Neutrophils as Prognostic Factors in Granulomatosis With Polyangiitis (Formerly Named Wegener's Granulomatosis) (NeutroVasc)
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|ClinicalTrials.gov Identifier: NCT01862068|
Recruitment Status : Unknown
Verified November 2017 by Assistance Publique - Hôpitaux de Paris.
Recruitment status was: Active, not recruiting
First Posted : May 24, 2013
Last Update Posted : November 9, 2017
|First Submitted Date||January 1, 2013|
|First Posted Date||May 24, 2013|
|Last Update Posted Date||November 9, 2017|
|Actual Study Start Date||May 25, 2012|
|Estimated Primary Completion Date||May 2018 (Final data collection date for primary outcome measure)|
|Current Primary Outcome Measures
||level of neutrophils [ Time Frame: At Day 0 ]
Proteomic analysis of neutrophils compared to healthy donors and MPA (microscopic polyangiitis) patients
|Original Primary Outcome Measures||Same as current|
|Current Secondary Outcome Measures||Not Provided|
|Original Secondary Outcome Measures||Not Provided|
|Current Other Pre-specified Outcome Measures||Not Provided|
|Original Other Pre-specified Outcome Measures||Not Provided|
|Brief Title||Neutrophils as Prognostic Factors in Granulomatosis With Polyangiitis (Formerly Named Wegener's Granulomatosis)|
|Official Title||Neutrophils Function and Identification of Prognostic Factors in Granulomatosis With Polyangiitis (Formerly Named Wegener's Granulomatosis).|
Anti-neutrophil cytoplasm antibodies (ANCA)-associated vasculitis are rare diseases characterized by inflammation of blood vessels. Among the numerous cell types that play a role in vasculitis, one of the key actors is the neutrophil. Neutrophils are equipped with very powerful molecules that they use to destroy the invading microbes. Therefore, the mechanisms controlling neutrophil activation should be tightly controlled. If that is not the case, neutrophils may destroy the tissues of the host. This is what happens during chronic inflammation in vasculitis. Autoantibodies directed against neutrophils, ANCA, produced thus demonstrating that neutrophils are also targets of the immune system in these diseases. In addition, molecular studies provided evidence that genes normally silenced in mature neutrophils under normal conditions can be re-expressed in neutrophils from patients with ANCA-associated vasculitis thus strongly suggesting a profound deregulation of neutrophil functions in these conditions. Notably, the investigators have preliminary data showing that neutrophils from patients with granulomatosis with polyangiitis (GPA, formerly Wegener's granulomatosis), an ANCA-associated vasculitis, interfere with the normal phase of resolution of inflammation.
The objective of the investigators' study is to understand the mechanisms underlying this increased activation state and determine if neutrophils could be used to define prognostic markers by clinicians to optimize patients' care. Therefore, the investigators plan to study the expression of proteins implicated in GPA pathophysiology at the membrane of neutrophils when they undergo apoptosis. The investigators will also study the deregulation of protein expression in neutrophils. This point will be the molecular translation of neutrophil deregulation. This technique is powerful and well adapted to identify by mass spectrometry the proteins that will be differentially expressed between the control and the disease state. After identification of proteins differentially expressed in patients with GPA, the investigators will further investigate whether their expression is modulated during the disease course and/or modified by the treatment.
The investigators believe that understanding these neutrophil perturbations can lead to better monitoring of disease activity. Ultimately, the investigators may propose more targeted anti-inflammatory therapies which would be better tolerated by patients. the investigators also can identify new markers for disease activity which allow clinicians to define a better therapeutic strategy.
A - Specific Aims General aim: This project is designed to study physiopathological mechanisms involved in GPA at the cellular and molecular level. the investigators want to study neutrophils as prognostic factors and determine the implication of PR3, the major autoantigen in the resolution phase of inflammation.
the investigators' working hypothesis is that structural and functional analysis of PR3 in neutrophils will help the investigators to unravel its role in GPA. Hence, our long-term objective is to better understand the mechanisms involved in the modulation of PR3 expression at the membrane of neutrophils in order to inhibit their activation and propose novel anti-inflammatory therapeutic strategies adapted to GPA.
Present project: The investigators recently demonstrated that in human neutrophils i) PR3 can be expressed at the membrane of neutrophils during apoptosis in the absence of degranulation, ii) PR3 is a member of a molecular scaffold comprising phospholipidscramblase1 (scramblase) and calreticulin externalized during apoptosis, iii) apoptosis-induced PR3 membrane expression impairs macrophage reprogramming after phagocytosis of apoptotic neutrophils The investigators' hypothesis is that PR3 might have specific molecular partners which can facilitate its externalization and/or its persistence at the plasma membrane during neutrophil apoptosis thus potentiating its auto-antigenic capacity.
The specific aims are:
B. Background and Significance 1.1. Introduction GPA is a systemic vasculitis which is mainly associated with anti-PR3 ANCA. The role of activated neutrophils in vasculitis lesions is suggested by their presence in perivascular infiltrates, especially in kidney glomeruli and in the lung.
PR3 is a serine protease first described within azurophilic granules of neutrophils which are NOT supposed to fuse with the plasma membrane but are released within the phagolysosome after phagocytosis of the pathogen. We have shown for the first time that the subcellular localization of PR3 was not restricted to azurophilic granules, as previously thought, but that PR3 was localized in secretory vesicles, a highly mobilizable compartment, thus explaining the presence of PR3 at the surface of resting isolated neutrophils. Moreover, a high percentage of neutrophils expressing PR3 observed in vasculitis patients could be considered as a risk factor for vasculitis. This high expression of membrane PR3 in patients with GPA resulted in an increase in ANCA-induced respiratory burst in neutrophils thus enhancing the inflammatory process. In vitro the binding of ANCA to PR3 trigger the respiratory burst, degranulation and the release of pro-inflammatory cytokines.
Because PR3 membrane expression is an important pathophysiological event in GPA since it is a prerequisite for the subsequent activation of neutrophils by ANCA, we sought to determine mechanisms involved in PR3 membrane expression. In neutrophils, we demonstrated that apoptosis triggered a significantly increased membrane PR3 expression without degranulation. In these experiments, we failed to detect MPO at the membrane of neutrophils. Our results differ from previously reported data showing that both MPO and PR3 were externalized during apoptosis because of the translocation of azurophilic granules to the plasma membrane. This phenomenon seems to occur when neutrophils undergo late apoptosis and secondary necrosis, leading to expression of all granule proteins on the cell surface.
Apoptosis-induced PR3 externalization could be of great relevance in the pathophysiology of GPA because PR3, which was supposed to be an intracellular antigen, is thus exposed at the plasma membrane of apoptotic cells thus amplifying its pro-inflammatory potential, its immunogenic properties and interfering with macrophage phagocytosis. We have also demonstrated that during apoptosis PR3 expression is associated with the expression of calreticulin, a well-known "eat-me signal". We have finally evidenced that the presence of PR3 impairs the reprogramming of macrophage to an anti-inflammatory profile after phagocytosis of apoptotic cells through the CRT/LRP pathway.
It can be hypothesized that the expression of the complex PR3/scramblase/CRT and other partners (CD11b, CD16) at the surface of apoptotic neutrophils could constitute good prognosis markers.. Gene array studies have pointed out a deregulation of neutrophils in GPA. We make the hypothesis that the proteome of neutrophils of GPA patients would be very informative of its activation state, survival state and that it could be a biological marker of clinical severity.
It is thus pertinent to study the deregulation of neutrophils during their all life cycle: their oxidative burst under activation, their physiologic apoptosis, the expression of PR3 partners. This study will be performed in neutrophils from patients with GPA compared to age and sex adjusted healthy controls.
C. Preliminary studies Analysis of apoptosis in neutrophils from GPA patients In the literature, neutrophils from patients with ANCA associated vasculitis (AAV) were found to have a disturbed TNFα-induced apoptosis and tends to have a higher rate of apoptosis than controls. However, published data on physiologic apoptosis which is the spontaneous apoptosis that neutrophils undergo in the absence of exogenous stimuli is contradictory. When calf foetal serum is added in medium, neutrophils from GPA patients display a similar apoptosis rate than controls. When the serum of patients is used, neutrophils from AAV patients in remission have a higher survival rate in vivo than controls, and this difference disappear when normal serum is used. These results are in agreement with the discovery that GPA patients have a high expression of PCNA which is associated with survival. Therefore, we want to study the apoptosis rate of neutrophils in GPA at diagnosis, during relapse and after remission on whole blood. These results will be compared to age and sex matched controls. Apoptosis will be evaluated by the phosphatidylserine externalization measured by flow cytometry after annexin-V labelling and 7-AAD staining on CD15+ cells. PCNA expression will be also investigated at the different clinical stage and proteomic profile of apoptotic neutrophils will be performed.
Analysis of scramblase/ Calreticulin expression in neutrophils from GPA patients In a cellular model (RBL cell line), we have demonstrated that scramblase extinction by shRNA was associated with a decreased expression of phosphatidylserine during apoptosis and that PR3 apoptosis induced expression was also impaired (unpublished data). We were also able to demonstrate that in vitro scramblase and PR3 physically interact. During apoptosis, PR3 is also associated with calreticulin at the membrane of neutrophils. These data suggest that the association PR3/Scramblase/Calreticulin form a molecular scaffold which is a functional platform which could interfere with recognition pathways (like CRT/LRP) between macrophages and apoptotic neutrophils. The expression of these partners at the neutrophil membrane during apoptosis will be investigated in patients at diagnosis and when remission is obtained.
Research design and Methods:
To study the proteome of GPA patient when active and in remission compared to healthy controls we will use a differential in gel electrophoresis of cytosolic samples of neutrophils obtained by sonication. Identification of proteins of interest will be done by mass spectrometry MALDI-TOF-TOF. Functional validation of these proteins by over-expression or siRNA extinction will be performed.
|Study Design||Observational Model: Cohort
Time Perspective: Prospective
|Target Follow-Up Duration||Not Provided|
|Biospecimen||Retention: Samples Without DNA
|Sampling Method||Non-Probability Sample|
|Study Population||GPA (Granulomatosis with polyangiitis) patients and MPA ( Microscopic Polyangiitis) with an active disease at inclusion|
|Condition||Granulomatosis With Polyangiitis|
|Publications *||Not Provided|
* Includes publications given by the data provider as well as publications identified by ClinicalTrials.gov Identifier (NCT Number) in Medline.
|Recruitment Status||Unknown status|
|Original Estimated Enrollment
|Estimated Study Completion Date||November 2018|
|Estimated Primary Completion Date||May 2018 (Final data collection date for primary outcome measure)|
For patients with GPA
For patients with MPA
For patients with EGPA
For atherosclerotic patients
|Ages||18 Years and older (Adult, Older Adult)|
|Accepts Healthy Volunteers||Yes|
|Contacts||Contact information is only displayed when the study is recruiting subjects|
|Listed Location Countries||France|
|Removed Location Countries|
|Other Study ID Numbers||NI 10017|
|Has Data Monitoring Committee||No|
|U.S. FDA-regulated Product||Not Provided|
|IPD Sharing Statement||Not Provided|
|Responsible Party||Assistance Publique - Hôpitaux de Paris|
|Study Sponsor||Assistance Publique - Hôpitaux de Paris|
|PRS Account||Assistance Publique - Hôpitaux de Paris|
|Verification Date||November 2017|