Research


ESR 1: Fatemeh Asgari

The role of TIR8 and PTX3 in sepsis induced immunosuppression

Objectives: PTX3 is a key component of the humoral innate immunity identified by ICH, induced by cytokines and microbes at sites of infection by stromal and immune cells. TIR8/SIGIRR is a member of the IL-1R like receptor family acting intracellularly to inhibit IL-1R like receptors and TLR signalling, cloned by ICH and associated with infections and inflammation.

At Humanitas Research Institute, we will evaluate the involvement of TIR8 and PTX3 in the pathogenesis of sepsis, taking advantage of gene-targeted mice generated by ICH. Available models include murine sepsis associated with infections caused by diverse microbes, e.g. Pseudomonas aeruginosa, Streptococcus pneumoniae, Aspergillus fumigatus, Klebsiella pnaumoniae, and Staphylococcus aureus, and tolerance models. These models will be used to compare the susceptibility and the inflammatory response of immunocompetent, PTX3- and TIR8-deficient mice, and to address the mechanisms underlying defective resistance to infections and exacerbated inflammation or immunosuppression. Recombinant PTX3 will be used to perform treatment. Moreover, we have two separate domains of the molecule and mutants to investigate their interaction with microbial components and address the functional parts of PTX3. TIR8 activating and inhibitory antibodies are under development and will be used to modulate the functional activity of TIR8, through in vitro and in vivo studies.

Host: Instituto Clinico Humanitas, Milan, Italy

Supervisor: Prof. A. Mantovani, MD, PhD; Prof. Cecilia Garlanda, DVM, PhD (cecilia.garlanda@humanitasresearch.it)

 

ESR2: Individualized modulation of immune response

Objectives: This project will combine research in mouse models and clinical samples and will try to elaborate which biomarker can be used as an indicator of the pro-inflammatory and of the anti-inflammatory nature of each patient. As a next step a randomized pro-of-concept clinical trial will run using these biomarkers as a tool for decision making. Before progression into the clinical stage, the hypothesis will be validated into distinct animal models with predominance of the pro-inflammatory or of the anti-inflammatory response. For the trial, two main drugs will be tested: anakinra as an inhibitor of the pro-inflammatory response and recombinant interferon-gamma as an enhancer of the anti-inflammatory response. The end result of this project is how biomarkers will be used to indicate the most appropriate therapy of the host.

Host: National and Kapodistrian University of Athens, 4th Department of Internal Medicine

Supervisor: Evangelos J. Giamarellos-Bourboulis, MD, PhD

 

ESR3: Berke Gürkan

The role of IRAK-M in sepsis induced immunosuppression

 Objectives: IRAK-M has emerged as a key player in sepsis induced immunosuppression. ESR2 will (1) Obtain insight into the distribution and regulation of IRAK-M expression in vivo, (2) Determine the binding sites involved in the interactions of IRAK-M with IRAK-1 and IRAK-4, (3) Determine the impact of IRAK-M deficiency in sepsis and (4) Determine the effect of IRAK-M inhibition in sepsis. IRAK-M gene-targeted mice are available at the AMC. Murine models of sepsis of infectious origin will be used. The approach will include the study of the role of IRAK-M in the following aspects: inflammatory response to the pathogen; the interplay of both molecules with cellular receptors; the prophylactic and therapeutic potential of IRAK-M in murine models of sepsis (B. pseudomallei, S. pneumoniae, E. coli). The project will generate insight in the role of IRAK-M in different infection models and thus identify potential pathologies that may benefit from treatment with IRAK-M inhibitors. A plural competence in cellular and molecular biology is  highly desirable for this position.

Host: Academic Medical Center, University of Amsterdam (AMC, UvA), Center for Experimental and Molecular Medicine (CEMM).

Supervisors: Dr. Kees van ‘t Veer, Dr. Joost Wiersinga

 

ESR4: Nora Wolff

Design novel molecular tools to develop potential novel diagnostics and therapeutics strategies for sepsis

Objectives: ESR4 work will combine wet lab. experiments and in silico duties together with murine models of sepsis (B. pseudomallei, S. pneumoniae, K. pneumoniae). Studies will be performed in a translational fashion combing patient related studies in mice. The objective of ESR4 will be to develop potential novel diagnostics and therapeutics strategies for sepsis. Special focus will be on the role of the gut microbiota and the lung microbiota in models of pneumonia-derived sepsis. This is a whole new area of biomarker development in the sepsis research arena. The potential relevance of genes of interest will be tested using in vitro and in vivo models. A plural competence in cellular, molecular and computational  biology and affinity with the clinic is  highly desirable for this position.

Host: Academic Medical Center, University of Amsterdam (AMC, UvA), Center for Experimental and Molecular Medicine (CEMM).

Supervisors: Dr. Joost Wiersinga, Prof. Tom van der Poll

 

ESR5: Elene Karakike

Development of microRNAs as biomarkers in sepsis

 Objectives: The aim of this project is to link the microRNAome with distinct septic transcription patterns characteristic of sepsis immunosuppression. More precisely, at a first stage analysis will run in samples available from the large biobank of the Hellenic Sepsis Study Group. Analysis will run to well-characterize which microRNAs can be used as indicators of the anti-inflammatory nature of the host taking into consideration the specific patient phenotype. Then a prospective study will run at a second stage with two aims: a) to validate the findings of the first stage particularly as related to specific phenotypes; and b) daily collection of samples trying to identify how the change of the kinetics of circulating microRNAs is related with final outcome in specific sepsis phenotypes and how it can develop to improve decision making.

Host: National and Kapodistrian University of Athens, 4th Department of Internal Medicine

Supervisor: Evangelos J. Giamarellos-Bourboulis, MD, PhD

 

ESR 6: Irene Schrijver

Dynamic profiling of MDSC in sepsis

 Objectives: To assess the expression and heterogeneity of myeloid-derived suppressor cells (MDSC) in sepsis patients using most up-to-date technologies in multiparameter flow cytometry and mass cell cytometry (CyTOF). Analyses will be done using peripheral blood from healthy subjects and from septic patients, obtained at admission in ICUs and subsequent days. The simultaneous measurement of 30-40 cellular parameters (surface markers, signaling molecules and cytokines) by CyTOF will provide a unique protein cartography of the distinct MDSC subpopulations in healthy and septic patients.

Host: Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

Supervisor: PD-Dr Thierry Roger; Prof Thierry Calandra

 

ESR 7: Charlotte Théroude

Functional studies of MDSC in sepsis

Objectives: To evaluate the biological functions of myeloid-derived suppressor cells (MDSC) subpopulations cell-sorted based either on established immunophenotyping or using markers identified in collaboration with ESR6. Analyses will be performed using MDSC, from healthy donors and sepsis patients, stimulated or not with microbial products or pathogens acting through various classes of pattern recognition receptors. All parameters will be compared to biological and clinical parameters, the occurrence of nosocomial infections and the outcome of septic patients.

 Host: Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

Supervisor: PD-Dr Thierry Roger; Prof Thierry Calandra

 

ESR 8: Sadaf Davoudian

Evaluation and validation of discovered biomarkers for early prognostic stratification of septic patients.

Objectives: PTX3 is an acute phase protein identified by ICH, induced by cytokines and microbes at sites of infection by stromal and immune cells. TIR8/SIGIRR and IL-1R2 are negative regulators of the IL-1R like receptor family, involved in tuning infections and inflammation. MSF is a novel marker of M2 macrophages.

(ICH) PTX3 and IL-1R2 levels will be analysed in plasma of septic patients enrolled by clinical partners by ELISA assays developed in house. The study will validate PTX3 and IL-1R2 as markers for early prognostic stratification of septic patients. Data obtained will be correlated to clinical parameters and to other classical biomarkers. TIR8 expression will be analysed by FACS analysis on leukocytes from septic patients. To this aim, a prospective study will be conducted at ICH after approval from the Institutional Review Board. The project will validate the hypothesis that TIR8 expressed by leukocytes and platelets may be a marker of the anti-inflammatory phase of the septic response. MSF has emerged as a novel marker associated to M2 macrophages. The project consists in testing the relevance of this molecule as marker of regulatory macrophages in sepsis. Tools under development (antibodies, ELISA) will be used to analyse MSF levels in plasma of septic patients, which will be available from ICH and from the network.

ESR8 will validate a) the diagnostic and prognostic potential of PTX3, stratification of patients and monitoring stage of sepsis; b) the hypothesis that TIR8 is a marker of the anti-inflammatory phase of the septic response; c) the hypothesis that IL-1R2 is a marker of the anti-inflammatory phase of the septic response; d) to evaluate the relevance of MSF as marker of regulatory monocytes/macrophages. ESR8 will gain experience in circulating cell isolation, purification and handling coupled with FACS analysis and biochemical assays.

Host: Instituto Clinico Humanitas, Milan, Italy

Supervisor: Prof. A. Mantovani, MD, PhD; Dr Barbara Bottazzi, PhD

 

ESR 9

Evaluation of theranostic biomarkers in sepsis.

 Objectives: The failure of multiple immunomodulatory trials in sepsis is arguably due to a one-size-fits-all strategy. A personalized approach, whereby the right patient can be targeted to receive the drug at optimal dose and dosing period, offers a far better chance of success. The lab has developed reproducible and sophisticated long-term rat models of peritonitis with advanced monitoring that allow early prognostication by a variety of physiological or biological tests. Modulation of the abnormal poor-prognosis signature through targeted intervention by a number of putative agents modulating immune response, adrenergic stress, or metabolism may result in improved outcomes. ESR 9 will test such approaches and, on identification of suitable candidates, assist in developing point-of-care test panels using various platforms. The ESR will learn in vivo physiological techniques, including advanced instrumentation, echocardiography, indirect calorimetry and tissue perfusion monitoring, and a variety of standard (ELISA, Western, FACS) and advanced (such as nanotechnology, microdialysis) ex vivo techniques.

Host: University College London, UK

Supervisor: Prof. Mervyn Singer

 

ESR 10: Vera Peters

The role of bioenergetic dysfunction in sepsis-induced immunosuppression.

 Objectives: The pathophysiological mechanisms underlying immunosuppression in sepsis remain uncertain. Cellular energy failure is a plausible hypothesis; early studies show a strong association between bioenergetic dysfunction and decreased immune functionality though whether this is causal or epiphenomenal requires elucidation. Both non-specific and mitochnodrial-targetted therapies allow direct activation of biogenesis pathways to facilitate mitochondrial recovery, or protection against excessive oxidative or adrenergic stress, or against mitophagy. Studies will be made in samples taken temporally from long-term rat models of peritonitis and from critically ill septic patients in the intensive care unit. Interventions will be performed both in vitro and successful candidate strategies can then be tested in vivo. The ESR will learn in vivo physiological techniques, including advanced instrumentation and echocardiography, and a variety of tests assessing immune and mitochondrial functionality (e.g. FACS, spectrophotometry, respirometry, dual photon confocal microscopy)

Host: University College London, UK

Supervisor: Prof. Mervyn Singer

 

ESR11: Chloe Albert

New immune functional assay coupled with transcriptomic analysis for the characterization of the altered immune status observed in septic patients

Define a new immune functional assay (IFA) coupled with transcriptomic analysis to characterize the altered immune status observed in septic patients

The project consist of the development and optimization of an immune functional assay (IFA), in order to characterize the altered immune status observed in septic patients. Septic patients develop an early dominant hyper-inflammatory response followed by a partially overlapping immuno-suppressive phase. The hyper-inflammatory phase can lead to early death by organ dysfunction, resolved toward homeostasis or either enter a long-lasting immunosuppressive state. To drive therapeutic decisions, an IFA should be designed to deliver results in very short time and with limited sample handling. To meet these clinical constraints, such assay will be performed as follows i) use of whole blood to avoid the time-consuming isolation of a specific cell population, ii) stimulation with a yet to be determine stimuli within a 4 hour incubation time and iii) transcriptomic read-outs obtained with rapid and sensitive methods such as quantitative Polymerase Chain Reaction and RNA Sequencing.

First of all, a selection of stimuli will be performed based on immuno-modulatory properties, well-chemically defined characteristics and accessibility to the compound. Then a drug screening will be set up using several ex vivo models mimicking altered state of immunity, to choose the stimuli with the most discriminant immune-modulatory characteristics.
In a second step, RNA sequencing technique and/or alternative molecular techniques will be optimized and transcriptomic markers will be selected to define a confident immune response. The relevant read out able to discriminate fold-changes in the transcriptomic profile as well as differentially expressed markers, will be combined into a multiplexed PCR based assay compatible with clinical settings. The combination of stimuli and related read out will defined the IFA prototype.
In a third part, basic research will be performed to understand the mechanism of action of the compounds. This will be looked at the cellular level, i.e. which cell populations take part in the observed response to the drug, and at the molecular level, i.e. which receptors and associated pathways are involved in drug response.
Last, this prototype will be validated by assessing its ability to stratify septic patients compare to classical methods and that would allow to adapt a suitable treatment to each patient.

Host: bioMerieux, Medical Diagnostic Discovery Department, Marcy l’Etoile (Lyon) France.

Supervisors: François Mallet; Sophie Assant

 

ESR12: Dina Tawfik

Design of a fully integrated molecular tool to manage Intensive Care Unit patients based on their immune status

Objective: To identify the patients per their immune status, through identifying the whole blood transcriptome to intervene accordingly towards a customized treatment and to finally stratify the patients. Dina’s work will include both laboratory experiments and in silico duties, basically consisting in elementary transcriptome analysis, design and evaluation of PCR assays, definition of algorithms to ease physician’s interpretation of the results as well as the proposal of an adaptive methodology for clinical evaluations

  • Evaluate and refine the potentiality of a list of candidate markers (40-50 from literature and in-house studies) to delineate the host response to sepsis and their hyper- and hypo-inflammatory states using bioinformatics tools.
  • The impact of each gene within the signature will be determined, using expression data from
    1. Gene expression analyses previously performed in the lab using Affymetrix microarrays.
    2. Evaluate the applicability of each candidate by PCR multiplex Film Array platform
  • Design and implement an algorithm to interpret the gene expression data results to be easily utilized in clinical trials and for physician’s clinical interpretations.
  • Develop an adaptive methodology for evaluation/ follow-up of the clinical status of the immunocompromised patients.

Host: bioMerieux, Medical Diagnostic Discovery Department, Marcy l’Etoile (Lyon) France.

Supervisors: Julien  Textoris; Laurence  Ganée

 

ESR13: Harjeet Singh

Whole blood molecular assays to quantitate sepsis induced immunosuppression

Objectives: Molecular signatures derived from blood leukocytes provide more information about the nature and severity of the inflammatory response and the magnitude of organ injury than traditional single protein or gene biomarkers.  These signatures can be used for identifying patients at risk of dying and/or sepsis complications, thereby allowing individualised preventive or therapeutic interventions. The main objective of ESR13’s project is to identify molecular signatures within blood leukocytes that are able: (1) to quantitate sepsis induced immunosuppression, (2) to identify patients at high risk of developing sepsis and (3) to stratify the individual patient with respect to risk of dying. Work will include an -omnics approach: DNA methylation patterns, epigenetic signatures and metabolomics. Our previously identified whole blood leukocyte gene expression patterns of survivors and non-survivors of septic shock will be incorporated in a custom array and subsequently in a PCR based bedside assay will be tested in independent sepsis patient cohorts. Tentative projects include validating previous findings obtained from our adults sepsis cohorts in pediatric sepsis and in tropical sepsis (e.g. in patients with melioidosis). Affinity with clinical work and competence in molecular biology as well as computational biology is desirable for this position.

Host: Academic Medical Center, University of Amsterdam (AMC, UvA), Center for Experimental and Molecular Medicine (CEMM).

Supervisors: Dr. Brendon Scicluna, Prof. Tom van der Poll,  Dr. Joost Wiersinga

 

ESR 14: Andres Alban

Sequential sampling for efficient clinical trials with biomarker covariates

Objectives: The objective is to improve the cost and time required on average to perform health technology assessments of new clinical treatments or processes by developing adaptive sequential sampling algorithms for medical trials. The goal is to make better use of patient covariate and health outcome data in order to more quickly and safely identify which treatments work better as a function of patient parameters. The work is methodological and the ESR will learn and develop theory in the area of applied probability, Bayesian statistics, kriging metamodelling, and application to sepsis and cost benefit analysis for health innovation. The project will involve the development of mathematics and implementation of computer code to simulate the performance of the new trial designs.

Host: INSEAD, Fontainebleau, France

Supervisor: Prof. Stephen Chick, PhD.

 

ESR 15: Analytics to identify biomarkers for sepsis diagnosis or treatment

Objectives: This work will focus on the exploration of sepsis data, such as from the MARS study, to identify factors which seem to be more diagnostic of the onset of sepsis or benefits of treatment. This will involve the study, implementation, and application of a variety of data analytics tools which do statistical factor identification. Other more novel machine learning tools will also be explored for their usefulness in the sepsis context. The project will include a theoretical and empirical assessment with computer experiment of the comparative benefits of several analytics methods for their appropriateness for their use with data related to sepsis diagnosis and treatment.

Host: INSEAD, Fontainebleau, France

Supervisor: Prof. Stephen Chick, PhD