<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT03233659</url>
  </required_header>
  <id_info>
    <org_study_id>IMM-2011-01</org_study_id>
    <nct_id>NCT03233659</nct_id>
  </id_info>
  <brief_title>Immune Monitoring After Allogeneic Hematopoietic Stem Cell Transplantation</brief_title>
  <official_title>Study of the Function of Immune System in Patients Undergoing Allogeneic Stem Cell Transplantation</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>IRCCS Azienda Ospedaliero-Universitaria di Bologna</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>IRCCS Azienda Ospedaliero-Universitaria di Bologna</source>
  <oversight_info>
    <has_dmc>Yes</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      Graft versus Host disease ( GVHD) is one of the major complications of Allogeneic Stem Cell&#xD;
      Transplantation. Acute GVHD develops early ( within 2to 3 months) after transplantation and&#xD;
      is the leading cause of death of transplanted patients. The pathogenesis of Chronic GVHD is&#xD;
      still little known. Chronic GVHD is caused by donor T lymphocytes, but we have no precise&#xD;
      knowledge on the participation of specific subsets of immune system cells to chronic GVHD. In&#xD;
      general, chronic GVHD is associated with an increase in the number of T effector lymphocytes,&#xD;
      both helper type 2 and cytotoxic.&#xD;
&#xD;
      Recently, also antigen presenting cells (APCs) have been implicated in pathogenesis of&#xD;
      chronic GVHD in studies performed on animal models. T lymphocyte responses that characterize&#xD;
      chronic GVHD require that recipient antigens are submitted by APCs which originate from the&#xD;
      donor's HSC ( Hematopoietic Stem Cells) APCs are heterogeneous population that includes&#xD;
      dendritic cells (DCs) ,monocytes, activated B lymphocytes and CD34+ cell subpopulations.&#xD;
      These cells can be identified by cytometry.&#xD;
&#xD;
      The data about APCs role in chronic GVHD are preliminary and often discordant. Seemingly,&#xD;
      there isn't correlation between circulating APCs number and risk of cGVHD. However, recent&#xD;
      data of our group show that patients with cGVHD could have higher number of monocytes in bone&#xD;
      marrow than transplanted patients without cGVHD.&#xD;
&#xD;
      The aim of study is to measure the number of circulating immune cells in the PB (peripherical&#xD;
      blood) before and after Allogeneic Hematopoietic Stem Cell Transplantation by flow cytometry.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Study Endpoints&#xD;
&#xD;
      The primary endpoint of study is detection of circulating cells by flow cytometry before and&#xD;
      after Allogeneic Stem Cell Transplantation. We analyze the following cells: Myeloid Dendritic&#xD;
      Cells, Plasmacytoid Dendritic cells, CD16+ Dendritic cells, CD14+ monocytes, CD14+/CD16+&#xD;
      monocytes, total T lymphocytes, total T helper lymphocytes, central memory, naive, effector&#xD;
      memory and effector T helper lymphocytes, total cytotoxic lymphocytes, central memory, naive,&#xD;
      effector memory and effector T cytotoxic lymphocytes, CD16+/CD56+ NK cells, B lymphocytes, T&#xD;
      regulatory lymphocytes.&#xD;
&#xD;
      The secondary endpoints are:&#xD;
&#xD;
        -  Determination of the serum concentration of Cytokines (TNFα, IFNγ, IL-4) and Chemokines&#xD;
           ( MIP and MCP group).&#xD;
&#xD;
        -  Determination of autoantibodies in the serum. In the first phase, screening analysis is&#xD;
           performed by searching anti nuclear antibodies (ANA) and antibodies anti-ENA&#xD;
           (Extractable Nuclear Antigens). Positive results are subjected to second level analysis.&#xD;
           Next, we are proceed to the analysis of different Organ-Specific Autoantibodies,&#xD;
           previously associated with chronic GVHD, like liver antibodies (SMA, AMA, SLA),&#xD;
           anti-cardiolipin and beta2 microglobulin antibodies, Antibodies against thyroid and&#xD;
           gastric parietal cells.&#xD;
&#xD;
        -  Study of CD86, CXCR4, CCR2, CCR5, CD11a and CD49d molecules expression in Monocytes and&#xD;
           Dendritic Cells.&#xD;
&#xD;
        -  Study of TNF and IL-12 production by purified monocytes&#xD;
&#xD;
        -  Study of Allostimulatory activity of purified monocytes against Allogeneic CD4+ T cells.&#xD;
&#xD;
        -  Study of antigen capture and processing by phagocytosis, macropinocytosis and&#xD;
           endocytosis mediated by purified monocytes receptors.&#xD;
&#xD;
        -  Study of CD134 (0X40L), CD154 (CD40L) molecules expression, study of differentiation&#xD;
           molecules type1 and type 2 T helper cells and T regulatory Cells associated, study of&#xD;
           molecules Cellular Death associated.&#xD;
&#xD;
        -  Study of Cytokines proliferation and production (including IL-2, IL-5, IFNγ and IL-10)&#xD;
           and release of perforin and granzyme after stimulation with mitogens.&#xD;
&#xD;
        -  Study of antigen-specific T lymphocytes response by pentamers for detection of HY&#xD;
           antigen by ELISPOT assay. Recipient's cells are collected and stored before the&#xD;
           transplantation&#xD;
&#xD;
        -  PCR study of micro (mi) RNA in patient's serum and cells.&#xD;
&#xD;
        -  PCR and Microarray study of gene expression profile of transplanted monocytes&#xD;
&#xD;
      Study design&#xD;
&#xD;
      Observational single center prospective study employing human tissues for in vitro study&#xD;
      after Allogeneic Stem Cell Transplantation. The study includes a retrospective phase. The&#xD;
      study requires that patient's Peripheral blood samples are harvested :&#xD;
&#xD;
        -  on entering the department&#xD;
&#xD;
        -  after 1 month of transplantation&#xD;
&#xD;
        -  after 3, 6, 9,12 months of transplantation&#xD;
&#xD;
      Study Population&#xD;
&#xD;
      All patients undergoing Allogeneic Transplantation in our Institute are included.&#xD;
&#xD;
      Collection and processing of blood samples&#xD;
&#xD;
      Each peripheral blood sample must be collected in three heparin tubes ( up to 18 ml of SP)&#xD;
      and a non-anticoagulant tube (6 ml for a total of at least 3 ml of serum)&#xD;
&#xD;
      Each sample is processed as follows:&#xD;
&#xD;
      Blood sample taken without anticoagulant will be centrifuged to obtain serum, the aliquots of&#xD;
      serum are frozen and stored at -20°C The immunophenotypic analysis for number and function of&#xD;
      the mentioned cells are performed on fresh PB samples within 72 hours of collection.&#xD;
&#xD;
      Mononuclear cells and monocytes are purified by standard procedures (MNCs by density gradient&#xD;
      centrifugation, monocytes by immunomagnetic selection) and then stored in liquid nitrogen&#xD;
      until they are used for expected functional assays. The negative CD14 fraction are frozen and&#xD;
      used as the source of T lymphocytes.&#xD;
&#xD;
      Flow cytometry analysis&#xD;
&#xD;
      The number and phenotype of the immune system circulating cells are determinated by flow&#xD;
      cytometry using the specific monoclonal antibodies.We are considered the following immune&#xD;
      cells :&#xD;
&#xD;
        -  CD4+ and CD8+ T lymphocytes including T naïve lymphocytes (CD45RA + CCR7 +) &quot;effector&quot;&#xD;
           memory lymphocytes (CD45RA-CCR7-), &quot;central&quot; memory lymphocytes (CD45RA-CCR7 +) and&#xD;
           effector lymphocytes (CD45RA-CCR7 +).&#xD;
&#xD;
        -  T regulatory lymphocytes (CD4 + CD25 + CD127-FoxP3 +).&#xD;
&#xD;
        -  B Lymphocytes (CD19 +)&#xD;
&#xD;
        -  NK cells (CD56 + CD16-) and cytotoxic (CD56 + CD16 +) cells.&#xD;
&#xD;
        -  Myeloid Dendritic Cells CD11c + (lineage-, DR +, CD33 +), Plasmacytoid Dendritic Cells&#xD;
           (lineage-, DR +, CD33- and CD123 +) and Monocytoid (CD33 +, CD14-, CD16 +) DCs.&#xD;
&#xD;
        -  Inflammatory (CD14 + CD16 +) and Constituents (CD14 + CD16-) Monocytes&#xD;
&#xD;
      Functional studies on purified monocytes&#xD;
&#xD;
        -  TNF alpha and IL12 production are measured in flow cytometry.&#xD;
&#xD;
        -  The allostimulatory function is determined in mixed leukocyte cultures using HLA&#xD;
           different CD4 + T lymphocytes as &quot;responders&quot;.&#xD;
&#xD;
        -  The ability to capture antigens is measured in flow cytometry on individual cells by&#xD;
           fluorescence antigens, such as FITC conjugated albumin or fluorescent beads.&#xD;
&#xD;
        -  The gene expression profile is performed using a MICROARRAY study&#xD;
&#xD;
      Functional studies on purified T lymphocytes&#xD;
&#xD;
        -  The expression of activation, differentiation and apoptosis molecules is determinated by&#xD;
           cytometry&#xD;
&#xD;
        -  Cytokine production is determined in single-color flow cytometry after stimulation with&#xD;
           anti-CD3 and anti-CD28.&#xD;
&#xD;
        -  In positive male HLA-A2 patients receiving female donor transplantation, the frequency&#xD;
           of T anti-HY lymphocytes is measured by fluorochrome-conjugated pentamers.&#xD;
&#xD;
        -  In all other patients, T-cell donor response to receptor antigens is measured in&#xD;
           ELISPOT.&#xD;
&#xD;
      Serum cytokines Study&#xD;
&#xD;
      The concentration of serum cytokines is determined in cytometry using CBA (Cytokine Bead&#xD;
      Assay, CBA, Becton Dickinson, Mountian View, CA, USA). We analyze the following cytokines:&#xD;
&#xD;
        -  T-helper 1 cytokines: TNF-alpha, IFN-gamma and IL-12;&#xD;
&#xD;
        -  T-helper 2 and regulators cytokines (IL-10, IL-5), and TGF-beta&#xD;
&#xD;
        -  Chemokines: MCP1, MCP2, RANTES, MIP1-alpha and MIP1-beta; IL-8, IP-10.&#xD;
&#xD;
      Serum autoantibodies Study&#xD;
&#xD;
      The concentration of the antinuclear antibodies is measured by indirect immunofluorescence on&#xD;
      HEp2 cells (a cell line of hepatocarcinoma). The antibodies against extractable nuclear&#xD;
      antigens (ENA) are measured by ELISA (ENA SCREENING). In case of positivity, the presence of&#xD;
      antibodies against specific antigens is determined by DOT BLOT.&#xD;
&#xD;
      micro(mi) RNA PCR study in patient's serum and cells&#xD;
&#xD;
      The total RNA is extracted from human serum samples and stored at -80 ° C. A part of RNA (&#xD;
      3µl) is processed for reverse transcription and pre-amplification with a pool of primers&#xD;
      according to the supplier's description. 664 human miRNA, 6 small human RNA and 1 control&#xD;
      miRNA are processed in parallel.&#xD;
&#xD;
      Gene expression profile Study of transplanted patient's monocytes by PCR and MICROARRAY&#xD;
&#xD;
      The monocyte RNA messenger is extracted from the cells, converted into cDNA by reverse&#xD;
      transcriptase and at the same time labeled with a fluorescent probe. The hybridization&#xD;
      between the nucleic acids (probes) present on the matrix and the cDNA target will remain&#xD;
      hybridized and can then be identified by detecting the location where it is bound. At the end&#xD;
      of study, all samples will be destroyed.&#xD;
&#xD;
      Concomitant treatments&#xD;
&#xD;
      Patients will receive or have received chemotherapy for transplant preparation according to&#xD;
      current clinical practice. After transplantation, patients will receive medication for&#xD;
      prophylaxis of GVHD and infections according to clinical practice. Concomitant therapies&#xD;
      possibly administered according to clinical practice are documented in CRF.&#xD;
&#xD;
      Schedule of visits and evaluations&#xD;
&#xD;
      Patient visit is expected and CRF is completed at the enrollment. There are 6 blood samples&#xD;
      per patient. Any blood sample after the enrollment can be delayed or anticipated by 14 days&#xD;
&#xD;
      Laboratory Tests&#xD;
&#xD;
      Tests are performed under the normal care pathway and are not study-specific. Blood exam&#xD;
      informations are reported on CRFs.&#xD;
&#xD;
      Statistical analysis&#xD;
&#xD;
      The primary endpoint of the study is the measure of circulating immune cell number in PB&#xD;
      before and after Allogeneic Hematopoietic Stem Cell Transplantation. Statistical analysis&#xD;
      consists of comparison analysis between averages using GRAPH PAD PRISM version 4.02 program.&#xD;
      The level of significance is defined as &lt;.05 Analysis of secondary endpoints is performed&#xD;
      using the same methods.&#xD;
&#xD;
      Sample Size&#xD;
&#xD;
      The study is proposed to all patients undergoing allogeneic transplantation. The number of&#xD;
      transplants performed at the Seràgnoli Institute is on average between 50 and 60 per year. It&#xD;
      is therefore estimated that 300 patients will be enrolled in the study.&#xD;
&#xD;
      Administrative procedures&#xD;
&#xD;
      Any modification to the protocol will be made in the form of the amendment. No other types of&#xD;
      modification to the protocol are allowed during the study period. Any unexpected changes in&#xD;
      the study will be recorded in the Clinical Study Report.&#xD;
&#xD;
      Management of informed consent&#xD;
&#xD;
      All patients date and write the Informed Consent within one of the visits provided by the&#xD;
      normal care path. For dead patients, it is considered that the data processing is authorized&#xD;
      with the approval of the study by the Ethics Committee, according to general authorization&#xD;
      (Official Journal 72 of 26/03/2012)&#xD;
&#xD;
      Documentation archive&#xD;
&#xD;
      The principal Investigator is responsible for storing of the essential documents of the study&#xD;
      before, during, and after the completion or termination of the study, in accordance with the&#xD;
      time required by the applicable laws and GCPs.&#xD;
&#xD;
      The data collected in the CRF are strictly anonymous and the subject is only identified with&#xD;
      a number and initials.&#xD;
&#xD;
      The Investigator will have to keep the patient's original data and informed written consent.&#xD;
&#xD;
      Inspections / Audits&#xD;
&#xD;
      If a Regulatory Authority requires an inspection, the Investigator must immediately inform&#xD;
      the Ethics Committee.&#xD;
&#xD;
      Publication of results&#xD;
&#xD;
      The results of the experiment will be published within twelve months of the conclusion.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">August 12, 2014</start_date>
  <completion_date type="Actual">June 2018</completion_date>
  <primary_completion_date type="Actual">June 2018</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Cohort</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>The primary endpoint of study is detection of circulating cells by flow cytometry before and after Allogeneic Stem Cell Transplantation</measure>
    <time_frame>Patient's PB samples are harvested : on entering the department;1 month after transplantation ; 3, 6, 9,12 months after transplantation</time_frame>
    <description>We analyze the following cells:Myeloid Dendritic Cells, Plasmacytoid Dendritic cells, CD16+ Dendritic cells, CD14+ monocytes, CD14+/CD16+ monocytes, total T lymphocytes, total T helper lymphocytes, central memory, naive, effector memory and effector T helper lymphocytes, total cytotoxic lymphocytes, central memory, naive, effector memory and effector cytotoxic lymphocytes, CD16+/CD56+ NK cells, B lymphocytes, T regulatory lymphocytes.</description>
  </primary_outcome>
  <number_of_groups>1</number_of_groups>
  <enrollment type="Actual">177</enrollment>
  <condition>Allogeneic Stem Cell Transplantation</condition>
  <condition>GVHD</condition>
  <arm_group>
    <arm_group_label>BMT patients</arm_group_label>
    <description>All patients undergoing Allogeneic Stem Cell Transplantation are enrolled in the study.</description>
  </arm_group>
  <biospec_retention>Samples With DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      Samples of serum and PBMC&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        All patients undergoing Allogeneic Stem Cell Transplantation are enrolled in the study.&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
        All patients undergoing Allogeneic hemapoietic Stem Cell Transplantation&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
        Patient not undergoing allogeneic hematopoietic stem cell transplantation&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>N/A</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Mario Arpinati, MD</last_name>
    <role>Principal Investigator</role>
    <affiliation>St. Orsola-Malpighi University Hospital Bologna, Italy</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>Mario Arpinati</name>
      <address>
        <city>Bologna</city>
        <country>Italy</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Italy</country>
  </location_countries>
  <reference>
    <citation>Nakamura K, Amakawa R, Takebayashi M, Son Y, Miyaji M, Tajima K, Nakai K, Ito T, Matsumoto N, Zen K, Kishimoto Y, Fukuhara S. IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD. Bone Marrow Transplant. 2005 Oct;36(7):639-47. doi: 10.1038/sj.bmt.1705107.</citation>
    <PMID>16044136</PMID>
  </reference>
  <reference>
    <citation>D' Asaro M, Salerno A, Dieli F, Caccamo N. Analysis of memory and effector CD8+ T cell subsets in chronic graft-versus-host disease. Int J Immunopathol Pharmacol. 2009 Jan-Mar;22(1):195-205. doi: 10.1177/039463200902200122.</citation>
    <PMID>19309567</PMID>
  </reference>
  <reference>
    <citation>Arpinati M, Chirumbolo G, Saunthararajah Y, Stanzani M, Bonifazi F, Bandini G, Baccarani M, Rondelli D. Higher numbers of blood CD14+ cells before starting conditioning regimen correlate with greater risk of acute graft-versus-host disease in allogeneic stem cell transplantation from related donors. Biol Blood Marrow Transplant. 2007 Feb;13(2):228-34. doi: 10.1016/j.bbmt.2006.10.004.</citation>
    <PMID>17241928</PMID>
  </reference>
  <reference>
    <citation>Anderson BE, McNiff JM, Jain D, Blazar BR, Shlomchik WD, Shlomchik MJ. Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine chronic graft-versus-host disease: requirements depend on target organ. Blood. 2005 Mar 1;105(5):2227-34. doi: 10.1182/blood-2004-08-3032. Epub 2004 Nov 2.</citation>
    <PMID>15522961</PMID>
  </reference>
  <reference>
    <citation>Arpinati M, Chirumbolo G, Marzocchi G, Baccarani M, Rondelli D. Increased donor CD86+CD14+ cells in the bone marrow and peripheral blood of patients with chronic graft-versus-host disease. Transplantation. 2008 Jun 27;85(12):1826-32. doi: 10.1097/TP.0b013e3181788a84.</citation>
    <PMID>18580477</PMID>
  </reference>
  <verification_date>January 2023</verification_date>
  <study_first_submitted>July 26, 2017</study_first_submitted>
  <study_first_submitted_qc>July 26, 2017</study_first_submitted_qc>
  <study_first_posted type="Actual">July 28, 2017</study_first_posted>
  <last_update_submitted>January 31, 2023</last_update_submitted>
  <last_update_submitted_qc>January 31, 2023</last_update_submitted_qc>
  <last_update_posted type="Actual">February 1, 2023</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>IRCCS Azienda Ospedaliero-Universitaria di Bologna</investigator_affiliation>
    <investigator_full_name>Mario Arpinati</investigator_full_name>
    <investigator_title>MD</investigator_title>
  </responsible_party>
  <keyword>Allogeneic Stem Cell Transplantation, APCs, GVHD</keyword>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT03233659
org study id: IMM-2011-01
nct id: NCT03233659
lead sponsor: 
has dmc: Yes
is fda regulated drug: No
is fda regulated device: No
Graft versus Host disease ( GVHD) is one of the major complications of Allogeneic Stem Cell
      Transplantation. Acute GVHD develops early ( within 2to 3 months) after transplantation and
      is the leading cause of death of transplanted patients. The pathogenesis of Chronic GVHD is
      still little known. Chronic GVHD is caused by donor T lymphocytes, but we have no precise
      knowledge on the participation of specific subsets of immune system cells to chronic GVHD. In
      general, chronic GVHD is associated with an increase in the number of T effector lymphocytes,
      both helper type 2 and cytotoxic.

      Recently, also antigen presenting cells (APCs) have been implicated in pathogenesis of
      chronic GVHD in studies performed on animal models. T lymphocyte responses that characterize
      chronic GVHD require that recipient antigens are submitted by APCs which originate from the
      donor's HSC ( Hematopoietic Stem Cells) APCs are heterogeneous population that includes
      dendritic cells (DCs) ,monocytes, activated B lymphocytes and CD34+ cell subpopulations.
      These cells can be identified by cytometry.

      The data about APCs role in chronic GVHD are preliminary and often discordant. Seemingly,
      there isn't correlation between circulating APCs number and risk of cGVHD. However, recent
      data of our group show that patients with cGVHD could have higher number of monocytes in bone
      marrow than transplanted patients without cGVHD.

      The aim of study is to measure the number of circulating immune cells in the PB (peripherical
      blood) before and after Allogeneic Hematopoietic Stem Cell Transplantation by flow cytometry.
Study Endpoints

      The primary endpoint of study is detection of circulating cells by flow cytometry before and
      after Allogeneic Stem Cell Transplantation. We analyze the following cells: Myeloid Dendritic
      Cells, Plasmacytoid Dendritic cells, CD16+ Dendritic cells, CD14+ monocytes, CD14+/CD16+
      monocytes, total T lymphocytes, total T helper lymphocytes, central memory, naive, effector
      memory and effector T helper lymphocytes, total cytotoxic lymphocytes, central memory, naive,
      effector memory and effector T cytotoxic lymphocytes, CD16+/CD56+ NK cells, B lymphocytes, T
      regulatory lymphocytes.

      The secondary endpoints are:

        -  Determination of the serum concentration of Cytokines (TNFα, IFNγ, IL-4) and Chemokines
           ( MIP and MCP group).

        -  Determination of autoantibodies in the serum. In the first phase, screening analysis is
           performed by searching anti nuclear antibodies (ANA) and antibodies anti-ENA
           (Extractable Nuclear Antigens). Positive results are subjected to second level analysis.
           Next, we are proceed to the analysis of different Organ-Specific Autoantibodies,
           previously associated with chronic GVHD, like liver antibodies (SMA, AMA, SLA),
           anti-cardiolipin and beta2 microglobulin antibodies, Antibodies against thyroid and
           gastric parietal cells.

        -  Study of CD86, CXCR4, CCR2, CCR5, CD11a and CD49d molecules expression in Monocytes and
           Dendritic Cells.

        -  Study of TNF and IL-12 production by purified monocytes

        -  Study of Allostimulatory activity of purified monocytes against Allogeneic CD4+ T cells.

        -  Study of antigen capture and processing by phagocytosis, macropinocytosis and
           endocytosis mediated by purified monocytes receptors.

        -  Study of CD134 (0X40L), CD154 (CD40L) molecules expression, study of differentiation
           molecules type1 and type 2 T helper cells and T regulatory Cells associated, study of
           molecules Cellular Death associated.

        -  Study of Cytokines proliferation and production (including IL-2, IL-5, IFNγ and IL-10)
           and release of perforin and granzyme after stimulation with mitogens.

        -  Study of antigen-specific T lymphocytes response by pentamers for detection of HY
           antigen by ELISPOT assay. Recipient's cells are collected and stored before the
           transplantation

        -  PCR study of micro (mi) RNA in patient's serum and cells.

        -  PCR and Microarray study of gene expression profile of transplanted monocytes

      Study design

      Observational single center prospective study employing human tissues for in vitro study
      after Allogeneic Stem Cell Transplantation. The study includes a retrospective phase. The
      study requires that patient's Peripheral blood samples are harvested :

        -  on entering the department

        -  after 1 month of transplantation

        -  after 3, 6, 9,12 months of transplantation

      Study Population

      All patients undergoing Allogeneic Transplantation in our Institute are included.

      Collection and processing of blood samples

      Each peripheral blood sample must be collected in three heparin tubes ( up to 18 ml of SP)
      and a non-anticoagulant tube (6 ml for a total of at least 3 ml of serum)

      Each sample is processed as follows:

      Blood sample taken without anticoagulant will be centrifuged to obtain serum, the aliquots of
      serum are frozen and stored at -20°C The immunophenotypic analysis for number and function of
      the mentioned cells are performed on fresh PB samples within 72 hours of collection.

      Mononuclear cells and monocytes are purified by standard procedures (MNCs by density gradient
      centrifugation, monocytes by immunomagnetic selection) and then stored in liquid nitrogen
      until they are used for expected functional assays. The negative CD14 fraction are frozen and
      used as the source of T lymphocytes.

      Flow cytometry analysis

      The number and phenotype of the immune system circulating cells are determinated by flow
      cytometry using the specific monoclonal antibodies.We are considered the following immune
      cells :

        -  CD4+ and CD8+ T lymphocytes including T naïve lymphocytes (CD45RA + CCR7 +) "effector"
           memory lymphocytes (CD45RA-CCR7-), "central" memory lymphocytes (CD45RA-CCR7 +) and
           effector lymphocytes (CD45RA-CCR7 +).

        -  T regulatory lymphocytes (CD4 + CD25 + CD127-FoxP3 +).

        -  B Lymphocytes (CD19 +)

        -  NK cells (CD56 + CD16-) and cytotoxic (CD56 + CD16 +) cells.

        -  Myeloid Dendritic Cells CD11c + (lineage-, DR +, CD33 +), Plasmacytoid Dendritic Cells
           (lineage-, DR +, CD33- and CD123 +) and Monocytoid (CD33 +, CD14-, CD16 +) DCs.

        -  Inflammatory (CD14 + CD16 +) and Constituents (CD14 + CD16-) Monocytes

      Functional studies on purified monocytes

        -  TNF alpha and IL12 production are measured in flow cytometry.

        -  The allostimulatory function is determined in mixed leukocyte cultures using HLA
           different CD4 + T lymphocytes as "responders".

        -  The ability to capture antigens is measured in flow cytometry on individual cells by
           fluorescence antigens, such as FITC conjugated albumin or fluorescent beads.

        -  The gene expression profile is performed using a MICROARRAY study

      Functional studies on purified T lymphocytes

        -  The expression of activation, differentiation and apoptosis molecules is determinated by
           cytometry

        -  Cytokine production is determined in single-color flow cytometry after stimulation with
           anti-CD3 and anti-CD28.

        -  In positive male HLA-A2 patients receiving female donor transplantation, the frequency
           of T anti-HY lymphocytes is measured by fluorochrome-conjugated pentamers.

        -  In all other patients, T-cell donor response to receptor antigens is measured in
           ELISPOT.

      Serum cytokines Study

      The concentration of serum cytokines is determined in cytometry using CBA (Cytokine Bead
      Assay, CBA, Becton Dickinson, Mountian View, CA, USA). We analyze the following cytokines:

        -  T-helper 1 cytokines: TNF-alpha, IFN-gamma and IL-12;

        -  T-helper 2 and regulators cytokines (IL-10, IL-5), and TGF-beta

        -  Chemokines: MCP1, MCP2, RANTES, MIP1-alpha and MIP1-beta; IL-8, IP-10.

      Serum autoantibodies Study

      The concentration of the antinuclear antibodies is measured by indirect immunofluorescence on
      HEp2 cells (a cell line of hepatocarcinoma). The antibodies against extractable nuclear
      antigens (ENA) are measured by ELISA (ENA SCREENING). In case of positivity, the presence of
      antibodies against specific antigens is determined by DOT BLOT.

      micro(mi) RNA PCR study in patient's serum and cells

      The total RNA is extracted from human serum samples and stored at -80 ° C. A part of RNA (
      3µl) is processed for reverse transcription and pre-amplification with a pool of primers
      according to the supplier's description. 664 human miRNA, 6 small human RNA and 1 control
      miRNA are processed in parallel.

      Gene expression profile Study of transplanted patient's monocytes by PCR and MICROARRAY

      The monocyte RNA messenger is extracted from the cells, converted into cDNA by reverse
      transcriptase and at the same time labeled with a fluorescent probe. The hybridization
      between the nucleic acids (probes) present on the matrix and the cDNA target will remain
      hybridized and can then be identified by detecting the location where it is bound. At the end
      of study, all samples will be destroyed.

      Concomitant treatments

      Patients will receive or have received chemotherapy for transplant preparation according to
      current clinical practice. After transplantation, patients will receive medication for
      prophylaxis of GVHD and infections according to clinical practice. Concomitant therapies
      possibly administered according to clinical practice are documented in CRF.

      Schedule of visits and evaluations

      Patient visit is expected and CRF is completed at the enrollment. There are 6 blood samples
      per patient. Any blood sample after the enrollment can be delayed or anticipated by 14 days

      Laboratory Tests

      Tests are performed under the normal care pathway and are not study-specific. Blood exam
      informations are reported on CRFs.

      Statistical analysis

      The primary endpoint of the study is the measure of circulating immune cell number in PB
      before and after Allogeneic Hematopoietic Stem Cell Transplantation. Statistical analysis
      consists of comparison analysis between averages using GRAPH PAD PRISM version 4.02 program.
      The level of significance is defined as <.05 Analysis of secondary endpoints is performed
      using the same methods.

      Sample Size

      The study is proposed to all patients undergoing allogeneic transplantation. The number of
      transplants performed at the Seràgnoli Institute is on average between 50 and 60 per year. It
      is therefore estimated that 300 patients will be enrolled in the study.

      Administrative procedures

      Any modification to the protocol will be made in the form of the amendment. No other types of
      modification to the protocol are allowed during the study period. Any unexpected changes in
      the study will be recorded in the Clinical Study Report.

      Management of informed consent

      All patients date and write the Informed Consent within one of the visits provided by the
      normal care path. For dead patients, it is considered that the data processing is authorized
      with the approval of the study by the Ethics Committee, according to general authorization
      (Official Journal 72 of 26/03/2012)

      Documentation archive

      The principal Investigator is responsible for storing of the essential documents of the study
      before, during, and after the completion or termination of the study, in accordance with the
      time required by the applicable laws and GCPs.

      The data collected in the CRF are strictly anonymous and the subject is only identified with
      a number and initials.

      The Investigator will have to keep the patient's original data and informed written consent.

      Inspections / Audits

      If a Regulatory Authority requires an inspection, the Investigator must immediately inform
      the Ethics Committee.

      Publication of results

      The results of the experiment will be published within twelve months of the conclusion.
observational model: Cohort
time perspective: Prospective
measure: The primary endpoint of study is detection of circulating cells by flow cytometry before and after Allogeneic Stem Cell Transplantation
time frame: Patient's PB samples are harvested : on entering the department;1 month after transplantation ; 3, 6, 9,12 months after transplantation
description: We analyze the following cells:Myeloid Dendritic Cells, Plasmacytoid Dendritic cells, CD16+ Dendritic cells, CD14+ monocytes, CD14+/CD16+ monocytes, total T lymphocytes, total T helper lymphocytes, central memory, naive, effector memory and effector T helper lymphocytes, total cytotoxic lymphocytes, central memory, naive, effector memory and effector cytotoxic lymphocytes, CD16+/CD56+ NK cells, B lymphocytes, T regulatory lymphocytes.
arm group label: BMT patients
description: All patients undergoing Allogeneic Stem Cell Transplantation are enrolled in the study.
Samples of serum and PBMC
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 18 Years
maximum age: N/A
healthy volunteers: No
last name: Mario Arpinati, MD
role: Principal Investigator
affiliation: St. Orsola-Malpighi University Hospital Bologna, Italy
facility: 
country: Italy
citation: Nakamura K, Amakawa R, Takebayashi M, Son Y, Miyaji M, Tajima K, Nakai K, Ito T, Matsumoto N, Zen K, Kishimoto Y, Fukuhara S. IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD. Bone Marrow Transplant. 2005 Oct;36(7):639-47. doi: 10.1038/sj.bmt.1705107.
PMID: 16044136
citation: D' Asaro M, Salerno A, Dieli F, Caccamo N. Analysis of memory and effector CD8+ T cell subsets in chronic graft-versus-host disease. Int J Immunopathol Pharmacol. 2009 Jan-Mar;22(1):195-205. doi: 10.1177/039463200902200122.
PMID: 19309567
citation: Arpinati M, Chirumbolo G, Saunthararajah Y, Stanzani M, Bonifazi F, Bandini G, Baccarani M, Rondelli D. Higher numbers of blood CD14+ cells before starting conditioning regimen correlate with greater risk of acute graft-versus-host disease in allogeneic stem cell transplantation from related donors. Biol Blood Marrow Transplant. 2007 Feb;13(2):228-34. doi: 10.1016/j.bbmt.2006.10.004.
PMID: 17241928
citation: Anderson BE, McNiff JM, Jain D, Blazar BR, Shlomchik WD, Shlomchik MJ. Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine chronic graft-versus-host disease: requirements depend on target organ. Blood. 2005 Mar 1;105(5):2227-34. doi: 10.1182/blood-2004-08-3032. Epub 2004 Nov 2.
PMID: 15522961
citation: Arpinati M, Chirumbolo G, Marzocchi G, Baccarani M, Rondelli D. Increased donor CD86+CD14+ cells in the bone marrow and peripheral blood of patients with chronic graft-versus-host disease. Transplantation. 2008 Jun 27;85(12):1826-32. doi: 10.1097/TP.0b013e3181788a84.
PMID: 18580477
responsible party type: Principal Investigator
investigator affiliation: IRCCS Azienda Ospedaliero-Universitaria di Bologna
investigator full name: Mario Arpinati
investigator title: MD