Correlation of B cell activating factor, B cell maturation antigen, and other cytokines profile with inhibitor status in hemophilia A

Sanya Arshad; Vandana Tiwari; Nuzhat Husain; Azfar Neyaz; Namrata P Awasthi

doi:10.4103/ejh.ejh_6_19

ORIGINAL ARTICLE

Year : 2019 | Volume : 44 | Issue : 2 | Page : 149-156

Correlation of B cell activating factor, B cell maturation antigen, and other cytokines profile with inhibitor status in hemophilia A

Sanya Arshad¹, Vandana Tiwari², Nuzhat Husain MD ¹, Azfar Neyaz¹, Namrata P Awasthi¹
¹ Department of Pathology, Dr Ram Manohar Lohia Institute of Medical Sciences, Gomti Nagar, Lucknow, Uttar Pradesh, India
² Department of Biochemistry, Dr Ram Manohar Lohia Institute of Medical Sciences, Gomti Nagar, Lucknow, Uttar Pradesh, India

Date of Submission	10-Feb-2019
Date of Acceptance	04-Apr-2019
Date of Web Publication	15-Nov-2019

Correspondence Address:
Nuzhat Husain
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Gomti Nagar, Lucknow 226010, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_6_19

Abstract

Background The role of cytokines in inhibitor-positive versus inhibitor-negative patients with hemophilia A (PWHA) is controversial. Raised levels of B cell activating factor (BAFF) and B cell maturation antigen (BCMA) have been recently reported and have been linked to presence of inhibitors.
Aim To directly quantify serum BAFF and BCMA along with interleukin (IL)-10, IL-5, and tumor necrosis factor-α (TNF-α) in PWHA following an on-demand schedule for factor VIII replacement, and to correlate these with demographic, clinicopathological, and treatment parameters.
Patients and methods The study group included 35 inhibitor-positive, 131 inhibitor-negative PWHA, and 20 healthy controls. Serum levels of BAFF, BCMA, IL-10, IL-5, and TNF-α were determined by enzyme-linked immunosorbent assay.
Results The mean serum levels in inhibitor positive, inhibitor negative, and healthy controls for BAFF was 0.346, 0.218, and 0.118 pg/ml (P=0.280), for BCMA was 0.266, 0.176, and 0.187 pg/ml (P=0.298), for IL-10 was 13.71, 5.72, and 2.45 pg/ml (P≤0.001), for IL-5 was 2.90, 2.74, and 2.50 pg/ml (P=0.205) and for TNF-α was 5.20, 6.78, and 4.0 pg/ml (P=0.558), respectively. Six (30%) inhibitor-positive PWHA had elevated BCMA levels while none of the inhibitor-negative PWHA showed increased values.
Conclusion The current study has, for the first time, analyzed BAFF and BCMA in PWHA with inhibitor status in a clinical setting of on-demand therapy. As a preliminary conclusion we feel that, while cytokines play a role in pathogenesis they do not seem to trigger inhibitor development in PWHA.

Keywords: B cell activating factor, B cell maturation antigen, cytokines profile, factor VIII inhibitors, hemophilia A, interleukin-10, on-demand therapy

How to cite this article:
Arshad S, Tiwari V, Husain N, Neyaz A, Awasthi NP. Correlation of B cell activating factor, B cell maturation antigen, and other cytokines profile with inhibitor status in hemophilia A. Egypt J Haematol 2019;44:149-56

How to cite this URL:
Arshad S, Tiwari V, Husain N, Neyaz A, Awasthi NP. Correlation of B cell activating factor, B cell maturation antigen, and other cytokines profile with inhibitor status in hemophilia A. Egypt J Haematol [serial online] 2019 [cited 2023 Jun 5];44:149-56. Available from: http://www.ehj.eg.net/text.asp?2019/44/2/149/271083

Background

Factor VIII (FVIII) inhibitor development in patients with hemophilia A (PWHA), is a frequent complication which affects their treatment and prognosis, and is a complex, multifactorial immune response to replacement therapy with FVIII, involving both genetic and nongenetic factors. Several studies in both animal and human models have explored links between specific polymorphisms of cytokine genes, especially those of interleukin (IL)-10, tumor necrosis factor (TNF), interferon (IFN), and transforming growth factor, and their association with risk of developing inhibitors in PWHA [1],[2],[3],[4],[5],[6]. A recent study by Doshi and colleagues done on small group of hemophilia A patients and mice reported that B cell activating factor (BAFF) may allow for the persistence of anti-FVIII antibodies during immune tolerance induction and after rituximab therapy in hemophilia A inhibitor patients. In their study, soluble B cell maturation antigen (BCMA) levels correlated with inhibitor titer, suggesting that it plays a role in anti-FVIII plasma cell survival. BAFF levels were also found to increase in hemophilia A mice with inhibitor formation [7].

The role of cytokines is controversial, and their reported high to low levels in inhibitor-positive PWHA may be etiological or related to the pathogenesis. The direct estimation of these cytokine and changes in their levels in PWHA receiving on-demand therapy has not been studied. The current study was hence done with the objectives to evaluate the levels of several cytokines BAFF and BCMA along with IL-10, IL-5, and TNF-α in PWHA following an on-demand schedule for FVIII replacement therapy. These cytokine levels were further correlated with the clinicopathological parameters.

Patients and methods

Patients and controls

The study group comprised of a series of 166 PWHA from North India. The subgroups evaluated included: (a) healthy controls (n=20), (b) inhibitor-negative PWHA (n=131), and (c) inhibitor-positive PWHA (n=35).

Collection of samples

The blood samples from PWHA were collected through direct reference to our laboratory as well as in outreach camps held in various cities of North India in association with the local chapters of the Haemophilia Federation of India. Ethical clearance was obtained from the institutional ethical committee. All the patients received on-demand therapy and were treated with plasma-derived FVIII (Hemofil M; Baxalta US Inc., USA). For coagulation studies and cytokine analysis, peripheral blood was collected from PWHA in 3.2% sodium citrate and plain vials respectively (Becton & Dickinson, Franklin Lakes, New Jersey, USA) and samples were processed within 1 h of collection for plasma and serum separation by centrifugation at 4000 rpm for 10 min at 4°C and stored at −80°C until further testing.

Inclusion/exclusion criteria

PWHA giving informed consent to enter the study were included. In case of minors informed consent from guardians was obtained. Patients with other bleeding disorders or active infection were excluded.

Clinical assessment

A detailed clinical history was recorded including age at onset, age at diagnosis, disability profile, number of bleeding episodes, FVIII requirement per annum in terms of IU/kg/year in each PWHA, and frequency of whole blood and cryoprecipitate transfusions was recorded. Pedigree was charted on the basis of family history. Intron 22 inversion mutation was tested by inverse PCR method [8].

Phenotype analysis

The PWHA were categorized into severe, moderate, and mild on the basis of FVIII activity less than 1%, 1–5%, more than 5–30%, respectively.

Inhibitor assay

Inhibitor assay was performed by Classical Bethesda assay in all PWHA and Nijmegen modified Bethesda assay in PWHA negative for Classical Bethesda assay. Methods have been detailed in our earlier publication [9].

Measurement of serum cytokine levels including B cell activating factor, TNFRSF17 (B cell maturation antigen), interleukin-5, interleukin-10, and tumor necrosis factor-α

Serum levels of BAFF, BCMA, IL-10, IL-5, and TNF-α were determined by enzyme-linked immunosorbent assay. Commercially available research kits for TNF-α, IL-5, and IL-10 from Diaclone (France) and for BCMA and BAFF from Finetest (Wuhan Fine Biotech Co. Ltd, China) were used. The tests were run according to the recommended protocol in the manufacturer’s instructions. All tests were performed in duplicate wells. A standard curve was plotted by using absorbance on x axis and given standard concentration on y axis. Values of samples were derived using the standard plot. Concentrations which were below the lower limit of detection (LOD), were substituted with a median constant value between 0 and LOD for the purpose of statistical analyses [10]. The LOD of IL-5, IL-10, and TNF-α was 5, 4.9, and 8 pg/ml, respectively.

Cut-off values

Cut-off values for were obtained from studies of normal values in literature; cut offs for IL-10 was taken at 13.7 pg/ml, IL-5 4.4 pg/ml, and TNF-α at 18.5 pg/ml [11],[12],[13]. For BAFF and BCMA, we could find not large reference value studies, and cut off was obtained from mean±SD2 of the normal controls included in the analysis. Cut off above which elevated values of BAFF were taken was 0.278 pg/ml and for BCMA was 0.239 pg/ml.

Statistical analysis

The data and results were compiled using Microsoft Excel software. The results were presented in frequencies, percentages, mean±SD, and median. Association between various cytokine levels and clinicopathological parameters were analyzed by the Mann–Whitney U test and Kruskal–Wallis test. Association between categorical variables was analyzed by χ² and Fisher exact test. A P value of less than 0.05 was considered significant. All the analysis was carried out on SPSS 16.0 version (SPSS Inc., Chicago, Illinois, USA).

Results

Clinical characteristics

The mean age of PWHA was 17.81±11.21 years with a family history present in 103 (62.04%) PWHA. The mean age at onset of bleeding and age at first transfusion were 33.6 months and 6.2 years, respectively. All patients received an on-demand therapy with FVIII given to 39.8%, only blood products in 5.4% and combination therapy in 54.8%. Intron 22 inversion was present in 48.1% cases.

Inhibitor levels

The median Bethesda titer in inhibitor-positive group was 7.2 BU/ml (range, 0.7–96 BU/ml; mean, 15.07 BU/ml; SD, 19.6 BU/ml). Inhibitors were classified as low responder (≤5 BU/ml) and high responders (>5 BU/ml). Twenty-two PWHA had a Bethesda titer of more than 5 BU/ml (62.9%) whereas 13 PWHA (37.1%) had a titer of less than or equal to 5 BU/ml.

Serum cytokine levels

IL-10, IL-5, TNF-α were quantified in serum of PWHA along with a group of normal controls. IL-10 was quantified in 145 PWHA, IL-5 in 146 PWHA, TNF-α in 143 PWHA, BAFF in 34 PWHA, and BCMA in 34 PWHA. All five cytokines were studied across 34 PWHA. Cytokine levels in inhibitor-positive, inhibitor-negative PWHA, and healthy controls are depicted in [Table 1] in terms of mean values ([Table 1]a) and frequency of elevated and low levels of serum cytokines in inhibitor-positive and inhibitor-negative group ([Table 1]b). IL-10 values across study groups were significantly elevated in inhibitor-positive PWHA. The mean cytokine levels of inhibitor-positive PWHA was higher IL-5, BAFF, and BCMA. This difference was, however, not statistically significant. Cytokines profiles were compared between PWHA and healthy controls ([Table 1]c). A detailed correlation of various clinicopathological parameters with cytokines are mentioned in [Table 2].

Table 1 Mean difference of serum cytokines between inhibitor positive, inhibitor negative, and healthy control groups; number of cases with elevated and low levels of serum cytokines in inhibitor-positive and inhibitor-negative group; mean difference of serum cytokines between patients with hemophilia A and healthy control groups

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Table 2 Correlation of cytokines B cell activating factor, B cell maturation antigen, interleukin-10, interleukin-5, and tumor necrosis factor-α with clinicopathological parameters

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B cell activating factor

The mean serum BAFF was 0.293 pg/ml (median, 0.104; SD, 0.42; range, 0.054–1.939 pg/ml), in 20 inhibitor-positive PWHA 0.346±0.505 pg/ml and 14 inhibitor-negative was 0.218±0.263 pg/ml. All healthy control (n=20) had values ranging from 0.061 to 0.351 pg/ml (median : SD, 0.083 pg/ml). There was no significant statistical difference in means between serum BAFF levels of inhibitor positive, inhibitor negative, and healthy control group but there was a considerable difference in means of the three groups (0.346, 0.218, and 0.118, respectively) ([Figure 1]a). The mean values in PWHA was higher (0.293 pg/ml) as compared with normal controls (0.118 pg/ml) but this was not statistically significantly ([Table 1]c). The relationship of elevated BAFF levels to clinicopathological parameters was analyzed and statistical analysis did not reveal any other relevant correlates.

Figure 1 Distribution of serum (A) BAFF, (B) BCMA, (C) IL-10, (D) IL-5 and (E) TNF-α levels in healthy controls, inhibitor-negative PWHA and inhibitor-positive PWHA.

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B cell maturation antigen

BCMA levels were measured in serum of 34 PWHA. The mean serum BCMA was 0.229 pg/ml (median, 0.168; SD, 0.167; range, 0.153–1.102 pg/ml), in 20 inhibitor-positive PWHA 0.266±0.212 pg/ml and 14 inhibitor-negative was 0.176±0.021 pg/ml. All healthy control (n=20) had values ranging from 0.155 to 0.229 pg/ml (SD, 0.026 pg/ml) ([Figure 1]b). Six out of 20 inhibitor-positive PWHA (30%) had elevated values, whereas among the inhibitor-negative group none had elevated values thereby giving a P value of 0.031. There was no statistical significance between mean serum BCMA levels of inhibitor-positive, inhibitor-negative, and health control group, but there was a considerable difference in means of the three groups (0.266, 0.176, and 0.187, respectively). The mean values in PWHA was higher (0.229 pg/ml) as compared with normal controls (0.187 pg/ml) but this was not statistically significant ([Table 1]c). The relationship of elevated BCMA levels to clinicopathological parameters was analyzed and statistical analysis did not reveal any other relevant correlates.

Interleukin-10

IL-10 levels were measured in serum of 145 PWHA. The mean serum IL-10 was 7.65 pg/ml (median, 2.45; SD, 24.16; range, 2.45–289.06 pg/ml), in 35 inhibitor-positive PWHA 13.71±48.35 pg/ml and 111 inhibitor-negative was 5.72±5.15 pg/ml. All healthy controls (n=20) had values less than 2.45 pg/ml, which was lower LOD for IL-10. Four out of 35 inhibitor-positive PWHA (11.4%) had elevated values, whereas among the inhibitor-negative group 10 out 110 (9.1%) had elevated values. A statistical significance was observed in difference of means between serum IL-10 levels in inhibitor-positive, inhibitor-negative, and healthy controls with means of 13.71, 5.72, and 2.45 (P<0.001) ([Figure 1]c). The mean values in PWHA was 7.65 pg/ml and this was significantly (P<0.001) different from normal controls (2.45 pg/ml) ([Table 1]c). The relationship of elevated IL-10 levels to clinicopathological parameters was analyzed and an elevated levels were observed in the pediatric age group (<18 years) (P<0.001). Patients receiving FVIII had an elevated IL-10 level in comparison with patients receiving blood products and combination therapy (P=0.055) ([Table 2]).

Interleukin-5

IL-5 levels were measured in serum of 146 PWHA. The mean serum IL-5 in study group was 2.78 pg/ml (median, 2.5; SD, 1.33; range, 2.50–12.05 pg/ml), in 35 inhibitor-positive PWHA 2.90±1.30 pg/ml and 111 inhibitor-negative PWHA was 2.74±1.35 pg/ml. All healthy controls (n=20) had values less than 2.5 pg/ml, which was lower LOD for IL-5. There was no significant difference in means between serum IL-5 levels of inhibitor-positive, inhibitor-negative, and healthy control group ([Figure 1]d). The mean values in PWHA was 2.78 pg/ml and this was not significantly different from normal controls (2.50 pg/ml). The relationship of elevated IL-5 levels to clinicopathological parameters was analyzed and statistical analysis did not reveal any relevant correlates.

Tumor necrosis factor-α

TNF-α levels were measured in serum of 143 PWHA. The mean serum TNF-α in study group was 6.40 pg/ml (median, 4.0; SD, 13.07; range, 4.0–115.96 pg/ml), in 35 inhibitor-positive PWHA 5.20±5.66 pg/ml and 108 inhibitor-negative PWHA was 6.78±14.7 pg/ml. All healthy controls (n=20) had values less than 4.0 pg/ml, which was lower LOD for TNF-α. There was no significant difference in means between serum TNF-α levels of inhibitor-positive, inhibitor-negative, and healthy control group ([Figure 1]e). The mean value in PWHA was higher (6.39 pg/ml) as compared with normal controls (4.0 pg/ml) but this was not statistically significant. The relationship of elevated TNF-α levels to clinicopathological parameters was analyzed and a higher levels were observed in the pediatric age group (<18 years) (P=0.028). Statistical analysis did not reveal any other relevant correlates.

Analysis of patients with hemophilia A with raised B cell activating factor and B cell maturation antigen

Elevated BAFF levels were evident in 11 PWHA inhibitors were evident in seven PWHA with titers varying from 0.7 to 48.2 BU/ml. These PWHA had a severe phenotype, 10/11 had received FVIII while one has been managed on blood products only and was negative for inhibitors. Mean annual factor intake in inhibitor-positive PWHA with elevated BAFF was 5964.28 and in inhibitor-negative PWHA was 1750 IU/kg/year. Frequency of bleeding episodes varied from 2 to 30, with inhibitor-negative PWHA showing a lower range from 2 to 12/year and inhibitor-positive PWHA in a higher range of 8–30/year.

Elevated BCMA levels were evident in six PWHA ranging from 0.316 to 1.1018 pg/ml. Inhibitors were present in all six PWHA and their titers varied from 2 to 35.2 BU/ml. These PWHA had a severe phenotype and were inversion 22 positive, age range 5–32 (mean, 15.66 years), mean annual factor intake of 5250 IU/kg/year, bleeding episodes 3–30/year with a mean of 15 bleeds per year.

Discussion and conclusion

The current study has, for the first time, analyzed BAFF and BCMA in PWHA with and without inhibitors a clinical setting of on-demand therapy. Further cytokines frequently reported to be related to inhibitor development that is IL-10, IL-5, and TNF-α levels were also assessed. The immune system is activated after FVIII treatment, because the infused FVIII is recognized as a foreign protein. In India, an on-demand protocol is widely followed, unlike developed countries, where prophylactic infusions are a choice for management of PWHA. All the patients recruited in the current study were on an on-demand protocol and were managed by infusion of plasma-derived FVIII concentrates as well as blood products during uncontrolled bleeding episodes.

BAFF and BCMA, a relatively new class of cytokines belonging to TNF family, has been reported to play a role in inhibitor development in small study groups receiving prophylactic therapy [7],[14]. BAFF has been explored as a cytokine involved in the humoral immunity affecting survival and maturation of B cells [15]. Takeda and colleagues reported BAFF levels to be significantly higher in PWHA with inhibitors compared with healthy controls or PWHA without inhibitors. They suggested that elevated BAFF levels allow anti-FVIII antibody-secreting plasma cells to survive and produce inhibitors [14]. Some elevation in mean BAFF and BCMA values in inhibitor-positive PWHA were observed in our study as compared with inhibitor-negative PWHA, but were not statistically significant. It appears that elevated cytokine levels are an effect of antigenic FVIII infusions and do not appear to be a risk factor for development of inhibitors. A detailed analysis of treatment history revealed that higher IL-10 levels in serum was present in where FVIII and combined therapy was given as compared with those receiving only blood products. The mean cytokine level for IL-10 were significantly higher in PWHA as a whole as compared with healthy controls and across the study groups (P<0.001).

A large repertoire of cytokines has been incriminated in the process of inhibitor development. Polymorphisms in different cytokine genes (ILs, TNF-α) and correlated these with formation of inhibitors [1],[2],[16]. The cause or effect relationship of the occurrence of cytokines in generation of inhibitors is debated. Some investigators are of the opinion that that cytokine gene polymorphisms and high levels of cytokines play a critical role. The Malmo International Brother Study demonstrated that polymorphisms of the TNF-α gene and IL-10 are associated with an increased risk of inhibitor formation [1],[2]. Additional studies on IL-10 polymorphisms have revealed inconsistent results [6],[17],[18]. In studies defining a causal role in relation to gene polymorphisms the cytokine dependent mechanism does not explain the development of inhibitors in all PWHA but has been observed in about one-third PWHA [19].

The immune mechanism which is involved in inhibitor formation involves the Th1 T cells which release proinflammatory cytokines such as IL-2, IL-12, and IFN-γ. The Th2 cells release anti-inflammatory cytokines such as IL-4 and IL-10 that inhibit Th1 cells and induce B cell activation [20]. Oliveira and colleagues have proposed a model to explain the immune activation directed against FVIII. They propose that inhibitor-negative PWHA prevent inhibitors development due to a dual pathway: (i) modulating the proinflammatory immune response by the recruitment of IL-4+, CD4+, and CD8+ T cells and (ii) up-regulating the production of IL-10 by B cells in a T cell-dependent manner promoting the synthesis of anti-FVIII IgG1 antibodies. Second, the anti-inflammatory regulatory role of neutrophils and monocytes, with the basal levels of cytokines produced by T cells and B lymphocytes, may favor formation of anti-FVIII IgG4 inhibitors in inhibitor-positive PWHA [19].

We have done direct measurement of cytokines in PWHA without using any stimulatory response measures. Studies such as one by Towfighi et al. [21] have measured cytokine response following stimulation of PBMCs with rFVIII and phytohemagglutinin mitogen. In direct assays of plasma cytokines in normal and inhibitor-negative PWHA, Oliveira and colleagues observed that both groups had similar levels of proinflammatory cytokines (IFNc, TNF-α, and IL-2), while inhibitor-positive PWHA had significant lower levels of these cytokines. The anti-inflammatory/regulatory cytokines showed higher levels of IL-5 in the normal and inhibitor-negative groups, IL-4, and IL-10 did not vary in inhibitor-positive, inhibitor-negative, and normal groups. It appears direct measurements may not reflect changes in immune mechanisms believed to support inhibitor formation.

Studies have attempted to establish correlation between FVIII inhibitor development, FVIII gene mutations, HLA haplotypes, and polymorphisms in cytokine genes, and other clinicopathological parameters and ethnicity [3],[22],[23],[24],[25],[26]. However, a clear connection between these variables and development of an immune response against FVIII replacement has not been established [19]. Bleeding into joints is also know to produce a proinflammatory reaction such as IL-1, IL-6, and TNF-α [27]. This may be a confounder when analyzing proinflammatory cytokine increase. Most of our cases had hemarthrosis.

Doshi and colleagues have published interesting preliminary data exploring the role of BAFF and BCMA as serum markers of inhibitor development, however this does not seem to apply to our patients receiving an on-demand protocol. A future including targeting of BAFF as a therapeutic strategy has also been suggested as a promising treatment in a subset of PWHA [7],[14]. Our study is limited by numbers in the BAFF and BCMA quantification. Further cytokine gene polymorphisms have not been correlated in our PWHA. Further studies are necessary before conclusions can be drawn on the role of these cytokines in PWHA in prophylactic versus on-demand therapy and its application in the clinical settings. As a preliminary conclusion we feel cytokines do not seem to be triggers for inhibitor development. They have a role in pathogenesis but have no diagnostic value in PWHA receiving on-demand therapy.

Acknowledgments

The authors wish to acknowledge Department of Science and Technology (DST), New Delhi, India for providing SERB grant support (Ref. no. SB/SO/HS/025/2014)..

Sanya Arshad: collection of cases, acquisition of data, interpretation of data, drafting of the paper, writing, and final approval of the paper. Vandana Tiwari: acquisition of data, interpretation of data, and final approval of the paper. Nuzhat Husain: conception and design of the research study, analysis and interpretation of data, writing, and final approval of the paper. Azfar Neyaz: analysis and interpretation of data, final approval of the paper. Namrata P. Awasthi: conception and design of the research study, writing, and final approval of the paper.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Astermark J, Oldenburg J, Carlson J, Pavlova A, Kavakli K, Berntorp E, Lefvert AK. Polymorphisms in the TNFA gene and the risk of inhibitor development in patients with hemophilia A. Blood 2006; 108:3739–3745.
2.	Astermark J, Oldenburg J, Pavlova A, Berntorp E, Lefvert AK. MIBS Study Group. Polymorphisms in the IL10 but not in the IL1beta and IL4 genes are associated with inhibitor development in patients with hemophilia A. Blood 2006; 107:3167–3172.
3.	Chaves D, Belisário A, Castro G, Santoro M, Rodrigues C. Analysis of cytokine genes polymorphism as markers for inhibitor development in haemophilia A. Int J Immunogenet 2010; 37:79–82.
4.	de Alencar JB, Macedo LC, de Barros MF, Rodrigues C, Shinzato AH, Pelissari CB et al. New associations: INFG and TGFB1 genes and the inhibitor development in severe haemophilia A. Haemophilia 2015; 21:e312–e316.
5.	Gaitonde P, Peng A, Straubinger RM, Bankert RB, Balu-Iyer SV. Downregulation of CD40 signal and induction of TGF-β by phosphatidylinositol mediates reduction in immunogenicity against recombinant human Factor VIII. J Pharm Sci 2012; 101:48–55.
6.	Pavlova A, Delev D, Lacroix-Desmazes S, Schwaab R, Mende M, Fimmers R et al. Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10,tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A. J Thromb Haemost 2009; 7:2006–2015.
7.	Doshi BS, Lacey SF, Chen F, Raffini LJ, Arruda VR. Novel correlation between B cell survival cytokines and inhibitors in hemophilia A. Blood 2017; 130(Suppl 1):3677.
8.	Liu Q, Sommer SS. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene. Biotechniques 1998; 25:1022–1028.
9.	Arshad S, Singh A, Awasthi NP, Kumari S, Husain N. Clinicopathological parameters influencing inhibitor development in patients with hemophilia A receiving on-demand therapy. Ther Adv Hematol 2018; 9:213–226.
10.	Croghan CW, Egeghy PP. Methods of dealing with values below the limit of detection using SAS. St. Petersburg, FL: Lecture Note Presented at Southeastern SAS User Group; 2003.
11.	Kleiner G, Marcuzzi A, Zanin V, Monasta L, Zauli G. Cytokine levels in the serum of healthy subjects. Mediators Inflamm 2013; 2013:434010.
12.	Joseph J, Benedict S, Safa W, Joseph M. Serum interleukin-5 levels are elevated in mild and moderate persistent asthma irrespective of regular inhaled glucocorticoid therapy. BMC Pulm Med 2004; 4:2.
13.	Arican O, Aral M, Sasmaz S, Ciragil P. Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity. Mediators Inflamm 2005; 2005:273–279.
14.	Takeda T, Sakurai Y, Tatsumi K, Kato J, Kasuda S, Yoshioka A, Shima M. Elevation of B cell-activating factor belonging to the tumour necrosis factor [corrected] family (BAFF) in haemophilia A patients with inhibitor. Thromb Haemost 2009; 101:408–410.
15.	Rolink AG, Tschopp J, Schneider P, Melchers F. BAFF is a survival and maturation factor for mouse B cells. Eur J Immunol 2002; 32:2004–2010.
16.	Visentainer JE, Sell AM, da Silva GC, Cavichioli AD, Franceschi DS, Lieber SR, de Souza CA. TNF, IFNG, IL6, IL10 and TGFB1 gene polymorphisms in South and Southeast Brazil. Int J Immunogenet 2008; 35:287–293.
17.	Bafunno V, Santacroce R, Chetta M, D’Andrea G, Pisanelli D, Sessa F et al. Polymorphisms in genes involved in autoimmune disease and the risk of FVIII inhibitor development in Italian patients with haemophilia A. Haemophilia 2010; 16:469–473.
18.	Lozier JN, Rosenberg PS, Goedert JJ, Menashe I. A case-control study reveals immunoregulatory gene haplotypes that influence inhibitor risk in severe haemophilia A. Haemophilia 2011; 17:641–649.
19.	Oliveira CA, Velloso-Rodrigues C, Machado FC, Carvalho BN, Gentz SH, Martins-Filho OA, Chaves DG. Cytokine profile and FVIII inhibitors development in haemophilia A. Haemophilia 2013; 19:e139–e142.
20.	Murphy KM. T lymphocyte differentiation in the periphery. Curr Opin Immunol 1998; 10:226–232.
21.	Towfighi F, Gharagozlou S, Kardar GA, Sharifian RA, Karimi K, Lak M et al. Assessment of in vitro cytokine response in hemophilia A patients with or without factor VIII inhibitory antibody. J Interferon Cytokine Res 2007; 27:665–674.
22.	Pinto P, Ghosh K, Shetty S. Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients. Haemophilia 2012; 18:794–797.
23.	Carpenter SL, Michael Soucie J, Sterner S, Presley R; Hemophilia Treatment Center Network (HTCN) Investigators. Increased prevalence of inhibitors in Hispanic patients with severe haemophilia A enrolled in the Universal Data Collection database. Haemophilia 2012; 18:e260–e265.
24.	Peyvandi F, Mannucci PM, Garagiola I, El-Beshlawy A, Elalfy M, Ramanan V et al. A randomized trial of factor VIII and neutralizing antibodies in hemophilia A. N Engl J Med 2016; 374:2054–2064.
25.	Oldenburg J, Schröder J, Brackmann HH, Müller-Reible C, Schwaab R, Tuddenham E. Environmental and genetic factors influencing inhibitor development. Semin Hematol 2004; 41(Suppl 1):82–88.
26.	Ragni MV, Ojeifo O, Feng J, Yan J, Hill KA, Sommer SS et al. Risk factors for inhibitor formation in haemophilia: a prevalent case-control study. Haemophilia 2009; 15:1074–1082.
27.	Roosendaal G, Vianen ME, Wenting MJ, van Rinsum AC, van den Berg HM, Lafeber FP, Bijlsma JW. Iron deposits and catabolic properties of synovial tissue from patients with haemophilia. J Bone Joint Surg Br 1998; 80:540–545.