Investigating a model cell line for tumour stem cells in colorectal cancer

Laut der Tumorstammzelltheorie ist nur ein Bruchteil der Zellen eines Tumors verantwortlich für Tumorbildung, -erhalt und -wachstum. Die Tumorstammzellen besitzen Eigenschaften von Stammzellen wie Langlebigkeit, Selbsterneuerung, ein hohes Differenzierungspotential, Schutz vor Apoptose und sie sind resistent gegenüber herkömmlichen Therapieansätzen. Im Darmkrebs kann man sich das so vorstellen, dass die initiale Mutation im APC-Gen, welche in den meisten Fällen beobachtet werden kann, zu einem konstitutiv aktiven Wnt Signaltransduktionsweg führt. Diese Signalkaskade ist auch in normalen adulten Stammzellen, die für den hohen Zellumsatz in der Darmschleimhaut verantwortlich sind, aktiv. Vorangegangene Studien zeigten, dass adenome Darmkrebszellen der Zellinie LT97 eine Unterpopulation besitzen, welche das Wnt- Zielprotein CD44 exprimieren und Stammzelleigenschaften zeigten, sowohl in der Genexpression (musashi, Telomerase), als auch in Kultur (erhöhte Wachstums- und Überlebensfähigkeit verglichen mit den CD44- Zellen). In der vorliegenden Diplomarbeit wurden die grundlegenden Mechanismen, die zu der verstärkten Wachstums- und Überlebensfähigkeit der CD44+ Population führen, auf RNA-Ebene mittels Standard- und RealTime PCR, und auf Protein-Ebene mittels Western blot untersucht. Das Ergebnis zeigt (1) Schutz vor Apoptose durch Expression des Wnt-Zielgens survivin in CD44+ Zellen, (2) Herunterregulierung des IGF1 Überlebenssignals durch IGFBP3 Expression in CD44- Zellen, und (3) Hochregulierung der FGFR3IIIc Splice Variante in CD44+ Zellen, welche ein FGF18 abhängiges Überlebenssignal andeuten. Analyse des downstream signalling durch Western blot zeigte reduzierte Phosphorylierung von IRS1, GSK3, ERK1/2 und S6 und verringerte Expression von IRS1, GSK3 und S6 in CD44- Zellen. Sowohl Hemmung des Wnt Signalwegs mit Sulindac als auch des IGF Signalwegs mit PPP führten zu verringertem Wachstum und Überleben von CD44+ Zellen, was die Bedeutung dieser Signalwege für die Ausbildung von Darmtumoren aufzeigt. Expression eines dominant negativen FGFR3 Konstrukts erhöhte die Effizienz des Ausplattierens, aber führte zu verringertem Wachstum in CD44+ und CD44- Zellen, während die Einführung eines FGF18 überexprimierenden Konstrukts zu erhöhtem Wachstum und Überleben in CD44+ Zellen führte. In einer vergleichenden Studie von Kandidaten für Stammzellmarker wurde eine umgekehrte Beziehung zwischen CD44 und CD133 sowie eine direkte Beziehung zwischen CD44 und CD166 sowohl in Zellinien als auch Primärkulturen von kolorektalen Tumoren entdeckt. Musashi und Lgr5 wurden hauptsächlich in Normalgewebe und kaum in Tumorgewebe gefunden. Zusammenfassend kann man sagen, dass drei für verstärktes Wachstum und Überleben von CD44+ Zellen verantwortliche und somit für therapeutische Ansätze vielversprechende Signalwege bestätigt wurden, wohingegen der Stammzell-Zustand der CD44+ LT97 Zellen nicht verifiziert werden konnte.According to the cancer stem cell theory, only a small subset of cells in a tumour is responsible for tumour initiation, maintenance and growth. The cancer stem cells (CSC) display stem cell characteristics like longevity, self-renewal, a high differentiation potential and protection from apoptosis and are resistant to conventional therapy approaches. For colon cancer this phenomenon can easily be envisioned as the initial APC-mutation observed in most colon cancers leads to a constitutively active Wnt pathway. This signalling cascade is also active in the normal adult stem cells that drive cell turn over in the colonic mucosa and are situated at the bottom of the colonic crypt. Previous studies revealed a subpopulation in LT97 colon adenoma cells positive for the Wnt target protein CD44 that also displayed stem cell characteristics in gene expression (musashi, telomerase) and behaviour (enhanced growth and survival abilities compared to CD44- cells). In the present thesis the underlying mechanisms that lead to enhanced growth and survival of the CD44+ subpopulation were investigated on RNA level by standard and RealTime PCR and on protein level by western blot. The results indicate (1) protection from apoptosis by expression of the Wnt target gene survivin in the CD44+ cells; (2) down modulation of IGF1 survival signalling by IGFBP3 expression in CD44- cells. and (3) up regulation of the FGFR3IIIc splice variant CD44+ cells indicating a FGF18 dependent survival signal. Analysis of downstream signalling by western blot revealed reduced phosphorylation of IRS1, GSK3, ERK1/2 and S6 and decreased protein expression of IRS1, GSK and S6 in CD44- cells. Both inhibition of Wnt signalling by Sulindac and IGF signalling by PPP led to decreased growth and survival of CD44+ cells, indicating the importance of these pathways for colon tumour formation. Expression of a dominant negative FGFR3 construct increased plating efficiency, but led to decreased growth of CD44+ and CD44- cells, while introduction of a FGF18 over expressing construct led to enhanced growth and survival in CD44+ cells. In a comparative study of candidate stem cell markers a reverse relationship between CD44 and CD133 as well as a direct relationship between CD44 and CD166 was found in both cell lines and primary cultures obtained from colorectal tumours. Musashi and Lgr5 were found more in normal tissue than in tumour sections. In summary, 3 signalling pathways responsible for enhanced growth and survival of CD44+ LT97 cells and promising for therapeutic targeting have been verified; however the stem cell state of CD44+ LT97 has not been appraised

IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients

Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic. publisher: Elsevier articletitle: IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients journaltitle: Journal of Autoimmunity articlelink: http://dx.doi.org/10.1016/j.jaut.2016.11.005 content_type: article copyright: © 2016 The Authors. Published by Elsevier Ltd. ispartof: Journal of Autoimmunity vol:77 pages:104-115 ispartof: location:England status: publishe

Antibodies to nodal/paranodal proteins in paediatric immune-mediated neuropathy

Altres ajuts: This work was partly supported by grants from the "Jubiläumsfonds der Österreichischen Nationalbank," project 16919 (R. Höftberger), the GBS/CIDP Foundation International (J. Wanschitz), Austrian Science Fund FWF, DOC 33-B27 (R. Höftberger, M. Winklehner) and I3334-B27 (R. Höftberger), Hertha Firnberg project number T996-B30 (I. Koneczny), the grant of the Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III (fondos FEDER) (L. Querol), personal grant of the Pla estratègic de Recerca i Innovació en Salut (PERIS), Departament de Salut, Generalitat de Catalunya (L. Querol), and the German Ministry of Education and Research (BMBF, 01 GM1908A)

A New Classification System for IgG4 Autoantibodies

IgG4 autoimmune diseases are characterized by the presence of antigen-specific autoantibodies of the IgG4 subclass and contain well-characterized diseases such as muscle-specific kinase myasthenia gravis, pemphigus, and thrombotic thrombocytopenic purpura. In recent years, several new diseases were identified, and by now 14 antigens targeted by IgG4 autoantibodies have been described. The IgG4 subclass is considered immunologically inert and functionally monovalent due to structural differences compared to other IgG subclasses. IgG4 usually arises after chronic exposure to antigen and competes with other antibody species, thus “blocking” their pathogenic effector mechanisms. Accordingly, in the context of IgG4 autoimmunity, the pathogenicity of IgG4 is associated with blocking of enzymatic activity or protein–protein interactions of the target antigen. Pathogenicity of IgG4 autoantibodies has not yet been systematically analyzed in IgG4 autoimmune diseases. Here, we establish a modified classification system based on Witebsky’s postulates to determine IgG4 pathogenicity in IgG4 autoimmune diseases, review characteristics and pathogenic mechanisms of IgG4 in these disorders, and also investigate the contribution of other antibody entities to pathophysiology by additional mechanisms. As a result, three classes of IgG4 autoimmune diseases emerge: class I where IgG4 pathogenicity is validated by the use of subclass-specific autoantibodies in animal models and/or in vitro models of pathogenicity; class II where IgG4 pathogenicity is highly suspected but lack validation by the use of subclass specific antibodies in in vitro models of pathogenicity or animal models; and class III with insufficient data or a pathogenic mechanism associated with multivalent antigen binding. Five out of the 14 IgG4 antigens were validated as class I, five as class II, and four as class III. Antibodies of other IgG subclasses or immunoglobulin classes were present in several diseases and could contribute additional pathogenic mechanisms

Editorial: Molecular Mechanisms Underlying Assembly and Maintenance of the Neuromuscular Junction

International audienceNo abstract availabl

Characterisation of antibodies from MuSK-MG patients.

<p>(A) MuSK antibody titres of 14 MuSK MG patients, 30 healthy controls and 5 AChR MG patients as determined by RIA. Cut-off for MuSK antibody positivity was the mean titre of pooled healthy controls + 3x standard deviation. (B) Example of IgG subclass profiles from 3 MuSK patients. Patient plasma was incubated with HEK293 cells expressing MuSK, or to mock-transfected HEK293 cells. A subclass-specific secondary antibody was added, followed by a fluorescent tertiary antibody, and the amount of binding (mean fluorescence intensity, mfi) was measured by flow cytometry. Δmfi is the mfi obtained with cells expressing MuSK minus the mfi with mock-transfected cells. (C) Correlation between IgG subclasses, measured by flow cytometry as for (B), and MuSK antibody titres measured by RIA of 14 MuSK patient plasmas. Statistic analysis: linear regression followed by Pearson correlation (Gaussian distribution was determined by D’Agostino and Pearson test). (D) IgG1-3 and IgG4 subclasses were purified from MuSK-MG patients 9 and 12. The purity of each of these two subclass groups was analysed by flow cytometry as for (B). The IgG4 fraction for both patients contains some contamination of IgG2, but the IgG1-3 fraction is devoid of IgG4 for both patients.</p

Patient derived Fab fragments reduce agrin-induced AChR clustering as well as purified IgG.

<p>Whole IgG was purified from plasma from seven MuSK-MG patients and digested to Fab fragments with papain. (A) Example of purified Fab fragments analyzed by non-denaturing gel electrophoresis and coomassie stain, showing starting material containing whole IgG, papain-digested IgG (dig.) and purified Fab fragments (Fab). Note the absence of whole IgG (IgG) in the lane containing purified Fab. Further bands represent the presence of light chain (Lc) or the Fc fragment (Fc). The absence of heavy chain, which would migrate as indicated (Hc), demonstrates complete digestion by the papain. (B) Binding of IgG and Fab fragments to radiolabelled MuSK plotted against quantity of proteins used. (C-E) The effect of Fab fragments on agrin-induced AChR clustering on C2C12 myotubes was tested. Myotubes were incubated overnight in the presence of agrin and patient or healthy control derived purified IgG or Fab fragments. For each patient, Fab fragments and IgG were used at final MuSK antibody concentrations: patient 2=0.81nM, patient 3=4.57nM, patient 6=1.37nM, patient 8=0.75nM, patient 9= 1.13nM, patient 10=0.21nM, patient 11=0.11nM. AChR clusters were visualised with Alexa Fluor 594-conjugated α-bungarotoxin, and 30 microscopic images were acquired and analyzed blinded. (C) Example images showing myotubes that were incubated with healthy control (HC) or patient 2 IgG or Fab fragments, scale bar= 50µm. (D, E) Pooled data from three experiments showing the effect of IgG or Fab fragments on AChR clustering for all patients tested. Statistical analysis: one way ANOVA followed by Bonferroni post test, each column was compared to pooled healthy control column. **** p≤0.0001 for (D) and (E).</p

MuSK patient IgG4 or IgG1-3 do not induce endocytosis of MuSK.

<p>Patient plasma or purified IgG1-3 or IgG4 were applied at 0.17nM final concentration of MuSK antibody to HEK293 cells expressing MuSK at the cell surface. Cells were either incubated at 4°C to prevent, or at 37°C to allow, endocytosis. Patient antibody binding was visualised by addition of a secondary fluorescent anti-human antibody at the end of the experiment. (A) An example of cells that were incubated with IgG1-3 from patient 9. Robust staining was observed after 6 hours incubation at both temperatures. Scale bar=25µm. (B) Staining was scored by two individuals as described in methods, and the scores were normalised to the score at 0 hours for each condition. A slight decrease in staining was observed for cells incubated at 37°C, and pre-fixed cells also showed this reduction. (C) As a positive control for endocytosis, IgG1-3 or IgG4 was cross-linked by addition of Alexa Fluor 568-conjugated anti-human IgG prior to incubation. Example images show cells treated with patient 9 IgG4 and IgG1-3 in the presence and absence of cross-linking anti-human IgG after 6 hours incubation at 37°C. There is a clear difference when the cross-linking secondary antibody is present. Scale bar =50µm (D) Cross-linking by the secondary antibody induced internalisation of human anti-MuSK antibodies as well as MuSK-EGFP as early as 30 minutes, as observed by confocal microscopy. An example image of a cell from a confocal z-stack with orthogonal side-views is shown. The arrow shows internalised secondary Alexa Fluor 568-conjugated anti-human IgG (red) colocalised with EGFP-tagged MuSK (green). Scale bar =5µm.</p