Pathomechanisms of Immunological Disturbances in ß-Thalassemia.

Thalassemia, a chronic disease with chronic anemia, is caused by mutations in the ß-globin gene, leading to reduced levels or complete deficiency of ß-globin chain synthesis. Patients with ß-thalassemia display variable clinical severity which ranges from asymptomatic features to severe transfusion-dependent anemia and complications in multiple organs. They not only are at increased risk of blood-borne infections resulting from multiple transfusions, but they also show enhanced susceptibility to infections as a consequence of coexistent immune deficiency. Enhanced susceptibility to infections in ß-thalassemia patients is associated with the interplay of several complex biological processes. ß-thalassemia-related abnormalities of the innate immune system include decreased levels of complement, properdin, and lysozyme, reduced absorption and phagocytic ability of polymorphonuclear neutrophils, disturbed chemotaxis, and altered intracellular metabolism processes. According to available literature data, immunological abnormalities observed in patients with thalassemia can be caused by both the disease itself as well as therapies. The most important factors promoting such alterations involve iron overload, phenotypical and functional abnormalities of immune system cells resulting from chronic inflammation oxidative stress, multiple blood transfusion, iron chelation therapy, and splenectomy. Unravelling the mechanisms underlying immune deficiency in ß-thalassemia patients may enable the designing of appropriate therapies for this group of patients.

Phenotypical and functional abnormalities of circulating neutrophils in patients with ß-thalassemia.

ß-Thalassemia is an inherited single gene disorder related to reduced synthesis of the ß-globin chain of hemoglobin. Patients with ß-thalassemia present variable clinical severity ranging from asymptomatic trait to severe transfusion-dependent anemia and multiple organs complications. Moreover, multiple immune abnormalities are a major concern in ß-thalassemia patients. Aberrant neutrophil effector function plays a pivotal role in infection susceptibility in these patients. In severe and persistent inflammation, immature neutrophils are released from the bone marrow and are functionally different compared with mature ones. Despite some abnormalities reported for thalassemia patient's immune system, few data exist on the characterization of human neutrophils in ß-thalassemia. The aim of this study was to investigate the phenotype and function of circulating neutrophil subsets in patients with ß-thalassemia major and with ß-thalassemia intermedia divided in transfusion-dependent and non-transfusion-dependent. By the use of immunochemical and cytofluorimetric analyses, we observed that patients' CD16+ neutrophils exhibit abnormalities in their phenotype and functions and the abnormalities vary according to the clinical form of the disease and to the neutrophil subset (CD16bright and CD16dim). Abnormalities include altered surface expression of the innate immune receptor CD45, Toll-like receptor 4, and CD32, reduced ability to produce an oxidative burst, and elevated levels of membrane lipid peroxidation, especially in patients with a more severe form of the disease. Overall, our results indicating the occurrence of an immuno-senescent phenotype on circulating neutrophils from thalassemia patients suggest the usefulness of neutrophil feature assessment as a tool for better clinical management of ß-thalassemia.

Major Depression in Children with Transfusion-Dependent Thalassemia Is Strongly Associated with the Combined Effects of Blood Transfusion Rate, Iron Overload, and Increased Pro-inflammatory Cytokines.

Beta-thalassemia major patients are treated with repeated blood transfusions, which may cause iron overload, which in turn may induce immune aberrations, and show an increased risk of depression. The aim of the present study is to examine whether repeated blood transfusions, iron overload, and immune-inflammatory responses are associated with depression in children (6-12 years) with transfusion-dependent thalassemia (TDT). The Children's Depression Inventory (CDI), iron status (serum iron, ferritin, transferrin, TS%), and serum levels of CCL11, IL-1ß, IL-10, and TNF-α were measured in TDT with (n = 54) and without (n = 57) a major depression-like episode (MDLE) and in healthy children (n = 55). The results show that MDLE due to TDT is associated with a greater number of blood transfusions and increased iron overload and IL-1ß levels. Partial least squares path analysis shows that 68.8% of the variance in the CDI score is explained by the number of blood transfusions, iron overload, and increased levels of IL-1ß and TNF-α. The latter two cytokines partly mediate the effects of iron overload on the CDI score, while the effects of blood transfusions on the CDI score are partly mediated by iron overload and the path from iron overload to immune activation. Iron overload is also associated with increased IL-10 and lower CCL11 levels, but these alterations are not significantly associated with depression. In conclusion, blood transfusions may be causally related to MDLE in TDT children and their effects are in part mediated by increased iron overload and the consequent immune-inflammatory response. The results suggest that effects of iron overload and its consequences including inflammation and oxidative stress toxicity may cause MDLE. Current treatment modalities with folic acid and vitamin C are insufficient to attenuate iron overload and immune-inflammatory responses and to prevent MDLE in children with TDT.

Defective cytokine production from monocytes/macrophages of E-beta thalassemia patients in response to Pythium insidiosum infection.

BACKGROUND: Pythium insidiosum has been mainly reported to cause morbidity and mortality in thalassemia patients. P. insidiosum zoospores can germinate to be hyphae within a few hours; therefore, it is difficult to study the initial immune response that P. insidiosum zoospores induce. The present study aims to compare immune responses against P. insidiosum zoospore infection by comparing monocytes/macrophages from thalassemia patients with those from non-thalassemia controls. METHODS: In order to keepP. insidiosum in the zoospore stage in vitro for inoculation, the P. insidiosum zoospores were preserved without germination by treatment with inorganic hypochlorite solution. CD14+ cells were isolated from peripheral blood mononuclear cells of thalassemia and non-thalassemia donors and then left to transition to macrophages. Monocytes/macrophage culture was infected with P. insidiosum zoospores and culture supernatants were subjected to Th1/Th2 multiplex cytokine detection. RESULTS: Our study of cytokine production revealed that the basal level of GM-CSF produced by thalassemia monocytes/macrophages was lower than that observed in monocytes/macrophages of non-thalassemia individuals. Higher GM-CSF and IFN-γ response was also found when cells from non-thalassemia people were stimulated with P. insidiosum zoospores compared to thalassemia cells. It was also found that TNF-α, GM-CSF and IFN-γ productions from monocytes/macrophages of thalassemia patients who received iron chelator treatment were significantly higher than those produced from thalassemia patients without iron chelator treatment. CONCLUSION: For the first time, the present study demonstrates defective immune responses in monocytes/macrophages derived from thalassemia patients in response toP. insidiosum zoospore infection. The results also show an inverse correlation between iron overload and cytokine production in monocytes/macrophages of thalassemia patients. This finding could explain why thalassemia patients are susceptible to P. insidiosum infection.

Pneumococcal vaccination for splenectomized patients with thalassemia major in Indonesia.

INTRODUCTION: Streptococcus pneumoniae is a capsulated bacterium that can cause severe infection in patients with thalassemia major, particularly those who have undergone splenectomy. The absence of the spleen as well as zinc deficiency in splenectomized patients with thalassemia major increases the possibility of developing invasive pneumococcal infection. The aims of this study are to evaluate pneumococcal IgG levels following PCV and PPV immunizations and the effect of zinc supplementation on qualitative specific immune responses in splenectomized patients with thalassemia. METHODS: Splenectomized patients with thalassemia major were administered a PCV pneumococcal vaccine (Prevenar 13®) at the start of the trial, after which they were randomly assigned to 2 groups (zinc and placebo group). After 8weeks, the patients received a PPV pneumococcal vaccine (Pneumovax®). Zinc syrup was provided to the zinc group at a dose of 1.5mg/kg/day (maximum of 50mg/day). Pneumococcal IgG examinations were conducted at the start of the trial and after 12weeks. RESULTS: In the group without PPV, the median initial pneumococcal IgG value was 315 (ranging from 65 to 1419) mU/mL for the zinc group and 338.5 (ranging from 82 to 1648) mU/mL for the placebo group. The median final pneumococcal IgG value was 1812.5 (ranging from 834 to 2444) mU/mL for the zinc group and 2857.5 (ranging from 834 to 2624) for the placebo group. The increase in the pneumococcal IgG value between the two groups was comparable (p=0.642). In the group with previous PPV, the median initial pneumococcal IgG value was 1333 (ranging from 793 to 2031) mU/mL for the zinc group and 880 (ranging from 74 to 1686) mU/mL for the placebo group. The median final pneumococcal IgG value was 1487 (ranging from 635 to 1757) mU/mL for the zinc group and 1012 (ranging from 292 to 1732) mU/mL for the placebo group. The increase in the pneumococcal IgG value between the two groups was comparable (p=0.528). CONCLUSION: There is no difference in the increase in pneumococcal IgG level in splenectomized patients with thalassemia major prior to and after receiving PPV. There were no differences observed in the development of pneumococcal IgG following zinc supplementation.

Anti-Thyroid Peroxidase Antibodies and Male Gender Are Associated with Diabetes Occurrence in Patients with Beta-Thalassemia Major.

BACKGROUND: Intensive transfusion schedule and iron-chelating therapy prolonged and improved quality of life in patients with ß-thalassemia (ß-T) major. However, this led to an increased risk of developing impaired glucose tolerance or diabetes. In this study we analyzed variables associated with the occurrence of impaired glucose tolerance or diabetes in patients with ß-T major. METHODS: 388 Sardinian patients were included. Age, gender, duration of chelation therapy, body mass index, and markers of pancreatic and extrapancreatic autoimmunity were analyzed. RESULTS: Multiple logistic regression analysis showed that anti-thyroid peroxidase (TPO) antibodies (Ab) (OR = 3.36; p = 0.008) and male gender (OR = 1.98; p = 0.025) were significantly associated with glucose impairment, while the other variables were not. Ferritin levels were significantly higher in TPOAb positive compared to TPOAb negative patients (4870 ± 1665 µg/L versus 2922 ± 2773 µg/L; p < 0.0001). CONCLUSIONS: In patients with ß-T major a progressive damage of insulin-producing cells due to secondary hemosiderosis appears to be the most reasonable mechanism associated with glucose metabolism disorders. The findings need to be confirmed with additional well designed studies to address the question of whether TPOAb may have a role in the management of these patients.

Evaluating the safety and efficacy of silymarin in ß-thalassemia patients: a review.

ß-Thalassemia (ß-thal) is a type of hereditary anemia affecting hemoglobin (Hb) synthesis causing severe chronic anemia in homozygous patients. Regular blood transfusions are the mainstay treatment for this type of anemia. In turn, this leads to iron overload which is responsible for the formation of reactive oxygen species (ROS), oxidative stress and organ damage. Deferoxamine (DFO) is the standard of treatment for iron overload but regular painful subcutaneous administration of this medication prevents optimal compliance. Oral chelators, such as deferasirox (DFX) and deferiprone (DFP), are also effective and safe. Deferiprone is most effective in combination therapy with DFO rather than monotherapy; however, DFX is very expensive and the cost is a significant new burden for patients. Recently, researchers have proposed an iron chelating effect for silymarin that is a flavonoid extract from the milk thistle plant. This extract has different properties and has long been used for its antioxidant and hepatoprotective effects. In this review we assess different aspects of silymarin's potential effects and compare them to the profile of thalassemic patients.

[Clinico-biological and immunohaematological profile of patients with ß-thalassemia in Tunisia: about 26 cases]. / Profil clinico-biologique et immunohématologique des patients atteints de ß-thalassémie en Tunisie : à propos de 26 cas.

AIM OF THE STUDY: To study the clinical and biological profile of ß-thalassemic patients in our region, reflecting the quality of their care. PATIENTS AND METHODS: A retrospective study (2010-2011) on 26 ß-thalassemic patients followed in the pediatrics service at CHU Farhat Hached Sousse, Tunisia. Epidemiological, clinical and biological data were collected from medical records and transfusion files of patients. The transfusion protocol adopted was to maintain a hemoglobin level>10g/dL by regular transfusions every 3-4 weeks. Iron chelation therapy, in order to maintain serum ferritin<1500ng/mL, was introduced when serum ferritin exceeded 800-1000ng/mL. RESULTS: The mean age of patients at diagnosis was 15 months. The clinical impact of anemia had resulted in failure to thrive in 54% of patients and facial dysmorphism in 23%. The average transfusion requirement was estimated at 311.02mL/kg/year with 6 cases of hyperconsumption. The immunohaematological monitoring showed the appearance of anti-RBC alloimmunization in one patient and 4 cases of autoimmunization. Poor adherence of chelation therapy was 62% and causing 5 cases of cardiac complications, 4 cases of liver injury and 14 cases of endocrine complications. CONCLUSION: Improving the therapeutic care of ß-thalassemic children requires better monitoring of transfusion recovery and improved adherence to chelation therapy.

Serum levels of TGFß, IL-10, IL-17, and IL-23 cytokines in ß-thalassemia major patients: the impact of silymarin therapy.

Abstract Several immunological abnormalities have been characterized in ß-thalassemia, many of which are linked to or identified with cytokines. In this study, we investigated the serum levels of TGF-ß, IL-10, IL-17 and IL-23 in ß-thalassemia major patients in comparison with healthy controls. The immunomodulatory effect of silymarin (a flavonoid complex obtained from Silybum marinum) on the serum levels of cytokines was further evaluated in thalassemia patients receiving silymarin (420 mg/day) and compared with patients treated with placebo for 6-month. Serum cytokines levels were measured by enzyme linked immunosorbent assay (ELISA). The results showed a significant higher concentration of TGF-ß and IL-23 in the patient group than control group. Among studied cytokines, a significant reduction in serum IL-10 levels was found in patients treated with silymarin when compared with IL-10 values at baseline. However, no significant difference was observed between baseline values of cytokine compared with end values in placebo group. Our data suggest the presence of imbalanced immune condition involving inflammation and immunosuppression in thalassemia patients, which could be modulated to a more effective immune response by silymarin.

Hepatitis B surface antigen carrier rate in unvaccinated and vaccinated children with thalassaemia major at Bahawal Victoria Hospital, Bahawalpur, Pakistan.

Screening of blood reduces but does not eliminate the risk of hepatitis B virus (HBV) infection in multi-transfused thalassaemia patients. This study was done to evaluate efficacy of HBV vaccination on hepatitis B virus surface antigen (HBsAg) carrier rate in children with thalassaemia major receiving multiple blood transfusions. In a cohort study conducted at a hospital in Bahawalpur, Pakistan, during 2009-10, children with thalassaemia major aged < 60 months who received more than 24 blood transfusions and were HBsAg negative at the time of first blood transfusion were included. Of 196 unvaccinated children, the seropositive rate was 12.2%; while among 218 children vaccinated during the first year of life via the Pakistan Expanded Programme on Immunization, the seropositive rate was only 0.9%. The HBV vaccine was highly effective in reducing the HBsAg carrier rate in hildren with thalassaemia aged < 5 years.

Investigation of the distribution of lymphocyte subsets and zinc levels in multitransfused beta-thalassemia major patients.

Homozygous beta-thalassemia is a common genetic disorder in the Arabian Peninsula and an important cause of morbidity in Kuwait. The anemia is so severe that chronic blood transfusions, and the resulting iron overload, cause a shift in immunoregulatory balances and a deficiency in zinc. It was reported that individual immunological profile of CD8+ T-lymphocytes may have a modifying effect on the severity of iron overload in HFE homozygous hemochromatosis patients, with low numbers being negatively correlated with the total amount of body iron stores. This has not been tested in thalassemia major patients. This study was designed to utilize flow cytometric immunophenotyping to characterize effects of regular blood transfusion, and high serum ferritin levels because of irregular use of iron chelation therapy on T lymphocytes (CD2, CD3, CD4 and CD8), B lymphocytes (CD19) and natural killer cells (CD56) and zinc levels in the blood of patients with thalassemia major (n = 49) and healthy normal controls (n = 60) in Kuwait. None of the patients had active infections. T-cell markers' percentage levels were comparable between patients and controls (P > 0.05), while B cell marker (CD19) was significantly higher in patients (P = 0.007). Patients had lower percentage levels of CD56 cells (P = 0.007) and normal serum zinc. All patients had high serum ferritin levels with no significant correlation to CD8+ T lymphocytes (P > 0.05). High iron stores did not have an effect on T lymphocytes' profile, with normal zinc levels perhaps related to non compliance with chelation therapy. The high B cell marker may be indicative of stimulation of antibody producing cells as a result of regular blood transfusions.

Immune and neural status of thalassemic patients receiving deferiprone or combined deferiprone and deferoxamine chelation treatment.

Deferiprone (L1), has previously been reported to be associated with immunological abnormalities in iron loaded thalassemia patients. However, other factors may also have similar effects such as the level of iron overload, chronic immuno-stimulation due to transfusions, splenectomy and deferoxamine (DFO). During chelation therapy with DFO, several complications have been reported, which were due to pharmacological activity and high dose toxicity with regard to both acoustic and visual effects, as well as peripheral nerve disorders that were measured by nerve conduction velocities. The immune and neural status of 44 beta-thalassemic patients, aged 10-30 years (mean 19.4 +/- 4.9), receiving L1 as a monotherapy (n = 21), or in combination with DFO (n = 23), has been followed for 2 years by monitoring the level of immunoglobulins (IgG, IgM, IgA), the level of T and B lymphocytes (CD4/CD8), the auto antibodies: anti nuclear (ANA), anti-double-stranded (anti-ds DNA), anti reticulin (anti-R1), anti-extra nuclear (anti-ENA), anti histone (anti-AHA), anti liver-kidney-muscle (anti-LKM), anti-smooth muscle (anti-SMA), anti-thyroid (anti-ATA), anti-mitochondrial (anti-AMA) antibodies and the C-reactive protein. The percentage of patients with disorders of the immune and nervous system concerned very few cases. None of our patients with pathological findings in their immunological or neurophysiological examinations presented any signs or symptoms of involvement of the immune or nervous system. Further advantages have been identified for the oral use of L1 and its combination with DFO, including synergistic efficacy and lower dosing with limited toxicity.

In vitro effects of vitamin C and selenium on NK activity of patients with beta-thalassemia major.

In this study, the in vitro effects of vitamin C and selenium on natural killer (NK) cell activity of ss-thalassemia major patients was investigated. At first, significant decreased NK activity was found at E:T ratios of 1:1, 5:1, and 10:1 in whole thalassemia patients. Low-dose selenium treatment enhanced NK activity in patients but there was no change in the control group. High-dose selenium decreased NK activity significantly in splenectomized patients. Different doses vitamin C enhanced NK activity significantly in both splenectomized and unsplenectomized patients. According to these results, selenium dosage should be arranged carefully in thalassemia patients, whereas vitamin C can be used confidently.

Pathogenetic aspects of immune deficiency associated with beta-thalassemia.

Infectious complications constitute the second most common cause of mortality and a main cause of morbidity in beta-thalassemia. Besides the high risk of blood-borne infections associated with multiple transfusions, the increased susceptibility of these patients to infectious diseases has been attributed to a coexistent immune deficiency. Immune abnormalities have also been held responsible for the frequent occurrence of malignancies in beta-thalassemia, especially leukemia and lymphomas. Recent studies on immune competence in beta-thalassemia have revealed numerous quantitative and functional defects, involving T and B lymphocytes, immunoglobulin production, neutrophils and macrophages, chemotaxis, and phagocytosis, as well as the complement system. Regarding pathogenesis, iron overload, a primary complication of both thalassemia itself and transfusion therapy, is thought to be the main precipitating mechanism, due to the important immunoregulatory properties of iron and its binding proteins; iron excess may derange the immune balance in favor of the growth of infectious organisms. Other factors include multiple transfusions, associated with constant allo-antigenic stimulation, as well as with transmission of immunosuppressive viruses; splenectomy, resulting in increased susceptibility to infections by encapsulated bacteria and to immune system modifications; low levels of zinc, another immune regulator; iron chelation therapy, which predisposes to serious infections by yersinia species; and the circulation of abnormal native thalassemic erythrocytes, forming another permanent immune stimulus. Thus surveillance for infections in patients with beta-thalassemia is crucial, while further studies are warranted on immune function abnormalities and the implicated mechanisms.

CsA-associated neurotoxicity and ineffective prophylaxis with clonazepam in patients transplanted for thalassemia major: analysis of risk factors.

Cyclosporin A (CsA) has been shown to be useful in the prophylaxis of acute graft-versus-host-disease (GVHD). However, this immunosuppressive agent produces multiple side-effects including nephrotoxicity, hypertension, hypertricosis, gum hyperplasia, infections, and neurotoxicity. We report a retrospective analysis of neurotoxicity in 625 recipients transplanted for thalassemia and given CsA as part of GVHD prophylaxis. Neurotoxicity consisted in mental status changes, tremor, headache (grade 1), visual disturbance and cortical blindness (grade 2) and seizures and coma (grade 3). The overall toxicity was 28.8% and the incidence of convulsions was 10.1%. Neurological findings were reversible after temporary reduction or discontinuation of CsA. Class 3 patients, when prepared with protocol 6 (Bu 14 + Cy 200 and CsA for GVHD) or when they developed acute GVHD, had the highest risk of convulsions. Age, sex, different conditioning regimens, different anticonvulsive prophylaxis, liver damage due to iron-overload and/or to chronic inflammation did not influence the occurrence of CsA-related CNS toxicity. The occurrence of acute GVHD with concomitant use of high-dose corticosteroids is the single significant predisposing factor in the occurrence of convulsions. Grades 1 and 2 of neurotoxicity occurred earlier and were not influenced even by acute GVHD.