that are re-

sponsible for oxidative modifications in plasma and cell mem-

brane constituents.[28,29] We observed that lipid peroxidation

levels were significantly increased in psoriatic patients, as

compared with the control group, and the increased ratio of

TBA/TAS suggests the development of an oxidative stress

condition.

During treatment of psoriasis a progressive reduction in the

body surface area covered by lesions and an improvement in the

severity of the lesions were observed, as shown by the signif-

icant reduction in PASI score. This improvement of the disease

was linked to a reduction in the inflammatory process, as shown

by the significant decrease in CRP, although values remained

higher than in the control group, suggesting that a low-grade

inflammation still persists after therapy. Total leukocytes also

showed a significant decrease, and the same occurred with

neutrophils; this reduction in circulating neutrophils was linked

to significantly reduced levels of their activation products,

elastase and lactoferrin, even though lactoferrin values re-

mained higher than the control group. The markers of redox

status studied, TBA and TBA/TAS, were also reduced during

treatment of psoriasis. These data support the theory that

psoriasis therapy leads to a reduction in inflammation and

oxidative stress. Therefore, it would be reasonable to assume

that these changes could reduce RBC damage/aging, resultingin a longer life span for RBCs and, therefore, an increase in

circulating RBCs. However, we found that with treatment,

patients presented with only a trend towards higher values of

RBCs and similar levels of bilirubin, suggesting that neither the

reduced inflammation and oxidative stress nor the treatment

contributed to significantly alter the number of circulating

RBCs. However, a rise in erythropoiesis, as shown by the sig-

nificant increase in RPI and in the number of circulating re-

ticulocytes, seems to occur alongside treatment. As this increase

is not triggered by enhanced RBC damage and removal, we

believe that this is due to the reduction in inflammation, which

is probably associated with a reduction in inflammatory cyto-

kines released by the inflammatory cells that are known to in-

hibit erythropoiesis.Tab

leV

.F

ollo

w-u

pof

leuko

cyte

activation

and

redox

sta

tus

inpsoriatic

patients

receiv

ing

photo

chem

oth

era

py

(psora

len

plu

sU

VA

[PU

VA

]),

narr

ow

-band

UV

B(N

B-U

VB

),and

topic

al

thera

pya

Para

mete

rP

UV

A(n

=20)

NB

-UV

B(n

=17)

Topic

alt

hera

py

(n=

10)

baselin

e(T

0)

12th

week

(T12)

p-v

alu

ebaselin

e(T

0)

12th

week

(T12)

p-v

alu

ebaselin

e(T

0)

12th

week

(T12)

p-v

alu

e

WB

Cs

(·10

9/L

)7.1

0[5

.65–7.9

8]

6.2

5[5

.25–7.0

5]

0.0

42

6.9

0[6

.05–9.3

5]

6.4

0[5

.70–7.3

5]

0.2

75

6.7

0[5

.60–8.0

5]

6.3

5[5

.88–7.8

8]

1.0

00

Neutr

ophils

(·10

9/L

)4.2

5[3

.43–5.9

0]

3.9

0[3

.03–4.6

5]

0.0

26

4.0

0[3

.35–5.5

0]

4.1

0[3

.35–4.7

0]

0.2

24

4.0

5[3

.18–5.3

0]

3.6

5[3

.08–5.1

8]

0.9

19

Ela

sta

se

(ng/m

L)

49.7

[40.0

–58.5

]39.7

[34.1

–52.7

]0.0

01

49.6

[37.5

–58.1

]37.3

[32.1

–40.9

]<0

.001

42.9

[34.1

–57.8

]34.4

[28.4

–52.4

]0.0

59

Lacto

ferr

in(n

g/m

L)

230.0

[180.2

–269.0

]190.5

[153.2

–274.0

]0.0

30

259.0

[227.5

–359.0

]195.0

[149.0

–259.0

]<0

.001

211.0

[170.2

–355.5

]230.5

[174.2

–267.7

]0.2

03

CR

P(m

g/L

)3.7

6[1

.32–10.6

6]

2.4

3[1

.20–5.2

2]

0.0

04

3.1

5[1

.64–7.8

5]

2.3

2[1

.10–5.5

0]

0.0

15

2.1

3[0

.91–5.7

2]

2.9

6[1

.05–6.4

7]

0.2

08

TB

A(m

mol/L

)1.3

6[1

.14–1.6

2]

1.2

0[0

.89–1.2

8]

<0.0

01

1.4

4[1

.27–1.6

2]

1.2

8[1

.04–1.4

6]

0.0

02

1.3

4[1

.16–1.6

6]

1.1

3[0

.90–1.5

0]

0.0

46

TA

S(m

mol/L

)1.2

8[1

.21–1.3

9]

1.2

6[1

.18–1.3

7]

0.2

44

1.3

0[1

.22–1.3

8]

1.3

0[1

.30–1.3

9]

0.5

31

1.2

8[1

.20–1.4

0]

1.3

3[1

.17–1.4

2]

0.7

33

TB

A/T

AS

(·10

-3)

1.0

4[0

.81–1.3

6]

0.9

2[0

.69–1.1

3]

0.0

02

1.0

8[0

.96–1.2

7]

0.9

3[0

.80–1.0

7]

0.0

04

1.0

4[0

.79–1.4

6]

0.7

9[0

.68–1.3

6]

0.1

44

aV

alu

es

expre

ssed

as

media

n[inte

rquart

ilera

nge].

CR

P=

C-r

eactive

pro

tein

;T

AS

=to

tala

ntioxid

antsta

tus;T

BA

=th

iobarb

ituric

acid

reactivity

;W

BC

s=

white

blo

od

cells

.

8 Coimbra et al.


AU

TH

OR

PR

OO

F

The increase in circulating reticulocytes after treatment ex-

plains the values observed for the MBH and in the band 3

profile. Indeed, the linkage of denatured hemoglobin is sig-

nificantly reduced and, therefore, the aggregation of band 3 is

also significantly reduced. Both changes are in agreement with

a younger RBC population.

We must notice, however, that after treatment the patients

still presented with values suggestive of accelerated RBC

damage, removal and production, as most of the studied pa-

rameters, i.e. RBC count, hematocrit, bilirubin, reticulocytes,

RPI, and MBH were still higher than those presented by the

control group. Considering that the same occurred with CRP, it

seems that an inflammation status persists in these patients that

may underlie those changes in erythroid cells.

PUVA and NB-UVB therapies, usually used to treat mod-

erate to severe forms of psoriasis vulgaris, are known to have

anti-inflammatory and immunomodulatory actions.[52,53] The

topical agents used for the mildest forms of the disease act

locally and present a less marked anti-inflammatory activity.

When analyzing the results according to the type of treat-

ment, the only significant change with topical therapy was a

significant reduction in TBA, suggesting that the reduction in

the hyperproliferative process within the lesions is very im-

portant for the redox status; actually, this is a common finding

in the course of the three therapies. With topical therapy no

other significant changes were observed, probably due to a less

marked anti-inflammatory response induced by these agents.

Some studies have suggested that PUVA has no significant

effect on neutrophils, while others suggest that PUVA enhances

neutrophil activity;[54-56] UVB exposure of psoriatic skin ap-

pears to induce IL-4 expression by neutrophils.[57] After treat-

ment with PUVA, we found that the patients presented with the

same changes described for the total patient group. For NB-

UVB, the same changes were observed, except there was no

significant reduction in total leukocytes, neutrophils, RPI or

reticulocytes. Thus, clearance of psoriasis, during and after

PUVA treatment, was associated with a significant decrease in

leukocyte and neutrophil counts, neutrophil activation prod-

ucts, CRP, and markers of redox status, as well as a rise in

erythropoiesis and a younger/less damaged RBC population.

Since patients submitted to PUVA and NB-UVB therapies,

before commencing therapy, did not present significantly dif-

ferent characteristics, specifically in PASI scores, it is possible

to say that the improvements seem to be more pronounced in

patients treated with PUVA. Patients treated with PUVA

presented at the end of treatment a trend towards a lower PASI

score than those treated with NB-UVB (and lower PASI score

than that presented by patients treated with topical agents),Tab

leV

I.F

ollo

w-u

pstu

dy

ofP

soriasi

sA

rea

and

Severity

Index

(PA

SI)

,ery

thro

cyte

para

mete

rs,t

ota

lbili

rubin

,mem

bra

ne-b

ound

hem

oglo

bin

(MB

H),

and

band

3pro

file

inpsoriatic

patients

receiv

ing

photo

chem

oth

era

py

(psora

len

plu

sU

VA

[PU

VA

]),narr

ow

-band

UV

B(N

B-U

VB

),and

topic

alt

hera

py

a

Para

mete

rP

UV

A(n

=20)

NB

-UV

B(n

=17)

Topic

alth

era

py

(n=

10)

baselin

e(T

0)

12th

week

(T12)

p-v

alu

ebaselin

e(T

0)

12th

week

(T12)

p-v

alu

ebaselin

e(T

0)

12th

week

(T12)

p-v

alu

e

PA

SI

24.8

[13.2

–33.4

]1.5

[0.7

–3.8

]<0

.001

17.2

[12.6

–25.0

]3.1

[1.6

–6.6

]<0

.001

8.6

[6.9

–10.5

]4.2

[2.6

–6.2

]0.0

05

RB

Cs

(·10

12/L

)4.5

8[4

.30–5.0

9]

4.7

6[4

.30–4.9

8]

0.2

83

4.5

5[4

.36–4.9

9]

4.5

7[4

.22–4.8

0]

0.2

88

4.6

6[4

.23–5.2

7]

4.7

3[4

.16–5.1

2]

0.2

26

Hem

ato

crit(%

)41.9

[38.7

–44.2

]41.9

[39.9

–44.2

]0.7

74

40.5

[39.2

–42.3

]40.1

[37.5

–42.1

]0.1

03

40.9

[36.8

–45.0

]43.0

[36.7

–43.9

]0.4

32

Hem

oglo

bin

(g/d

L)

14.5

[13.5

–15.2

]14.4

[13.8

–15.3

]0.6

86

14.1

[13.5

–14.7

]13.9

[13.2

–14.2

]0.0

49

14.2

[12.8

–15.6

]14.6

[12.5

–15.0

]0.1

53

MC

V(f

L)

88.9

[86.9

–95.0

]88.5

[85.8

–94.5

]0.0

23

88.9

[86.7

–90.4

]87.9

[86.1

–90.2

]0.2

76

87.9

[84.0

–91.5

]88.9

[83.6

–91.2

]0.4

47

MC

H(p

g)

31.1

[29.9

–33.5

]31.2

[29.8

–32.6

]0.0

47

30.7

[30.0

–31.2

]30.3

[29.7

–31.1

]0.8

66

30.7

[28.7

–31.6

]30.7

[28.7

–31.9

]0.6

77

MC

HC

(g/d

L)

34.7

[34.4

–35.2

]34.8

[34.3

–35.3

]0.4

92

34.5

[34.0

–35.0

]34.5

[34.0

–34.9

]0.4

84

34.6

[34.1

–34.9

]34.5

[33.5

–34.7

]0.2

01

Reticulo

cyte

s(·

10

9/L

)57.1

[41.3

–91.5

]82.5

[54.2

–113.8

]0.0

15

57.9

[39.0

–77.3

]50.2

[39.5

–90.8

]0.1

93

42.0

[30.5

–55.7

]45.5

[31.1

–60.8

]0.3

86

RP

I1.1

7[0

.82–1.8

5]

1.6

7[1

.04–2.2

7]

0.0

21

1.1

4[0

.69–1.5

7]

0.9

8[0

.74–1.7

5]

0.1

70

0.8

2[0

.59–1.0

8]

0.9

7[0

.49–1.1

4]

0.4

44

Tota

lbili

rubin

(mg/d

L)

0.5

5[0

.38–0.6

9]

0.4

6[0

.34–0.7

5]

0.6

23

0.8

0[0

.40–0.8

2]

0.5

4[0

.41–0.8

0]

0.8

01

0.8

0[0

.53–0.8

0]

0.8

0[0

.54–0.8

5]

0.6

71

MB

H(%

·10

-2)

0.6

9[0

.58–0.9

0]

0.4

6[0

.34–0.6

3]

<0.0

01

0.8

5[0

.56–0.9

2]

0.5

3[0

.49–0.7

4]

0.0

07

0.7

1[0

.51–0.8

3]

0.6

4[0

.46–0.7

7]

0.3

86

Band

3pro

file

(%)

HM

WA

g19.3

[16.0

–21.4

]13.3

[10.7

–17.4

]<0

.001

18.0

[13.3

–19.7

]11.6

[8.7

–18.3

]0.0

02

16.1

[11.1

–19.1

]15.0

[11.8

–16.6

]0.3

86

band

361.3

[54.2

–66.2

]61.2

[56.1

–65.8

]0.6

54

57.0

[54.1

–62.9

]60.2

[55.0

–66.3

]0.0

11

60.5

[57.1

–64.5

]63.2

[57.3

–65.6

]0.9

59

tota

lP

frag

20.9

[15.0

–25.5

]25.1

[20.7

–28.6

]0.0

02

24.3

[21.5

–26.1

]26.7

[22.5

–33.6

]0.0

44

23.0

[21.8

–23.8

]23.1

[17.3

–30.6

]0.9

59

aV

alu

es

expre

ssed

as

media

n[inte

rquart

ilera

nge].

HM

WA

g=

hig

h-m

ole

cula

r-w

eig

hta

ggre

gate

s;M

CH

=m

ean

cell

hem

oglo

bin

;MC

HC

=M

CH

concentr

ation;M

CV

=m

ean

cell

volu

me;P

frag

=pro

teoly

ticfr

agm

ents

;RB

Cs

=re

dblo

od

cells

;

RP

I=re

ticu

locyte

pro

duction

index.



AU

TH

OR

PR

OO

F

suggesting that a better improvement of the disease might ex-

plain these differences.

Conclusions

Our data suggest that psoriasis vulgaris triggers an inflam-

matory response with release of acute-phase reactants, reactive

oxygen species, cationic proteins, and proteases, leading to

enhanced RBC damage/aging and, ultimately, to enhanced

RBC removal. These assumptions were strengthened by the

observation that with the treatment of psoriasis some of these

changes reversed, the inflammation was reduced, the pro-

duction of reticulocytes was increased, and the RBCs showed

changes usually observed in younger/less damaged RBCs.

These erythroid changes were enhanced with PUVA therapy,

probably due to the more pronounced clearing of the lesions, as

suggested by PASI scores. Finally, after treatment, a residual

inflammation still persisted that might contribute to the ob-

served erythroid disturbances.

Acknowledgments

This study was supported by FCT (POCI/SAU –OBS/58600/2004) andby Fundacao para a Ciencia e Tecnologia (FCT: POCI/SAU – OBS/58600/2004) and Fundo Europeu de Desenvolvimento Regional (FEDER).

The authors have no conflicts of interest that are directly relevant to the

content of this study.

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51-3

Correspondence: Susana Coimbra, PhD, or Alice Santos-Silva, PhD, Departa-

mento de Ciencias Biologicas, Laboratorio de Bioquımica, Faculdade de

Farmacia da Universidade do Porto, R Anıbal Cunha 164, 4050-047 Porto,

Portugal.

E-mail: [email protected]; [email protected]



mailto:[email protected]; [email protected]

Erythroid Disturbances Before and After Treatment of Portuguese Psoriasis Vulgaris Patients

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