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Lymphoid aggregates in the CNS of progressive multiple sclerosis patients lack regulatory T cells
(2020)
In gray matter pathology of multiple sclerosis, neurodegeneration associates with a high degree of meningeal inflammatory activity. Importantly, ectopic lymphoid follicles (eLFs) were identified at the inflamed meninges of patients with progressive multiple sclerosis. Besides T lymphocytes, they comprise B cells and might elicit germinal center (GC)-like reactions. GC reactions are controlled by FOXP3+ T-follicular regulatory cells (TFR), but it is unknown if they participate in autoantibody production in eLFs. Receiving human post-mortem material, gathered from autopsies of progressive multiple sclerosis patients, indeed, distinct inflammatory infiltrates enriched with B cells could be detected in perivascular areas and deep sulci. CD35+ cells, parafollicular CD138+ plasma cells, and abundant expression of the homing receptor for GCs, CXCR5, on lymphocytes defined some of them as eLFs. However, they resembled GCs only in varying extent, as T cells did not express PD-1, only few cells were positive for the key transcriptional regulator BCL-6 and ongoing proliferation, whereas a substantial number of T cells expressed high NFATc1 like GC-follicular T cells. Then again, predominant cytoplasmic NFATc1 and an enrichment with CD3+CD27+ memory and CD4+CD69+ tissue-resident cells implied a chronic state, very much in line with PD-1 and BCL-6 downregulation. Intriguingly, FOXP3+ cells were almost absent in the whole brain sections and CD3+FOXP3+ TFRs were never found in the lymphoid aggregates. This also points to less controlled humoral immune responses in those lymphoid aggregates possibly enabling the occurrence of CNS-specific autoantibodies in multiple sclerosis patients.
Intrakranielle Blutungen sind im Säuglingsalter seltene, aber lebensbedrohende Ereignisse. Neben Gefäßmissbildungen, Stoffwechseldefekten sowie Störungen der Blutgerinnung kommen v. a. nichtakzidentielle Traumata, Schütteltrauma in Betracht. Die klinische Diagnostik umfasst hinsichtlich der Blutungsgenese neben Sonographie und MRT als apparatives Verfahren auch eine Fundoskopie sowie laborchemische Analysen, insbesondere der Gerinnungsparameter. Für die Blutgerinnung ist das fettlösliche Vitamin K essenziell: Frühe, klassische und späte Vitamin-K-Mangel-Blutungen werden dabei unterschieden. Um ein gehäuftes Wiederauftreten von Vitamin-K-Mangel-Blutungen bei Neugeborenen und jungen Säuglingen zu verhindern, bedarf es einer hinreichenden Aufklärung der Eltern. Eine Verweigerung der Prophylaxe scheint Folge einer weltanschaulich begründeten Ablehnung der Schulmedizin und ein zunehmendes Phänomen in wohlhabenden Industrieländern zu sein.
The aim of the present study was a refined analysis of neuroinflammation including TMEM119 as a useful microglia-specific marker in forensic assessments of traumatic causes of death, e.g., traumatic brain injury (TBI). Human brain tissue samples were obtained from autopsies and divided into cases with lethal TBI (n = 25) and subdivided into three groups according to their trauma survival time and compared with an age-, gender-, and postmortem interval-matched cohort of sudden cardiovascular fatalities as controls (n = 23). Brain tissue samples next to cortex contusions and surrounding white matter as well as samples of the ipsilateral uninjured brain stem and cerebellum were collected and stained immunohistochemically with antibodies against TMEM119, CD206, and CCR2. We could document the highest number of TMEM119-positive cells in acute TBI death with highly significant differences to the control numbers. CCR2-positive monocytes showed a significantly higher cell count in the cortex samples of TBI cases than in the controls with an increasing number of immunopositive cells over time. The number of CD206-positive M2 microglial cells increased survival time-dependent. After 3 days of survival, the cell number increased significantly in all four regions investigated compared with controls. In sum, we validate a specific and robustly expressed as well as fast reacting microglia marker, TMEM119, which distinguishes microglia from resident and infiltrating macrophages and thus offers a great potential for the estimation of the minimum survival time after TBI.
Impact of the Chemokine Receptors CXCR4 and CXCR7 on Clinical Outcome in Adrenocortical Carcinoma
(2020)
Chemokine receptors have a negative impact on tumor progression in several human cancers and have therefore been of interest for molecular imaging and targeted therapy. However, their clinical and prognostic significance in adrenocortical carcinoma (ACC) is unknown. The aim of this study was to evaluate the chemokine receptor profile in ACC and to analyse its association with clinicopathological characteristics and clinical outcome. A chemokine receptor profile was initially evaluated by quantitative PCR in 4 normal adrenals, 18 ACC samples and human ACC cell line NCI-H295. High expression of CXCR4 and CXCR7 in both healthy and malignant adrenal tissue and ACC cells was confirmed. In the next step, we analyzed the expression and cellular localization of CXCR4 and CXCR7 in ACC by immunohistochemistry in 187 and 84 samples, respectively. These results were correlated with clinicopathological parameters and survival outcome. We detected strong membrane expression of CXCR4 and CXCR7 in 50% of ACC samples. Strong cytoplasmic CXCR4 staining was more frequent among samples derived from metastases compared to primaries (p=0.01) and local recurrences (p=0.04). CXCR4 membrane staining positively correlated with proliferation index Ki67 (r=0.17, p=0.028). CXCR7 membrane staining negatively correlated with Ki67 (r=−0.254, p=0.03) but positively with tumor size (r=0.3, p=0.02). No differences in progression-free or overall survival were observed between patients with strong and weak staining intensities for CXCR4 or CXCR7. Taken together, high expression of CXCR4 and CXCR7 in both local tumors and metastases suggests that some ACC patients might benefit from CXCR4/CXCR7-targeted therapy.
Despite its significant overexpression in several malignant neoplasms, the expression of RPS27 in the central nervous system (CNS) is widely unknown. We identified the cell types expressing RPS27 in the CNS under normal and disease conditions. We acquired specimens of healthy brain (NB), adult pilocytic astrocytoma (PA) World Health Organization (WHO) grade I, anaplastic PA WHO grade III, gliomas WHO grade II/III with or without isocitrate dehydrogenase (IDH) mutation, and glioblastoma multiforme (GBM). RPS27 protein expression was examined by immunohistochemistry and double-fluorescence staining and its mRNA expression quantified by RT-PCR. Patients’ clinical and tumor characteristics were collected retrospectively. RPS27 protein was specifically expressed in tumor cells and neurons, but not in healthy astrocytes. In tumor tissue, most macrophages were positive, while this was rarely the case in inflamed tissue. Compared to NB, RPS27 mRNA was in mean 6.2- and 8.8-fold enhanced in gliomas WHO grade II/III with (p < 0.01) and without IDH mutation (p = 0.01), respectively. GBM displayed a 4.6-fold increased mean expression (p = 0.02). Although RPS27 expression levels did not affect the patients’ survival, their association with tumor cells and tumor-associated macrophages provides a rationale for a future investigation of a potential function during gliomagenesis and tumor immune response.
Multiple myeloma (MM) is a disease of terminally differentiated B-cells which accumulate in the bone marrow leading to bone lesions, hematopoietic insufficiency and hypercalcemia. Genetically, MM is characterized by a great heterogeneity. A recent next-generation sequencing approach resulted in the identification of a signaling network with an accumulation of mutations in receptor-tyrosine kinases (RTKs), adhesion molecules and downstream effectors. A deep-sequencing amplicon approach of the coding DNA sequence of the six RTKs EPHA2, EGFR, ERBB3, IGF1R, NTRK1 and NTRK2 was conducted in a patient cohort (75 MM samples and 68 corresponding normal samples) of the “Deutsche Studiengruppe Multiples Myelom (DSMM)” to further elucidate the role of RTKs in MM. As an initial approach the detected mutations were correlated with cytogenetic abnormalities and clinical data in the course of this thesis. RTK mutations were present in 13% of MM patients of the DSMM XI trial and accumulated in the ligand-binding and tyrosine-kinase domain. The newly identified mutations were associated with an adverse patient survival, but not with any cytogenetic abnormality common in MM. Especially rare patient-specific SNPs (single nucleotide polymorphism) had a negative impact on patient survival. For a more comprehensive understanding of the role of rare RTK SNPs in MM, a second amplicon sequencing approach was performed in a patient cohort of the DSMM XII trial that included 75 tumor and 184 normal samples. This approach identified a total of 23 different mutations in the six RTKs EPHA2, EGFR, ERBB3, IGF1R, NTRK1 and NTRK2 affecting 24 patients. These mutations could furthermore be divided into 20 rare SNPs and 3 SNVs (single nucleotide variant). In contrast to the first study, the rare SNPs were significantly associated with the adverse prognostic factor del17p.
IGF1R was among the most commonly mutated RTKs in the first amplicon sequencing approach and is known to play an important role in diverse cellular processes such as cell proliferation and survival. To study the role of IGF1R mutations in the hard-to-transfect MM cells, stable IGF1R-knockdown MM cell lines were established. One of the knockdown cell lines (L363-C/C9) as well as a IGF1R-WT MM cell line (AMO1) were subsequently used for the stable overexpression of WT IGF1R and mutant IGF1R (N1129S, D1146N). Overall, an impact on the MAPK and PI3K/AKT signaling pathways was observed upon the IGF1R knockdown as well as upon WT and mutant IGF1R overexpression. The resulting signaling pattern, however, differed between different MM cell lines used in this thesis as well as in a parallel performed master thesis which further demonstrates the great heterogeneity described in MM.
Taken together, the conducted sequencing and functional studies illustrate the importance of RTKs and especially of IGF1R and its mutants in the pathogenesis of MM. Moreover, the results support the potential role of IGF1R as a therapeutic target for a subset of MM patients with mutated IGF1R and/or IGF1R overexpression.
Inhibition of the protein kinase MPS1, a mitotic spindle-checkpoint regulator, reinforces the effects of multiple therapies against glioblastoma multiforme (GBM) in experimental settings. We analyzed MPS1 mRNA-expression in gliomas WHO grade II, III and in clinical subgroups of GBM. Data were obtained by qPCR analysis of tumor and healthy brain specimens and correlated with the patients’ clinical data. MPS1 was overexpressed in all gliomas on an mRNA level (ANOVA, p < 0.01) and correlated with tumor aggressiveness. We explain previously published conflicting results on survival: high MPS1 was associated with poorer long term survival when all gliomas were analyzed combined in one group (Cox regression: t < 24 months, p = 0.009, Hazard ratio: 8.0, 95% CI: 1.7–38.4), with poorer survival solely in low-grade gliomas (LogRank: p = 0.02, Cox regression: p = 0.06, Hazard-Ratio: 8.0, 95% CI: 0.9–66.7), but not in GBM (LogRank: p > 0.05). This might be due to their lower tumor volume at the therapy start. GBM patients with high MPS1 mRNA-expression developed clinical symptoms at an earlier stage. This, however, did not benefit their overall survival, most likely due to the more aggressive tumor growth. Since MPS1 mRNA-expression in gliomas was enhanced with increasing tumor aggressiveness, patients with the worst outcome might benefit best from a treatment directed against MPS1.
Downregulation of miR-221-3p expression in prostate cancer (PCa) predicted overall and cancer-specific survival of high-risk PCa patients. Apart from PCa, miR-221-3p expression levels predicted a response to tyrosine kinase inhibitors (TKI) in clear cell renal cell carcinoma (ccRCC) patients. Since this role of miR-221-3p was explained with a specific targeting of VEGFR2, we examined whether miR-221-3p regulated VEGFR2 in PCa. First, we confirmed VEGFR2/KDR as a target gene of miR-221-3p in PCa cells by applying Luciferase reporter assays and Western blotting experiments. Although VEGFR2 was mainly downregulated in the PCa cohort of the TCGA (The Cancer Genome Atlas) database, VEGFR2 was upregulated in our high-risk PCa cohort (n = 142) and predicted clinical progression. In vitro miR-221-3p acted as an escape mechanism from TKI in PC3 cells, as displayed by proliferation and apoptosis assays. Moreover, we confirmed that Sunitinib induced an interferon-related gene signature in PC3 cells by analyzing external microarray data and by demonstrating a significant upregulation of miR-221-3p/miR-222-3p after Sunitinib exposure. Our findings bear a clinical perspective for high-risk PCa patients with low miR-221-3p levels since this could predict a favorable TKI response. Apart from this therapeutic niche, we identified a partially oncogenic function of miR-221-3p as an escape mechanism from VEGFR2 inhibition.
Acetylcholine (ACh) mediates transmission at vertebrate neuromuscular junctions and many other synapses. The postsynaptic ACh receptors at neuromuscular junctions are of the nicotinic subtype (nAChRs). They are among the best studied receptor channels and often serve as models or receptor prototypes. Despite a wealth of information on muscle type nAChRs so far little is known about species specific functional differences. In this work, mouse and human adult muscle type nAChRs are investigated.
Cell attached recordings in the HEK293T heterologous expression system provided evidence that the ACh affinity of recombinant mouse and human adult muscle type nAChRs are different. To clarify this, I compared these receptors in outside-out patches employing a system for fast agonist application. Thus, the individual membrane patches with receptors can be exposed to various ligand concentrations. In response to 10 and 30 µM ACh normalized peak currents (î) were significantly larger and current rise-time (tr) shorter in human than in mouse receptors. Analyzing dose-response curves of î and tr and fitting them with a two-step equivalent binding-site kinetic mechanism revealed a two-fold higher ACh association rate constant in human compared to mouse receptors. Furthermore, human nAChRs were blocked faster in outside-out patches by superfusion of 300 nM α-Bungarotoxin (α-Bgtx) than mouse nAChRs. Finally, human nAChRs in outside-out patches showed higher affinity at 3 µM ACh than chimeric receptors consisting of mouse α- and human β-, γ- and ε-subunits. The higher affinity of human than mouse receptors for ACh and α-Bgtx is thus at least in part due to sequence difference in their α-subunits.
Objective
Recently, we described a disintegrin and metalloproteinase 9 (ADAM9) overexpression by Schwann cells of vestibular schwannoma (VS) and suggested that it might be a marker for VS tumor growth and invasiveness. This research note provides additional data utilizing a small cohort of VS primary cultures and tissue samples. We examined whether reconstitution of Merlin expression in VS cells regulates ADAM9 protein expression and performed lentiviral ADAM9 knock down to investigate possible effects on VS cells numbers. Moreover, the co-localization of ADAM9 and Integrins α6 and α2β1, respectively, was examined by immunofluorescence double staining.
Results
ADAM9 expression was not regulated by Merlin in VS. However, ADAM9 knock down led to 58% reduction in cell numbers in VS primary cell cultures (p < 0.0001). While ADAM9 and Integrin α2β1 were co-localized in only 22% (2 of 9) of VS, ADAM9 and Integrin α6 were co-localized in 91% (10 of 11) of VS. Therefore, we provide first observations on possible regulatory functions of ADAM9 expression in VS.