Interaktionen von Lymphendothelzellen und mesenchymalen Stammzellen in vitro

The formation of distant metastasis is the main cause of morbidity and mortality for patients with cancer. Cancer cells can disseminate from the primary site via hematogenous and lymphatic routes, which is the main obstacle for the development of effective cancer therapies. Although surgical and radio therapy are able to control many primary tumors, the occurrence of metastasis still means a bad prognosis for the patients. A better understanding of the underlying mechanisms of metastasis could help to develop more effective therapies. In the future, an approach for prevention or reduction of lymphatic metastasis could aim the lymphatic vessels themselves. However, little is known so far about development, function and molecular mechanisms of lymphangiogenesis. Therefore the aim of the present study was the evaluation of the in vitro interactions of human mesenchymal stem cells and lymphatic endothelial cells, which is the basis for ongoing experiments to understand the formation of lymphatic vessels in pathological diseases as well as the lymphatic metastasis more detailed. MSC are an interesting candidate for studying these processes, because there is already evidence from blood- angiogenesis that MSC and blood endothelial cells interact with each other and thus contribute to neoangiogenesis. In the first part of the study, the interactions of MSC and LEC by cytokines or cell-cell contacts were analyzed by in vitro angiogenesis assays. Processes of lymphangiogenesis like proliferation, migration, transmigration and sprouting were examined by adding the known pro(lymph)angiogenic growth factors VEGF-C, bFGF, HGF or MSC conditioned medium to the cells or by combination of the two cell types. Furthermore, the contribution of MSC to the formation of new vessels by forming tubular structures (tube formation) in vitro was analyzed. In the second part of the study the results of the growth factor experiments were validated by adding specific neutralizing antibodies to the single growth factors or the MSC CM. However, their influence on lymphatic endothelial cells is largely unexplored. Lymphangiogenic processes like proliferation, migration, transmigration and tube formation could be potently enhanced both by the single growth factors VEGF-C, bFGF, HGF and MSC secreted factors. This stimulative effect was confirmed by addition of specific neutralizing antibodies inhibiting the lymphangiogenic response. It became clear that the examined factors are involved differently in the individual processes of lymphangiogenesis. Stimulation of LEC with the single growth factors in basal medium in combination with the corresponding antibodies elucidated that VEGF-C as well as bFGF and HGF directly act on lymphangiogenesis, because the lymphatic response of LEC was completely silenced afterwards. Addition of the antibodies to MSC CM showed that only VEGF-C and its receptor VEGFR-3 are essential for all of the examined processes (proliferation, migration, transmigration, tube formation). There is also an essential role of bFGF and HGF for proliferation and tube formation but not for the migratory behavior of LEC. To clarify the exact roles of the different growth factors further experiments, also with alternative factors, as well as a detailed analysis of the composition of the MSC CM are necessary. In contrast to the processes of proliferation, migration and tube formation the present results have shown that stimulation of LEC sprouting was not as much as expected both in fibrin and collagen gels. This study presents for the first time the positive influence of conditioned medium of primary human MSC on the lymphangiogenic response of primary human LEC. The results from the in vitro experiments are the basis for the following in vivo establishment of a lymphatic vessel network. Understanding the mechanisms of lymphangiogenesis and the role of growth factors involved could help to get deeper insights into the mechanisms of lymphangiogenesis in pathological processes as well as lymphatic metastasis. Stimulation with growth factors and modification by the use of transfected LEC serve to further investigate manipulation of the lymphangiogenic cascade with the aim to validate novel therapeutic strategies.Neben der Metastasierung über das Blutgefäßsystem metastasieren maligne Tumore in andere Organe über die Lymphgefäße. Dies wird als die Haupttodesursache bei Krebspatienten und als größtes Hindernis bei der Entwicklung effektiver Krebstherapien angesehen. Obwohl die chirurgische Therapie und Radiotherapie heute in der Lage ist, viele Primärtumoren zu kontrollieren, bedeutet das Auftreten von Metastasen immer noch eine schlechte Prognose. Ein besseres Verständnis über die Mechanismen der Metastasierung könnte helfen, effektivere Therapien zu entwickeln. Ein Ansatz zur Verhinderung oder Reduktion der lymphogenen Metastasierung könnte zukünftig auf die Lymphgefäße selbst zielen. Allerdings ist bisher nur wenig über die Entwicklung, Funktion und die molekularen Mechanismen der Lymphangiogenese bekannt. Ziel der vorliegenden Arbeit war es, die Interaktion von humanen mesenchymalen Stammzellen und Lymphgefäßendothelzellen näher zu untersuchen, um später die Neubildung von Lymphgefäßen in pathologischen Vorgängen sowie die lymphogene Metastasierung besser verstehen zu können. MSC stellen dabei einen interessanten Kandidaten für die Untersuchung dieser Prozesse dar, da es bereits Hinweise aus der Blutangiogenese gibt, dass MSC und Blutendothelzellen miteinander interagieren und so zur Neoangiogenese beitragen. Ihr Einfluss auf Lymphendothelzellen ist jedoch weitestgehend unerforscht. Es wurden zunächst die Interaktionen von MSC und Lymphgefäßendothelzellen über Zytokine oder Zell-Zellkontakte mittels klassischer in vitro Angiogeneseversuche analysiert. Durch Zugabe der bekannten pro-(lymph)angiogenen Wachstumsfaktoren VEGF-C, bFGF und HGF bzw. MSC konditioniertem Medium oder dem Zusammenführen der beiden Zellpopulationen wurden Komponenten der Lymphangiogenese wie Proliferation, Migration, Transmigration und Sprossung untersucht. Des Weiteren wurde die Beteiligung bzw. der Einbau der MSC am Gefäßaufbau durch das Bilden röhrenförmiger Strukturen in vitro genauer betrachtet. In einem weiteren Versuchsabschnitt wurden die Ergebnisse der Wachstumsfaktorversuche mit spezifischen, neutralisierenden Antikörpern gegen die Wachstumsfaktoren überprüft. Mit Hilfe der durchgeführten Wachstumsfaktor- und Antikörperversuche konnte gezeigt werden, dass sowohl die einzeln zugegebenen Wachstumsfaktoren VEGF-C, bFGF und HGF als auch die durch MSC sekretierten Faktoren im MSC CM eine stimulierende Wirkung auf die Prozesse der Proliferation, Migration, Transmigration und Bildung röhrenförmiger Strukturen haben. Dies konnte zudem durch die Zugabe spezifischer, neutralisierender Antikörper bestätigt werden, da es hier zu einer Hemmung der stimulierenden Wirkung kam. Dabei wurde deutlich, dass die untersuchten Faktoren unterschiedlich an den einzelnen Prozessen der Lymphangiogenese beteiligt sind. So konnte zunächst nach Zugabe der einzelnen Faktoren festgestellt werden, dass VEGF-C, bFGF und HGF direkt auf die Lymphangiogenese wirken, da eine Neutralisierung durch die entsprechenden Antikörper eine lymphangiogene Antwort der LEC unterdrückte. Die Ergebnisse nach Zugabe der Antikörper zum MSC CM zeigten jedoch, dass nur VEGF-C und sein Rezeptor VEGFR-3 essentiell für alle untersuchten Prozesse (Proliferation, Migration, Transmigration, Bildung röhrenförmiger Strukturen) sind, da deren Wirkung nicht durch andere, im MSC CM enthaltene Faktoren kompensiert werden konnte. Für bFGF und HGF konnte ebenfalls eine essentielle Rolle für die Proliferation und Bildung röhrenförmiger Strukturen durch die LEC nachgewiesen werden, nicht jedoch für das migratorische Verhalten der Zellen. Um den Einfluss dieser Faktoren genauer aufzuklären, sind weitere Versuche, unter anderem auch mit alternativen pro-lymphangiogenen Faktoren, notwendig. Hierfür ist eine detailierte Analyse der Zusammensetzung des MSC CM unumgänglich. Im Gegensatz zu den Prozessen der Proliferation, Migration und der Bildung röhrenförmiger Strukturen, welche durch MSC sekretierte Faktoren deutlich stimuliert werden konnten, war ein derartiger Effekt für die Sprossung der LEC Sphäroide sowohl nach Einbettung in Kollagen- als auch Fibringele nicht zu beobachten. Mit der vorliegenden Arbeit konnte erstmals eine pro-lymphangiogene Wirkung von humanen MSC auf humane LEC nachgewiesen werden. Dies schafft die Grundlage für die Etablierung eines Lymphgefäßnetzwerkes in vivo, mit Hilfe dessen die Neubildung von Lymphgefäßen in pathologischen Vorgängen wie z. B. Tumorleiden sowie die lymphangiogene Metastasierung genauer untersucht werden kann. Diese Erkenntnisse können dann für eine möglicherweise therapeutische Hemmung der Lymphgefäßnetzwerkbildung und somit vermindertes Tumorwachstum und Metastasierung genutzt werden

Direct Substrate Delivery into Mitochondrial-Fission Deficient Pancreatic Islets Rescues Insulin Secretion

In pancreatic beta cells, mitochondrial bioenergetics control glucose-stimulated insulin secretion (GSIS). Mitochondrial dynamics are generally associated with quality control, maintaining the functionality of bioenergetics. By acute pharmacological inhibition of mitochondrial fission protein Drp1, we here demonstrate that mitochondrial fission is necessary for GSIS in mouse and human islets. We confirm that genetic silencing of Drp1 increases mitochondrial proton leak in MIN6 cells. However, our comprehensive analysis of pancreatic islet bioenergetics reveals that Drp1 does not control insulin secretion via its effect on proton leak but instead via modulation of glucose-fuelled respiration. Notably, pyruvate fully rescues the impaired insulin secretion of fission-deficient beta cells, demonstrating that defective mitochondrial dynamics solely impact substrate supply upstream of oxidative phosphorylation. The present findings provide novel insights in how mitochondrial dysfunction may cause pancreatic beta cell failure. In addition, the results will stimulate new thinking in the intersecting fields of mitochondrial dynamics and bioenergetics, as treatment of defective dynamics in mitochondrial diseases appears to be possible by improving metabolism upstream of mitochondria

Is Ankyrin a genetic risk factor for psychiatric phenotypes?

Background Genome wide association studies reported two single nucleotide polymorphisms in ANK3 (rs9804190 and rs10994336) as independent genetic risk factors for bipolar disorder. Another SNP in ANK3 (rs10761482) was associated with schizophrenia in a large European sample. Within the debate on common susceptibility genes for schizophrenia and bipolar disorder, we tried to investigate common findings by analyzing association of ANK3 with schizophrenia, bipolar disorder and unipolar depression. Methods We genotyped three single nucleotide polymorphisms (SNPs) in ANK3 (rs9804190, rs10994336, and rs10761482) in a case-control sample of German descent including 920 patients with schizophrenia, 400 with bipolar affective disorder, 220 patients with unipolar depression according to ICD 10 and 480 healthy controls. Sample was further differentiated according to Leonhard's classification featuring disease entities with specific combination of bipolar and psychotic syndromes. Results We found no association of rs9804190 and rs10994336 with bipolar disorder, unipolar depression or schizophrenia. In contrast to previous findings rs10761482 was associated with bipolar disorder (p = 0.015) but not with schizophrenia or unipolar depression. We observed no association with disease entities according to Leonhard's classification. Conclusion Our results support a specific genetic contribution of ANK3 to bipolar disorder though we failed to replicate findings for schizophrenia. We cannot confirm ANK3 as a common risk factor for different diseases

Clozapine, neutropenia and Covid-19: should clinicians be concerned? 3 months report

© 2021 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Background: Clozapine is among the most effective antipsychotics used for treatment resistant schizophrenia. Adverse reactions to clozapine include neutropenia. In March 2020, at the start of the Coronavirus -19 pandemic, clinicians raised concerns regarding continuation of antipsychotic treatment, and specifically of clozapine, in patients with coronavirus disease. We aimed here at providing a short report focusing on the association between neutropenia and clozapine in a case series of psychiatric inpatients diagnosed with COVID-19.Patients & methods: We retrospectively inspected data of 10 patients on clozapine, admitted to Highgate Mental Health Centre, Camden & Islington NHS Foundation Trust, between March and July 2020; selection was based on their COVID-19 positive PCR test. We used a linear regression model to estimate whether there was a significant drop in the neutrophil count during SARS-CoV-2 infection.The analysis was done in R using a linear regression to the origin.Results: Data were collected on 10 patients, of which 7 were males. During COVID-19 infection, neutrophils' count (ANC) was 4.13 ​× ​10 9/l (SD ​= ​2.70) which constituted a significant drop from a baseline value of 5.2 ​× ​10 9/l (SD ​= ​2.24). The mean relative reduction in ANC was -0.2729 (SD ​= ​0.1666). The beta value of 0.8377 obtained with the linear regression showed that ANC values during SARS-CoV-2 infection were 83.77% of the baseline ANC showing that within the two time points there was a decrease of 16.23%. The linear regression had a pvalue ​= ​8.96 ​× ​10 -8 and an adjusted R 2 of 95.94% which shows that the variability of the data is very well explained by the model. We also compared baseline ANC with ANC values approximately a month after resolution of the infection and results indicate that ANC values return to a 95% of baseline. Conclusions: Clinicians should bear in mind that a significant drop in neutrophils' count may occur in patients taking clozapine and affected from a SARS-CoV-2 infectionand that this drop is only transitory.Peer reviewe

ZDHHC8 as a candidate gene for schizophrenia: Analysis of a putative functional intronic marker in case-control and family-based association studies

BACKGROUND: The chromosome 22q11 region is proposed as a major candidate locus for susceptibility genes to schizophrenia. Recently, the gene ZDHHC8 encoding a putative palmitoyltransferase at 22q11 was proposed to increase liability to schizophrenia based on both animal models and human association studies by significant over-transmission of allele rs175174A in female, but not male subjects with schizophrenia. METHODS: Given the genetic complexity of schizophrenia and the potential genetic heterogeneity in different populations, we examined rs175174 in 204 German proband-parent triads and in an independent case-control study (schizophrenic cases: n = 433; controls: n = 186). RESULTS: In the triads heterozygous parents transmitted allele G preferentially to females, and allele A to males (heterogeneity χ(2 )= 4.43; p = 0.035). The case-control sample provided no further evidence for overall or gender-specific effects regarding allele and genotype frequency distributions. CONCLUSION: The findings on rs175174 at ZDHHC8 are still far from being conclusive, but evidence for sexual dimorphism is moderate, and our data do not support a significant genetic contribution of rs175174 to the aetiopathogenesis of schizophrenia

Wernicke-Kleist-Leonhard phenotypes of endogenous psychoses: a review of their validity .

While the ICD-DSM paradigm has been a major advance in clinical psychiatry, its usefulness for biological psychiatry is debated. By defining consensus-based disorders rather than empirically driven phenotypes, consensus classifications were not an implementation of the biomedical paradigm. In the field of endogenous psychoses, the Wernicke-Kleist-Leonhard (WKL) pathway has optimized the descriptions of 35 major phenotypes using common medical heuristics on lifelong diachronic observations. Regarding their construct validity, WKL phenotypes have good reliability and predictive and face validity. WKL phenotypes come with remarkable evidence for differential validity on age of onset, familiality, pregnancy complications, precipitating factors, and treatment response. Most impressive is the replicated separation of high- and low-familiality phenotypes. Created in the purest tradition of the biomedical paradigm, the WKL phenotypes deserve to be contrasted as credible alternatives with other approaches currently under discussion.

Is FKBP5 a genetic marker of affective psychosis? A case control study and analysis of disease related traits

BACKGROUND: A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been proposed as an important pathogenic factor in depression. Genetic variants of FKBP5, a protein of the HPA system modulating the glucocorticoid receptor, have been reported to be genetically associated with improved response to medical treatment and an increase of depressive episodes. METHODS: We examined three single nucleotide polymorphisms (SNPs) in FKBP5, rs4713916 in the proposed promoter region, rs1360780 in the second intron and rs3800373 in the 3'-untranslated region (3'-UTR), in a case-control study of Caucasian origin (affective psychosis: n = 248; controls: n = 188) for genetic association and association with disease related traits. RESULTS: Allele and genotype frequencies of rs4713916, rs1360780 and rs3800373 were not significantly different between cases and controls. Two three-locus haplotypes, G-C-T and A-T-G, accounted for 86.2% in controls. Odds ratios were not increased between cases and controls, except the rare haplotype G-C-G (OR 6.81), representing 2.1% of cases and 0.3% of controls. The frequency of rs4713916AG in patients deviated from expected Hardy-Weinberg equilibrium, the genotype AA at rs4713916 in monopolar depression (P = 0.011), and the two-locus haplotype rs1360780T – rs3800373T in the total sample (overall P = 0.045) were nominally associated with longer continuance of disease. CONCLUSION: Our data do not support a significant genetic contribution of FKBP5 polymorphisms and haplotypes to affective psychosis, and the findings are inconclusive regarding their contribution to disease-related traits

Dual-specificity MAP kinase phosphatases in health and disease

Source at https://doi.org/10.1016/j.bbamcr.2018.09.002.It is well established that a family of dual-specificity MAP kinase phosphatases (MKPs) play key roles in the regulated dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues. MKPs provide a mechanism of spatiotemporal feedback control of these key signalling pathways, but can also mediate crosstalk between distinct MAP kinase cascades and facilitate interactions between MAP kinase pathways and other key signalling modules. As our knowledge of the regulation, substrate specificity and catalytic mechanisms of MKPs has matured, more recent work using genetic models has revealed key physiological functions for MKPs and also uncovered potentially important roles in regulating the pathophysiological outcome of signalling with relevance to human diseases. These include cancer, diabetes, inflammatory and neurodegenerative disorders. It is hoped that this understanding will reveal novel therapeutic targets and biomarkers for disease, thus contributing to more effective diagnosis and treatment for these debilitating and often fatal conditions