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.

• ClinicalTrials.gov Identifier: NCT03111589
• Recruitment Status: Completed
• First Posted: April 13, 2017
• Last Update Posted: October 18, 2018
• Sponsor: Francis Corazza
• Information provided by (Responsible Party): Francis Corazza, Brugmann University Hospital

Tracking Information

• First Submitted Date: April 7, 2017
• First Posted Date: April 13, 2017
• Last Update Posted Date: October 18, 2018
• Actual Study Start Date: October 2016
• Actual Primary Completion Date: October 2018 (Final data collection date for primary outcome measure)

Current Primary Outcome Measures (submitted: April 7, 2017)

• Intracellular HO-1 expression in monocytes [ Time Frame: 1 month post vaccination ]
  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.
• HO-1 level in serum [ Time Frame: 1 month post vaccination ]
  Monocyte heme oxidase-1 (HO-1) level in serum will be measured by a commercial ELISA kit
• Cytokines levels measurement [ Time Frame: 1 month post vaccination ]
  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.
• Identification of T regulatory cells [ Time Frame: 1 month post vaccination ]
  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
• Immune response to vaccination [ Time Frame: 1 month post vaccination ]
  Post-vaccination serum H1N1 antibodies titers (IgG and IgM) will be measured by an ELISA kit

• Original Primary Outcome Measures: Same as current

Current Secondary Outcome Measures (submitted: April 7, 2017)

• Intracellular HO-1 expression in monocytes [ Time Frame: Baseline: at vaccination ]
  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 in monocytes [ Time Frame: 3 months post vaccination ]
  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 in monocytes [ Time Frame: 6 months post vaccination ]
  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.
• HO-1 level in serum [ Time Frame: Baseline: at vaccination ]
  HO-1 level in serum will be measured by a commercial ELISA kit
• HO-1 level in serum [ Time Frame: 3 months post vaccination ]
  HO-1 level in serum will be measured by a commercial ELISA kit
• HO-1 level in serum [ Time Frame: 6 months post vaccination ]
  HO-1 level in serum will be measured by a commercial ELISA kit
• Cytokines levels measurement [ Time Frame: Baseline: at vaccination ]
  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.
• Cytokines levels measurement [ Time Frame: 3 months post vaccination ]
  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.
• Cytokines levels measurement [ Time Frame: 6 months post vaccination ]
  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.
• Identification of T regulatory cells [ Time Frame: Baseline: at vaccination ]
  Evaluation of Treg cells in PBMC will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers
• Identification of T regulatory cells [ Time Frame: 3 months post vaccination ]
  Evaluation of Treg cells in PBMC will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers
• Identification of T regulatory cells [ Time Frame: 6 months post vaccination ]
  Evaluation of Treg cells in PBMC will be performed by flow cytometry using appropriate fluorochrome conjugated monoclonal antibodies for CD25 and FoxP3 markers
• Immune response to vaccination [ Time Frame: Baseline: at vaccination ]
  Post-vaccination serum H1N1 antibodies titers (IgG and IgM) will be measured by an ELISA kit
• Immune response to vaccination [ Time Frame: 3 months post vaccination ]
  Post-vaccination serum H1N1 antibodies titers (IgG and IgM) will be measured by an ELISA kit
• Immune response to vaccination [ Time Frame: 6 months post vaccination ]
  Post-vaccination serum H1N1 antibodies titers (IgG and IgM) will be measured by an ELISA kit

• Original Secondary Outcome Measures: Same as current
• Current Other Pre-specified Outcome Measures: Not Provided
• Original Other Pre-specified Outcome Measures: Not Provided

Descriptive Information

• Brief Title: 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.
• Official Title: 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

Brief Summary

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.

• Detailed Description: Not Provided
• Study Type: Interventional
• Study Phase: Not Applicable
• Study Design: Allocation: Non-Randomized; Intervention Model: Parallel Assignment; Masking: None (Open Label); Primary Purpose: Basic Science
• Condition: Sickle Cell Disease

Intervention

• Biological: Inactivated influenza A (H1N1) virus vaccine
  All groups of patients and the control group will receive the new annually recommended inactivated influenza A (H1N1) virus vaccine.
• Diagnostic Test: Blood sampling
  Testing of the different humoral immune responses to vaccines or to erythrocyte alloantigens.

Study Arms

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

  • Biological: Inactivated influenza A (H1N1) virus vaccine
  • Diagnostic Test: Blood sampling
• Experimental: SCD patients under HU treatment alone
  Sickle cell disease patients (SCD) under hydroxyurea (HU) alone. Pediatric and adult patients from the HUDERF and CHU-Brugmann Hospitals.
  Interventions:

  • Biological: Inactivated influenza A (H1N1) virus vaccine
  • Diagnostic Test: Blood sampling
• Experimental: SCD patients under HU treatment+sporadic transfusion
  Sickle cell disease patients (SCD) under hydroxyurea (HU) and receiving sporadic transfusions.Pediatric and adult patients from the HUDERF and CHU-Brugmann Hospitals.
  Interventions:

  • Biological: Inactivated influenza A (H1N1) virus vaccine
  • Diagnostic Test: Blood sampling
• Active Comparator: Control group
  Pediatric and adult patients from the HUDERF and CHU-Brugmann Hospitals.
  Interventions:

  • Biological: Inactivated influenza A (H1N1) virus vaccine
  • Diagnostic Test: Blood sampling

Publications *

• Chintagari NR, Nguyen J, Belcher JD, Vercellotti GM, Alayash AI. Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease. Blood Cells Mol Dis. 2015 Mar;54(3):302-6. doi: 10.1016/j.bcmd.2014.12.001. Epub 2014 Dec 22.
• Cunnington AJ, Njie M, Correa S, Takem EN, Riley EM, Walther M. Prolonged neutrophil dysfunction after Plasmodium falciparum malaria is related to hemolysis and heme oxygenase-1 induction. J Immunol. 2012 Dec 1;189(11):5336-46. doi: 10.4049/jimmunol.1201028. Epub 2012 Oct 24.
• De Montalembert M, Abboud MR, Fiquet A, Inati A, Lebensburger JD, Kaddah N, Mokhtar G, Piga A, Halasa N, Inusa B, Rees DC, Heath PT, Telfer P, Driscoll C, Al Hajjar S, Tozzi A, Jiang Q, Emini EA, Gruber WC, Gurtman A, Scott DA. 13-valent pneumococcal conjugate vaccine (PCV13) is immunogenic and safe in children 6-17 years of age with sickle cell disease previously vaccinated with 23-valent pneumococcal polysaccharide vaccine (PPSV23): Results of a phase 3 study. Pediatr Blood Cancer. 2015 Aug;62(8):1427-36. doi: 10.1002/pbc.25502. Epub 2015 Mar 23. Erratum In: Pediatr Blood Cancer. 2019 Feb;66(2):e27554.
• Hankins J, Jeng M, Harris S, Li CS, Liu T, Wang W. Chronic transfusion therapy for children with sickle cell disease and recurrent acute chest syndrome. J Pediatr Hematol Oncol. 2005 Mar;27(3):158-61. doi: 10.1097/01.mph.0000157789.73706.53.
• Lanaro C, Franco-Penteado CF, Albuqueque DM, Saad ST, Conran N, Costa FF. Altered levels of cytokines and inflammatory mediators in plasma and leukocytes of sickle cell anemia patients and effects of hydroxyurea therapy. J Leukoc Biol. 2009 Feb;85(2):235-42. doi: 10.1189/jlb.0708445. Epub 2008 Nov 12.
• Lederman HM, Connolly MA, Kalpatthi R, Ware RE, Wang WC, Luchtman-Jones L, Waclawiw M, Goldsmith JC, Swift A, Casella JF; BABY HUG Investigators. Immunologic effects of hydroxyurea in sickle cell anemia. Pediatrics. 2014 Oct;134(4):686-95. doi: 10.1542/peds.2014-0571. Epub 2014 Sep 1.
• Mohri T, Ogura H, Koh T, Fujita K, Sumi Y, Yoshiya K, Matsushima A, Hosotsubo H, Kuwagata Y, Tanaka H, Shimazu T, Sugimoto H. Enhanced expression of intracellular heme oxygenase-1 in deactivated monocytes from patients with severe systemic inflammatory response syndrome. J Trauma. 2006 Sep;61(3):616-23; discussion 623. doi: 10.1097/01.ta.0000238228.67894.d7.
• Nickel RS, Osunkwo I, Garrett A, Robertson J, Archer DR, Promislow DE, Horan JT, Hendrickson JE, Kean LS. Immune parameter analysis of children with sickle cell disease on hydroxycarbamide or chronic transfusion therapy. Br J Haematol. 2015 May;169(4):574-83. doi: 10.1111/bjh.13326. Epub 2015 Mar 5.
• Pathare A, Al Kindi S, Alnaqdy AA, Daar S, Knox-Macaulay H, Dennison D. Cytokine profile of sickle cell disease in Oman. Am J Hematol. 2004 Dec;77(4):323-8. doi: 10.1002/ajh.20196.
• Purohit S, Alvarez O, O'Brien R, Andreansky S. Durable immune response to inactivated H1N1 vaccine is less likely in children with sickle cell anemia receiving chronic transfusions. Pediatr Blood Cancer. 2012 Dec 15;59(7):1280-3. doi: 10.1002/pbc.24206. Epub 2012 May 24.
• Estcourt LJ, Fortin PM, Hopewell S, Trivella M, Wang WC. Blood transfusion for preventing primary and secondary stroke in people with sickle cell disease. Cochrane Database Syst Rev. 2017 Jan 17;1(1):CD003146. doi: 10.1002/14651858.CD003146.pub3.
• Yawn BP, Buchanan GR, Afenyi-Annan AN, Ballas SK, Hassell KL, James AH, Jordan L, Lanzkron SM, Lottenberg R, Savage WJ, Tanabe PJ, Ware RE, Murad MH, Goldsmith JC, Ortiz E, Fulwood R, Horton A, John-Sowah J. Management of sickle cell disease: summary of the 2014 evidence-based report by expert panel members. JAMA. 2014 Sep 10;312(10):1033-48. doi: 10.1001/jama.2014.10517. Erratum In: JAMA. 2014 Nov 12;312(18):1932. JAMA. 2015 Feb 17;313(7):729.
• Yazdanbakhsh K. Mechanisms of sickle cell alloimmunization. Transfus Clin Biol. 2015 Aug;22(3):178-81. doi: 10.1016/j.tracli.2015.05.005. Epub 2015 Jun 6.

Recruitment Information

• Recruitment Status: Completed
• Actual Enrollment (submitted: October 17, 2018): 102
• Original Estimated Enrollment (submitted: April 7, 2017): 120
• Actual Study Completion Date: October 2018
• Actual Primary Completion Date: October 2018 (Final data collection date for primary outcome measure)

Eligibility Criteria

Inclusion Criteria:

- Pediatric and adult patients with sickle cell disease from the HUDERF and CHU-Brugmann Hospital

Exclusion Criteria:

• None

Sex/Gender

• Sexes Eligible for Study: All

• Ages: Child, Adult, Older Adult
• Accepts Healthy Volunteers: No
• Contacts: Contact information is only displayed when the study is recruiting subjects
• Listed Location Countries: Belgium

Administrative Information

• NCT Number: NCT03111589
• Other Study ID Numbers: CHUB-HO1 sickle cell
• Has Data Monitoring Committee: No

U.S. FDA-regulated Product

• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No

IPD Sharing Statement

• Plan to Share IPD: No

• Current Responsible Party: Francis Corazza, Brugmann University Hospital
• Original Responsible Party: Same as current
• Current Study Sponsor: Francis Corazza
• Original Study Sponsor: Same as current
• Collaborators: Not Provided

Investigators

• Principal Investigator: Francis Corazza, MD; CHU Brugmann

• PRS Account: Brugmann University Hospital
• Verification Date: October 2018