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Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

IMMUNOLOGY – LABROTATIONS & RESEARCH TOPICS

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: (Immuno)pathogenesis of chronic lymphocytic leukemia

Workgroupleader:
dr. A.W. Langerak
T: 010-704 4089
E: [email protected]
W: http://www.erasmusmc.nl/immunologie/onderzoek/moleculaireimmunologie/mid/?lang=en
Background
Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemic proliferation in the
Western world. CLL is found in adults and typically associated with age. The majority of CLL
cases is of B-cell type, while a minority derives from T lymphocytes (also called T-cell large
granular lymphocyte leukemia, T-LGL). Over the last years it has become increasingly clear
that CLL is a heterogeneous disease, with a variable clinical course and differences in
survival. CLL is an example of a multi-factorial disease, in which both genetic and micro-
environmental factors contribute to leukemogenesis. Although in recent years many studies
have focused on prognostic markers, there is still no complete picture of the factors that are
involved in the (immuno)pathogenesis and that are determining for the prognosis.
Research topics
One of the research lines in the MID (Molecular Immunodiagnostics) research group
(Immunology department, Erasmus MC) focuses on the (immuno)pathogenesis of CLL (both
B- and T-cell types), in particular on antigenic stimulation (autoantigens and/or microbial
antigens) and on genetic aberrations. For this we use state-of-the-art cellular (multi-colour flow
cytometry, cell sorting) and molecular (sequencing, spectratyping, array) techniques.
Our specific research questions are:
1. What is the role of antigenic stimulation via Ig / TCR molecules in the pathogenesis
of CLL and what are the possible auto- or exoantigens that are involved?
2. What is the composition of T lymphocytes in CLL and to what extent is that
correlated to clinical and/or biological parameters?
3. What are the molecular genetic aberrations that are involved in CLL pathogenesis?

The MID research group has an (inter)national reputation with fruitful collaborations within
Erasmus MC (Hematology, Virology) and outside Erasmus MC (European Research Initiative
on CLL, ERIC; University of Salamanca; University of Belfast).
Related publications
 Sandberg Y, Almeida J, Gonzalez M, Lima M, Barcena P, Szczepański T, Van Gastel-Mol EJ, Wind HK, Balanzategui A, Van Dongen JJM, San Miguel JF, Orfao A, Langerak AW. TCR+ large granular
lymphocyte leukemias reflect the spectrum of antigen-selected normal TCR+ T-cells in peripheral
blood. Leukemia 2006; 20:503-513.
 Rodriguez-Caballero A, Garcia-Montero AC, Barcena P, Almeida J, Ruiz-Cabello F, Taberno MD, Garrido P, Munoz-Criado S, Sandberg Y, Langerak AW, Balanzategui A, Orfao A. Expanded cells in
monoclonal TCR+/CD4+/Nka+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood
2008;112:4609-4616.
 Dijkstra MK, Van Lom K, Tielemans D, Langerak AW, Van 't Veer MB, Jongen-Lavrencic M. 17p deletion / TP53 deletion in B-CLL patients is associated with microRNA-34a downregulation. Leukemia
2009;23:625-627.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Characterization of acute leukemia

Workgroupleader:
Dr. V.H.J. (Vincent) van der Velden
T: 010-704 4253
E: [email protected]
Background
Acute leukemias are malignancies of immature hematopoietic cells. Depending on the
involved cell type, two main categories of acute leukemia can be distinguished: acute myeloid
leukemia (AML) and acute lymphoblastic leukemia (ALL). Further subdivision can be made,
dependent on immunophenotype and genetic aberrations. Such subdivision is necessary
because the different subgroups have different clinical outcomes and consequently require
different therapeutic approaches.
ALL is most prominent in childhood and, using current chemotherapeutic regimens, has a 5-
year event free survival up to 85%. For this group of patients it will especially be important to
recognize those patients with a high risk of relapse, who may benefit from treatment
intensification, and those with a low-risk of relapse, who may benefit from treatment reduction.
In contrast, AML is most frequent in adults (especially those >60 years of age) and although
chemotherapy induces clinical remission in the vast majority of patients, most of them
ultimately relapse, resulting in a 5-year survival of 40%. It is therefore clear that new
therapeutic modalities are required for this type of leukemia.
We and others have recently shown that detection of low numbers of leukemic cells during
and after treatment, so called detection of minimal residual disease (MRD), is of prognostic
value in acute leukemia. In our laboratory, flowcytometric immunophenotyping, PCR analysis
of chromosome aberrations, and PCR analysis of immunoglobulin (Ig) and T cell receptor
(TCR) gene rearrangements are used for MRD detection in several patient groups to
investigate the kinetics of tumor cell disappearance and thereby define the in vivo treatment
effectiveness and their risk of relapse. In addition, MRD information is used to determine the
efficacy of new therapies, including antibody-mediated chemotherapy, and to provide insight
into the immunobiology of acute leukemia (e.g. Ig/TCR gene rearrangement patterns in
leukemia subtypes).
Research topics
The main aim of our translational research is to improve our understanding on the
immunobiology of acute leukemias and to evaluate their response to treatment in order to
improve diagnostics and to optimize treatment protocols. To this end, we use start-of-the-art
technologies, like 8-color floccytometric immunophenotyping, cell sorting, sequencing, and
micro-array technology. The LLD research group coordinates and participates in several
national and international networks.
Our specific aims are: 1. To obtain insight in the immunobiological characteristics of childhood acute leukemia. This includes a.o.: a. Analysis of extramedullary acute leukemia for identifying markers that may predict extramedullary localization and that may be used as new therapeutic targets; b. Analysis of acute leukemia at the myeloid-lymphoid interface for understanding which molecules play a role in lineage specification and commitment. 2. To analyze Minimal Residual disease: a. Development and standardization of sensitive techniques to measure MRD in acute leukemia patients; this concerns both cellular and molecular methods; b. Recognition of MRD-based subgroups of patients that may benefit from treatment intensification or treatment reduction. 3. To identify parameters which affect the efficacy of antibody-based chemotherapy in acute Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Immune regulation diagnostics (IRD)
Workgroupleader:
dr. W.A. Dik (+31 (0)10 70.43528, [email protected]).
Background
The immune system is a complex and tightly regulated network in which several different cell
types, signalling molecules (cytokines) and receptors collaborate to fulfill its function.
Disordered regulation of the immune system is associated with a variety of diseases and
inflammatory conditions.
Research
We investigate regulatory mechanisms involved in inflammation and fibrosis and aim to
develop novel therapies to treat these conditions. Diseases of special interest are shock
related syndromes, systemic sclerosis, Graves' disease and uveitis. Our research is
multidisciplinary with an optimal integration of both laboratory and clinical expertise from
various departments within the Erasmus MC and the Rotterdam Eye Hospital.
Major achievements in the past years
Evaluation of CD103 as a cellular marker for the diagnosis of pulmonary sarcoidosis. Clinical
Immunology. 2008 126:338-344.
Is Imatinib mesylate a promising drug in systemic sclerosis? Arthritis and Rheumatism. 2008;
58:2549-52.
Synthetic oligopeptides related to the b-subunit of human chorionic gonadotropin attenuate
inframmation and liver damage after (trauma) hemorrhagic shock and resuscitation. Shock.
2009; 31:285-91
Imatinib mesylate and AMN107 inhibit PDFG-signaling in orbital fibroblasts: a potential
treatment for Graves' ophthalmopathy. Invest Ophtalmol. Vis. Sci; 2009; 50:3091-8
Training options
Interested and motivated students are encouraged to contact us about possibilities to do their
lab internship in our team. Together with the student we will define a project that is in line with
ongoing projects.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Immunopathogenesis of the Guillain-Barré syndrome

Workgroupleaders:
Dr. B.C. Jacobs, [email protected]
Dr. R. Huizinga, [email protected]
Background
Guillain-Barré syndrome (GBS) is a post-infectious polyneuropathy and the most common
cause of acute neuromuscular paralysis. In its most severe form GBS is a life-threatening
disease in which patients develop a rapidly progressive paresis of limb and respiratory
musculature for which they are treated at the ICU. Campylobacter jejuni, identified in 30-50%
of the GBS patients, is the predominant preceding infection and especially associated with
these most severe forms. Other common types of preceding infections include Mycoplasma
pneunomiae
, Epstein-Barr virus and cytomegalovirus.
The neurological symptoms in GBS are caused by autoantibodies which bind to glycolipid
antigens in the peripheral nervous system (gangliosides). The binding of antibodies results in
complement activation and subsequently damaging of nerve fibers that innervate the muscles.
Infection with C. jejuni triggers the production of pathogenic antibodies. As the lipo-
oligosaccharide (LOS) proportion of C. jejuni is highly similar to gangliosides (so-called
molecular mimicry), antibodies induced against LOS will cross-react with gangliosides in the
nervous system. The disease is monophasic and the recovery can be accelerated by
treatment with intravenous immunoglobulins (IVIg), a preparation made from serum of
thousands of healthy individuals.
Research topics
The GBS research group from the Erasmus MC is a multidisciplinary team that addresses
research questions ranging from basic science to clinical trials. Research projects for the
Infection & Immunity MSc students focus on the following projects:
1.
What is the cellular mechanism of pathogenic antibody formation?
It is known that preceding infections trigger the production of cross-reactive antibodies,
but how exactly the B cells are activated is still unclear. We hypothesize that in GBS
patients dendritic cells are stimulated by the microbial antigens, mimicking human
nerve target structures, and directly activate B-cells in absence of T-cells. We are
addressing this issue using an in vitro dendritic cell – B cell model and will perform in
vivo studies (mouse) to determine which cells are involved in the antibody response to
C. jejuni.
What are the target antigens in the GBS patients without anti-ganglioside
antibodies?

These antibodies are absent in at least half of the patients and our hypothesis is that
these patients have antibodies to other neural structures. Also in other autoimmune
disorders of the peripheral nervous system, such as chronic inflammatory
demyelinating neuropathy the target antigens remain to be elucidated. Various new
techniques for antibody detection have been developed, including dotblot arrays that
can be applied to identify the targets in seronegative patients.
What is the mechanism of action of IVIg in GBS?
IVIg consists of a complex mixture of donor immunoglobulins that is used to treat
patients with a spectrum of autoimmune disorders, including GBS. Although IVIg is an
established treatment, surprisingly little is known about the modes of action that
explain the therapeutic effects. IVIg could scavenge pathogenic antibodies or may
inhibit the production of pathogenic antibodies by plasma cells. GBS may be an useful
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
model disease to elucidate the working mechanism of GBS, since the disease is monophasic and the immune targets in half of the patients have been indentified. These insights will be highly valuable in clinical practice to improve treatment of patients with GBS and other IVIg-responsive disorders.
Principal techniques
Primary cell isolation and culture, proliferation assays, ELISA, FACS, antibody purification,
immunohistochemistry, Western blotting, quantitative PCR, management of patient cohorts.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Immunoglobulin repertoire generation and selection for protective immunity

Workgroupleader:
Dr. Menno C. van Zelm
T. 010-7043038
E. [email protected]
W. http://www.erasmusmc.nl/immunologie/onderzoek/moleculaireimmunologie/1705787/?
lang=en
Background
• B-cells are critical in the immune system, mainly by their unique capability to produce
enormous amounts of specific antibodies against pathogens. The B-cell response is an adaptive response and memory is maintained in the form of antibody-producing plasma cells and resting memory-B-cells. • During the humoral immune response, activated B-cells are selected for high affinity antibodies that show no cross reactivity with self antigens. Therefore, the Ig repertoire of memory-B-cells is a direct reflection of what the individual was allowed to produce to prevent disease: both infections AND adverse autoimmune phenomena.
Research
• The research lines in the work group ‘B-cell differentiation' (BCD) in the dept. Immunology
of the Erasmus MC are focused on the generation of a large Ig repertoire in bone marrow and the selection of this repertoire in immune responses and found in memory-B cells and plasma cells in peripheral lymphoid organs. • For studies addressing B-cell antigen repertoire selection, we use multi-color flow cytometric analysis to identify different types of (antigen-specific) memory-B-cells in peripheral blood of human subjects. Thus, we can purify these cell populations to study the Ig repertoire selection in memory-B-cells of healthy individuals and patients with an immune disease (e.g.primary antibody deficiency, autoimmunity, allergy, persistent infection). This should provide insight into:  The regulation of Ig gene rearrangements by the 3D spatial reorganization of Ig loci in precursor-B-cell subsets; How the CD19-CD21-CD81 complex functions to lowers the threshold for BCR signaling and subsequent memory-B-cell formation; Functional properties of memory-B-cells generated by different defined antigen Related publications  MC van Zelm, I Reisli, M van der Burg et al. (2006) An Antibody-Deficiency Syndrome Due to Mutations in the CD19 Gene. N. Engl. J. Med. 354:1901-1912. MC van Zelm, T Szczepański, M van der Burg, and JJM van Dongen. (2007) Replication History of B-lymphocytes Reveals Homeostatic Proliferation and Extensive Antigen-induced B-cell Expansion. J. Exp. Med. 204(3):645-655. MC van Zelm, J Smet, F Mascart et al. (2010) CD81 Gene Defect in an Antibody- deficient Patient Disrupts CD19-complex Formation and Impairs Terminal B-cell Differentiation. J. Clin. Invest. 120(4):1265-1274. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Antibody deficiencies: searching for the unknown

Workgroupleader:
dr. Mirjam van der Burg
T: 010-704 4089
E: [email protected]
W:
http://www.erasmusmc.nl/immunologie/onderzoek/moleculaireimmunologie/1269162/?lang=en
Background
Antibody deficiencies form the largest group of primary immunodeficiencies. Patients can
present either in early childhood or in adulthood with increased susceptibility to mainly
bacterial infections and hypogammaglobulinemia or agammaglobulinemia. A subdivision into
several categories can be made based on the presence of B-lymphocytes and serum
immunoglobulin (Ig) levels. Identification of genetic defects in patients with an antibody
deficiency requires thorough understanding of normal B-cell differentiation and its cellular and
molecular processes. Flow cytometric immunophenotyping is an important and powerful tool in
the diagnostic process to study B-cell subsets in peripheral blood and, in case of strongly
reduced peripheral B-cell numbers, precursor B-cells in bone marrow. However, in some
patients this is insufficient to unravel the underlying pathophysiology and immunogenetic
defect. Functional assays are a valuable contribution and can include measurement of
replication history, B-cell antigen receptor signaling, somatic hypermutations in Ig-class
switched transcripts, and in vivo response to vaccination. However, we expect that in vitro cell
systems, cytokine arrays, and genotyping using SNP arrays are also required to get new
insights.
Research topics
One of the research lines of the Workgroup Primary Immunodeficiencies (PID) of the Dept. of
Immunology, Erasmus MC focuses on antibody deficiencies. At least three categories of
antibody deficiencies can be identified in which the underlying immunogenetic defect has not
yet been unraveled:
1. Agammaglobulinemia patients with absence of precursor B-cells
2. CVID patient with thymoma (Good syndrome) with complete suppression of the
B-cell system
3. Serum IgM deficiency
The aim of the project is to unravel the underlying pathophysiological and immunogenetic defect in these three categories of antibody deficiencies. The PID group is a national and internal reference center for agammaglobulinemia and severe combined immunodeficiency (SCID). The field of research of the PID workgroup is normal (precursor) B-cell differentiation and defective precursor B-cell differentiation resulting in antibody deficiencies as well as V(D)J recombination and its regulation, which is affected in patients with T-B- (SCID). The project on antibody deficiencies is embedded in a longstanding collaboration with the Dept. of Pediatrics and Dept. of Internal Medicine of the Erasmus MC and several international research centers. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
Title: Bipolar disorder, major depressive disorder and schizophrenia viewed as
consequences of an abnormal set point of the immune system. Relations to autoimmunity
and chronic inflammatory conditions
.
Workgroupleader:
Prof. dr H.A. Drexhage (Autoimmune Unit).
T : 010-7044093
E : [email protected]
W: www.moodinflame.eu
Background
Our previous studies point in the direction of the existence of a shared vulnerability factor for bipolar
disorder (BD), major depressive disorder (MDD), schizophrenia (SCZ), autoimmune thyroiditis
(AIT), diabetes and atherosclerosis This shared vulnerability factor is – in our opinion - an abnormal
inflammatory set point of the immune system, in particular of the mononuclear phagocyte (MPS:
monocytes/macrophages/dendritic cells) and the T cell system (Th1/Th2/Th17 and T regulator
cells).
Our premise is that an activated inflammatory response system (IRS) drives all the above
mentioned pathologic processes, yet differences occur between these complex diseases due to
differences in eliciting or protecting co-factors of genetic and environmental character, such as e.g.
polymorphisms in the HLA system, infections with commensal micro-organisms, dietary habits
leading to low serum HDL, smoking, and gender.
In our research over the past 5-10 years we have identified a plethora of functional inflammatory
abnormalities of cells of the MPS and T cell system in BD, MDD, SCZ, AIT, diabetes and
atherosclerosis. This research was performed on patient material (serum and leukocyte
preparations) and – in parallel – in animal models of these diseases, in particular the NOD mouse
and the BB-DP rat.
At present we focus on molecular studies in a NWO-TOP grant and a large scale EU program (19
partners from 10 EU countries, acronym MOODINFLAME), which our group coordinates.
Present Research
Our research presently involves studies on
1. An aberrant pro-inflammatory gene expression of cells of the MPS as one of the important
reflections of the activation of the IRS. To approach the problem on a molecular level we have identified different inflammatory gene fingerprints aberrantly expressed in monocytes of BD, MDD, SCZ, AIT and diabetes patients, as well as in the NOD mouse. We have designed custom made (RQ-PCR) TLDA arrays for these genes and test the ability of these arrays to identify individuals at risk for the development of the mentioned diseases and to distinguish various subtypes of the diseases. 2. Abnormal ratio's between Th1, Th17 and Th2 cells on the one hand and regulatory T cells on the other hand. For these analyses we make use of FACS. 3. Systems biology approaches to link the pro-inflammatory states of the MPS and T cell system in these diseases to concomitant abnormalities in the stress system (glucocorticoid responsiveness) and epigenetic changes (early and even peri-natal infections). For these studies we make use of micro-RNA approaches, Chip-SEQ and we plan methylation arrays.
Reference: Drexhage RC, Knijff EM, Padmos RC, Heul-Nieuwenhuijzen L, Beumer W, Versnel MA, Drexhage HA. The
mononuclear phagocyte system and its cytokine inflammatory networks in schizophrenia and bipolar disorder. Expert Rev
Neurother. 2010 Jan;10(1):59-76. Review.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Dysregulated myelomonocytic development in inflammatory disease : the role of
micro RNAs

Workgroup Autoimmune Diseases III
Workgroupleader:
dr. P.J.M. Leenen, workgroup leader Autoimmune Diseases III (AIP)
T: 010-704 3171
E: [email protected]
W: http://www.erasmusmc.nl/immunologie/onderzoek/autoimmuunziekten/
Background
The central theme in our research is the developmental biology of myelomonocytic cells under
normal conditions and in inflammatory diseases where deviations in development and function
of these cells putatively underlie pathological
bone marrow states (Fig. 1). The myelomonocytic cells of interest comprise monocytes, various types of macrophages and dendritic cells, and their immediate precursors in hematopoietic organs. Despite their extensive functional and phenotypic heterogeneity, these cells are related via common developmental pathways as well as shared functions and regulatory Fig.  1.  Central  research  theme:  developmental  biology  of  On the basis of significant progress made in the
past decades, it is our conviction that tuning of the activation stage of cells of the
myelomonocytic lineage is crucial not only for the appropriate function of host defense
systems, but for organ and tissue homeostasis in general. In line with this notion, aberrant
function of these cells has been shown in a wide variety of diseases, including autoimmune
diseases such as type 1 diabetes, atherosclerosis, cancer, obesity and psychiatric diseases
such as bipolar disorder. In particular the inflammatory nature of the myelomonocytic cells in
these conditions may underlie many of the pathological signs and symptoms.
Differentiation and activation of hematopoietic cells is regulated at different levels. Recently,
microRNAs (non-translated oligonucleotides of 17-23 nt) have been identified as important
post-transcriptional regulators of mRNA translation and stability. These microRNAs appear to
be highly conserved during evolution, indicating their fundamental role in regulation.
Interestingly, microRNAs have important roles in the control of normal differentiation and
functional activation of cells in the hematopoietic system.
Research focus
Our current research projects are centered around the working hypothesis that microRNAs are
important molecular regulators of myelomonocytic cells, and thus represent potential
diagnostic and therapeutic targets. Therefore, we investigate the involvement of microRNAs in
various inflammatory conditions in patients and animal models of human disease. These
include diabetes, psychiatric disease, histiocytosis, bacterial infection and atherosclerosis. In
these projects, we collaborate with various local, national and international research groups
and clinical partners.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: The Interferon type I signature in monocytes in primary Sjögren's Syndrome:
A novel biomarker

Workgroupleader:
Dr. M.A. Versnel, workgroupleader Autoimmune Diseases II
T: 010-7048086
E: [email protected]
W: http://www.erasmusmc.nl/immunologie/onderzoek/autoimmuunziekten/1269072/
Background
Sjögren's Syndrome (SjS) is an autoimmune disease characterized by focal lymphocytic
infiltrations in the salivary and lachrymal glands. Patients mainly suffer from dry mouth
(xerostomia) and dry eyes (keratoconjunctivitis sicca), while multiple extraglandular
manifestations such as fatigue, myalgia and arthralgia are frequently present and form a major
cause of morbidity. Diagnosis of SjS is difficult due to lack of diseases specific markers and
the heterogeneity of the disease.
Dendritic cells (DC) play an important role in the initiation of pSjS (van Blokland et al., 2000).
Monocytes migrate through the blood to the peripheral tissues, where they can develop into
dendritic cells (DC). Using microarray analysis we detected an Interferon (IFN) Type I induced
gene expression signature in monocytes of pSjS patients (Wildenberg et al., 2008). The
presence of the IFN type I signature in a subgroup of patients points towards a pathogenic
pathway contributing to the development of pSjS.

Research
The aim of this project is to determine the prevalence of the systemic interferon type I
signature, to relate the presence of the signature to clinical symptoms and to therapeutically
target IFN type I in patients with Sjögren's Syndrome.
This aim will be approached by the following specific questions:
1. What is the prevalence of the IFN type I signature in a cohort of pSjS patients?
2. Is the presence of the signature related to the clinical manifestation of the

disease?
3. What is the efficacy of an anti-IFN alpha monoclonal antibody treatment in pSjS
patients?
4. Is there a correlation between the efficacy of anti-IFN type I therapy with the
presence of the INF type I signature?
5. Can the IFN type I signature be used to monitor treatment?

To approach these questions the following techniques will be used: Isolation of monocytes from peripheral blood, RNA isolation, quantitive PCR, cell culture. The project will be carried out in collaboration with national and international research groups (France, Italy). Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: The role of Th17 cells and cytokines in the development of autoimmune arthritis

Workgroupleader:
Dr. E. Lubberts, workgroup leader Rheumatology Research Lab.
Tel. 010-7044309
e-mail: [email protected]
Background
Rheumatoid arthritis (RA) is a chronic systemic disorder characterized by autoimmunity,
infiltration of joint synovium by activated inflammatory cells, synovial hyperplasia,
neoangiogenesis, and progressive destruction of cartilage and bone. This disease affects 1-
2% of the population worldwide, most commonly middle-aged women. The etiology of RA is
unknown but pro-inflammatory cytokines play a central role in RA. Recognition of RA early in
the disease course is becoming increasingly important since early treatment has been shown
to prevent joint damage and to preserve function and work participation. Understanding the
immunological pathways involved in this process is of crucial importance to further improve
current therapy.
T cells represent a large proportion of the inflammatory cells invading the synovial tissue. T
cell activation and migration into the synovium occurs as an early consequence of disease,
and these cells adopt a pro-inflammatory phenotype. Considerable evidence now supports a
role for T cells in the initiation and perpetuation of the chronic inflammation prevalent in RA.
Interestingly, the vast majority of these are (memory) T cells producing IL-17 that is up
regulated in early disease and is thought to contribute to the inflammation associated with RA.
The research of the Department of Rheumatology/Immunology is focused on early diagnosis
of RA and to understand the immunological mechanism(s) critical in the development of
chronic destructive arthritis (RA).
Research
The workgroup Rheumatology focuses on three interrelated subjects:
1. The role and modulation of lymphocytes in the development of
autoimmunity, with focus on the IL-23/Th17 immune pathway in different
arthropathies (RA, SLE, psoriatic arthritis) and in collaboration in other
chronic inflammatory diseases such as MS, psoriasis, and COPD;

2. The role of T and B cell cytokines / cytokine signaling in different
arthropathies (RA, SLE, psoriatic arthritis);
3. Unraveling the immunological profile responsible for the development of
RA and inflammatory OA;
To gain insight into these processes, we use human material from two RA patient cohorts, the REACH (Rotterdam Early Arthritis Cohort) and the PARA (Pregnancy Amelioration of Rheumatoid Arthritis) as well as various (humanized) animal models for RA. Flow cytometry, in situ analysis, and several molecular methods are employed. Fruitful collaborations exist with research groups within the Erasmus MC (Internal Medicine, Pulmonary Medicine, Dermatology, Immunology) and outside Erasmus MC (University of Utrecht, Leiden, Pittsburgh, Boston, New Orleans). Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Multiple sclerosis
Workgroupleaders:
Prof. dr. J.D. Laman (Group leader)
T: 010-7043796
E: [email protected]
Prof.dr. R.Q. Hintzen (head MS Centre ErasMS)
E: [email protected]
W: http://www.erasmusmc.nl/immunologie/onderzoek/immuunregulatie/1269120/?lang=en
Background

Multiple sclerosis (MS) is the most common neurological disorder affecting young adults. The
disease is largely characterized by demyelination and eventually by neuronal loss leading to
neurological disability. MS disease course is highly variable with the relapsing remitting form
being the most frequent in which episodes of disease are followed by recovery. This phase is
often followed by the progressive form characterized by severe paralytic disability. MS is a
complex disease of which the underlying mechanisms remain poorly understood. The most
pivotal players in MS disease are autoagressive T cells and antigen presenting cells in
conjunction with genetic traits and environmental factors.
Research
The work of the MS research group mainly focuses on translational research. The MS
workgroup in the Dept. Immunology is part of the MS Centre ErasMS, headed by Prof. Hintzen
of the Dept. Neurology of Erasmus MC. The research theme of ErasMS is Biological
determinants of disease course: genetics, microbiology and immunology.
To perform our research we make use of state of the art research techniques (quantitative
PCR, multi-colour flowcytometry and immunocytochemistry/fluorescence). As the research
has a large translational component we have collected very exclusive patient material (such
as cells and serum from pregnant MS patients) and for other studies we use material provided
by the Netherlands Brain Bank. In addition, classic and innovative animal models for MS are
employed.
Research lines:
1. (Immuno) genetics  Identification of gene polymorphisms involved in MS disease
susceptibility and analysis of their possible function in MS
2. Immunology  involvement of microbiological components in MS disease activation,
immunosuppression of inflammation by foam cells, effect of environmental factors on
inflammation, analysis of immunological parameters during MS and pregnancy
3. Biological disease marker identification  State of the art proteomics analysis of
cerebrospinal fluid and subsequent validation of found target proteins.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

MICROBIOLOGY – LAB ROTATIONS

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
LABORATORY ROTATIONS
DEPARTMENT OF MEDICAL MICROBIOLOGY AND INFECTIOUS DISEASES (MMIZ)
We offer four different options for three weeks' laboratory rotations which will be explained below. Students selecting one of these rotations will be embedded in small research units and will participate in the ongoing research projects within that unit. Students will assist in experiments, be involved in research discussions and journal clubs. A package of relevant scientific papers authored by the research team will be handed for self-study. In addition, during the individual rotations there are opportunities for several half-day instruction episodes relating to specific research topics within the MMIZ department. 1. Molecular microbiological investigations into the pathogenicity of
Campylobacter jejuni: C. jejuni is an enteric pathogen primarily causing diarrhoea. In
addition, C. jejuni may also cause an auto-immune disease named the Guillain Barré
Syndrome (GBS). GBS is caused by autoimmunity towards human nerve gangliosides
which is primed by structural homology between these gangliosides and the bacterial
surface moiety lipo-oligosaccharide (LOS). Within the unit, detailed research is
performed on the nature of C. jejuni virulence factors involved in GBS, the host
response and mechanisms of immune priming. Technologies used include bacterial
culture, molecular biology techniques (PCR, cloning, in vitro protein expression and
purification, knock out mutagenesis, genomics and transcriptomics) and cell biological
procedures (bacterial adhesion and invasion, immunohistochemistry, microscopy). For
further information contact Rogier Louwen ([email protected]) or Astrid
Heikema ([email protected]) (PhD students).
2. Nasal carriage of Staphylococcus aureus: Approximately 30% of humankind carries
S. aureus in the vestibulum nasi, the foremost site of the interior nose. It is unknown why humans carry in this particular location, but it has been firmly established that such carriage predisposes to (auto-)infection. Infections can be mild (boils, skin infections) but also life threatening (sepsis, endocarditis). Within the unit the search is on for human and bacterial determinants of S. aureus nasal carriage. Bacteriological technologies used comprise in vivo human nasal inoculation with S. aureus, bacterial transcriptomics and comparative genomics. At the host end we investigate genetic predisposition through genome wide association studies and we participate in extensive longitudinal cohort studies. The humoral antibody responses in carriers, non-carriers and infected individuals are determined using bead-based flow cytometry. For further information contact Nelianne Verkaik ([email protected]), Lonneke Bode ([email protected]) (PhD students) or Willem van Wamel [email protected]) (assist. professor) 3. Fungal infections and therapy: Various fungal species cause invasive infections.
This happens in immuno-compromised patients mainly, but also apparently healthy individuals can be infected. We study two fungal pathogens: one is Aspergillus fumigatus, an airborne spore-producer that can cause massive pulmonary infections; the second is Madurella mycetomatis, a soil-borne pathogen that causes massive lesions in people living in tropical endemic areas. For both pathogens we develop diagnostic tools, human and fungal epidemiological surveillance markers and we study anti-fungal resistance and routes of pathogenicity. Technologies used vary from fungal culture and molecular diagnostics, antifungal susceptibility testing (ELISA format, XTT testing) and fungal genome sequencing to hosts susceptibility testing by genome polymorphism assessment. For further information contact Patricia Verwer Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
([email protected]) (PhD student), Alieke Vonk ([email protected]) or Wendy van de Sande ([email protected]) (senior post doc researchers). 4. Innovative microbiological diagnostics: Medical microbiology has been
revolutionised by, first, the introduction of immunological testing and, second, the rise of molecular, nucleic acid-mediated diagnostics. Both technologies are part of the current standard of practice, but still innovation continues. Within this unit, novel diagnostic tools are developed, evaluated and finally translated into day-to-day practice. Technologies are compared with respect to sensitivity and specificity with the current Gold Standard procedures. Novel methods are applied and these include Raman spectroscopy, multiplex ligation probe mediated amplification (MLPA), mass spectrometry, electronic noses, straightforward PCR and TMA testing and many others. For further information contact Rene te Witt ([email protected]), Marcel Bruins ([email protected]) (PhD students) or Willem van Leeuwen [email protected]) (molecular medical microbiologist). Half day activities to be included in the laboratory rotations are: 1. Visiting the Animal Experimentation Centre of Erasmus MC and attending activities. 2. Getting acquainted with infection control policies of Erasmus MC. 3. Getting to know the routine diagnostic microbiology laboratory of Erasmus MC. 4. Perform routine molecular diagnostics of sexually transmitted diseases. 5. Join an Infectious Diseases specialist-in-action. For general information contact Alex van Belkum ([email protected]) or Jan Nouwen ([email protected]). Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

MICROBIOLOGY – RESEARCH TOPICS
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Mycetoma caused by Madurella mycetomatis

Workgroupleader:
Dr. W. van de Sande
Tel. 010-7035225
e-mail: [email protected]
Background
Mycetoma is a chronic subcutaneous fungal infection characterized by the discharge of grains
and purulent material through sinuses. This infection can be caused by fungi and bacteria, but
the most common causative agent is the fungus Madurella mycetomatis. This disease is
endemic in tropic and subtropical countries, and countries with a high incidence include
Mexico, India and Sudan. Overall, mycetoma is found in immunocompetent individuals and
the predisposing factor for developing mycetoma is still unkown. Strikingly, mycetoma is more
often found in young males.
Diagnosis and therapy for mycetoma are still in their infancy. Diagnoses of eumycetoma
depends mainly on culture, histopathology and imaging techniques. Downsides of these
techniques are that they are invasive, take long culture times and are not very accurate.
Therapy of mycetoma still depends on surgery combined with prolonged therapy with
ketoconazole. Unfortunately, success rates vary considerably and recurrent infections are
common.
The research of the mycetoma working group is focused on early diagnosis and improvement
of therapy for mycetoma. Furthermore, we try to gain insight in the interaction between fungus
and host during mycetoma development.
Research
The working group mycetoma aims to
1. Improve the current diagnostic tools for the endemic areas
2. Improve the therapy for mycetoma
3. Establish the role of the fungus in the development of mycetoma
4. Establish the role of the host in the development of mycetoma

To gain insight into these processes, we use fungal isolates and human material from mycetoma patients seen in the mycetoma clinic in Sudan. These materials were obtained during our long-standing cooperation between the Mycetoma Research Centre in Sudan and the ErasmusMC. Various novel DNA manipulation techniques such as cloning and protein expression, immunologic assays and in vitro susceptibility assays were developed for M. mycetomatis. In addition, a mouse model of M. mycetomatis infection was developed. These techniques will be used to address our research questions. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Invasive pulmonary aspergillosis caused by Aspergillus fumigatus

Workgroupleaders:
Dr. I.A.J.M Bakker-woudenberg
Dr. W. van de Sande
Tel. 010-7035225
e-mail: [email protected]
Background
In a retrospective study in a medical intensive care unit an incidence of invasive aspergillosis
of 5.8% was found, with an observed mortality of 91%. Since there are only three classes of
antifungal agents in use for Invasive Pulmonary Aspergillosis (IPA), therapeutic options are
limited and the therapeutic response rates are relatively low. In addition, toxic side effects
often occur and a rise in resistance to the most commonly used antifungal agent voriconazole
is reported. Furthermore, the number of immunocompromised patients at risk for developing
IPA is still rising. This confirms the need for the development of new therapeutic strategies for
IPA.
In our laboratory we developed a model of aerogenic left-sided invasive pulmonary
aspergillosis model in neutropenic rats which resembles the infection found in
immunocompromised patients. This model is appropriate to study new therapeutic strategies
for IPA.
Research
The current research aims to expand the novel therapeutic options for IPA, with:
1. Combination
2. The development of new antifungal agents
3. Employment of molecular tools

In order to design these therapeutic strategies for investigation in our animal model, in vitro methods are used as well. For the development of new antifungal agents, we employ various molecular methods. Furthermore we cooperate with various partners inside the Netherlands (Utrecht) and outside the Netherlands (USA and Germany). To determine the antifungal potential in vitro antifungal susceptibility assays are employed. Finally the most effective strategies are tested in our animal model. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

TITLE: Campylobacter jejuni antibody responses in enteritis and Guillain Barré
syndrome patients

Workgroupleader:
Astrid Heikema
Tel. 010-7035225
e-mail: [email protected]
Background
Campylobacter jejuni (C. jejuni) is a food born pathogen that, when digested, can induce mild
to severe enteritis in humans. In developing countries, symptomatic disease occurs in young
children and causes substantial morbidity and mortality. Because of high-level exposure to the
organism, there is a gradual development of protective immunity resulting in persistent,
asymptomatic carriage in adults. In developed countries, infections occur at all ages with peak
incidence in children below one year and young adults (15-24 years old).
A rare complication of an infection with C. jejuni is the Guillain-Barré syndrome where patient
develop paralysis caused by acute demyelination of peripheral nerves. Antibodies raised by
the host against C. jejuni, cross react with ganglioside structures on host nerve tissue causing
nerve damage. The attack is triggered by molecular mimicry between C. jejuni lipo-
oligosaccharides (LOS) and the neural gangliosides.

Research
To obtain more knowledge about anti-C. jejuni immune responses in human, we want to map
antibody responses in enteritis and Guillain-Barré syndrome patient after a C.jejuni infection.
Methods:
A panel of 40-50 C. jejuni surface proteins will be expressed and purified. These proteins will
be coupled to beads and, using the luminex system, will be used to screen patient serum for
antibodies against these surface proteins

The goals of this project are:
1) Searching for vaccine candidates by mapping antibody responses that lead to protective
immunity against C. jejuni infection in patients form developing countries.
2) Detecting additional antibody responses that lead to the development of the Guillain-Barré
syndrome
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

TITLE: Functional interaction between C. jejuni and siglecs

Workgroupleader:
Astrid Heikema
Tel. 010-7035225
e-mail: [email protected]
Background
The Guillain-Barré syndrome (GBS) and its variant the Miller Fisher syndrome (MFS) are post-
infectious diseases of the peripheral nerves. These syndromes can be triggered by an
infection with a bacterial species or virus that eventually may results in paralysis. Paralysis
can be systemic, in case of GBS, or it can be directed to the eyes muscles in MFS.
Campylobacter jejuni (C. jejuni), a bacterial species that can cause gastro-enteritis upon
ingestion of contaminated foods, is the main triggering agent of GBS and MFS. C. jejuni
expresses surface structures, lipooligosaccharides (LOS), which are highly similar to
structures, gangliosides, on human peripheral nerves. After the acute phase of the infection,
antibodies raised against the LOS of C. jejuni cross-react with gangliosides on the human
nerves and cause immune-activation and -attack of the nervous tissue resulting in paralysis.
Presence of sialylated LOS structures on C. jejuni is the main pathogenic factor for
development of GBS and MFS. However, 1 out of 2 C. jejuni strains isolated from enteritis
patients possess these sialylated structures as well and only 1/1000 enteritis patients develop
GBS/MFS. Other bacterial factors or host factors must contribute to the development of the
post-infections sequelae.
We recently found that especially GBS and MFS associated strains bind to members of the
siglec (sialic acid recognizing lectins) family. Siglec are present on a range of immune cells
and are involved in ligand binding and immune-regulation. We found that GBS associated
strains have a preference for binding to siglec-1, present on a subset of macrophages.
Further, we found that MFS associated strain predominantly interact with siglec-7, expressed
on i.e. monocytes and dendritic cells
Research
We hypnotize that binding of C. jejuni to either siglec-1 or siglec-7 skews the infection to a
situation that facilitates increased production of cross-reactive antibodies and subsequent
development of GBS or MFS. In a series of test we want to elucidate what the consequences
are of binding to siglecs-1 or -7. We want to determine if siglec binding affects the fate of the
bacteria and/or if it alters immune activation for example by production or down regulation of
specific cytokines. We will focus on binding, internalization trafficking and cytokine production
studies using primary cells isolated form peripheral blood or from the lung. We also want to
investigate the occurrence of single nucleotide polymorphisms (SNPs) in siglec-1 or siglec-7
genes that are related to GBS/MFS predisposition in patients.
Methods:
- Isolation CD14+ cells from peripheral blood
- Stimulation of these cells to siglec-1 expression macrophages or siglec-7 expressing
dendritic cells
- Binding and internalization assays
- Analysis of internal trafficking using immuno histochemistry
- Cytokine production assays
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
- Setting up a system that we can use to study the influence of siglec-1 binding on TLR-4 activation - SNP identification and analysis Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

INTERNAL MEDICINE INFECTIOUS DISEASES SECTION – LABROTATIONS & RESEARCH
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

General information
The scientific projects of the Infectious Diseases section of the department of Internal
Medicine described hereunder are patient based studies, ranging from epidemiologic to
clinical to laboratory studies. The Infectious Diseases section cooperates closely with the
departments of Medical Microbiology & Infectious Diseases, Virology, Immunology, Gastro-
Enterology, Haematology, Epidemiology & Biostatistics and Public Health.
During your laboratory rotations and research you will, depending on the topic, learn to use a
variety of laboratory techniques. Participation in clinical work like outpatients clinics, hospital
rounds and consults can be offered and is highly recommended during the rotation. You will
also participate in clinical and scientific meetings.
Duration of the Laboratory Rotation is 3 weeks. The Research Projects can either be 6, 12 or
18 months projects, with a preference for 18 months projects.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Pathogenesis of Extended Spectrum Beta-Lactamase Gram-negative bacteria in
infections presenting to the general practitioner.

Workgroupleaders:
Dr. A. Verbon ([email protected]) and
Dr. W. Goessens ([email protected])
Background
Ecological, experimental and observational evidence suggests that, on a population level,
antibiotic use is associated with colonization by antibiotic-resistant micro-organisms.
Emergence of antibiotic resistance may occur at the locus of infection but usually develops in
the gastrointestinal tract, harbouring many bacterial species.
Antibiotic treatment of urinary tract infections (UTI) increasingly result in antibiotic resistant
bacteria on a population level but also in individual patients. Emergence of resistance in
individual patients might either occur by selection and overgrowth of resistant strains from the
individual bacterial gene pool or by acquisition of resistant genes or strains from the
community. Even a transient effect of antibiotic use on the carriage of resistant bacteria by the
individual could have an impact on the level of resistance in the population due to
transmission between individuals. In the Netherlands an increase in CTX-M-positive E. coli
has been observed in urine specimens, especially from patients with complicated UTIs. In the
first reports of these isolates, the ESBL genes were mainly of polyclonal origin. Later reports
and their data show polyclonal as well as clonal spread. In particular, isolates in possession of
CTX-M-15 seem genetically related. The clonal relationship implies that people become
infected from a single source or sources. However, the source of the increasing amount of
ESBL positive Gram-negative bacteria is still unknown and warrants further research.
Research
 To investigate the proportion of ESBL in the digestive tract of persons treated for UTI and determine the percentage of CTX-M. Explore possible sources of CTX-M in household surroundings including family members, pets, food and surroundings such as water supply.  This is a clinically relevant study which combines epidemiological studies with laboratory techniques such as PCR, clonal typing, bacterial cultures, and susceptibility testing of bacteria. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Influence of dosing regimens on the development of antibiotic resistance.

Workgroupleaders:
Dr. A. Verbon ([email protected]) and
Dr. B. Rijnders ([email protected])
Background
Worldwide, antibiotic resistant micro-organisms are increasing. This increase is preceded by
an increase in use of a particular antibiotic or class of antibiotics. Infections with antibiotic
resistant bacteria often are difficult to treat and have a higher mortality. Emergence of
antibiotic resistance occurs usually not at the locus of infection but develops in the
gastrointestinal tract harbouring many bacterial species. Unfortunately, new antibiotic
medication has been sparsely developed. Therefore, optimal antibiotic dose regimens are
needed for maximal efficacy and prevention of antimicrobial resistance.
Scarce data suggest that continuous infusion (CI) may reduce the development of
antimicrobial resistance, but other preclinical studies show the opposite. To determine the
development of antimicrobial resistance at the infectious locus and in the gastrointestinal tract
as wel as the efficacy of Beta-lactam antibiotics patients with invasive methicillin sensitive
Staphylococcus aureus infections will be treated by either continuous infusion (CI) or
intermittent infusion (II) of flucloxacillin. Complete methicillin resistance is due to acquisition of
chromosomally located resistance cassettes and increases in MIC are not useful to measure.
However, using nose, and throat swabs, the presence of resistant streptococci will be
determined. This collateral damage of development of resistance as well as clinical outcome
will be associated with pharmacokinetics and-dynamics of CI and II of flucloxacillin.
Research
 To determine pharmacokinetic and- dynamic parameters and relate the outcome with bacterial cultures, susceptibility testing, and clinical outcome.  This is a clinically relevant multicenter study in which several different laboratory techniques will be learned. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
Title: Role of housekeeper genes in viral infections.

Workgroupleaders:
Dr. A. Verbon ([email protected]) and
Dr. J. Bierau ([email protected])
Background
Inosine triphosphatase is a housekeeping protein that catalyses the rapid conversion of the
non-canonical nucleotides ITP, dITP and XTP to the respective monophosphates and
inorganic pyrophosphate, thus maintaining low concentrations of these nucleotide
triphosphates. The protein is encoded by the gene ITPA, located on chromosome 20p13.
Deficiency of the enzyme leads to intracellular accumulation of ITP and dITP and was first
described in 1964. ITPase deficiency is considered to be benign. Several ITPA polymorphisms
are known, of which two (ITPA 94C>A and IVS 2+21 A>C) frequently occur in Asian and
Caucasian populations. In recent years the scientific discussion concerning the clinical
relevance of ITPase has focused on its putative role in mercaptopurine metabolism, but this is
not yet fully elucidated. To this day, the clinical relevance of ITPase deficiency and its genetic
polymorphisms remains an enigma. Recently, Fellay et al. have suggested a protective effect
of ITPA gene variants against ribavirin induced anemia in patients treated with ribavirin for
chronic hepatitis C. Although, these authors have not included measurement of inosine
triphosphatase (ITPase) in their study, the message is compelling. However, it is not clear
whether the assumption that ITPase genotype is directly correlated to phenotypic activity
during antiviral therapy is correct. In a preliminary study we showed that in HIV-seropositive
patients ITPA genotype was not correlated with phenotypic ITPase activity. The Hypothesis is
that extrapolation of genotype to phenotypic ITPase activity during viral infections and their
therapy is not as clear cut as in non-viral infected populations.
Research
 To establish the relation of genetic mutations of housekeeping genes, especially ITPA with HCV infection and adverse effects of antiviral therapy.  The study combines a clinical study with epidemiological data management and advanced laboratory techniques involving PCR, gene sequencing, measuring enzyme activity.  The clinical part of the study will take in Rotterdam, the laboratory part in Maastricht. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: HIV transmission networks in rural Zambia (CUTE).

Workgroupleaders:
Dr. J.L. Nouwen ([email protected])
Dr. D. van de Vijver ([email protected]) and
Prof. Dr. C.A.B. Boucher ([email protected])
Background
Currently an estimated 33 million people are living HIV/AIDS globally, 22 million living in Sub-
Sahara Africa. In 2007 2.7 million persons were newly infected with HIV (1.9 million in Sub-
Sahara) and 2.0 million died (1.5 million in Sub-Sahara). Sub-Sahara Africa harbours an even
more disproportionate number of children living with HIV/AIDS (1.8 million on a total of 2.0
million worldwide), newly infected children (330.000) and child deaths due to AIDS (240.000).
Although great efforts have been done to roll out antiretroviral treatment (ART) in Sub-Sahara
Africa and much has been achieved (more than 3.0 million HIV infected persons on ART, and
in some countries more than 50% of eligible HIV infected persons started on ART), still the
number of newly infected persons outnumbers the number of persons put on ART, leading to
an ongoing epidemic/pandemic.
To prevent the spread of HIV and reduce the number of newly infected persons, many
prevention interventions have been tried, e.g. education, advocation of condom use including
female condoms, microbicide trials, prevention of genital HSV infections and vaccine trials.
The sad conclusion so far is that all of these interventions have failed in significantly reducing
the spread of HIV. Only circumcision has been demonstrated to reduce HIV transmission and
spread, but the question is if this is an intervention which can be implemented on a huge scale
in already highly stressed health care systems. So now is the time to rethink the available
options to prevent the spread of HIV for the mid- and long-term future.
The roll-out of ART in Sub-Sahara Africa, although highly criticised at first, has clearly been
successful with many programs demonstrating efficacy comparable to those in Western
countries. Recent studies have demonstrated that earlier ART initiation is mandatory to reduce
mortality, and this does not only apply to Western countries. PMTCT programs have been
shown to be successful, although still not implemented at the scale needed. And also PrEP
(pre-exposure prophylaxis using tenofovir or tenofovir-emtricitabine) seems to be very
promising as a prevention intervention, and randomized controlled trials (RCT) are underway.
Furthermore, studies have clearly shown that HIV infected persons with a HIV viral load below
1000 copies per ml of blood are not ‘infectious' to their HIV-negative partner(s). All these data
together suggest that for the near future ART is the only, but promising option to prevent the
further spread of HIV.
Testing and treating as a prevention strategy
This project will investigate whether HIV-testing of targeted populations followed by treatment
can reduce new infections with HIV. Earlier identification of infected patients through HIV-
testing can reduce infections because of a change in risk behaviour. Treating infected
individuals is also beneficial as antiretroviral drugs can suppress the viral load, which is the
key factor driving transmission of HIV, to undetectable levels.
A recent model (Granich et al., Lancet 2009) already explored a strategy of annual universal
testing followed by immediate treatment regardless of CD4 count. The model found that the
pandemic can be reduced to less than 1 incident patient per 1000 people within a decade.
But this strategy does not seem feasible because:
1. Annual universal testing is a logistical challenge in sub-Saharan Africa. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
2. The window phase HIV tests will deny identification of many patients in the highly infectious acute stage of infection. 3. Start of ART regardless of CD4 count is not in line with treatment guidelines and cannot be achieved because of funding constraints. 4. The model assumed an unrealistically strong reduction in risk behavior after a positive 5. Retention of patients not yet on ART was not included but has reported to be poor. Any model is an approximation of reality. The predictions therefore strongly depend on the quality of data used for parameterization. We will address this issue by collecting realistic data in rural Africa. Using data from rural Africa we will: 1. Provide the urgently needed realistic estimate of the effectiveness of testing and treating as compared to the above mentioned model. 2. Evaluate a strategy of targeted testing of concurrent partnerships and targeted testing of communities with a high prevalence of HIV. 3. Study the impact of increased treating on the emergence of resistance and whether (more) transmission of drug resistant HIV will occur. 4. Study the impact of contact tracing?
Research
This project will determine the impact of targeted testing and treating strategies on the
incidence HIV infections in rural Africa. The objectives will be studied using mathematical
modelling which have contributed to the understanding of the spread of infectious disease and
the measures needed to contain or mitigate them. Data will be collected in rural Zambia to
ensure that the models provide realistic estimates. At the end of this project we will have
answered the following research questions:
1. Can a strategy of testing and treating of targeted individuals reduce new infections with HIV in rural Africa? 2. Can the HIV epidemic be reduced using realistic estimates regarding test rates, patient-retention, and availability of ART, treatment guidelines and resistance? 3. Can the effectiveness of HIV testing on prevention of new infections be improved by targeted testing of concurrent partnerships and communities with a high HIV prevalence? 4. Is an earlier start of treatment (CD4<500 as compared to a start at CD4<350) associated with a reduction in new infections with HIV? 5. What is the impact of tracing and testing of recent partners of patients newly diagnosed with HIV on the epidemic? Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Antibiotic resistance in rural Zambia (RESIST).

Workgroupleaders:
Dr. J.L. Nouwen ([email protected])
Background
Antibiotic resistance is on the rise globally, mainly driven by the selective pressure imposed by
(inappropriate) antibiotic use. While antibiotics are among the most commonly prescribed
drugs in hospitals, infections with resistant microorganisms increase the cost of health care,
length of hospital stay and mortality. Even in a country like the Netherlands, with its low
antibiotic use and resistance rates, antibiotic use is often not appropriate. The major cause of
inappropriate antibiotic use being lack of knowledge of infectious diseases and antimicrobial
therapy; antibiotics as the ‘drugs of fear'.
Resistance to antimicrobial drugs has been clearly linked to consumption of antibiotics. The
boundaries between community and hospital environments are becoming more blurred; as
patients might leave the hospital with resistant micro-organisms and can potentially further
spread these in the community. This may have consequences for the development of
resistance to antimicrobial drugs. Strategies to limit the spread of resistant strains should
include the prudent use of antimicrobial agents. Antibiotic treatment guidelines should be
based on results derived from well-designed surveillance studies.
In developing countries like Zambia, not only HIV, TB and Malaria but also bacterial gastro-
intestinal and respiratory infections are a major cause of morbidity and mortality, especially in
children. Diagnostic and treatment options are often limited and specifically targeted at the big
three (HIV-TB-Malaria). Increasing antibiotic resistance in bacterial pathogens will further
hamper adequate and appropriate antibacterial treatment.
Currently, local and national data on bacterial pathogens involved in bacterial infections and
their antibiotic resistance patterns are largely lacking in resource-poor settings. National and
international (WHO) guidelines depend on scarce data and on adaptation of guidelines from
developed countries to the resource-poor setting and therefore are not evidence-based. As
recent studies in Zambia have demonstrated, the spectrum of bacterial pathogens involved in
for instance pneumonia is often very different from what is seen in Western Europe and the
USA. Also, antibiotic resistance in these bacterial pathogens was demonstrated to be quite
significant, leading to inappropriate antibiotic treatment in as high as 30-50% of patients.
Research
 To study the prevalence of bacterial pathogens and their antibiotic resistance patterns involved in specific infections such as respiratory tract infections, urinary tract infections, meningitis, abscesses and wound infections in rural Zambia.  To study the prevalence of carriage of specific bacterial pathogens and their antibiotic resistance patterns in healthy persons in rural Zambia.  To study the impact of providing local health care providers with real-time adequate information on causative bacteria and on patient morbidity and mortality, and hospital costs.  To develop evidence-based local guidelines for antibiotic treatment.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
VIROLOGY – LABROTATIONS & RESEARCH TOPICS
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title Research line:
The genetic and antigenic evolution of influenza A (H3N2) virus neuraminidase
Workgroup leader: Dr. K. Westgeest
[email protected]
Description
Each winter, the highly contagious respiratory influenza virus infects approximately 5-15% of
the world population, resulting in an estimated three to five million hospitalization cases and
between 250,000 and 500,000 deaths every year. To enhance accuracy of the vaccine strain
selection, thorough analysis of the genetic and antigenic differences within virus strains is
essential. In 2004, Smith et al. presented ‘antigenic cartography', a technique to study and
monitor viral evolution. By using hemagglutination inhibition (HI) assay data, the antigenic
evolution of hemagglutinin of influenza A (H3N2) virus was mapped from its introduction in
humans in 1968 up to 2003. However, mutations in the hemagglutinin glycoprotein are not
solely responsible for antigenic drift and antigenic data covering neuraminidase must therefore
be considered. This study includes the investigation of the evolution of influenza A (H3N2)
virus neuraminidase on genetic (through sequence and phylogenetic analysis), and antigenic
level (by using neuraminidase inhibition assays).
A master student that will do a 3-week lab rotation in my group
Can choose from the following research topics
- The genetic and antigenic evolution of influenza A (H3N2) virus neuraminidase
will learn the following techniques:
- Cell culturing
- Virus culturing
- RNA isolation
- Virus neutralization assays
- Sequencing
- Building phylogenetic trees
- Setting up and optimizing neuraminidase (inhibition) assays
and will participate in the following seminars/research/literature meetings/discussions
- Monday/Wednesday research meeting (whole department)
- Thursday lab meeting (Flu/hMPV group)
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title Research line: Pathogenesis of measles and RSV
Workgroup leader: Dr. Rik L. de Swart
Email address: [email protected]
A master student that will do a 3-week lab rotation in my group will work on:
Infection of different cells or cell lines with measles virus and/or respiratory syncytial virus, and detection of virus-specific antibodies. We are studying the enhancing or inhibiting effect of different lipopeptides on infection with these paramyxoviruses. The effect is both dependent on the structure of the lipopeptide and on properties of the host cell. The student will participate in the experiments that are ongoing at that moment. will learn the following techniques: Cell culture, virus infection and –titration, flow cytometry, ELISA. and will participate in the following seminars/research/literature meetings/discussions Department of Virology seminars (twice a week) Workgroup meetings (both daily informal meetings and monthly formal meeting) Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Interaction between respiratory viruses and the innate immune system.
Workgroup leader: Bernadette van den Hoogen
[email protected]
Description
Rapid induction of type I interferon expression is a central event in the establishment of the
innate immune response against viral infection, and requires the activation of multiple
transcriptional proteins following engagement and signalling through Toll-like receptor-
dependent and –independent pathways. Many viruses therefore encode factors that subvert
the IFN system to enhance their virulence. Within our group, we study how respiratory viruses,
especially the human metapneumovirus (hMPV), subvert the innate immune system. In this
study we use recombinant viruses with deletions for particular genes, genomic tools,
molecular tools, microscopic tools and bioassays to study the interaction between the virus
and the innate immune system. Gaining more knowledge on both the innate immune system
itself and how the virus deals with this will result in a more rational design for vaccine and
therapeutic development.
A master student that will do a 3-week lab rotation in my group
Can choose from the following research topics
1). Detection of interferon production by respiratory viruses.
Does hMPV block the IFN production induced by Sendai virus virus? Data so far
demonstrate that hMPV does not induce the production of type I interferon. If hMPV is actively
blocking a step in this production pathway, the virus should be able to block induction induced
by a control virus.
2) Does hMPV induce activation of the transcription factor IRF3?
IRF3 nuclear translocation is necessary for the production of type I interferon. IRF3
translocates to the nucleus upon activation and dimerization. HMPV does not induce nuclear
translocation. This study will focus on the influence of hMPV on the activation of IRF3 (is IRF3
phosphorylated, does it form dimmers etc). This will be studied with aid of PCR assays and
western blot techniques.
3) Is hMPV able to block the interferon induced Jak/Stat signalling pathway?
If cells are treated with exogenous type I interferon, antiviral proteins can be detected in the
supernatant of the treated cells. In case hMPV is able to block this pathway, an infection
preceding the interferon treatment should result in a decrease in the production of antiviral
proteins. This can be detected by bioassays, western blots and PCR assays.
Will learn the following techniques:
- cell culture, virus culture
- molecular techniques: western blotting, PCR
- bioassays : transfection procedures, fluorescence microscopy, FACS, IFA
and will participate in the following seminars/research/literature meetings/discussions
- weekly labmeetings with whole department of Virology
- weekly labmeeting with workgroup hMPV/Influenza with Prof. R. Fouchier
- one on one interviews/discussion with group leaders within the department of Virology, to
learn about other projects within the department
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

A master student that will do a 6 month Master of Science project in my group
Can choose from the following research topics
1.) Construction of recombinant RSV and hMPV viruses and deletion mutants thereof
and study their behaviour in innate immunity with classical tools and genomic tools.
Both RSV and hMPV do not induce the production of interferon. RSV uses the NS1 and NS2
proteins for this purpose and hMPV does not encode for these proteins. We want to use
recombinant RSV lacking the NS1 and NS2 genes to study whether hMPV proteins can
compensate for the lack of NS1 and NS2. The construction of recombinant RSV lacking these
genes is ongoing, in addition to recombinant RSV viruses in which NS1 and NS2 genes are
replaced with hMPV genes. After this reverse genetics part of the project, the viruses will be
tested for interferon production capacity, but also their influence on the innate immune system
will be studied with aid of genomic tools (in collaboration with Dr. A. Andeweg). This project
should lead to identifying the interferon-antagonistic protein encoded by hMPV.
2.) Blocking of IFN signalling by hMPV. Using recombinant viruses (and deletion
mutants), expression plasmids (transfection procedures) and bioassays.

If cells are treated with exogenous type I interferon, antiviral proteins can be detected in the
supernatant of the treated cells. In case hMPV is able to block this pathway, an infection
preceding the interferon treatment should result in a decrease in the production of antiviral
proteins. This can be detected by bioassays, western blots and PCR assays.
Most paramyxoviruses block the Jak/Stat signalling pathway. HMPV, a paramyxovirus, does
not encode the genes used by other paramyxoviruses for this purpose. Preliminary data
indicate that hMPV does block the Jak/Stat signalling pathway. Additional studies have to
show what components of this pathway are targeted by hMPV, and which hMPV protein is
responsible.
3.) Detection of (the lack of) activation of transcription factors using recombinant
viruses, expression plasmids, PCR assays, confocal microscopy and bioassays.
HMPV and RSV do not induce the production of type I interferon. In addition, both viruses do
not induce IRF3 translocation to the nucleus. For nuclear translocation, IRF3 needs to be
activated. The focus of this study will be on activation of IRF3: does the virus inhibit
phosporylation of IRF3? Inhibit formation of heterodimers? Inhibit binding to other cofactors?
Or does the virus inhibit processes upstream of IRF3 activation?
will learn the following techniques:
- cell culture, virus culture
- molecular techniques: cloning, sequencing, western blotting, PCR
- bioassays : transfection, fluorescence microscopy, confocal microscopy, FACS, IFA
and will participate in the following seminars/research/literature meetings/discussions
- weekly labmeetings with whole department of Virology
- weekly labmeeting with workgroup hMPV/Influenza with Prof. R. Fouchier
- one on one interviews/discussion with group leaders within the department of Virology, to
learn about other projects within the department
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

A master student that will do a 12 or 18 month Master of Science project in my group
Can choose from the following research topics
1.) Construction of recombinant RSV and hMPV viruses and deletion mutants thereof
and study their behaviour in innate immunity with classical tools and genomic tools.
Both RSV and hMPV do not induce the production of interferon. RSV uses the NS1 and NS2
proteins for this purpose and hMPV does not encode for these proteins. We want to use
recombinant RSV lacking the NS1 and NS2 genes to study whether hMPV proteins can
compensate for the lack of NS1 and NS2. The construction of recombinant RSV lacking these
genes is ongoing, in addition to recombinant RSV viruses in which NS1 and NS2 genes are
replaced with hMPV genes. After this reverse genetics part of the project, the viruses will be
tested for interferon production capacity, but also their influence on the innate immune system
will be studied with aid of genomic tools (in collaboration with Dr. A. Andeweg). This project
should lead to identifying the interferon-antagonistic protein encoded by hMPV.
This project should lead to identifying the interferon-antagonistic protein encoded by hMPV.
2.) Blocking of IFN signalling by hMPV, RSV and PIV. Using recombinant viruses (and
deletion mutants), expression plasmids (transfection procedures) and bioassays.
Most paramyxoviruses block the Jak/Stat signalling pathway. HMPV, a paramyxovirus, does
not encode the genes used by other paramyxoviruses for this purpose. Preliminary data
indicate that hMPV does block the Jak/Stat signalling pathway. Additional studies have to
show what component of this pathway is targeted by hMPV, and which protein is responsible.
3.) Detection of (the lack of) activation of transcription factors using recombinant
viruses, expression plasmids, PCR assays, confocal microscopy and bioassays.
HMPV and RSV do not induce the production of type I interferon. In addition, both viruses do
not induce IRF3 translocation to the nucleus. For nuclear translocation, IRF3 needs to be
activated (eg. Phosporylated). In this project the interaction between hMPV and kinases
(responsible for activation of transcription factors) such as TBK, IKK etc, will be studied. This
project occurs in close collaborations with researchgroups in New York and Montreal.
4). How does the innate immune system recognize hMPV? Focussing on (a lack of)
sensing of viral RNA by RIG-I, TLRs and MDA-5.

HMPV and RSV do not induce the production of type I interferon. One possibility for these phenomena might be that the innate immune system does not sense the presence of hMPV. RIG-I and MDA-5, cytosolic RNA sensors, are main targets for other respiratory viruses, while other viruses have capped their RNA in order not to be sensed by RIG-I. The interaction between hMPV and the sensors of innate immune system will be investigated in close collaboration with research groups in New York and Montreal. will learn the following techniques:
- cell culture, virus culture, genomic tools
- molecular techniques: cloning, sequencing, western blotting, PCR, reverse genetics
- bioassays : transfection, fluorescence microscopy, confocal microscopy, FACS, IFA
will participate in the following seminars/research/literature meetings/discussions:
- weekly labmeetings with whole department of Virology
- weekly labmeeting with workgroup hMPV/Influenza with Prof. R. Fouchier
AND: possibility to spend a longer period with the research group of A.Garcia-Sastre,
Mount Sinai, New York, USA or Prof. J. Hiscott, McGill University, Montreal, Canada as
part of the ongoing collaboration with these groups.

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Pathobiology of Varicella Zoster Virus

Workgroup leader:
Georges M.G.M. Verjans PhD ([email protected])

Outline research:
Varicella Zoster Virus (VZV) is an endemic human neurotropic alpha-herpesvirus. VZV causes
varicella (chickenpox; "waterpokken") as a primary infection, establishes latency in the
sensory ganglia of the host and may reactivate later in life to cause herpes zoster (shingles;
"gordelroos") [1]. Both varicella and herpes zoster are generally considered as benign
diseases. However, neonates, elderly, and particularly immunecompromised individuals are at
risk of severe morbidity and occasionally die from disseminated infection [2,3]. The highly
restricted host range and cell-associated nature of the virus have seriously hampered studies
on the pathogenesis and molecular biology of VZV. Currently, the virus is propagated in cell
culture by co-cultivation of VZV-infected cells with uninfected cells. However, this results in
non-synchronized infections and does not mimic VZV pathogenesis in vivo. Recently, it was
shown that high titres of cell-free and cryostable VZV can be obtained using clinical virus
isolates cultured on human retinal pigmented epithelial cells (RPE) [4]. Our group has
confirmed the applicability of this strategy to generate high-titre cell-free VZV stocks. The VZV
stocks generated provide the unique opportunity to synchronize VZV infection in vitro. This is
a prerequisite to study - in detail - the molecular biology of VZV cell entry, replication and
spread, as well as the virus-mediated immune evasion strategies to prevent immune-mediated
control of VZV.
1. Three-week lab rotation:
 A master student interested in a 3-week lab rotation in the group will participate in the
research program of the graduate student Werner Ouwendijk. He/she will be involved
(hands-on) in pilot experiments to determine the cell tropism of VZV to human peripheral
blood mononuclear cell (PBMC) subsets in vitro.
 The master student will learn the following techniques: PBMC isolation from heparinized blood samples (Ficoll density gradient centrifugation), cell culture and virus culture.  The master student will participate in the department Virology research meetings every Monday and Wednesday (12:00-12:45 hr) as well as the 2-weekly meetings of the herpesvirus group.
2. Six-month Master of Science project:
 The master student interested in a 6-month Master of Science project will participate in the
research program of the graduate student Werner Ouwendijk and can choose from the two
following research topics: (1) to determine the cell tropism of VZV to human PBMC
subsets in vitro, or (2) to determine the effect of cellular polarization on VZV entry,
replication and spread. Both projects are more in-depth studies compared to the 3-week
lab rotation and will be performed together with the graduate student.
 The master student will learn the following techniques: human PBMC isolation, cell culture, virus culture, isolation of cell-free VZV and virus titration, flow cytometry, immunocytochemistry and culture of polarized epithelial cells.  The master student will participate in the department Virology research meetings every Monday and Wednesday (12:00-12:45 hr) as well as the 2-weekly meetings of the herpesvirus group. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

3. Twelve-month Master of Science project:
 The master student interested in a 12-month Master of Science project will be embedded
in the research program of the graduate student Werner Ouwendijk and can choose from
the two following research topics: (1) to determine the cell tropism of VZV to human
PBMC subsets in vitro, or (2) to determine the effect of cellular polarization on VZV
entry, replication and spread. Both projects are more in-depth studies compared to the 6-
month Master of Science project. During the first months, the master student will be
introduced into the theory and practical work by the graduate student. Subsequently,
he/she will perform the project as independently as possible and supervised by the
graduate student. The master student will learn to be an independent researcher: literature
survey, generation ideas, experimental design, interpretation and presentation of
experimental data.
 The master student will learn the following techniques: human PBMC isolation, cell culture, virus culture, isolation of cell-free VZV and virus titration, flow cytometry, immunocytochemistry and culture of polarized epithelial cells.  The master student will participate in the department Virology research meetings every Monday and Wednesday (12:00-12:45 hr) as well as the 2-weekly meetings of the herpesvirus group.
3. Eighteen-month Master of Science project:
 The master student interested in an 18-month Master of Science project will be embedded
in the research program of the graduate student Werner Ouwendijk and will perform
his/her own research project. The aim of this project is determine the cell tropism and
modulatory effect of VZV
on the functional properties of human mononuclear cells. The
project involves three phases. First, determine the cell tropism of VZV to human
mononuclear cells including B and T lymphocytes as well as the professional antigen
presenting cells like macrophages and dendritic cells. Second, determine the involvement
of the immunological synapse in the transfer of VZV from infected human
macrophages/dendritic cells, epithelial cells and B lymphoblastoid cell lines to VZV-
specific T lymphocytes. Third, determine the ability and subsequently the mechanisms
employed by VZV to downregulate immunologic relevant molecules, like major
histocompatibility and co-stimulatory molecules, on the respective VZV-susceptible human
mononuclear cell types. Likewise the 12-month Master of Science project, the master
student will learn and stongly encouraged becoming an independent researcher: literature
survey, generation ideas, experimental design, interpretation and presentation of
experimental data.
 The master student will learn the following techniques: human PBMC isolation, cell culture, virus culture, isolation of cell-free VZV and virus titration, flow cytometry, immunocytochemistry and culture of polarized epithelial cells.  The master student will participate in the department Virology research meetings every Monday and Wednesday (12:00-12:45 hr) as well as the 2-weekly meetings of the herpesvirus group.
References
1. Verjans GM, Hintzen RQ, van Dun JM, et al. Selective retention of herpes simplex virus-
specific T cells in latently infected human trigeminal ganglia. Proc Natl Acad Sci U S A 2007;104:3496-501. 2. Gilden DH, Kleinschmidt-DeMaster BK, LaGuardia JJ, Mahalingam R, Cohrs, RJ. Neurologic complications of the reactivation of varicella-zoster virus. N Engl J Med 2000;342:635-45. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Characterization of the immune response to Chikungunya virus infection in
C57BL/6 mice

Background
Chikungunya virus (CHIKV) belongs to the Family Togaviridae, genus Alphavirus. The virus is
endemic in Africa and Asia, where it is transmitted to humans by the bite of an infected Aedes
aegypti
mosquito. CHIKV was first isolated during an epidemic of dengue-like disease in
Tanzania in 1952. In 2006 CHIKV emerged for the first time in the Seychelles, Mauritius, and
Reunion islands, resulting in ten of thousands of infections. The virus has a broader tropism,
able to infect both Aedes aegypti to Aedes albopictus. Clinical evidence also suggests that the
new variant may be more virulent in humans. Infection with CHIKV results in fever, malaise,
headache and a debilitating and prolonged arthralgic syndrome that primarily affects the
peripheral small joints. The name Chikungunya was used by the indigenous people of the
region to describe the characteristic posture assumed by patients afflicted with the severe
muscle and joint pains associated with CHIKV infection. There is no specific vaccine or
antiviral treatment for Chikungunya. In our department we are working on two vaccine
strategies: the vectored and subunit. Modified Vaccinia Ankara (MVA) virus is used to clone
the glycoproteins of CHIKV and the baculovirus is used to produce the recombinant forms of
the glycoproteins. After selection of a stable MVA clone, and purification of large quantities of
the recombinant proteins, the different vaccine candidates will be tested in several animal
models, among them the C57BL/6 mouse model. In order to understand correlates of
protection and immunopathogenesis, both the antibody and T cell responses to CHIKV must
be characterized.
Aim
To characterize the antibody and T cell responses to CHIKV on several time points after
CHIKV infection and vaccination candidate vaccines in C57BL/6 mice
Techniques
ELISA and ELISPOT - Flowcytometry - Cell - Bioinformatics
Experimental animal work and training for art12

Suitability for type of project
12 or 18 months
Contact details
Dr. Byron E.E Martina
Erasmus MC Rotterdam
Department of Virology, Room Ee 1714a
s'Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands
Phone: +31 10 7044279
Fax: +31 10 7044760
e-mail: [email protected]
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

TITLE: Characterization of antibody responses to epidemic norovirus variants
Background
In the Netherlands, the number of consultations for acute gastro-enteritis (AGE) almost
doubled between 1996 and 2006, the number of hospitalizations increased by over 50% and
mortality by 20% (van Pelt et al., 2006). This increase coincides with emergence of new
norovirus variants belonging to genotypes GII.4 and GIIb/GII.3. Both lineages can be
considered novel strains resulting from evolutionary changes that influence both antigenicity
and host receptor binding (Siebenga et al., 2007; Lindesmith et al., 2008; Bok et al., 2009). In
2002, a particularly successful strain of GII.4 emerged. Advanced phylodynamic analyses
have confirmed that since 2002 the virus population has diverged from the stable equilibrium
that one would expect to see for an endemic disease, and that the number of effective
infections has increased (Siebenga et al., in press). Recent variants have caused mortality in
elderly, and an unexpectedly high proportion of cases infected with novel norovirus strains
developed chronic illness and shedding (Beersma et al., 2009). Evolution of strains within
such chronic shedders may result in novel epidemic variants (Siebenga et al., 2008).
The size of the population at risk for prolonged and severe norovirus infection will grow
significantly over the coming years. Therefore, without effective counter measures the impact
of noroviruses is likely to increase over time. As a consequence, candidate vaccines, based
on virus-like particles, are being developed and have entered phase III trials (Glass et al., ).
However, immunity to noroviruses is poorly understood, and the rapid changes of the viruses
observed bear resemblance to influenza immunity (Bush et al., 1999). Mutations in the viral
capsid gene that affect binding to the host receptor and antigenicity of the viruses have been
identified, and suggest that the P2 domain is an important antigen for studies of specific
antibody responses (Cao et al., 2007; Allen et al.2008; Siebenga et al.2008 and in press;
Canon et al., 2009). We will develop microarray based serological assays to be able to
measure incidence of infection with new variants at the population level in different age
groups. Changes in this incidence will be studied by comparative testing of serum samples of
randomly selected persons from 2 population serosurveys available at RIVM (1996 and 2006)
and from a historic serumbank available in Rotterdam (1970-1980).
Aim and specific objectives
 To develop assays for specific measurement of immune responses to the global norovirus variants  To do comparative evaluation of seroprevalence of new norovirus variants before and after a " pandemic" wave (defined as a global epidemic wave)
Techniques
Protein expression, micro-array production, validation, serology, epidemiology, PCR and
sequencing, bio-informatics
Type of project
The minimum duration for this project is 6 months, which will probably only provide a first
generation microarray. A minimum of 12 months is preferable.
Contact details: Prof dr. Marion Koopmans Tel 31.6.5209.8601
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

NOTE: THIS WORK WILL BE DONE MOSTLY AT THE NATIONAL INSTITUE OF PUBLIC
HEALTH IN BILTHOVEN www.rivm.nl

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

PULMONARY MEDICINE – LABROTATIONS & RESEARCH TOPICS

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Molecular Immunology and immunopathology of the lung
Department of Pulmonary Medicine
Further information
Dr. Rudi Hendriks, Head of the Research Laboratory, Department of Pulmonary Medicine
Tel. 010-7043700, and e-mail: [email protected]
Background
The adaptive immune response provides humans with B and T lymphocytes that carry
immunoreceptors that are highly specific for antigens that these cells have not encountered
before. Dendritic cells (DCs) are professional antigen-presenting cells (APC) that are located
at sites where maximal microbial encounter occurs. They are essential for the transport of
antigens (from the airway mucosa and interstitium to the draining lymph nodes) and thereby
initiate the activation of lymphocytes. In this way, the adaptive immune system has the
capacity to strengthen and to regulate the innate defense mechanisms and to build specific
immunological memory so that subsequent challenges with the same pathogen are efficiently
overcome. The lung is continuously exposed to the outside world and is a portal of entry for
viral, bacterial, and fungal infection. Throughout evolution, an extensive defense mechanism
has been developed that protects humans from these potentially lethal assaults and at the
same prevents reactivity to harmless antigens. Nevertheless, the incidence of lung diseases
such as asthma and sarcoidosis has risen dramatically over the last 50 years and pneumonia
is a leading cause of death among young children and the elderly worldwide.
Research
The research aims in the Department of Pulmonary Medicine comprise:
 To elucidate the role of DCs in directing and maintaining an acute or chronic localized immune response in the lung, e.g. in influenza virus infection, in asthma or in sarcoidosis.  To develop DC-based immunotherapy with tumor-pulsed autologous DCs to prevent mesothelioma recurrences. This approach is based on the observation that DCs are able to induce protective immunity in a mouse model of mesothelioma. In this context, we also aim to investigate how the effects of negative regulators of these responses, including myeloid-derived suppressor cells and regulatory T cells can be dampened.  To study the role of effector T cells in various pathological conditions, including asthma, sarcoidosis, chronic obstructive pulmonary disease or community acquired pneumonia. In particular, we focus on regulatory T cells and Th17 cells, a quite recently identified population of effector T cells that produce pro-inflammatory cytokines and is essential for mucosal immunity.  To identify the molecular mechanisms involved in B cell repertoire selection and B cell activation, both in autoimmunity and in infection, e.g. with influenza virus. Molecular approaches become increasingly important to unravel the pathogenesis of pulmonary diseases. Over the last years technical expertise has been acquired in mouse models for various diseases, including asthma, influenza and lung berylliosis. Numerous animal models have been generated with specific immune defects (in DC, or activation or development of B or T lymphocytes). In addition, several projects in the lab include human studies, using patient material. We make use of various assays for humoral and cellular immunity, multi-color flow cytometry and cell sorting, immunohistochemistry, molecular biology, genome-wide expression analysis, chromatin immunoprecipitation, protein biochemistry and retroviral transduction. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Collaborations
We have fruitful collaborations with various research groups at the Erasmus MC (Dept. Cell
Biology and Genetics, Virology, Immunology, Pediatrics) and outside the Erasmus MC
(University of Ghent, Belgium, University of Freiburg, Germany, Max-Planck Institute for
Infection Biology, Berlin, Institute for Molecular Pathology, Vienna).
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Subject for lab rotation and project:
The role of T helper 17 cells in the pathogenesis of COPD, community-acquired
pneumonia and cystic fibrosis
Department of Pulmonary Medicine

Contact information
Marthe Paats
Department of Pulmonary Medicine
Room Ee2263a
Tel. 010-7038016
e-mail: [email protected]
Workgroup leader
Dr. R.W. Hendriks (Head of research department)
Department of Pulmonary Medicine
Room Ee2251a
Telephone 010-7043700
e-mail: [email protected]

Background
Th17 cells are a newly identified population of effector T cells that produce pro-inflammatory
cytokines, in particular IL-17. They have been implicated in various autoimmune diseases and
control a wide range of infections at mucosal surfaces. In this research line, we focus on
various pulmonary diseases, including Chronic obstructive pulmonary disease (COPD),
Community-acquired pneumonia and cystic fibrosis (CF).
COPD is characterized by progressive development of airflow limitation and is attributed to long-term exposure to tobacco smoke. The cytokines IL-6 and IL-17 are central to mucus
production and goblet cell hyperplasia. Exacerbations of COPD are associated with increased
numbers of neutrophils in the airways, likely recruited by IL-6 and IL-17-dependent
mechanisms. COPD also shares similarities with autoimmune diseases, e.g. rheumatoid
arthritis, with a possible central role for Th17 cells to newly created or altered epitopes
induced by tobacco smoke.
Community-acquired pneumonia remains a common and serious illness, despite
antimicrobial therapy. Major gaps remain in our understanding of the pathogenesis of this
infectious disease. Emerging data have suggested that Th17 cells play an essential role in the
host defense against extracellular bacteria by induction of neutrophil-mediated protective
immune response and through regulation of cell-mediated immunity.
The pathophysiology of cystic fibrosis (CF) is characterized by chronic airway infection and
inflammation starting early in life. The mechanisms that lead to the development of a
sustained and predominantly neutrophilic inflammatory response remain unclear, but recent
data have indicated a possible role for IL-17 producing cells.
Research
In this research line we aim to define the role of Th17 cells in COPD, pneumonia and CF
pathogenesis. To this end we collect blood and broncho-alveolair lavage (BAL) fluid from
COPD, pneumonia and CF patients. We aim to:
a) Quantify the presence of lymphocyte and myeloid subpopulations in both BAL fluid and peripheral blood, using multi-color flow cytometry. b) Analyze cytokine production by both activated and memory T cells present in the lungs and peripheral blood of patients, using intracellular flow cytometry and ELISA. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
c) Analyze whether memory (antigen experienced) T cells of patients are more likely to develop a Th17 phenotype, as compared with healthy subjects. In order to assess this, we will purify memory T cell fractions from peripheral blood samples by cell sorting. Using these cell fractions, we will perform in vitro polarization cultures of anti-CD3/CD28-activated T cells under neutral and "memory" Th17 conditions (+IL-6, IL-1β, IL-23). Cytokine production will be evaluated by intracellular flow cytometry, ELISA and quantitative RT-PCR. We will also quantify the expression of subset-specific transcription factors. d) In parallel, we will sort naïve T cells from peripheral blood and perform in vitro polarization cultures of anti-CD3/CD28 activated T cells under Th0 and "naïve" Th17 conditions (+IL-21 and TGFβ). These analyses should show whether naïve T cells from patients have an increased propensity to differentiate into Th17 cells compared to naïve T cells from healthy subjects. e) In addition, lung biopsy material from various patients will be analyzed for the expression of various markers, including cytokines and their receptors, by immunohistochemistry.
Three-week lab rotation
A master student interested in a three-week lab rotation will be involved in the experiments
described above. In this rotation the student will be trained into peripheral blood mononuclear
cell isolation from heparinized blood samples using Ficoll density gradient centrifugation and T
cell culture, multi-color flow cytometric analysis (including working with FACS analysis
software programs) and ELISA for various cytokines.
In addition, students will obtain an overview of all current research projects at the
Department of Pulmonary Medicine. You will attend weekly work meetings and journal clubs.
You will have meetings with all researchers, who will give overviews of their scientific
activities. You will also be introduced into most of the ongoing molecular, cellular and
immunological research techniques in the lab.
Master of science project (6/12/18 months)
The master student will participate in the project described above in which the main focus will
be on the mechanism of the inflammatory response and the role of Th17 differentiation in the
pathogenesis of community-acquired pneumonia. This project involves collaboration with the
Dept. Microbiology.
• The following questions will be addressed using techniques described above:
(1) Is there a strong inflammatory response in the early course of pneumonia? (2) Is there a difference in inflammatory responses between different pathogens involved (3) What is the role of Th17 differentiation in the early course of pneumonia? Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Subject for lab rotation and project:
Dendritic cell-based personalized cancer immunotherapy
Department of Pulmonary Medicine
Contact information
Dr. J.P.J.J. Hegmans (UD)
Room Ee2253a
Tel: 010-7043697
e-mail: [email protected]

Workgroup leader
Dr. R.W. Hendriks (Head of research department)
Room Ee2251a
Telephone 010-7043700
e-mail: [email protected]
Background
Immunotherapy is a very promising approach in the treatment of cancer. It tries to harness the potency and specificity of the immune system to attack cancer cells, aiming for a non-toxic treatment with minor side-effects and a long-lasting immunological memory. One approach of immunotherapy uses dendritic cells (DC) to present tumour-associated antigens (TAA) and thereby generate tumour-specific immunity. DC are extremely potent antigen-presenting cells specialized for inducing activation and proliferation of CD8+ cytotoxic T lymphocytes (CTL) and helper CD4+ lymphocytes. We study malignant pleural mesothelioma (MM) as a model. This fatal disease has a median survival of < 1 year. The incidence of mesothelioma is closely associated to the exposure to airborne asbestos fibers. Although in most developed countries the production and usage of asbestos is prohibited, due to the long interval between exposure to asbestos and the onset of disease, incidences of mesothelioma is still increasing. For the Netherlands and most other European countries, the peak is predicted in the period 2010-2020. Earlier we investigated the effect of DC-based immunotherapy on the outgrowth of mesothelioma in a murine model. It was established that DC-based immunotherapy induced strong tumour-specific CTL responses leading to prolonged survival. However, the effect of immunotherapy was dependent of the tumour load. The most beneficial effects were established at early stages of tumour evolution. This is in agreement with our current knowledge of the effect of immunotherapy in other tumour types. On the basis of these preclinical animal studies, we completed a clinical trial of the intradermal and intravenous administration of autologous tumour lysate-pulsed DC in mesothelioma patients after cytoreductive therapy with chemotherapy. Patients received 50x106 DC pulsed with tumour lysate and KLH every two weeks for a total of three injections. It was shown that DC vaccination in patients was safe with moderate fever as the only side effect. There were no grade 3 or 4 toxicities associated with the vaccines or any evidence of autoimmunity. Local accumulations of infiltrating T cells were found at the site of vaccination. Importantly, distinct immunological responses on the surrogate marker KLH were induced by the vaccinations, both in vitro as in vivo as well as anti-tumour activity against autologous tumour cells in the blood of some patients after the vaccinations. Currently we started a follow-up study in patients using DC-immunotherapy in combination with regulatory T cell depletion (CCMO NL24050.000.08). Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Research
Tumour cells create an immunosuppressive environment that can lead to a down-regulation of
the anti-tumour immunity. Our research lines focus on different types of suppressive cells
present in the tumour microenvironment that seems instrumental in allowing a growing tumour
to evade immunological attack. Important cells in these are the regulatory T cells, tumour-
associated macrophages and myeloid derived suppressor cells. Measures that can overcome
the suppressive function of these cells might be used in combination with DC immunotherapy
to increase the success rate of tumour eradication. For this we use state of the art techniques
(e.g. multi-colour flow cytometry), both in-vitro (cell culture) and in-vivo (mice and human)
studies.
Three-week lab rotation
During a laboratory rotation of 3 weeks, you will be able to familiarize yourself with the
research project described above and you will be trained into various techniques.
In addition, you will obtain an overview of all current research projects at the Department of Pulmonary Medicine. You will attend weekly work meetings and journal clubs. You will have
meetings with all researchers, who will give overviews of their scientific activities. You will also
be introduced into most of the ongoing molecular, cellular and immunological research
techniques in the lab.
Master of science project (6/12/18 months)
Students interested in direct translational research who would like to support us in gaining
more insight into the different aspects of cancer (in particular the immunosuppression and
immunotherapy treatment) are kindly invited to participate. Master students interested in a 12-
month and 18-month Master of Science project can take an animal course (Art. 9) and also
participate in the production of clinical-grade dendritic cell production in a cleanroom
environment for vaccination of mesothelioma patients. Different aspects and procedures for
patient tailored therapy with autologous cell products as Good Clinical Practice, CCMO/METC,
Investigator's Brochure (IB), Investigational Medical Product Dossier (IMPD) will be explained.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Subject for lab rotation and project:
Chronic Beryllium Disease as a mouse model for Sarcoidosis: the role of inflammatory
cells
Department of Pulmonary Medicine
Contact information
Dr. Alex KleinJan (UD)
Room Ee2257a
Tel: 010-7043701
e-mail: [email protected]

Workgroup leader
Dr. R.W. Hendriks (Head of research department)
Room Ee2251a
Telephone 010-7043700
e-mail: [email protected]
Background
Sarcoidosis is a granulomatous disesease manifesting predominantly in the lower airways and lesser in skin, eye and brains. The cause of sarcoidosis is still unknown and the diagnosis is mainly by exclusion of other diseases and by the presence of granuloma in affected tissue. Analysis of bronchial alveolar lavage (BAL) cells showed a CD4 T-cell lymphocytosis and a CD4:CD8 ratio higher than 3. Sarcoidosis in general is seen as a Th1 disease. In human sarcoidosis we recently investigated the role of Th17 cells and found evidence for an important role of these cells in granuloma formation. There is also a role for regulatory T cells, although the precise function in sarcoidosis is not clear. Sarcoidosis has similarities with Chronic Beryllium Disease (CBD) which is an occupational disease that may develop in people who are exposed to Beryllium (Be). Avoiding the exposition to Be (fumes and dust) abrogates the disease. Be is a metal which is widely used in electronic and plane industry. Research
Currently, we are establishing a mouse model for CBD. Animals were exposed against Be or BeO by intratracheal injections during three weeks. Six - eight weeks after the first exposure mice developed CBD. This was confirmed by the identification of granuloma formation, focal dense infiltrates and follicles in the lungs around the large airways. Analysis of the lungs and BAL cells of mice with CBD showed the involvement B-cells, T-cells (including CD4, Th17 and T-reg), macrophages, DCs and neutrophils. The next step in this project is to investigate the role of each particular cell types in CBD in in vivo experiments. To approach this research question we will use (conditional) knock-out mice, and cell-specific depleting antibodies. Immunohistochemistry will be performed to analyze the role of particular cells in CBD. Furthermore ELISA and quantitative RT-PCR analysis will be performed to investigate the role of cytokines and chemokines. Spleen cells obtained from mice with CBD will be phenotyped and analyzed in Beryllium lymphocyte proliferation test (BeLPT). A reliable BeLPT needs to be developed and validated first. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Three-week lab rotation
A master student interested in a three-week lab rotation will be involved in the experiments
described above. In this rotation, the student will be trained into running project techniques:
immunohistochemistry, Flow cytometry, Q-PCR, ELISA and introduced into animal work.
In addition, students will obtain an overview of all current research projects at the Department of Pulmonary Medicine. You will attend weekly work meetings and journal clubs.
You will have meetings with all researchers, who will give overviews of their scientific
activities. You will also be introduced into most of the ongoing molecular, cellular and
immunological research techniques in the lab.
Master of science project (6/12/18 months)
Development of a reliable Be lymphocyte proliferation test and involved in the running project
techniques: immunohistochemistry, flow cytometry, Q-PCR, ELISA, animal work.
For students with an 12/18 months project it is possible to take an animal course (Art. 9) to
independently perform in vivo experimental work, including cell depletion experiments (either
by depleting antibodies or conditional knock-out animals).
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Subject for lab rotation and project:
B cell receptor (BCR) signaling in auto-immune disease
Department of Pulmonary Medicine

Contact information

Laurens Kil (PhD student)
Room Ee2251
Telephone: 010-7043698
Email: [email protected]
Workgroup leader
Dr. R.W. Hendriks (Head of research department)
Room Ee2251a
Telephone 010-7043700
Email: [email protected]

Background
B cells are key players in the pathogenesis of rheumatic auto-immune diseases including
rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Upon differentiation into
plasma cells, B cells contribute to auto-immune pathology in these diseases by producing
highly pathogenic auto-antibodies directed against IgG (rheumatoid factor), citrullinated
proteins (-ccp), and nuclear antigens (ANAs). In addition, B cells augment auto-immunity by
presenting self-antigens to T cells and by producing cytokines that polarize and enhance
autoreactive T cell responses. The central role of B cells in establishing auto-immune
diseases like RA and SLE has overtly been demonstrated by the highly effective treatment of
RA and SLE patients with B cell depletion therapy (Rituximab).
In healthy individuals, the appearance of fully differentiated B cells that express self- reactive B cell receptors (BCRs) is mostly prohibited by means of negative selection at multiple stages during B cell differentiation. In RA and SLE patients however, differentiating self-reactive B cells fail to be removed or regulated at these counterselection checkpoints, leading to increased numbers of mature autoreactive B cells and plasma cells that constitute auto-immune disease. In search for B cell populations from which autoreactive B cells possibly originate, a new subset of circulating B cells with prototypic SLE-like autoreactive BCRs has been identified that co-expresses rearranged immunoglobulin light chains (IgL) and (constituents of) surrogate light chains (SLC). Since the SLC serves as an immunoglobulin heavy chain (IgH) binding partner in large pre-B cells prior to productive recombination of an IgL allele, expression of the SLC is normally terminated beyond the large pre-B cell stage. These particular SLC+IgL+ double-expressing B cells occur at a very low frequency in the blood of healthy individuals, but these SLC+IgL+ B cells are greatly enriched in the synovial fluid of inflamed joints of RA patients, implicating a causative role of these cells in the pathogenesis of RA. At present, it is still unknown whether these cells are involved in the pathogenesis of rheumatic auto-immune diseases. Apart from defects in the counterselection and regulation of autoreactive B cells in SLE and RA patients, it has become increasingly clear that circulating B cells in SLE patients exhibit enhanced BCR signaling. Both in transgenic SLE mouse models and in SLE patients similar genetic defects have been identified that impair the signaling of several inhibitory receptors in B cells, leading indirectly to B cell hyperresponsiveness upon antigen binding to the BCR. Remarkably, it is largely unknown whether genetic defects that directly affect signaling molecules downstream of the BCR are involved in the pathogenesis of SLE or RA. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
Especially the possible effects of overexpression of Bruton's tyrosine kinase (BTK), a critical BCR signaling molecule, on systemic auto-immunity remain to be elucidated. Research
1. The role of SLC+IgL+ B cells in SLE and RA pathogenesis
We are currently investigating the role of SLC+IgL+ double-expressing B cells in the
pathogenesis of SLE and RA. To this aim, we have generated transgenic mice that
constitutively express SLC components throughout B cell differentiation, thereby creating a
large pool of mature SLC+IgL+ double-expressing B cells in these mice. Initial characterization
of these mice revealed strongly increased differentiation of SLC+IgL+ B cells into IgM
producing plasma cells. Importantly, these plasma cells produce high levels of IgM auto-
antibodies leading to the formation of circulating immune complexes that deposit in the
glomeruli of the kidneys in SLC-transgenic mice. When these mice are crossed onto other
SLE-prone genetic backgrounds, an aggravation of the auto-immune phenotype can be
observed, as demonstrated by increased numbers of activated B cells, germinal centers and
plasma cells, as well as increased IgG auto-antibody production.
We aim to elucidate the mechanism(s) by which enforced expression of SLC components in
mature B cells attributes to the development of auto-immune disease. Therefore we will
specifically investigate:
- What the exact effects of SLC expression in mature B cells are on the spontaneous development of SLE and on the development of collagen induced arthritis (CIA). - Whether sustained SLC expression throughout B cell differentiation affects B cell repertoire selection, both in non-immunized mice and in mice developing CIA. - Whether expression of SLC in mature B cells affects B cell activation. - Whether SLC+IgL+ B cells can initiate or sustain the formation of autoreactive germinal 2. The effect of BTK overexpression on auto-immune disease
To study the effects of B cell specific overexpression of BTK on mature B cell differentiation
and on the development of auto-immunity, transgenic mice have been generated that
overexpress BTK either throughout B cell differentiation or only during final B cell maturation.
Mature B cells that overexpress BTK display enhanced Ca2+ influx and increased membrane
expression of activation markers upon BCR stimulation in vitro. Moreover, BTK-transgenic
mice exhibit increased numbers of activated B cells, germinal center B cells and plasma cells,
and aging BTK-transgenic mice spontaneously develop an SLE-like auto-immune disease
characterized by high anti-nuclear auto-antibody levels, immune complex mediated
glomerulonephritis, and systemic vasculitis affecting lungs and kidneys.
Three-week lab rotation
During a laboratory rotation of 3 weeks, you will be able to familiarize yourself with the
research project described above. You will participate in this project and perform
immunological characterizations of mice described above. To this end, you will be trained into
various techniques, including multi-color flow cytometric analysis (including working with
FACS analysis software programs), ELISA for (auto)antibodies, and immunohistochemical
analysis of lymphoid organs, lung and/or kidney.
In addition, you will obtain an overview of all current research projects at the Department of Pulmonary Medicine. You will attend weekly work meetings and journal clubs. You will have meetings with all researchers, who will give overviews of their scientific activities. You will also be introduced into most of the ongoing molecular, cellular and immunological research techniques in the lab. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Master of Science projects (6/12/18 months)
As a Master student, you will be working at the Department of Pulmonary Medicine on the
research projects outlined above under the supervision of a PhD student and the head of the
research department (see ‘Contact details' below). As these research projects comprise
multiple research (sub)questions, you will be focusing during your research project mostly on
a well-defined part of the project. Especially, during a 12 or 18 months project, you will have
the opportunity to perform in-depth research in a more independent way, e.g.
1. Generation of transgenic mice that express SLC components under the
control of the MHC class II promoter. This includes cloning of the transgenic
construct, Southern blot screening for transgene transmission and the
characterization of the disease phenotype.
2. In vivo collagen-induced arthritis mouse model, to test the effects of SLC
expression on arthritis disease development. 3. Single-cell B cell sorting and cloning of Ig Heavy and Light chain, to
characterize the B cell repertoire in particular transgenic mice. As the project on BCR signaling in auto-immune disease demands an experimental approach that is both fundamental and disease-related, you will have the opportunity to learn and use a whole variety of techniques, including techniques for DNA analysis (e.g. cloning of BCR's, sequencing of BCR variable regions), RNA analysis (RT-PCR), protein analysis, ex vivo analysis of B lymphocytes using advanced flowcytometry (staining of extracellular markers, intracellular molecules such as cytokines, measuring of Ca2+ influx upon B cell stimulation, etcetera), immunohistochemistry on lymphoid organs and other tissues (kidneys, lungs), (auto-)antibody detection and quantification using different assays (HEp2 cell stainings, ELISA), B cell culture systems, and induced disease models such as CIA. During your project, you will be encouraged to follow educational courses on immunological topics or laboratory techniques. For students with an 12/18 months project it is possible to
take an animal course (Art. 9). Furthermore, you will be able to expand your knowledge on
immunology by participating in research and literature discussions which are organized at the
department on a weekly basis. In the fields of B cell immunology and rheumatic diseases,
close collaborations of the department exist with research groups at local, national and
international level, to ensure the exchange of the most up-to-date knowledge and techniques.
Concerning this research project, there is a close collaboration with the Department of
Rheumatology (with regular joint meetings). In addition, we collaborate with research groups
in the Departments of Immunology and Cell Biology at the Erasmus MC, the Department of
Human Genetics at the LUMC Leiden, the Max-Planck-Institute for Immunology in Freiburg
(Germany), and the Max-Planck-Institute for Infection Biology in Berlin (Germany).
Funding
This research project is financially supported by grants from the Reumafonds.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

PAEDIATRIC INFECTIOUS DISEASES – LABROTATIONS & RESEARCH TOPICS
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title:
Elucidation of the molecular mechanism underlying antigenic variation of, and immune
evasion by the respiratory pathogen Mycoplasma pneumoniae

Working group
Laboratory of Pediatric Infectious Diseases, Erasmus MC, Ee-1502a
Head of the laboratory and working group leader
Dr. C. Vink
Email: [email protected]
Tel: 010-7044224
Outline of the reseach line
Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory infections,
such as tracheobronchitis, pharyngitis, and atypical pneumonia. Up to 40% of the community-
acquired pneumonias and as many as 18% of cases requiring hospitalization in children are
caused by M. pneumoniae.
Mycoplasmas represent the smallest self-replicating organisms both regarding cellular
dimensions and genome size. The genome of M. pneumoniae was found to have a length of
only 816 kilobase pairs (kb), containing 688 open reading frames (ORFs). Despite its limited
size, it is interesting to note that a significant part ( 8%) of the M. pneumoniae genome
consists of 4 different types of repetitive DNA elements, which are referred to as RepMP1,
RepMP2/3, RepMP4 and RepMP5. Common features of these elements are that: (i) their
representatives (variants) are similar, but not identical in sequence, and (ii) they are also
contained in genes that encode surface-exposed, antigenic proteins. Among these proteins is
the M. pneumoniae P1 protein, which plays an essential role in bacterial adhesion to host
cells. The gene encoding the P1 protein, MPN141, contains both a RepMP4 and a RepMP2/3
element. Outside of the MPN141 gene, 9 variants of RepMP2/3 and 7 variants of RepMP4 are
found dispersed throughout the M. pneumoniae genome.
It has been hypothesized that homologous recombination between the RepMP elements
within MPN141 and elements elsewhere in the genome could generate sequence changes
within MPN141. These changes could lead to amino acid sequence variation of the antigenic
P1 protein, and thereby contribute to bacterial evasion from the host's immune system.
In our laboratory, we aim to study this hypothesis by pursuing the following research
objectives
:
1. To identify the M. pneumoniae proteins involved in recombination between repetitive
genomic elements. 2. To elucidate the mode of action of each of these proteins. 3. To elucidate the relationship between M. pneumoniae virulence and the presence of specific RepMP-sequences within the P1 gene.
Three-week lab rotations
Master students doing a 3-week lab rotation will be involved in the research projects described above, working side by side with PhD students and technicians. Goals will be to experience the wide variety of techniques that are used by our research group and get acquainted with a scientific research laboratory (Laboratory of Pediatrics) in which different disciplines from the medical sciences are combined. Students will participate in the weekly research meetings of the Laboratory of Pediatrics and of the infection and immunity group meetings. Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
The following techniques are extensively used in our group: • DNA • Protein expression and purification • (Real-time), quantitative PCR • DNA-protein interaction techniques • DNA recombination assays • Culturing Mycoplasma species Six-, 12- or 18-month Master of Science projects
Master students doing a 6-, 12- or 18-month Master of Science project will be involved in the research described under ‘research objectives' above. Various options for doing research are possible and the exact subject of any (sub)project will depend on the advances we will already have made at the time the student will start. In general, a student may choose to focus on: (1) Recombinant protein expression and purification, and designing functional assays to study the function of the purified protein, (2) Detection and typing of M. pneumoniae variants from different cohorts of e.g. children with respiratory infection, and (3) Determination of the genetic variability of M. pneumoniae strains. You can read the papers listed below for our former work on this subject. Please contact C. Vink for updated information on the possibilities for doing research. Our recent publications on this subject:
M. Sluijter, E. Kaptein, E.B.M. Spuesens, T. Hoogenboezem, N.G. Hartwig , A.M. van
Rossum, and C. Vink. The Mycoplasma genitalium MG352 gene encodes a Holliday
junction resolvase that has a non-functional ortholog in Mycoplasma pneumoniae. Mol.
Microbiol
, in press (doi:10.1111/j.1365-2958.2010.07288.x).
E.B.M. Spuesens, T. Hoogenboezem, M. Sluijter, N.G. Hartwig, A.M.C. van Rossum,
and C. Vink. Marcrolide resistance determination and molecular typing of Mycoplasma
pneumoniae
by pyrosequencing. J. Microbiol. Methods, 2010 Jun 11. [Epub ahead of print;
10.1016/j.mimet.2010.06.004].
E.B.M. Spuesens, N.G. Hartwig, A.M.C. van Rossum, and C. Vink. (2010).
Identification and classification of P1 variants of Mycoplasma pneumoniae. J. Clin.
Microbiol.
48:680.
M. Sluijter, E.B.M. Spuesens, N.G. Hartwig , A.M. van Rossum, and C. Vink. (2009).
The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes
encode RecA homologs that promote homologous DNA strand exchange. Infect. Immun.
77: 4905-4911.
E.B.M. Spuesens, M. Oduber, T. Hoogenboezem, M. Sluijter, N.G. Hartwig, A.M.C.
van Rossum, and C. Vink. (2009). Sequence variations in RepMP2/3 and RepMP4
elements reveal intragenomic homologous DNA recombination events in Mycoplasma
pneumoniae
. Microbiology 155: 2182-2196.
K. Maquelin, T. Hoogenboezem, J.-W. Jachtenberg, R. Dumke, E. Jacobs, G.J.
Puppels, N.G. Hartwig, and C. Vink. (2009). Raman spectroscopic typing reveals the
presence of carotenoids in Mycoplasma pneumoniae. Microbiology 155: 2068-2077.
Sluijter, M., T. Hoogenboezem, N.G. Hartwig, C. Vink. (2008). The Mycoplasma
pneumoniae MPN229 gene encodes a protein that selectively binds single-stranded DNA Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
and stimulates Recombinase A-mediated DNA strand exchange. BMC Microbiol. 8: 167
(1-13).
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
PAEDIATRIC GASTRO-ENTEROLOGY – LABROTATIONS & RESEARCH TOPICS
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title Research line: Mucosal immune regulation
Workgroup leader: Janneke Samsom (Department Pediatrics division Gastroenterology
and nutrition)
Email addresses: [email protected]
The aim of the research performed in the Samsom lab is to unravel the mechanisms that
underlie chronic intestinal disease such as celiac disease and inflammatory bowel disease.
The mucosal surfaces of our gastrointestinal tract are continuously exposed to a large variety
of foreign antigens, such as pathogens that enter the body at these sites but also harmless
antigens including food proteins and constituents of commensal bacteria. Although
inflammatory immune responses are needed to eliminate transient encounter with invasive
bacteria and protect the body from subsequent infection, active immunity to frequently
encountered harmless substances would be detrimental due to chronic tissue damage. To
avoid such damage the default immune response to harmless soluble proteins that are
encountered via the mucosa is mucosal tolerance. Loss of mucosal tolerance leads to chronic
inflammatory disorders of the intestine such as celiac disease (CD) caused by an intolerance
to the dietary protein gluten and inflammatory bowel diseases (IBD), that are driven by an
aberrant response to bacterial components. Both CD and IBD are characterized by the
activation of inflammatory effector T-cell (Te-cell) responses in the lamina propria of the gut
causing loss of barrier integrity and severe tissue destruction leading to loss of gut-function.
Unfortunately little is known of why these diseases occur. By combining basic science with
patient research we aim to identify initial immune mechanisms that are causative for the loss
of mucosal tolerance.
Master students doing a 3-week lab rotation will specifically be involved in the research on
mucosal immune regulation, working side by side with PhD students and technicians. In
addition, they will visit the clinic at the Sophia children's hospital and join the pathology
meetings where biopsies from patients are screened and diagnosed. Goals will be to
experience a wide variety of techniques used to study mouse and human mucosal
immunology, to become acquainted with the scientific approach that is taken to answer
research questions and to get an overview of the different lines of research that exist in the
laboratory.
The following techniques are in general use:
- Flow cytometry
- Real-time PCR
- Immunohistochemistry
- Cell-culture
- Magnetic and fluorescent based cell purification
- ELISA
- Animal models for intestinal disease
Students will participate in the weekly research meetings of the department of Pediatrics, the
mucosal immunology group meetings and the pathology meetings.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Master students doing a 6, 12 or 18 month Master of Science project will be able to choose
from different research topics, including:
- Understanding celiac disease pathogenesis
Celiac disease (CD) is an inflammatory disease of the small intestine caused by intolerance to
gluten-proteins that are constituents of wheat and rye. CD is a frequent disorder as it affects
between 0.5 and 1% of the western population. Due to severe inflammation small intestinal
villi, that are required for nutrient uptake, regress leading to malnutrition. Although CD
symptoms can be treated by avoiding gluten, the disease has increasingly recognized co-
morbidities such as diabetes and cancer leaving patients and clinicians with new therapeutic
problems. Although CD has a clear genetic predisposition: virtually all patients are HLADQ2 it
is unclear what causes CD development. Using patient biopsies and a unique murine model
for CD we are elucidating the first immune events that characterize CD. There is room to
choose an individual subject within this project. The project involves all techniques mentioned
above.
- Innate mucosal immune regulation in IBD
The two main forms of inflammatory bowel disease (IBD) are Crohn's disease (CD) and
ulcerative colitis (UC). Both diseases are characterized by a dysregulated mucosal immune
response to microbial products leading to chronic inflammation of the intestinal tract. To gain
better insight into the pathogenesis of these diseases it is critical to elucidate how the mucosal
immune system maintains a tolerogenic response to harmless antigens while continually being
challenged with microbial products. Recently, it has been shown that expression of the
intracellular immunoregulatory protein secretory leukocyte protease inhibitor (SLPI) is
decreased in colonic biopsies from patients with CD indicating a role for this protein in the IBD
pathogenesis. SLPI is a non-glycosylated serine protease inhibitor with well-described anti-
viral, microbicidal, and anti-inflammatory properties. Only recently, SLPI has also been
identified as a potent immunoregulatory protein, in particular capable of inhibiting NF-kappaB
mediated cellular activation by microbial stimuli. In this project we wish to investigate whether
SLPI plays a crucial role in regulating not only innate, but also adaptive tolerance to bacterial
products from normal flora. There is room to choose an individual subject within this project.
The project involves all techniques mentioned above and in addition requires western-blotting
and retroviral transduction.
Students will participate in the weekly research meetings of the department of Pediatrics, the
mucosal immunology group meetings and the pathology meetings.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

HEPATOLOGY – LABROTATIONS & RESEARCH TOPICS
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title Research line: Understanding, predicting and promoting tolerance to liver grafts
Workgroup leader:
Jaap Kwekkeboom, PhD. Laboratory for Gastroenterology and Hepatology, Erasmus MC.
Email address: [email protected]
Information about the lab and my research group: www.gastrolab.nl
Description
Liver transplantation (LTx) is the only adequate treatment for end-stage liver diseases. On the
short-term it is a successful treatment, but long-term patient survival and quality of life are
severely impaired by complications caused by life-long immunosuppressive treatment needed
to prevent rejection of the graft. However, there is clinical evidence that about 20% of LTx-
recipients become spontaneously tolerant to their graft, allowing safe withdrawal of
immunosuppressive medication. Our research aims to understand how liver grafts induce
tolerance in a subgroup of patients, to develop assays that accurately identify tolerant
patients, and to device strategies to promote graft acceptance in those patients that do not
spontaneously develop immunological tolerance to their liver graft.
All projects are performed in close interaction with liver transplant surgeons and physicians in the Erasmus MC under supervision of Prof. dr. Herold Metselaar and prof. dr. Hugo Tilanus, ensuring that our research has a translational character. In all our research project we use materials (blood, liver tissue, liver perfusates, liver biopsies etc.) from LTx-patients. Lab rotations
A master student that does a 3-week lab rotation in my group will:
- Participate in experiments that are ongoing in research project of one of the PhD-students
- Learn several of the following techniques: cell culture, immunomagnetic cell purification,
flowcytometry, ELISA, ELISPOT, immunohistochemistry.
- Participate in the weekly seminars of the Laboratory of Gastroenterology and Hepatalogy,
the weekly workgroup meetings, and in regular informal research project discussions.
- If wanted: attend a liver transplantation
Master of Science projects
A master student that will do a 6, 12 or 18 month Master of Science project in my group can
choose from the following research topics:
1. The role of migrating donor-derived leukocytes in immunological tolerance to liver
grafts

In contrast to other solid organ grafts, LTx results in leukocyte chimerism. We observed that
donor-derived dendritic cells, regulatory T-cells B-cells, NK-cells and NKT-cells detach from
human liver grafts after transplantation, and migrate via the blood circulation into the
recipients. By studying the characteristics of hepatic leukocytes that detach from human liver
grafts during pre-transplant perfusion, we aim to elucidate how these donor-derived hepatic
leukocytes may be involved in chimerism-associated transplant tolerance.
Collaboration: dr. I. Joosten, UMC St. Radboud, Nijmegen.
2. Identification of tolerant liver transplant recipients
Using an in-house developed highly sensitive assay, we study whether differences in
frequencies of recipient T-cells that recognize donor allo-antigens via the direct pathway, are
related to transplant tolerance. A sensitive assay for quantification of recipient T-cells which
react to indirectly presented donor HLA molecules is being developed, using mRNA-
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

electroporation of recipient Antigen-Presenting Cells. The predictive value of these assays will
be tested by withdrawal of immunosuppressive medication in patients that are identified as
low-responders.
Collaboration: Prof. K. Thielemans, Free University, Brussels

3. Safe immunosuppression

We found that treatment of LTx-recipients with Intravenous Immunoglobulins (IVIg) protects
against acute rejection. Basic research revealed that IVIg inhibit allogeneic T-cell activation by
at least three mechanisms: suppression of the maturation of dendritic cells (DC), stimulation of
killing of DC by NK-cells, and stimulation of the suppressive capacity of regulatory T-cells.
Currently, we aim to identify the active component(s) of IVIg that inhibit cellular immune
reactions. Biosynthetic compounds that mimic these components may be useful as natural
and safe immunosuppressive drugs.
Collaboration: dr. Th. Geijtenbeek, VUMC, Amsterdam, Prof. Dr. S. Kaveri, Paris.
4. Tolerance induction
We found that activated human plasmacytoid dendritic cells (PDC) are potent inducers of
anergy in allo-reactive memory T-cells. In addition, they induce the differentiation of CD8
regulatory T-cells that suppress allogeneic memory T-cell responses. Currently, we are
optimizing the protocol for generating CD8 regulatory T-cells by human PDC, and unravelling
the molecular mechanisms by which PDC induce these cells. Subsequently, we will study
whether administration of donor-derived PDC can induce long-lasting transplant tolerance in
experimental animals. The final aim of this project is to develop an immunotherapeutic
protocol for induction of transplant tolerance in LTx-patients.

During all Master of Science projects
:
- You will use the following techniques: cell culture, immunomagnetic cell purification,
flowcytometry, ELISA. Depending on the project you will use also: immunohistochemistry,
ELISPOT, Q-PCR, cloning, mRNA-electroporation, immunoprecipitation, westernblotting.
- Participate in the weekly seminars of the Laboratory of Gastroenterology and Hepatalogy,
the weekly workgroup meetings, regular informal research project discussions.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Immunology of persistent hepatitis C vius infections
Workgroup leader:
Andre Boonstra, PhD
[email protected]

Rotterdam Liver Unit
Dept. Gastroenterology & Hepatology, Room L-455
www.gastrolab.nl
Description
The hepatitis C virus (HCV) is an example of a human viral pathogen that is difficult to control by the immune system. As a result of this, about 80% of individuals infected with HCV become chronically infected. The long-term consequences of chronic HCV infections can be severe, since patients are at increased risk for developing liver fibrosis, cirrhosis and/or hepatocellular carcinoma. The research in our laboratory is aimed at understanding why the immune response to HCV is insufficient to clear the virus in infected patients. This knowledge is important and will be used to improve therapeutic strategies to treat chronic HCV patients. Our studies combine clinical and fundamental research. The fundamental immunological studies are directed by dr. André Boonstra, while clinical expertise is provided by prof. dr. Harry Janssen and dr. Rob de Knegt, both experts in the field of viral hepatitis. You can participate in one of the ongoing research lines as described below: Main research Lines

Regulation of the HCV-specific T cell responses
The HCV-specific T cell response in chronic HCV patients is not potent enough to eradicate
the virus. The project is aimed at characterizing the T cell response in these patients, with
special focus on the mechanisms of regulate and suppress this response. Besides examining
peripheral blood lymphocytes also the responses in the liver compartment are assessed, since
the local immune response in the liver is likely pivotal in the persistence of HCV.
The innate immune response to HCV
Impairment of the innate immune response to HCV is likely to have a major impact on the
development of HCV-specific T cell responses. Our previous studies have addressed
fundamental questions regarding the interaction of DC populations and T cells in the
regulation of immunity, as well as the effect of TLR agonist stimulation on cytokine production
by conventional DC, plasmacytoid DC and macrophages. Currently, detailed studies are
conducted on the impact of HCV on the innate immune system. Also in this project, the
intrahepatic immune response is a prime focus of research.

The intrahepatic immune response HCV
The local immune response to HCV in the liver is studied using a unique method to collect
lymphocytes directly from the liver from chronic HCV patients using fine needle aspiration
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics
biopsies. Using this technique, intrahepatic lymphocytes can be examined by flow-cytometry for their phenotype, but also functionally. Phenotypic studies using tetramer technology, as well as functional assays have already provided valuable information on the local immune response during viral hepatitis, and for evaluation of the effect of anti-viral therapy.  Study on the mechanism of action of current and novel therapies for HCV
The current therapy to treat chronic HCV patients is only partly effective, and elicits severe
adverse effects. A major focus of our laboratory in close interaction with the clinical branch of
the Rotterdam Liver Unit, is to examine the mechanisms of action of the current therapy based
on pegylated-interferon and ribavirin in patients with chronic HCV infections. In addition, we
perform investigator-initiated studies to assess the immunological effect of novel HCV
inhibitors in numerous clinical studies, performed to evaluate the efficacy and safety of novel
therapeutic strategies to treat HCV.
Techniques:
A broad range of immunological as well as molecular techniques are being used in the
laboratory, including cell culture, cell purification by cell sorting, multi-color flow cytometry,
various immunoassays, Q-PCR etc
The following meetings will take place weekly:
Department of Gastroenterology and Hepatology seminars Viral hepatitis meetings
Key publications:
Boonstra A,
van der Laan LJW, Vanwolleghem T, Janssen HLA.
Experimental models for hepatitis C viral infection.
Hepatology. Ahead of print. (2009).
Liu B, Janssen HLA, Boonstra A.
Modulation of dendritic cell function by persistent viruses.
J Leukoc Biol. 85: 205-214 (2009).

Boonstra A, Rasjbaum R, Holman M, Marques R, Asselin-Paturel C, Pereira J, Bates EEM,
Akira S, Vieira P, Liu Y-J, Trinchieri G, O'Garra A.
Macrophages and myeloid DC, but not plasmacytoid DC, produce IL-10 in response to
MyD88- and TRIF-dependent TLR signals, and TLR-independent signals.
J. Immunol. 177: 7551-7558 (2006)
Boonstra A
, Asselin-Paturel C, Gilliet M, Crain C, Trinchieri G, Liu YJ, O'Garra A.
Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper
type 1 and 2 cell development: dependency on antigen dose and differential Toll-like receptor
ligation.
J Exp Med. 197: 101-109 (2003).
Asselin-Paturel C, Boonstra A, Dalod M, Durand I, Yessaad N, Dezutter-Dambuyant C, Vicari
A, O'Garra A, Biron C, Brière F, Trinchieri G.
Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.
Nature Immunol. 2: 1144-1150 (2001).
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title: Influence of Hepatitis B virus (HBV) on dendritic cell development


Workgroup leader:
Dr. Andrea Woltman a.[email protected] 010-7032790

Contact:
Lianne van de Laar [email protected] 010-7033322
Nadine van Montfoort [email protected] 010-7035821
Department of Gastroenterology and Hepatology,
Erasmus MC, Rotterdam
L-458, 's-Gravendijkwal 230, 3015 CE Rotterdam
www.gastrolab.nl

Project
HBV infection is a worldwide problem, with about 1 million patients dying of chronic HBV-
related liver disease each year. An important cause of developing such a chronic infection is
the lack of an adequate immune response. The most important cells regulating immunity are
dendritic cells (DCs), professional antigen presenting cells that play key roles in the initiation
and regulation of immune responses, including anti-viral responses. The presence of sufficient
functional DCs is therefore crucial to develop adequate anti-HBV immunity. We and others
have reported that different DC subtypes are functionally impaired by HBV (1), suggesting that
HBV might inhibit DC to escape immunity.
The molecular mechanisms regulating human DC development and survival are relatively
unknown. Therefore, we have studied the intracellular signal transduction pathways and
transcription factors that control the differentiation and survival of distinct human DC subsets
from DC precursors from healthy individuals (2). In the current project, we plan to investigate
the effect of HBV on the development of functional DCs. We aim to study the phenotype,
survival and function of DC subsets that are differentiated from DC precursors in the presence
of HBV.
Research questions that can be addressed are:
Does HBV interfere with intracellular signal transduction pathways, transcription factors or genes crucial for DC development? What is the phenotype of DCs differentiated in the presence or absence of HBV? Are DCs differentiated in the presence of HBV fully functional in terms of antigen uptake, T cell activation and maturation?
Techniques:
Stem cell isolation from cord blood using magnetic beads (MACS), cell culture, production and
isolation of HBV, multicolour flow cytometry (FACS), cytokine analysis by ELISA and
intracellular FACS, T cell assays, fluorescence microscopy, RNA-isolation, RT-PCR, gel
electroforesis, western blot, molecular cloning
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Duration:
6-9 months
Key publications:
1. Woltman, A. M., A. Boonstra, and H. L. Janssen. 2010. Dendritic cells in chronic viral
hepatitis B and C: victims or guardian angels? Gut 59:115-125.
2. van de Laar, L., M. Buitenhuis, F. M. Wensveen, H. L. Janssen, P. J. Coffer, and A. M.
Woltman. 2010. Human CD34-derived myeloid dendritic cell development requires intact
phosphatidylinositol 3-kinase-protein kinase B-mammalian target of rapamycin signaling. J
Immunol 184:6600-6611.
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

HEMATOLOGY – LABROTATIONS & RESEARCH TOPICS

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Title Research line: Development of lymphoid organs
Workgroup leaders: Tom Cupedo, Jan Cornelissen (Department Hematology)
Email addresses: [email protected], [email protected]

Description
The aim of the research performed in the Cupedo and Cornelissen labs is to unravel the cells
and mechanisms that drive the development of lymph nodes and thymus.
The thymus is a primary lymphoid organ and T cell development occurs exclusively in this
organ. With increasing age, thymic function and the generation of new T cells decrease. In
healthy individuals this is countered by the longevity of naïve and memory T cells that
continue to provide sufficient protection against infections. However, when these peripheral T
cells are removed as a consequence of chemo and radiotherapy for hematopoietic
malignancies, the thymus is unable to swiftly recover the naïve T cell pool. As a result,
patients undergoing stem cell transplantation for hematological malignancies suffer from
therapy-induced immunodeficiencies that may last up to 2 years. Our research is aimed at
understanding thymus development and uses this knowledge to rejuvenate the adult thymus
in stem cell transplantation patients. This line of research is explicitly translational with
concurrent clinical studies in immunocompromised transplant recipients complementing our
more basic studies
The lymph nodes are secondary lymphoid organs and are the sites from which immune
responses are launched. All lymph nodes form during embryonic development, but our body
retains the capacity to form lymph node-like structures well into adulthood. These so-called
tertiary lymphoid organs form during several autoimmune diseases, chronic infections and
cancer and are the immune systems way of locally creating an optimal environment for
directing immune responses. While this may proof to be beneficial during cancer or chronic
infections, one can easily appreciate that enhancing the immune response against self in
autoimmune disease is likely to contribute to pathology. Our research is aimed at identifying
the cells and molecules essential for lymph node development with the ultimate goal of
controlling the formation of tertiary lymphoid organs during disease.
Master students doing a 3-week lab rotation will specifically be involved in the research on
thymus and lymph node development, working side by side with PhD students. In addition,
they will be offered a broader visit to the different working groups at the Hematology
department and will also be involved in the activities of our Transplantation Laboratory, which
selects and prepares hematopoietic stem cells from various sources for transplantation in
leukemia patients. Goals will be to experience a wide variety of techniques used to study
mouse and human organogenesis, to become acquainted with the selection and culture of
human hematopoietic stem cells, and to be informed about ongoing research in the fields of
organogenesis, hematopoiesis and leukemogenesis.
The following techniques are in general use:
- Flow cytometry
- Real-time PCR
- Fluorescent and confocal microscopy
- Advanced cell culture techniques including 3D cultures
Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics

Students will participate in the weekly research meetings of the department Hematology, the
Cupedo/Cornelissen groupmeetings, and will attend the grand rounds in the Erasmus MC-
Daniel Den Hoed Cancer center discussing the diagnosis and treatment of the various
opportunistic infections in our immunocompromised transplant recipients.
Master students doing a 6, 12 or 18 month Master of Science project will be able to choose
from different research topics, including:
- Identification of epithelial precursor cells in the human and mouse thymus
The aim of this project is to uncover the precursor cells that form the human and mouse
thymus. Research will include the use of in-vitro and in-vivo techniques to study functional
capabilities of epithelial cells. The student will make use of transgenic mouse systems as well
as human tissues.
- Stromal cells in normal and malignant B cell follicles
During several B cell malignancies novel B cell follicles are formed outside the lymph node.
The aim of this project is to discover whether the stromal cells that support B cell follicle
formation during embryonic development are also involved I the formation of ectopic follicles
during disease. If so, targeting the non-malignant microenvironment of these tumors could be
an attractive therapeutic target. The project involves fluorescent microscopy and 3D cell
culture techniques.
- Function of human lymphoid tissue inducer cells
We have recently identified the cells responsible for inducing lymph node formation in the
human fetus. Surprisingly, these cells remain present after birth and well into adulthood in
both tonsils and peripheral lymph nodes. Currently we are investigating the function of the
cells in adults and their possible role in formation of novel lymphoid organs during disease.
This project involves 3D cell culture, xeno-transplantation and confocal microscopy.
In addition to the above mentioned, both projects will involve:
- Flow cytometry
- Cell culture
- Magnetic and fluorescent based cell purification
- In-vivo animal models
- Fluorescence microscopy
Students will participate in the weekly research meetings of the department Hematology, the
Cupedo/Cornelissen groupmeetings, and will attend the grand rounds in the ErasmusMC-
Daniel Den Hoed Cancer center discussing the diagnosis and treatment of the various
opportunistic infections in our immunocompromised transplant recipients.

Source: http://www.infectionimmunity.nl/documents/Laboratory%20Rotations%20%20Researchtopics%20MSc%20IandI%20-%20100827.pdf

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2013 Health Insurance Trust Fund Annual Report Arizona Department of Administration Human Resources Division – Benefit Services Janice K. Brewer Brian C. McNeil Governor Director FOREWORD Benefit Options is the program name for the benefits offered to State of Arizona ("State") employees and retirees by the Arizona Department of Administration ("ADOA"). This report provides a broad overview of the Benefit Options program, and meets the requirements of A.R.S. §38-652 (G) and A.R.S. §38-658 (B). The data shown is presented for the period January 1, 2013 through December 31, 2013. The active and retiree plans were concurrent for this period. For this report, ADOA internally developed a consistent statistical model based on generally accepted actuarial principles and standards, including Milliman Health Cost Guidelines Commercial Rating Structures, July 1, 2012.