Cytokine 72 (2015) 43–47

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Serum IL-6, IL-10, and TNFa levels in pediatric sickle cell disease patients during vasoocclusive crisis and steady state condition Sameh Sarray a,⇑, Layal R. Saleh a, F. Lisa Saldanha a, Hebah H. Al-Habboubi a, Najat Mahdi b, Wassim Y. Almawi a a b

Department of Medical Biochemistry, Arabian Gulf University, Manama, Bahrain Department of Pediatrics, Salmaniya Medical Complex, Manama, Bahrain

a r t i c l e

i n f o

Article history: Received 18 July 2014 Received in revised form 20 November 2014 Accepted 27 November 2014

Keywords: Cytokine Inflammation Interleukin-10 Sickle cell disease Vaso-occlusive crisis

a b s t r a c t Vaso-occlusive crisis (VOC) is a significant complication of sickle cell disease (SCD), and altered production of pro-inflammatory and anti-inflammatory molecules contributed to its pathogenesis. In view of the association of chronic inflammation with VOC onset, and given the capacity of interleukin (IL)-10 as antiinflammatory, and IL-6, and TNFa as pro-inflammatory cytokines, we tested the association of altered IL10, IL-6, and TNFa secretion with VOC pathogenesis and its severity. Study subjects comprised 147 SCD patients with active VOC (VOC Group), and 63 pain-free SCD patients for at least 9 months before blood collection (Steady-state Group). Serum cytokine concentrations were determined by ELISA. IL-10 levels were significantly reduced, while IL-6 levels were increased in VOC compared to Steady-state groups; serum TNFa levels were comparable between both groups. There was enrichment of low IL-10, but high IL-6 and TNFa quartiles in VOC Group, which translated into increased VOC risk. In contrast, high IL-10, but low IL-6 and TNFa quartiles were seen in Steady-state Group. Correlation analysis demonstrated significant association between reduced IL-10 levels and the frequency, type, severity, and duration of VOC and requirement for hydroxyurea treatment, while IL-6 correlated with duration of VOC episodes. Our data support strong association of reduced IL-10 and increased IL-6 levels with VOC, and their modulation of VOC-related parameters. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Sickle cell disease (SCD) is an autosomal recessive hematological disorder caused by a single nucleotide substitution in the 6th codon of the b-globin gene, resulting in replacing the hydrophilic glutamate by the hydrophobic valine [1,2]. This amino acid change results in the polymerization of deoxygenated hemoglobin, and the typical distortion, decreased flexibility, and fragility of red blood cells, leading to microvascular occlusion, and hypoxia [2,3]. SCD is characterized by hemolytic anemia, occlusions of the microcirculation, frequent infections and fever, joint pain, lethargy, and weakness, resulting in painful vaso-occlusive crisis (VOC) and chronic

Abbreviations: CI, confidence interval; IL-10, interleukin-10; NSAID, non-steroidal anti-inflammatory drugs; OR, odds ratios; SCD, sickle cell disease; VOC, Vaso-occlusive crisis. ⇑ Corresponding author at: Department of Medical Biochemistry, College of Medicine & Medical Sciences, Arabian Gulf University, PO Box 22979, Manama, Bahrain. Tel.: +973 33340018; fax: +973 17271090. E-mail address: [email protected] (S. Sarray). http://dx.doi.org/10.1016/j.cyto.2014.11.030 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.

organ injury [4–7]. Several lines of evidence implicated a state of chronic inflammation with VOC pathogenesis, evidenced by the elevation in the levels of pro-inflammatory cytokines and acute phase proteins in the sera of SCD patients during VOC episodes [8–10]. Cytokines were implicated in several VOC processes, such as vascular endothelial activation, adhesion of erythrocytes and leukocytes to vascular endothelium, platelet activation, and deregulation of the apoptosis of endothelial cells [11,12]. Several studies reported on altered balance of inflammatory and anti-inflammatory cytokines in SCD patients during VOC, highlighted by elevation in pro-inflammatory cytokines [5,13,14], and reduction in anti-inflammatory cytokines levels [13,15] in SCD patients compared to healthy individuals. Among the anti-inflammatory cytokines, interleukin-10 (IL-10), a pleiotropic cytokine produced by T helper type 2 (Th2) and regulatory T cells (Treg), monocytes/macrophages, B cells, NK cells, and dendritic cells, and was described to be associated with SCD complications [16]. Several mechanisms were proposed for IL-10 suppression of inflammation, which included suppression of the production of inflammatory

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cytokines [17,18] and attenuation of the toxic effects of reactive oxygen species (ROS) and free radicals, such as nitrites [18,19]. However, the effects of IL-10 on vascular cells vary according to the cell origin [19], and the specific signaling pathways elicited by pro-inflammatory stimuli [17,19]. Cytokine imbalance was suggested to contribute to the pathogenesis of sickle cell pain crisis [20,21]. Reduced expression of anti-inflammatory cytokines, including IL-10, were reported in unselected SCD patients compared to healthy subjects [20–22], and in SCD patients with VOC compared to pain-free SCD patients [23]. However, contradictory findings were reported by other studies [8,24,25], principally due to the low number of subjects and selection criteria employed in those studies. We recently reported on the utility of analyzing changes in IL-10 serum levels in predicting VOC in SCD patients [26]. Here we extend these findings by testing the association of reduced IL-10 secretion with VOC and its associated features, by comparing its serum in levels in VOC cases and pain-free SCD control patients.

profile (HbS, HbF), and other hematological indices (Table 1). All subjects were Bahraini Arabs and non-Arabs or recently naturalized Bahrainis were excluded. The Arabian Gulf University Research and Ethics Committee approved the study protocol, which was in agreement with the Helsinki declaration of 2000. All participants (or guardians in the pediatric cases) gave written informed consent, after the purpose of the study was explained to them. 2.2. Serum IL-10 cytokine measurement Peripheral venous blood was collected into plain tubes (no anticoagulants). Serum was prepared by centrifugation of coagulated blood at 2000g for 10 min at 4 °C, and was stored in aliquots at or below 30 °C. Samples were tested for IL-10 (Cat. D1000B), IL6 (Cat. D6050), and TNFa (Cat. DTA00C) using human sandwich enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). Assay sensitivity for the cytokine ELISA kits ranged from 3.9 to 5.5 pg/ml, and inter-assay and intra-assay precision (CV%) ranged from 4.2–7.5% and 1.7–5.4%, respectively.

2. Patients and methods

2.3. Statistical analyses

2.1. Study subjects

Statistical analyses were performed on SPSS version 21 (Statistical Package for the Social Sciences). Categorical variables were expressed as percentages of total, while continuous variables were presented as mean ± SD. Student’s t-test was used to determine differences in means, and Pearson v2 or Fisher’s exact test was used to assess inter–group significance. For continuous variables that did not follow a normal distribution, we used nonparametric analysis: Mann–Whitney U-test for two group comparisons, or Kruskal–Wallis test for multiple group comparisons; quantitative data described as medians and range values. Scatter plot analyses were initially used to present the distribution of IL-10, IL-6, and TNFa among groups of individuals, with values out of percentiles 5–95 interval shown as individual points. Correlations among continuous variables were determined by Spearman correlation coefficient (r). The VOC risk was estimated in VOC patients relative to steady-state control patients by calculating the odds ratios (OR) and 95% confidence interval (CI), according to the method of Woolf. Cytokine serum levels were compared using comparison of quartiles technique to detect systematic switch of values toward one of the two groups. OR and 95% CI were also calculated for different cutoff points, based on the distribution in control subjects; IL-10, IL-6, and TNFa levels were used as continuous and then as categorized variables.

This was a retrospective case-control study, conducted between October 2011 and April 2013. Study subjects comprised 211 consecutively-recruited SCD patients, who were diagnosed with SCD according to hemoglobin profile (HbA, HbS, HbA2, and HbF), and were assigned to 1 of 2 groups: SCD patients who had any VOC event (VOC Group; n = 148), or clinically asymptomatic SCD control patients who reported no VOC events for the past 9 months (Steady-state Group; n = 63) (Table 1). VOC was defined as acute events characterized by diffuse pain occurring in the upper or lower extremities, back, chest, and abdomen, that was related to SCD, but not to SCD-unrelated cause such as trauma or cancer. Treatment of VOC consisted of oral (24.8%) or intravenous (42.9%) non-steroidal anti-inflammatory drugs (NSAID), narcotics alone (2.2%), and narcotics plus NSAID (20.7%). Comparable frequencies of hydroxyurea- (P = 0.80) and folic acid- (P = 0.32) treated patients were seen in both groups. SCD controls comprised patients presenting for routine followup in outpatient clinics. Inclusion criteria included afebrile state, no VOC episode, hospitalization or transfusion for at least 9 months, and were excluded if they were on pain medication, or presented with other coexisting condition. Steady-state SCD control patients were matched with VOC patients according to gender, hemoglobin Table 1 Characteristics of study participants.

3.1. Study subjects VOC Group

Age Gender (male:female) HbS (%) HbF (%) Total hemoglobin (g/dL) WBC (109/L) Platelets (109/L) Hematocrit (%) Mean corpuscular volume (fL) Mean corpuscular Hb (pg) Mean corpuscular Hb Conc (g/dL) Reticulocytes (%) Blood transfusion [number (%)] Hydroxyurea Rx [number (%)]

3. Results

a

11.4 ± 6.4 89:58 70.7 ± 9.0 19.2 ± 7.7 9.7 ± 1.4 9.6 ± 4.8 313.6 ± 177.5 26.6 ± 5.4 75.7 ± 10.0 25.8 ± 5.6 36.4 ± 2.7 5.0 ± 3.5 72 (56.3) 42 (28.7)

Steady-state

a

13.5 ± 11.8 36:27 70.8 ± 7.5 20.1 ± 6.5 10.7 ± 1.5 9.4 ± 2.0 332.7 ± 215.6 27.4 ± 4.9 77.1 ± 10.6 26.5 ± 4.8 35.3 ± 1.8 5.7 ± 3.9 20 (31.7) 24 (38.1)

P

b

0.208 0.649 0.956 0.464 0.277 0.516 0.556 0.257 0.419 0.465 0.403 0.392 0.002 0.250

a Study subjects comprised 147 SCA patients who had any VOC event during study (VOC Group) and 63 SCA patients who had no VOC events (Steady-state Group). b Pearson’s chi square test (categorical variables), 2-tailed t-test (continuous variables).

Table 1 summarizes the demographics and clinical characteristics of study participants. Both gender distribution (P = 0.649) and age at examination (P = 0.208) were comparable between VOC and steady-state SCD patients. HbS (P = 0.956), HbF (P = 0.464), along with other hematological and inflammatory indices, including total hemoglobin, hematocrit, WBC, platelet count, MCV, MCH, MCHC, and reticulocyte counts, were comparable between both SCD patients groups. VOC patients reported a mean 4.6 ± 2.2 VOC episodes/year, of mean duration of 4.6 ± 2.5 days/episode. The majority of VOC cases required blood transfusion (56.3%) and hospital admission (84.0%) principally to manage the painful crisis, with the pain score (on a scale of 0–10) being 7.1 ± 1.8. 3.2. Cytokine serum levels Mean serum IL-10 levels were reduced in VOC cases than in control SCD patients (P < 0.001) (Fig. 1). In contrast, serum IL-6 levels

S. Sarray et al. / Cytokine 72 (2015) 43–47

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Fig. 1. Serum TNFa, IL-10, and IL-6 IL-2 levels in SCD patients with vaso-occlusive crisis (VOC) (n = 147), and pain-free steady-state SCD patients (n = 63), were assessed by Mann–Whitney U-test for two group comparisons. Data are shown as boxplot of median with range.

were markedly elevated in patients with VOC compared to control SCD patients (P = 0.032), while serum TNFa levels were comparable between the two SCD patients groups (P = 0.107) (Fig. 1). Cytokine levels were categorized into 4 quartiles according to concentrations present in the control group. The association of IL-10, IL-6, and TNFa with VOC was further analyzed by examining the proportion of individuals with different IL-10, IL-6, and TNFa levels based on quartile distribution. Data from Table 2 demonstrated enrichment of low IL-10 producers (1st quartile) in VOC Group (P < 0.001), while increased prevalence of high IL-10 producers (4th quartile) was seen in the steady-state SCD control group (P < 0.001). In contrast, there was enrichment of high (4th quartile) IL-6 (P = 0.003) and TNFa (P = 0.005) producers in VOC Group, and increased prevalence of low (1st quartile) IL-6 (P = 0.021) and TNFa (P = 0.037) producers in the steady-state SCD control group. Given the shift in IL-10 values between VOC cases and steady-state controls, logistic regression analyses demonstrated a dose–effect relationship for altered cytokine levels with VOC. Setting the 1st quartile producers as reference (OR = 1.00), multivariate logistic regression analyses confirmed the association of low IL-10, but high IL-6, and TNFa levels as independent risk factors for VOC (Table 2). 3.3. Correlation studies Spearman correlation calculation demonstrated significant correlation between altered IL-10 levels and VOC frequency (number of episodes/year; r = 0.199; P = 0.008), VOC type (localized vs. generalized; r = 0.254; P = 0.001), duration of VOC episodes (r = 0.215; P = 0.018), and with pain scale (r = 0.164; P = 0.028), and weakly with hydroxyurea treatment (r = 0.142; P = 0.050) (Table 3). Apart from duration of VOC episodes (r = 0.247; P = 0.009), altered IL-6 production did not correlate with VOC-related parameters (Table 3). On the other hand, altered TNFa levels did not correlate with any of the VOC-related parameters examined (Table 3). 3.4. Influence of IL-10 Levels on VOC parameters In view of the correlation between reduced IL-10 levels and VOC parameters, we next examined the influence of reduced IL-10 secretion on VOC frequency (number of episodes/year), VOC type

(localized vs. generalized), and with pain scale. Taking steady state SCD patients as reference, results from Table 4 demonstrated significant association of reduced IL-10 secretion with the VOC type, which was more pronounced in generalized VOC. A step-wise reduction in IL-10 secretion was accompanied by increase in the number of VOC episodes/year, as well as increased VOC pain severity.

4. Discussion The release of cytokines in response to infections, endothelial cell activation and other insults plays a vital role in the pathophysiology of microvascular occlusion in SCD [8,23,25]. Previous studies evaluated changes in the levels of the anti-inflammatory cytokine IL-10 in patients with VOC, but with conflicting conclusions [12,22,25]. In this study, we investigated the association between serum levels of IL-6, TNFa, and IL-10 and VOC pathogenesis in pediatric SCD patients. Results obtained clearly demonstrated a significant association between reduced IL-10 levels, coupled with marginal association of elevated IL-6 levels, and the development of VOC and its severity. This was evidenced by the enrichment of low IL-10 producers in the VOC Group than in controls, and in the strong correlation between reduced IL-10 secretion and number of VOC parameters, namely severity, frequency and VOC type. A central role for chronic low-grade inflammation in the manifestation of recurrent VOC, hemolytic events, and endothelial cell activation in SCD patients was proposed [9,13–15], evidenced by the elevation in the levels of the pro-inflammatory cytokines TNF-a, IL-6 and IL-17 [11,23,27], along with acute phase proteins [9,28,29]. Other studies report normal, and even reduced levels of pro-inflammatory cytokines [30], and decreased expression of inflammatory mediators, such as nitric oxide, and oxidative stress [12,31]. Functionally, enhanced inflammatory cytokine expression may stimulate cellular adhesion, partly by upregulating the expression of vascular adhesion receptors, by inducing fibronectin and chemokine production, and also by increasing integrin avidity. Insofar as the incidence and severity of VOC and subsequent chronic organ damage varies between individuals [4,5], and as age influences the perception of, and reaction to painful episodes [32], we focused on pediatric SCD patients with age and ethnically matched pain-free steady-state SCD controls. Samples were collected during VOC episode to minimize the variability inherent in

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Table 2 Influence of cytokine cut-off levels on VOC risk. Cytokine IL-10

a b

VOC Groupa

Quartile (range) 1st (1.00–15.81) 2nd (16.05–18.24) 3rd (18.28–20.92) 4th (P21.15)

56 16 17 59

IL-6

1st (1.73–6.00) 2nd (6.02–6.91) 3rd (6.92–8.48) 4th (P8.48)

TNFa

1st (0.40–0.52) 2nd (0.53–0.64) 3rd (0.65–0.84) 4th (P0.84)

Steady-statea

b

(37.8) (10.8) (11.5) (39.9)

P

8 (12.7) 4 (6.3) 10 (15.9) 41 (65.1)

2.8  10 0.697 0.021 7.9  10

9 (6.1) 36 (24.5) 55 (37.4) 47 (32.0)

17 16 15 15

(27.0) (25.4) (23.8) (23.8)

10 18 15 20

53 42 29 23

(36.1) (28.6) (19.7) (15.6)

(15.9) (12.2) (23.8) (31.7)

OR (95% CI) 4

1.00 1.19 0.20 0.08

(Reference) (0.35–2.02) (0.05–0.78) (0.02–0.38)

0.021 0.687 0.574 0.003

1.00 0.84 1.29 5.51

(Reference) (0.35–1.98) (0.53–3.15) (1.79–16.94)

0.037 0.368 0.191 0.005

1.00 (Reference) 0.63 (0.23–1.72) 0.54 (0.21–1.36) 1.56–12.35)

4

Study subjects comprised 147 SCA patients who had any VOC event during study (VOC Group) and 63 SCA patients who had no VOC events (Steady-state Group). Number of subjects (percent total).

Table 3 Correlation between cytokine levels and VOC outcomes. Parameter

IL-10

Frequency (episodes/month) VOC type (generalized vs. local) Pain scale (1–10) Duration (days) Age at VOC onset (years) Need for hospitalization VOC treatment Hydroxyurea treatment HbS (%) HbF (%) a

TNFa

IL-6

ra

P

r

P

r

P

0.199 0.254 0.164 0.215 0.118 0.144 0.060 0.142 0.058 0.035

0.008 0.001 0.028 0.018 0.118 0.127 0.423 0.050 0.451 0.649

0.064 0.146 0.029 0.247 0.140 0.159 0.037 0.100 0.106 0.176

0.575 0.085 0.739 0.009 0.103 0.062 0.747 0.230 0.353 0.117

0.129 0.119 0.083 0.027 0.115 0.089 0.118 0.039 0.094 0.060

0.145 0.177 0.352 0.783 0.319 0.317 0.182 0.720 0.296 0.597

Spearman correlation index.

Table 4 Influence of sIL-10 levels on VOC parameters.

a

VOC parameter

Type

Median (range)

P

aOR (95% CI)a

VOC type

Steady-state Localized Generalized

24.93 (13.62–176.08) 21.28 (1.30–73.69) 14.84 (1.60–33.80)

0.017 0.001

0.97 (0.95–0.99) 0.87 (0.81–0.94)

Frequency (per year)

1–2 3–5 6–10 >11

25.17 20.47 18.48 16.10

0.908 0.033 0.013 0.007

0.99 0.96 0.95 0.90

Hospitalization

No Yes

23.67 (10.45–176.08) 18.15 (1.30–73.69)

0.321 0.003

0.98 (0.96–1.01) 0.86 (0.80–0.91)

Pain

Mild Moderate Severe

31.86 (13.62–176.08) 21.68 (130–117.71) 20.24 (1.30–73.69)

0.845 0.023 0.001

1.01 (0.96–1.05) 0.95 (0.90–0.98) 0.89 (0.84–0.93)

(14.59–46.04) (2.10–44.74) (1.60–61.71) (1.30–38.18)

(0.93–1.07) (0.90–0.96) (0.90–0.93) (0.81–0.93)

aOR = adjusted odds ratios; variables controlled for were HbS, HbF, and blood transfusion.

soluble cytokine determination. IL-10 levels in steady state SCD controls (median = 25.1 mg/L) were closely comparable to those of healthy non-SCD controls (24.24 mg/L; P = 0.609), both of which were significantly higher than that of VOC patients (18.6 mg/L). This supports the notion that reduced IL-10 availability results in increased inflammatory changes during VOC painful episodes. While IL-6 levels were significantly, but marginally, higher in VOC cases than steady-state control SCD patients (P = 0.032), it was linked with only the duration of VOC episodes, thereby questioning the role IL-6 plays in VOC pathogenesis. Our results were reminiscent of an earlier Nigerian study, in which steady-state SCD patients had higher serum (anti-inflammatory) IL-4 and IL-10 levels, when compared to SCD patients with VOC, or to healthy non-SCD controls [23]. This suggested

an obligatory role for IL-10 in countering the inflammatory mechanisms that accompany VOC. However, it was in apparent disagreement with American study, which showed that TNFa, IL-1b, IL-6, IL-8 and IL-10 were not elevated in asymptomatic SCD cases, or in patients with acute VOC [25]. They were also in disagreement with recent studies which reported elevated levels of TNFa, IFNc, IL-4, IL-5, IL-8, IL-10 and IL-13 in SCA Brazilian children of African descent origin [11,33]. However, SCD patients on hydroxyurea had elevated IL-10 levels [11], indicating a likely role for IL-10 in the clinical improvement of VOC outcome. These apparent inconsistencies are reconciled by the low number of subjects employed by these studies [21,33], lack of appropriate controls, and differences in ethics backgrounds [11,25,33].

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Results of this study indicate that severity, frequency, and type of VOC are due to imbalance between pro and anti-inflammatory mechanisms. It is tempting to speculate that this imbalance results from interference with a key intracellular target event associated with IL-10 signaling (phosphatidylinositol-3 kinase and downstream p70 S6-kinase and Akt/protein kinase B), previously shown to be required for IL-10 effect [34,35]. IL-10 may also modulate the translation of TNFa mRNA through interference with p38 MAPK activation, resulting in increased TNFa production [36]. Furthermore, iron overload due to RBC hemolysis as a consequence of SCD, or resulting from transfusion, leads to the generation of reactive oxygen species (ROS), and hence decreased IL-10 secretion and stimulation of TNFa secretion [22]. While our study clearly demonstrated an association between reduced IL-10 secretion and risk of VOC in SCD patients, some limitations to these findings are noteworthy. IL-10 levels were measured following VOC, which raises the possibility that they were the consequence, but not the cause of VOC. Another shortcoming was our selection of VOC patients, since only pediatric cases were included, which necessitates performing parallel determinations on adult SCD patients. Despite these shortcomings, our results support the notion of a covert inflammatory response in VOC, which will be instrumental in the future management strategies of VOC episodes. Acknowledgments The authors thank Dr. Khadija Al-Ola and Dr. Noor A. Redha for their helpful suggestions. The work was supported by Grants from Arabian Gulf University R&EC. References [1] Conran N, Franco-Penteado CF, Costa FF. Newer aspects of the pathophysiology of sickle cell disease vaso-occlusion. Hemoglobin 2009;33:1–16. [2] Mehanna AS. Sickle cell anemia and antisickling agents then and now. Curr Med Chem 2001;8:79–88. [3] Barabino GA, Platt MO, Kaul DK. Sickle cell biomechanics. Annu Rev Biomed Eng 2010;15(12):345–67. [4] Chiang EY, Frenette PS. Sickle cell vaso-occlusion. Hematol Oncol Clin North Am 2005;195:771–84. [5] Frenette PS. Sickle cell vaso-occlusion: multistep and multicellular paradigm. Curr Opin Hematol 2002;92:101–6. [6] Platt OS, Thorington B, Brambilla DJ, Milner PF, Rosse WF. Pain in sickle cell disease: rates and risk factors. N Engl J Med 1993;325:11–6. [7] Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, Steinberg MH. Klug PP. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med 1994;330:1639–44. [8] Mohammed FA, Mahdi N, Sater MA, Al-Ola K, Almawi WY. The relation of Creactive protein to vasoocclusive crisis in children with sickle cell disease. Blood Cells Mol Dis 2010;45:293–6. [9] Bourantas KL, Dalekos GN, Makis A, Chaidos A, Tsiara S, Mavridis A. Acute phase proteins and interleukins in steady state sickle cell disease. Eur J Haematol 1998;61:49–54. [10] Stuart J, Stone PCW, Akinola NO, Gallimore JR, Pepys MB. Monitoring the acute phase response to vaso-occlusive crisis in sickle cell disease. J Clin Pathol 1994;47:166–9. [11] Lanaro C, Franco-Penteado CF, Albuqueque DM, Saad ST, Conran N, Costa FF. Altered levels of cytokines and inflammatory mediators in plasma and leukocytes of sickle cell anemia patients and effects of hydroxyurea therapy. J Leukoc Biol 2009;85:235–42. [12] Makis AC, Hatzimichael EC, Bourantas KL. The role of cytokines in sickle cell disease. Ann Hematol 2000;79:407–13.

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