Monocytic Expression of Heme Oxidase-1 (HO-1) in Sickle Cell Patients and Correlation With the Humoral Immune Response to Vaccine and With Allo-immunization.

Monocytic Expression of Heme Oxidase-1 (HO-1) in Sickle Cell Patients and Correlation With the Humoral Immune Response to Vaccine and With Allo-immunization.

Monocytic Expression of Heme Oxidase-1 (HO-1) in Sickle Cell Patients and Correlation With the Humoral Immune Response to Vaccine and With Allo-immunization

Sickle cell disease (SCD) is an autosomal recessive disorder resulting from a substitution in the β chain of hemoglobin (Hb) which causes hemoglobin S to polymerize when deoxygenated. SCD patients present immune abnormalities that have always been attributed to functional asplenia. It it is now being recognized that patients with SCD have a pro-inflammatory condition with altered immune system activation contributing to the pathology of SCD. Increased levels of neutrophils, monocytes or cytokines have been reported in SCD patients. SCD is associated with many acute and chronic complications requiring immediate support. Actual strongly recommended therapies include chronic blood transfusions (CT) and hydroxyurea (HU). In addition, episodic transfusions are recommended and commonly used to manage many acute SCD complications.There is strong evidence to support the use of HU in adults with 3 or more severe vaso-occlusive crises during any 12-month period, with SCD pain or chronic anemia, or with severe or recurrent episodes of acute chest syndrome. HU use is now also common in children with SCD. Some patients receive chronic monthly RBC transfusion with the objective to reduce the proportion of HbS to < 30 %. Long-term RBC transfusions prevent and treat complications of SCD decreasing the risk of stroke and the incidence of acute chest syndrome (ACS). Therapeutic complications, such as alloimmunization against RBC in 20-50% of patients or hematopoietic stem cell transplantation (HSCT) graft rejection, constitute an immune-based clinical issue in SCD. Poorly understood RBC alloimmunization is responsible for serious hemolytic transfusion reaction associated with severe mortality and morbidity underlying the need for a better understanding of the immunology of SCD to improve SCD transfusion support/outcome. Little evidence exists about HU effects on immune functions in SCD. HU treatment doesn't appear to have deleterious effects on immune function and appears to decrease the abnormally elevated number of total WBC and lymphocytes, while CT does not. Patients with SCD are at higher risk of infections and prophylactic vaccination is strongly recommended. Recent data suggest that vaccinal response to pneumococcal antigens in SCD patients is identical to healthy control while controversy concern the stability of the immune protection after vaccination of SCD patient. Antibody levels declined over the year and the need for more frequent vaccination in SCD patient should be investigated. Currently, there is no evidence whether HU may interfere with pneumococcal immune response. Purohit showed that immune response to inactivated influenza A (H1N1) virus vaccine was altered in patient with SCD receiving CT but little is known on immune response to vaccination in patients with SCD receiving HU. Recent data suggest that not only inflammatory status but also humoral immune response to antigens in SCD patients may differ according to treatment. Yazdanbakhsh reported an imbalance between regulatory T cell (Treg) and effector T cell (Teff) in alloimmunized SCD patients with as consequence an increase in antibody production. In a model proposed by the authors, the balance between Treg and Teff is dictated by the monocyte control of cytokines expression. Altered activity of monocyte heme oxidase-1 (HO-1) would be responsible of a decrease in IL-12 and an increase in IL-10 cytokines secretion impacting the Treg/Teff cells ratio and promoting antibody production by B cells. The objectives of the project are to assess whether different humoral immune responses to vaccines or to erythrocyte alloantigens are related to the type of treatment administered to patients with SCD. We also aim to study if these differences might be related to different expressions of HO-1 by monocytes.

Sickle cell disease patients (SCD) under regular chronic exchange transfusions. Pediatric and adult patients from the HUDERF and CHU-Brugmann Hospitals.

Sickle cell disease patients (SCD) under hydroxyurea (HU) alone. Pediatric and adult patients from the HUDERF and CHU-Brugmann Hospitals.

Sickle cell disease patients (SCD) under hydroxyurea (HU) and receiving sporadic transfusions.Pediatric and adult patients from the HUDERF and CHU-Brugmann Hospitals.

All groups of patients and the control group will receive the new annually recommended inactivated influenza A (H1N1) virus vaccine.

Intracellular monocyte heme oxidase-1 (HO-1) expression will be measured by flow cytometry.The protein expression of HO-1 will be confirmed by Western blot. A commercial ELISA kit will be used in parallel to assess HO-1 levels in PBMC cell lysate.

Pro-inflammatory cytokine (IL-12) and anti-inflammatory cytokine (IL-10) levels will be evaluated in serum and in IL-1 stimulated whole blood supernatants using an ELISA assay.

Evaluation of Treg cells in peripheral blood mononuclear cells (PBMC) will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers

Intracellular HO-1 expression will be measured by flow cytometry.The protein expression of HO-1 will be confirmed by Western blot. A commercial ELISA kit will be used in parallel to assess HO-1 levels in PBMC cell lysate.

Intracellular HO-1 expression will be measured by flow cytometry.The protein expression of HO-1 will be confirmed by Western blot. A commercial ELISA kit will be used in parallel to assess HO-1 levels in PBMC cell lysate.

Intracellular HO-1 expression will be measured by flow cytometry.The protein expression of HO-1 will be confirmed by Western blot. A commercial ELISA kit will be used in parallel to assess HO-1 levels in PBMC cell lysate.

Pro-inflammatory cytokine (IL-12) and anti-inflammatory cytokine (IL-10) levels will be evaluated in serum and in IL-1 stimulated whole blood supernatants using an ELISA assay.

Pro-inflammatory cytokine (IL-12) and anti-inflammatory cytokine (IL-10) levels will be evaluated in serum and in IL-1 stimulated whole blood supernatants using an ELISA assay.

Pro-inflammatory cytokine (IL-12) and anti-inflammatory cytokine (IL-10) levels will be evaluated in serum and in IL-1 stimulated whole blood supernatants using an ELISA assay.

Evaluation of Treg cells in PBMC will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers

Evaluation of Treg cells in PBMC will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers

Evaluation of Treg cells in PBMC will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers

Inclusion Criteria: - Pediatric and adult patients with sickle cell disease from the HUDERF and CHU-Brugmann Hospital Exclusion Criteria: None

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