��-tocotrienol protects mouse and human hematopoietic progenitors from ��-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling

Background

Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments.

Design and Methods

CD2F1 mice and human hematopoietic progenitor CD34⁺ cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay.

Results

δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage⁻/Sca-1⁺/ckit⁺ stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34⁺ cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34⁺ cells.

Conclusions

Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34⁺ cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.     

Phenotypic and functional characterization of a mouse model of targeted Pig-a deletion in hematopoietic cells

Background

Somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) causes glycosyl phosphatidylinositol anchor deficiency in human patients with paroxysmal nocturnal hemoglobinuria.

Design and Methods

We produced an animal model of paroxysmal nocturnal hemoglobinuria by conditional Pig-a gene inactivation (Pig-a^−/−) in hematopoietic cells; mice carrying two lox sites flanking exon 6 of the Pig-a gene were bred with mice carrying the transgene Cre-recombinase under the human c-fes promoter. We characterized the phenotypic and functional properties of glycosyl phosphatidylinositol-deficient and glycosyl phosphatidylinositol-normal hematopoietic cells from these Pig-a^−/− mice using gene expression microarray, flow cytometry, bone marrow transplantation, spectratyping, and immunoblotting.

Results

In comparison to glycosyl phosphatidylinositol-normal bone marrow cells, glycosyl phosphatidylinositol-deficient bone marrow cells from the same Pig-a^−/− animals showed up-regulation of the expression of immune function genes and contained a significantly higher proportion of CD8 T cells. Both characteristics were maintained when glycosyl phosphatidylinositol-deficient cells were transplanted into lethally-irradiated recipients. Glycosyl phosphatidylinositol-deficient T cells were inactive, showed pronounced Vβ5.1/5.2 skewing, had fewer γ-interferon-producing cells after lectin stimulation, and contained fewer CD4⁺CD25⁺FoxP3⁺ regulatory T cells. However, the levels of T-cell receptor signaling proteins from glycosyl phosphatidylinositol-deficient cells were normal relative to glycosyl phosphatidylinositol-normal cells from wild type animals, and cells were capable of inducing target cell apoptosis in vitro.

Conclusions

Deletion of the Pig-a gene in hematopoietic cells does not cause frank marrow failure but leads to the appearance of clonally-restricted, inactive yet functionally competent CD8 T cells.     

Study of damage to red blood cells exposed to different doses of γ-ray irradiation

Background.

The aims of this research were to study alterations in the ultrastructure of red blood cells, the changes in concentrations of plasma electrolytes and the killing effect of lymphocytes in samples of blood exposed to different doses of γ-ray irradiation.

Materials and methods.

Blood samples were treated with different doses of γ-ray irradiation and then preserved for different periods. Specimens were prepared for standard electron microscopy and transmission electron microscopy. At the same time, changes in the concentrations of Na⁺, K⁺ and Cl⁻ and pH values in the plasma as well as Fas and FasL expression of lymphocytes before and after irradiation were determined.

Results.

The proportions of reversibly and irreversibly transformed cells, for example, echinocytes, sphero-echinocytes, and degenerated forms, increased with increasing doses of irradiation and storage period, while the number of discocyte shaped red blood cells decreased. The change in K⁺ concentration was greater than that of Na+ or Cl⁻ after irradiation and was dosage-dependent. Plasma pH was influenced by different doses of radiation and storage time. After exposure to ¹³⁷Cs γ-irradiation, the expression of both Fas and FasL in lymphocytes differed significantly from that in the control group: the expression was positively correlated with irradiation dose (r=0.95, 0.96), but no significant difference in the Fas/FasL ratio was observed (P>0.05).

Discussion.

We conclude that the ultrastructure of red blood cells is not changed obviously by irradiation with some doses of γ-rays and various periods of storage. However, irradiation does have some dose-dependent and time-dependent adverse effects on the erythrocytes.     

Plasma nitric oxide metabolite levels increase during successive exercise stress testing – A link to delayed ischemic preconditioning?

BACKGROUND:

Animal studies have shown that nitric oxide is involved in delayed ischemic preconditioning.

OBJECTIVES:

To determine whether plasma nitrates and nitrites (NO_x⁻, as measure of nitric oxide) are modified by two consecutive effort tests and whether these changes translate into clinical improvement

METHODS:

Twenty-two patients with ischemic heart disease each performed two effort tests at 24-h intervals. Plasma NO_x⁻ level was determined and compared before and after both stress tests. Peak effort, double product at peak effort and maximal ST segment depression were considered clinical endpoints and were compared between the two tests.

RESULTS:

Plasma NO_x⁻increased slightly after the first exercise test compared with pretest value (17.05±1.6 μmol/mL versus 15.38±1.4 μmol/mL). In turn, after the second test there was a significant rise in NO_x⁻ level (23.65±2.2 μmol/mL versus 15.10±1.3 μmol/mL, P<0.03). The pretest values were almost identical between the two tests. Peak effort and double product at peak effort remained unchanged between the two tests. Although ischemic stress was the same, ST depression was significantly lower (P<0.01) for the second test (0.85±0.06 mm versus 1.73±0.16 mm).

CONCLUSION:

Our study shows an increased plasma NO_x⁻level after the second of two consecutive exercise stress tests at 24-h intervals, along with a decrease of electrocardiographic consequences of approximately the same ischemic stress. These findings are consistent with experimental data in animals, which point to nitric oxide as a trigger and effector of ischemic preconditioning.     

Glomerular hyperfiltration and renal progression in children with autosomal dominant polycystic kidney disease

Summary

Background and objectives

The purpose of this study was to determine whether glomerular hyperfiltration (GH) occurring early in autosomal dominant polycystic kidney disease (ADPKD) is indicative of more rapid disease progression in children.

Design, setting, participants, & measurements

One hundred eighty children with ADPKD (ages 4 to 18 years) with normal renal function were examined by renal ultrasound. Renal volume was calculated using a standard formula for a modified ellipsoid. Creatinine clearance was calculated from serum creatinine and 24-hour urine creatinine. GH was defined as creatinine clearance ≥140 ml/min per 1.73 m².

Results

Thirty-two children had GH (mean age 11.4 ± 3.6 years) and 148 had normal renal function (mean age 10.8 ± 3.9 years). Patients with GH at baseline demonstrated an increased rate of total renal volume growth (β: rate of change = +19.3 ± 10.8 cm³/year) over 5 years compared with those without GH at baseline (β = −4.3 ± 7.7 cm³/year), P = 0.008. Those with GH at baseline experienced a faster decline in creatinine clearance in subsequent years (β = −5.0 ± 0.8 ml/min per 1.73 m² per year) compared with those without GH at baseline (β = +1.0 ± 0.4 ml/min per 1.73 m² per year), P < 0.0001.

Conclusions

This study revealed that occurrence of GH in ADPKD children is associated with a significantly faster decline in renal function and higher rate of kidney enlargement over time. GH combined with the increased renal volume may therefore be used as an early marker for a more severe progression of ADPKD in children.     

Imbalanced globin chain synthesis determines erythroid cell pathology in thalassemic mice

Background

β-thalassemia occurs from the imbalanced globin chain synthesis due to the absence or inadequate β-globin chain production. The excessive unbound α-globin chains precipitate in erythroid precursors and mature red blood cells leading to ineffective erythropoiesis and hemolysis.

Design and Methods

In vitro globin chain synthesis in reticulocytes from different types of thalassemic mice was performed. The effect of imbalanced globin chain synthesis was assessed from changes of red blood cell properties including increased numbers of red blood cells vesicles and apoptotic red blood cells, increased reactive oxygen species and decreased red blood cell survival.

Results

The α/β-globin chain ratio in β^IVSII-654-thalassemic mice, 1.26±0.03, was significantly higher than that of wild type mice, 0.96±0.05. The thalassemic mice show abnormal hematologic data and defective red blood cell properties. These values were improved significantly in doubly heterozygous thalassemic mice harboring 4 copies of human β^E-globin transgene, with a more balanced globin chain synthesis, 0.92±0.05. Moreover, transgenic mice harboring 8 extra copies of the human β^E-globin transgene showed inversely imbalanced α/β-globin synthesis ratio, 0.83±0.01, that resulted in a mild β-thalassemia phenotype due to the excessive β-globin chains. The degree of ineffective erythropoiesis also correlated with the degree of imbalanced globin chain synthesis. Bone marrow and splenic erythroid precursor cells of β^IVSII-654-thalassemic mice showed increased phosphatidylserine exposure in basophilic and polychromatophilic stages, which was restored to the normal level in doubly heterozygous mice.

Conclusions

Imbalanced α/β-globin chain as a consequence of either reduction or enhancement of β-globin chain synthesis can cause abnormal red blood cell properties in mouse models.     

Genetic modifiers of β-thalassemia and clinical severity as assessed by age at first transfusion

Background

The clinical and hematologic features of β-thalassemia are modulated by different factors, resulting in a wide range of clinical severity. The main factors are the type of disease-causing mutation and the ability to produce α-globin and γ-globin chains. In the present study we investigated the respective contributions of known modifiers to the prediction of the clinical severity of β-thalassemia as assessed by the patients’ age at first transfusion.

Design and Methods

We studied the effect of seven loci in a cohort of 316 Sardinian patients with β⁰-thalassemia. In addition to characterizing the β-globin gene mutations, α-globin gene defects and HBG2:g.−158C>T polymorphism, we genotyped two different markers in the BCL11A gene and three in the HBS1L-MYB intergenic region using single nucleotide polymorphism microarrays, imputation and direct genotyping. We performed Cox proportional hazard analysis of the time to first transfusion.

Results

According to the resulting model, we were able to explain phenotypic severity to a large extent (Harrell’s concordance index=0.72; Cox & Snell R²=0.394) and demonstrated that most of the model’s discriminatory ability is attributable to the genetic variants affecting fetal hemoglobin production (HBG2:g.−158C>T, BCL11A and HBS1L-MYB loci: C-index=0.68, R²=0.272), while the remaining is due to α-globin gene defects and gender. Consequently, significantly distinct survival curves can be described in our population.

Conclusions

This detailed analysis clarifies the impact of genetic modifiers on the clinical severity of the disease, measured by time to first transfusion, by determining their relative contributions in a homogeneous cohort of β⁰-thalassemia patients. It may also support clinical decisions regarding the beginning of transfusion therapy in patients with β-thalassemia.     

Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of α4 integrin- and CXCR4-mediated engraftment in NOD/SCID β2-microglobulin-null mice

Background

Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of α4 integrin, α5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34⁺ cells.

Design and Methods

Uncultured or expanded CD34⁺ cells were infused in NOD/SCID-β₂microglobulin-null mice. The function of α4, and α5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of α4 integrin was further tested by adhesion and migration assays.

Results

Neutralization of either α4 integrin or CXCR4 abolished engraftment of uncultured CD34⁺ cells at 6 week spost-transplant, while α5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking α4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of α5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that α4 integrin affinity in fresh CD34⁺ cells was low and susceptible to stimulation while in cultured CD34⁺ cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34⁺ cells but not in cultured CD34⁺ cells.

Conclusions

Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both α4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of α4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34⁺ cells.     

The ��-Specific Protein Kinase C Inhibitor Ruboxistaurin (LY333531) Suppresses Glucose-Induced Adhesion of Human Monocytes to Endothelial Cells in Vitro

Aims

Strong evidence shows that late diabetic complications in diabetes mellitus are substantially related to an increased synthesis of diacylglycerol with a subsequent activation of protein kinase C (PKC) β. Several studies have shown that specific inhibition of the PKC isoform β by ruboxistaurin is able to attenuate the development of microvascular complications under diabetic conditions. The aim of this in vitro study was to investigate the effect of ruboxistaurin on glucose-induced adhesion of monocytes to endothelial cells, representing one of the first pivotal steps in the course of atherogenesis.

Methods

Human umbilical venous endothelial cells were isolated and cultured to confluence in microtiter plates. After coincubation with monocytes in the presence of 0, 10, or 400 ng ruboxistaurin to achieve PKC β-specific and -unspecific PKC inhibition, cells were fixed and monocyte adhesion was determined by means of a standardized chemiluminescence assay. Expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin) was also measured by chemiluminescence methods.

Results

Adhesion of monocytes to endothelial cells cultured under hyperglycemic conditions (27.7 mM glucose) was increased by 30.9 ± 5.1% (p < 0.001) versus endothelial cells cultured under normoglycemic (NG) conditions (5.5 mM). Pretreatment of endothelial cells with 10 nM (PKC β-specific concentration) and 400 nM (PKC β-unspecific concentration) led to a significant reduction of glucose-induced adhesion of monocytes to endothelial cells that was statistically not different from endothelial adhesion under NG conditions (−7.2 ± 3.1 and −8.1 ± 2.6%, respectively; not significant vs NG). A nonsignificant tendency to lower the expression of adhesion molecules was seen with 10 ng of ruboxistaurin.

Conclusions

We conclude that monocyte adhesion to endothelial cells under hyperglycemic conditions is at least mediated by PKC β activation. Ruboxistaurin is able to suppress this monocyte adhesion even in a PKC β-specific concentration. Further studies should evaluate these potential effects of ruboxistaurin in vivo.     

Clinical and laboratory features of 103 patients from 42 Italian families with inherited thrombocytopenia derived from the monoallelic Ala156Val mutation of GPIbα (Bolzano mutation)

Background

Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients.

Design and Methods

Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin.

Results

We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases.

Conclusions

Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.     

Dominant inheritance of a novel integrin β3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families

Background

Defects of integrin α_IIbβ₃ are typical of Glanzmann’s thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann’s thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease.

Design and Methods

We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin β₃-gene (ITGB3) mutation.

Results

The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface α_Ib β₃, impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated α_IIbβ₃ upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective α_IIbβ₃-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647–686 of the βTD ectodomain of integrin β₃. Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome.

Conclusions

This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin β₃, and its βTD domain, in platelet formation and function.     

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

Background

Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor.

Design and Methods:

We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy.

Results

Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions.

Conclusions

We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.Key words: platelets, cold storage, glycoprotein Ibα, 14-3-3ζ, lipid raft, clustering     

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

Background

Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous β2‐microglubulin⁻/Thy1⁺ bone marrow derived cells.

Aim

To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia.

Methods and results

Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for β2‐microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non‐injured liver. Transplantation of bone marrow derived cells obtained from GFP‐transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP‐glucuronyltransferase enzyme evaluated by polymerase chain reaction.

Conclusions

I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte‐like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non‐affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.     

Association of late-life changes in blood pressure and cognitive status

Background Disagreement exists on the association between changes in blood pressure and cognitive impairment. We aimed to examine whether 4-year changes in systolic and diastolic blood pressure (SBP and DBP) are associated with cognitive status in a representative sample of older men and women. Methods Analysis of longitudinal data from 854 participants of a population-based German sample (aged 60-87 years) was performed with standard cognitive screening and blood pressure measurements. Effects of changes in SBP and DBP (10 mmHg and 5 mmHg respectively as unit of regression effect measure) on cognitive status were evaluated using non-parametric and linear regression modeling. Results No clear associations were seen between changes in SBP or in DBP and cognitive scores. Small effects were found after stratification for sex and hypertension awareness. Specifically, larger decreases in SBP were associated with higher cognitive scores in those men aware of their hypertension (10 mmHg decrease in SBP, β = -0.26, 95% CI: -0.51 to 0.02) and men with controlled hypertension (10 mmHg decrease in SBP, β = -0.44, 95% CI: -0.92 to -0.03). Additionally larger increases in DBP were associated with higher cognitive scores in men with controlled hypertension (5 mmHg increase in DBP, β = 0.67, 95% CI: 0.19-1.15). For women aware of their hypertension, larger decreases in DBP were associated with higher cognitive scores (5 mmHg decrease in DBP, β ?= -0.26; 95%CI: -0.51 to -0.01). Conclusions Changes in blood pressure were only weakly associated with cognitive status. Specifically, decreases in SBP were associated with higher cognitive scores in men aware of their hypertension and especially those that were medically controlled.     

The IL-10 polarized cytokine pattern in innate and adaptive immunity cells contribute to the development of FVIII inhibitors

Background

Hemophilia A (HA) is an X-linked inherited bleeding disorder, resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII). Antibodies against FVIII, also called inhibitors, block the procoagulant activity of FVIII; thus, impairing hemostatic activity in patients with HA. The exact mechanism underlying the immunological events behind the development of inhibitors remains unknown. This study aimed to understand immune response to FVIII in patients with HA who were either positive [HAα-FVIII(+)] or negative [HAα-FVIII(−)] for inhibitors.

Methods

Cytokine profiles [interferon-γ (IFN − γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-5, and IL-10] of innate and adaptive immune cells present in the peripheral blood of participants were characterized.

Results

Presence of inhibitors was significantly associated with decreased frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, IL-4-positive neutrophils, and increased frequencies of IL-10-positive neutrophils and T cells. T cells from HAα-FVIII(−) patients expressed increased levels of almost all cytokines. In contrast, HAα-FVIII(+) patients showed lower levels of all cytokines in CD4⁺ and CD8⁺ T cells, except IL-10. B cells from HAα-FVIII(−) patients expressed increased levels of IL-4 while those from HAα-FVIII(+) patients expressed increased levels of IL-10.

Conclusions

The global cytokine profiles of innate and adaptive immune cells showed an anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients and a mixed pattern, with a bias toward inflammatory cytokine profile, in HAα-FVIII(−) patients. The occurrence of these profiles seems to be associated with presence FVIII inhibitors.