Medizinerkolleg Münster
Liebe Teilnehmer*innen,
wir begrüßen Sie herzlich zum Zwischenkolloquium des Medizinerkollegs 2021_1.
Da wir das Kolloquium leider nicht als Präsenzveranstaltung, sondern als Zoom-Konferenz abhalten müssen, möchten wir Ihnen vorab ein paar Informationen zu diesem Format zukommen lassen:
Bei den Vorträgen ist eine Redezeit von 15 min und Diskussionszeit von 5 min vorgesehen; bei den Postern eine Redezeit von 5 Minuten und eine Diskussionszeit von 10 Minuten.
Sie sind alle herzlich eingeladen, sich aktiv an den Diskussionen zu beteiligen und gerne viele Fragen zu stellen. Wenn Sie gerade keinen Redebeitrag leisten möchten, bitten wir Sie das Mikrofon auszustellen.
Im Rahmen eines wertschätzenden Miteinanders während Zoom-Konferenzen möchten wir Sie bitten, ihre Kamera einzuschalten.
Neben den Vorträgen wird es ein weiteres Präsentationsformat geben, welches die in den vergange- nen Jahren durchgeführten Postersessions ersetzt. Diese alternativen Postersessions werden wie folgt ablaufen:
- Die Präsentierenden bekommen jeweils einen Breakoutraum zugeteilt und präsentieren ihr Thema in einer fünfminütigen Kurzpräsentation. Anschließend kann 10 min diskutiert werden.
Abstracts zu den Kurzpräsentationen finden Sie ab Seite 8 des Programmheftes
- Alle anderen Teilnehmer*innen können sich aussuchen, welchen Raum Sie besuchen und werden gebeten, sich möglichst gut auf die 5 angebotenen Räume zu verteilen.
- Nach 15 min werden die Breakouträume geschlossen und für eine weitere Runde erneut geöffnet. Sie haben die Möglichkeit, alle Räume zu besuchen.
Wir wünschen allen Teilnehmer*innen ein spannendes und erfolgreiches Kolloquium!
Das Organisations-Team
Anike Pohlmann (Kohortensprecherin 2021_1) Philipp Gräb (Kohortensprecher 2021_1) Prof. Dr. Rupert Hallmann (Sprecher des MedK) Melanie Wilbers (Studienkoordinatorin MedK)
Programm Freitag 21.1.2022:
8:30 Begrüßung Prof. Dr. Rupert Hallmann
Vorträge
8:40 "Characterisation of the toolbox for selective biotinylation of Neurexin1alpha”
Prof. Dr. med. Markus Missler Institut für Anatomie & Molekulare Neurobiologie
Madita Schulze Wehninck
9:00 “Comparative analysis of JAK/STAT-inhibitors on pro- and anti- inflammatory functions of monocytes”
PD Dr. Georg Varga Klinik für Pädiatrische Rheumatologie und Immunologie
Doreen Averbeck
9:20 “Elucidating the functional effect of gene variants in genes related to the piRNA biogenesis on male fertility”
Univ.-Prof. Dr. Frank Tüttelmann Institut für Reproduktionsgenetik
Clara Bühlmann
9:40 Pause 20 min
Postersession I
10:00 PSI-BR01: “Characterization of signal cascades as part of the HSP27-induced modulation of phagocyte functions “
Univ.-Prof. Dr. Johannes Roth Institut für Immunologie
Philipp Gräb
PSI-BR02: "Effect of Short-Chain Fatty Acids on the Expression and Assembly of the Polarity Complex and its Interaction with VE- Cadherin in Vascular Endothelial Cells"
Prof. Dr. Alexander Oberhuber
Klinik für Vaskuläre und Endovaskuläre Chirurgie
Inka Terhorst
PSI-BR03: "The effects of Pals1-knockout on cellular differentiation in intestinal organoids"
Prof. Dr. med. vet. Dr. rer. nat Michael Krahn Medizinische Klinik D
Julia Billian
PSI-BR04: “Evidence of neutrophil extracellular traps (NETs) in melanoma metastases and their impact on the response to immune checkpoint inhibitors (ICIs)“
PD Dr. Carsten Weishaupt Klinik für Hautkrankheiten – Hauttumorzentrum
Lennard Weide
11:00 Pause 20 min
Vorträge
11:20 “Mechanisms of BRD4-induced cell death in Early T-cell recursoracute lymphoblastic leukemia (ETP-ALL)“
PD Dr. med. Jan-Henrik Mikesch
Pädiatrische Hämatologie und Onkologie
Laureen Warncke
11:40 “Glucocorticoid-induced regulation of the pro-inflammatory immune response of murine monocytes and macrophages”
Univ.-Prof. Dr. Johannes Roth Institut für Immunologie
Franziska Krupp
12:00 Mittagspause 60 min
Postersession II
13:00 PSII-BR01: “Identification of pro-myelinating drugs using a humanized ex vivo cell culture model”
Prof. Dr. Tanja Kuhlmann Institut für Neuropathologie
Ferras Nanouh
PSII-BR02: “Titin’s M-band region is important for sarcomerogenesis and provides links to protein quality control pathways“
Prof. Dr. Wolfgang Linke Institut für Physiologie II
Josefine Bäßler
PSII-BR03: “Immunescape Strategies in a preclinical myeloma model”
Prof. Mathias Stelljes Med. Klinik A
Wiebke Begemann
13:45 Pause 5 Minuten
Vorträge
13:50 “Tumor-brain Organoids as a screeningplatform for pediatric brain tumors”
PD Dr. Kornelius Kerl Klinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie
Nicole Riedel
14:10 "Liquid Biopsy as a Tool for diagnostics, therapy Management and prognosis assesment in solid visceral tumours”
Priv. Doz. Dr. Juratli Klinik für Allgemein- Viszeral- und Transplantationschirurgie
Lena Kluck
14:30 "Identifying Mechanisms of Microglia-Tumor Cell Interactions in Retinoblastoma“
PD Dr. Kornelius Kerl Klinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie
Anike Pohlmann
14:50 Wrap-up Prof. Dr. Rupert
Hallmann
Programm Samstag 22.1.2022:
Vorträge
8:30 “‘Kinetic-CASA‘: Quantifying transient motility changes in sperm populations through stopped flow microscopy“
Prof. Timo Strünker
Centrum für Reproduktionsmedizin & Andrologie
Rebecca Tenambergen
8:50 “Regulation of the barrier function in epithelial cells through the RhoA regulator RhoGDI1”
Prof. Dr. Klaus Ebnet
Institut für medizinische Biochemie
Nicolina Wibbe
9:10 “IGF-1R Car T cell micropharmacy as potential treatment in Ewing sarcoma“
Univ.-Prof. Dr. Claudia Rössig
Klinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie
Johann von Buch
9:30 Pause 15 min
Postersession III
9:45 PSIII-BR01: “Modulation of osteoclastogenesis and angiogenesis via application of recombinant Syndecan-1 during fracture healing in mice“
Univ.-Prof. Dr. med. Richard Stange Institut für Muskuloskelettale Medizin
Nils Roters
PSIII-BR02: “Characterizing Outer Dynein Arm Defects In Respiratory Cilia by Immunofluorescence Microscopy”
Univ.-Prof. Dr. med. Heymut Omran Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
Kai Wetter
PSIII-BR03: “Modifications of the spermatogonial stem cell pool and its niche in infertile men”
PD Dr. Nina Neuhaus Centrum für Reproduktionsmedizin und Andrologie
Lea Mundt
PSIII-BR04: “Characterization of inducible neuronal cell culture with mutated CRLF1“
Prof. Dr. med. Frank Rutsch
Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
Lea Lange
10:45 Pause 15 min Vorträge
11:00 "The significance of cytoplasmic pre-assembling of axonemal components for primary ciliary dyskinesia"
Univ.-Prof. Dr. Heymut Omran
Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
Ina Baumann
11:20 “Environmental resistance and persistance of vancomycin-resistant enterococci in the hospital setting“
Prof. Dr. med. Stefanie Kampmeier Institut für Hygiene
Silke Härtner
11:40 “Establishment of an hiPSC-based stimulus transmission system and analysis of the pharmacology of connexins“
Prof. Dr. Guiscard Seebohm
fGH - Abteilung Zelluläre Elektrophysiologie
Paul Disse
12:00 Résumé und Verabschiedung Prof. Dr. Rupert
Hallmann
Poster-Abstracts:
PSI-BR01: “Characterization of signal cascades as part of the HSP27-induced modulation of phagocyte functions”
Philipp Gräb
Univ.-Prof. Dr. Johannes Roth, Institut für Immunologie
Sepsis is a disease that is still associated with high mortality rates worldwide. After an early strong inflammatory phase, sepsis patients might develop a secondary hypo-reactive state, called endotoxin- tolerance. In this case, invading pathogens cannot be recognized by the immune system and severe secondary infections can arise. Endotoxin tolerance of phagocytes is triggered by prolonged stimulation of Toll-like receptor 4 (TLR4) with low doses of pathogenic (e.g. lipopolysaccharide (LPS) stimuli). However, in 30% of sepsis patients an initial microbial trigger is missing. We recently demonstrated that under sterile conditions endogenous alarmins like S100A8/A9 or HSP27 are able to induce a hypo-responsiveness of phagocytes.
The goal of the present study was to analyze the molecular mechanisms underlying HSP27-induced immune tolerance. Thereby we had a special focus on the involved pattern recognition receptor (PRR) and the downstream pathways.
By using a TLR4 inhibitor in an invitro model, we could identify HSP27 as with a ligand of TLR4 and that the TLR4-pathway is involved in the induction of tolerance. This effect seemed to be dose- dependent and worked best at concentrations of 0,05 µg/ml. HSP27 was previous demonstrated as a potent activator of human monocyte IL-10 production. In this context, we presented that monocytes activated with HSP27, produce in a TLR4-dependend mechanism a higher amount of IL-10 than in a setting with a TLR4-inhibition. More experiments to define the downstream-pathway of a HSP27- activation and to clarify the interaction between the TLR4 and the IL-10 pathway are ongoing.
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PSI-BR02: "Effect of Short-Chain Fatty Acids on the Expression and Assembly of the Polarity Complex and its Interaction with VE-Cadherin in Vascular Endothelial Cells",
Inka Terhorst
Prof. Dr. Alexander Oberhuber, Klinik für Vaskuläre und Endovaskuläre Chirurgie
Aortic diseases remain a global challenge despite improvement in diagnostic and therapeutic methods. Acute aortic dissection is characterized by an intimal injury leading to the separation of the aortic wall layers. However, the exact pathophysiology of aortic dissection is still unclear. We hypothesize that alterations in endothelial cell-cell contacts might result in increased permeability leading to higher risk of aortic dissection.
Junctional proteins forming contacts between endothelial cells are fundamental for regulating permeability. Besides the tight junctions (TJ) comprising claudins, occludins and junctional adhesion
molecules (JAMs), the adherens junctions (AJ) play a major role for the barrier function. VE-Cadherin, a central protein forming AJs, is known to be linked to proteins of the polarity- or Par-complex, which consists of Par-3, Par-6 and the atypical protein kinase C (aPKC). These findings on the interaction between polarity proteins and VE-Cadherin are based on studies with venous endothelial cells or cells of small arteries. The role of Parproteins and the atypical protein kinase C concerning endothelial integrity in large arterial vessels remains unknown.
Short-chain fatty acids (SCFA) are produced by gut microbiota and are known to influence the barrier function of human venous endothelial cells, whereas knowledge about their role on aortic endothelial function is still limited.
Our study seeks to assess to what extend SCFA interact with the Par-complex and aPKCζ in conjunction with VE-Cadherin and influence aortic endothelial integrity
PSI-BR03: “PSI-BR03: "The effects of Pals1-knockout on cellular differentiation in intestinal organoids",
Julia Billian
Prof. Dr. med. vet. Dr. rer. nat Michael Krahn, Medizinische Klinik D
Maintenance of apical-basal polarity in epithelial cells is critical as its dysregulation is associated with the development of various pathologies such as cancer. One of the key modulators of apical polarity and several signaling pathways is Pals1, a scaffolding protein, which is part of the Crumbs-complex.
Although the function of Pals1 is well characterized in differentiated epithelial cells, little is known about the role of Pals1 in epithelial stem cells. Thus, our goal is to elucidate the effects of Pals1- knockout in stem cells and enterocytes in murine intestinal organoids.
For this we use adult stem cells from Cre-loxP-mice with Tamoxifen-inducible Pals1-excision. When cultured in Matrigel these cells form 3D-structures that mimic the intestinal epithelial tissue in vivo. By adding substances that target the Wnt- and Notch-pathway to our standard organoid-culture, we aimed to establish a pure-stem cell- or -enterocyte-culture. To verify the Pals1-knockout and characterize these cultures, immunofluorescence staining was performed. The development of the organoids was assessed using bright-field microscopy. As an indicator for apoptosis, TUNEL-staining was performed.
Confocal microscopy images confirmed that organoids treated with Tamoxifen show decreased Pals1-staining. TUNEL-staining and bright-field imaging revealed increased signs of apoptosis in these organoids compared to the wildtype. Our stem cell- and enterocyte-culture show morphological differences compared to the standard-culture, which need to be further validated.
Once established, this method will allow us to study the function of Pals1 in specific intestinal cell types. Thereby, we hope to better understand the role of Pals1 in intestinal stem cells and differentiated cells.
PSI-BR04: “Evidence of neutrophil extracellular traps (NETs) in melanoma metastases and their impact on the response to immune checkpoint inhibitors (ICIs)“,
Lennard Weide
PD Dr. Carsten Weishaupt, Klinik für Hautkrankheiten – Hauttumorzentrum
Neutrophils are able to release DNA equipped with lysosomal proteins into the extracellular space.
These neutrophil extracellular traps (NETs) can kill pathogens but are also found in association with several cancer types where they can function as either pro- or anti-tumorgenic (1,2). Additionally, NETs can reduce the response to immune checkpoint inhibitors (ICIs) in mice (3,4) which is especially interesting for melanoma treatment where ICIs are a standard treatment in adjuvant settings and advanced stages.
NETs had been documented in primary tumors of human melanomas and had shown anti-tumoral effects in vitro (5). We aim to investigate whether NETs are also present in melanoma metastases and seek to analyze their relevance for the patient´s response to ICIs.
Therefore, we screened the tissue registers of the UKM Departments of Pathology and Dermatology for melanoma metastases stored as paraffin embedded tissue blocks. Metastases of 199 patients were found of whom 99 have been treated with ICIs later on making a response evaluation possible.
Sections of these metastases were stained for DAPI, CD15 and citrullinated histones (H3Cit) to identify neutrophils and NETs. In addition the size of the tumor metastasis was measured either after immunohistochemical stainings with a panmelanoma marker or taken from the clinical records.
In the next step we will analyze the sections using ImageJ to measure the amount of NETs and neutrophils within the metastases so we can correlate both numbers. We will further correlate the relative amount of NETs with the patient´s response to ICIs objectified by RECIST 1.1
1) Millrud CR, Kågedal Å, Kumlien Georén S, et al. NET-producing CD16high CD62Ldim neutrophils migrate to tumor sites and predict improved survival in patients with HNSCC. Int J Cancer.
2017;140(11):2557-2567. doi:10.1002/ijc.30671
2) Erpenbeck L, Schön MP. Neutrophil extracellular traps: protagonists of cancer progression?.
Oncogene. 2017;36(18):2483-2490. doi:10.1038/onc.2016.406
3) Zhang Y, Chandra V, Riquelme Sanchez E, et al. Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer. J Exp Med.
2020;217(12):e20190354. doi:10.1084/jem.20190354
4) Zhang H, Wang Y, Onuma A, et al. Neutrophils Extracellular Traps Inhibition Improves PD-1 Blockade Immunotherapy in Colorectal Cancer. Cancers (Basel). 2021;13(21):5333. Published 2021 Oct 23. doi:10.3390/cancers13215333
5) Schedel F, Mayer-Hain S, Pappelbaum KI, et al. Evidence and impact of neutrophil extracellular traps in malignant melanoma. Pigment Cell Melanoma Res. 2020;33(1):63-73.
doi:10.1111/pcmr.12818
PSII-BR01: “Identification of pro-myelinating drugs using a humanized ex vivo cell culture model”, Nanouh Ferras
Prof. Dr. Tanja Kuhlmann, Institut für Neuropathologie
Oligodendrocytes play an important role in multiple sclerosis, which is characterized by episodes of demyelination in the central nervous system. Oligodendroglial precursor cells contribute to remyelination, an endogenous repair process of the CNS. However, the ability to repairdemyelination decreases with disease progression and the remyelination failure is at last partly caused by impaired oligodendroglial differrentiation.
In our study, we aim to validate compounds that have been shown to promote the differentiation of human induced pluripotent derived oligodendrocytes (hiOL)in 2D-cultures. To further validate the effect of the compounds on oligodendroglial proliferation, differentiation and myelination, I am establishing an ex-vivo cerebellar slice culture model in which the endogenous mouse oligodendrocytes are depleted and replaced by hi-OL.
PSII-BR02: “Titin’s M-band region is important for sarcomerogenesis and provides links to protein quality control pathways“,
Josefine Bäßler
Prof. Dr. Wolfgang Linke
TTN codes for the giant muscle protein titin. Heterozygous TTN truncating variants (TTNtv) cause up to 25% of all cases of dilated cardiomyopathy (DCM). The pathomechanisms of TTNtv-DCM have recently been shown to include titin haploinsufficiency and expression of potentially poisonous truncated titin proteins, causing contractile deficiency.
Here, we studied human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to obtain additional mechanistic insight into the pathomechanisms of TTNtv-DCM. We used CRISPR/Cas9-edited hiPSC-CMs with a heterozygous (HET) or homozygous (HOM) TTNtv in exon 359 of TTN, which codes for an M-band titin region; the isogenic wildtype (WT) line served as a control. Moreover, we examined tissue of an endstage failing DCM patient heart with a heterozygous M-band TTNtv in the same TTN location and heart tissue from a second patient with (another) M- band TTNtv. By electron microscopy, tissues of both patient hearts showed different degrees of damage of cardiomyocyte substructure. Intracellular titin-containing aggregates were frequent in both tissues. In hiPSC-CM lines, expression of titin protein was studied by western blot, demonstrating that HET contained less truncated protein than HOM and less wildtype protein than WT cells. We then performed immunofluorescence microscopy on hiPSC-CMs, focusing on titin and markers of the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathways. We observed no structural differences between HET and WT hiPSC-CMs. Even HOM cells could develop sarcomeres, although these were shorter and much less abundant. We used antibodies against titin (to epitopes just before and just after the truncation site), p62, and LC3B to stain hiPSC-CMs treated with UPS-inhibitor MG132 (or control vehicle). We found that p62 and LC3B co-localize with the M-band titin region and, in MG132-treated cells, also accumulate in perinuclear areas.
In conclusion, this work provides novel insight into titin protein expression and localisation in healthy and TTNtv cardiomyocytes and suggests a role for the protein quality control machinery in the pathomechanisms of TTNtv-DCM.
PSII-BR03: “Immunescape Strategies in a preclinical myeloma model”, Wiebke Begemann
Prof. Mathias Stelljes, Med. Klinik A
Multiple Myeloma is the most common type of B-Cell-Lymphoma with an incidence of 4-5/100000 per year.
Overexpression of SLAMF7, a protein belonging to the familiy of Signalling Lymphocyte Activation Molecules (SLAM), is typical for myeloma cells. It is a transmembrane protein and is also found on most NK-, B- and T-Cells. SLAMF7 works as a self-ligand either inhibiting or activating lymphocytes depending on the intracellular pathway.
This mechanism is used in antibody- and CAR-T-Cell-Therapy where SLAMF7 is a suitable targetprotein. In clinical practice, though, it has been a problem that these therapies are not always as efficient as expected.
In order to research whether immunescape of SLAMF7 is a possible reason for that in-vitro experiments are performed, representing a preclinical murine model. This includes T-Cell dependent Cytotoxicity Assays using a SLAMF7-targeted BiTE as well as CAR-T-Cytotoxicity Assays. Both are followed by FACS-Analysis and Western Blot Analysis of surviving cells.
First interim results show that tumor cells surviving a CAR-T-Cell Cytotoxicity Assay have a lower expression of SLAMF7 than control cells while other immunemodulating molecules like PD-L1 seem to be unaffected.
Analysis of Antibody-dependent Assays, however, shows less distinct resuts. Although a concentration-dependent cytotoxicity can be seen here as well, surviving cells show no significant difference regarding SLAMF7 expression.
Further research needs to adress the question how Immunescape in CAR-T-Cell Stimulation is more verifiable than in a TCDCC. Moreover, it is crucial to learn which mechanism leads to an immunescape via SLAMF7. This, as well as the more detailed interpretation of the results will be the upcoming tasks.
PSIII-BR01:“Modulation of osteoclastogenesis and angiogenesis via application of recombinant Syndecan-1 during fracture healing in mice“,
Nils Roters
Univ.-Prof. Dr. med. Richard Stange, Institut für Muskuloskelettale Medizin
Syndecan-1 is a heparan sulfate proteoglycan that is known to be a mediator of signals between extracellular matrix and cells. It takes influence on the osteoclastogenesis via the RANKL/OPG axis by interacting with OPG. A switch from OPG towards RANKL production increases the potential of osteblasts to induce osteoclastogenesis.
Our workinggroup has previously found that Syndecan-1 deficient mice show an impaired bone regeneration process. The aim of this new project is to investigate whether application of recombinant Syndecan-1 is able to negate this effect in Syndecan-1 deficient mice and even promote an increased bone regeneration in healthy mice.
So far we have performed osteotomies of the left femur on 10 female mice, amongst them wildtype and Syndecan-1 deficient mice. As part of the control group these did not receive recombinant Syndecan-1. We then euthanized the animals on day 7 post surgery and extracted the femora for µCT analysis.
Our next step is to perform cryosectioning to create thick and thin sections to analyse via immunofluoresence and conventional staining. After that we will perform osteotomies on another group of mice which will then receive the application of recombinant Syndecan-1 and analyse this group in the same way as explained above.
PSIII-BR02: „Characterizing Outer Dynein Arm Defects In Respiratory Cilia by Immunofluorescence Microscopy”
Kai Wetter
Univ.-Prof. Dr. med. Heymut Omran, Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
Primary Ciliary Dyskinesia (PCD, MIM #244400) is a genetically heterogenous disease characterized by ultrastructural and/or functional defects of respiratory cilia. Typical symptoms include recurrent upper and lower airway infections and lung damage (bronchiectasis) caused by an impaired mucociliary clearance.
Usually, ultrastructural defects of cilia underlie PCD and can be detected by transmission electron microscopy (TEM). However, about 30% of PCD cases have a normal ultrastructure by TEM.
Mutations in the ciliary dynein DNAH11 cause PCD with normal ultrastructure; the DNAH11 protein, which is located in the outer dynein arms (ODAs) of respiratory cilia, is only present in the proximal region of cilia in healthy patients. We have shown that DNAH11 mutations can be verified by immunofluorescence microscopy (IFM) using anti-DNAH11 antibodies (Dougherty et al. 2016). More recently, we have shown that the ciliary dynein DNAH9, which is also a component of ODAs and a paralog of DNAH11, is located in only the distal region of respiratory cilia in healthy patients (Loges et al, 2018). Both studies noted that DNAH9 is abnormal in DNAH11-mutant cilia, suggesting that DNAH9 IFM may be helpful to corroborate DNAH11 mutations.
In this work, we examined respiratory cilia from a larger sample size (>30 individuals confirmed with DNAH11 mutations) to establish a correlation, if any, between DNAH11 and DNAH9 immunoreactivity by IFM. To date, our data shows that DNAH9 is abnormal in several genetically distinct DNAH11- mutant cilia, providing a potential resource to validate PCD-causing DNAH11 mutations by IFM, which is a critical tool to support a genetic diagnosis.
PSIII-BR03: “Modifications of the spermatogonial stem cell pool and it's niche in infertile men”, Lea Mundt
PD Dr. Nina Neuhaus, Centre of reproductive medicine and andrology
Infertility affects approximately 10-15% of couples worldwide. 7% of men attending the CeRA for couple infertility are diagnosed with cryptozoospermia, a severe form of oligoasthenoteratozoospermia with a concentration of < 0.1 million sperm/ml ejaculate.
Different spermatogonial subpopulations constitute the beginning of the developmental process of spermatogenesis, and the extent of alterations in the spermatogonial compartment in cryptozoospermic patients still needs to be further examined.
Recently, by means of single-cell RNA sequencing analyses, 4 undifferentiated spermatogonial subpopulations were discovered, identifiable by the expression of specific marker genes. The cryptozoospermic patients showed an increased number of the most undifferentiated spermatogonia (PIWIL4+), and a decrease of cells only from the first meiotic division onwards.
To uncover possible reasons for these alterations in cryptozoospermic patients, this project focusses on two aims. Firstly, we will evaluate the proliferative activity of spermatogonia which may cause the enlargement of the PIWIL4+ stem cells in patients with severely impaired spermatogenesis. Secondly, we will assess whether alterations in the proliferative rate are associated with the location of the stem cells in relation to blood vessels.
By means of co‐immunofluorescence stainings for the proliferation marker Ki67 with markers for the different spermatogonial subpopulations (PIWIL4, GFRA1, FGFR3, KIT) the proliferative rate will be analysed in cryptozoospermic tissues and controls with normal spermatogenesis (n=6 each).
To address the second aim we will evaluate the location of spermatogonia in relation to blood vessels (marker SMA).
All marker stainings are established and quantifications regarding the proliferative rate of the spermatogonial subpopulations are currently ongoing.
PSIII-BR04: “Characterization of inducible neuronal cell culture with mutated CRLF1“
Lea Lange
Prof. Dr. med. Frank Rutsch,
Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
Crisponi Syndrome/ cold-inducing sweating syndrome (CS/CISS) is an autosomal recessive disorder.
The main symptoms are crisis of hyperthermia in the neonatal period and paradoxical sweating as an adult. Many patients die during their first year of live, because of hyperthermia. Extended levels of noradrenalin were measured in most of the patients. Noradrenaline is one of the main interactors when it comes to thermoregulation. Most of the Patients have swallowing, feeding and breathing problems, facial characteristics like large face, flat nose, contraction of the facial muscular, camptodactyly and scoliosis. Mutations in the cytokine-receptor like factor 1 (CRLF1) are the mainly reason for CS/CISS. So far, 40 different mutations of CRLF1 in 96 patients are known. CRLF1 builds a dimer with cardiotrophin-like cytokine factor 1 (CLCF1). This dimer acts as a ligand for the ciliary neurotrophic factor receptor (CNTFR). CNTFR is important for neuronal development. Although the genetics are known, the pathomechanism of CS/CISS remains unclear. The effects mutated CRLF1 has on thermoregulation need to be further researched. We established a cell culture model for this question. Stem cells, with a mutation in CRLF1, were cultured and induced to differentiate into sympathetic neurons. The differentiation takes place via stimulation with various factors. For example, the SMAD inhibition leads to the loss of pluripotency of the stem cells. Further experiments with the induced sympathetic neurons and noradrenalin interactions are planned.
